microbiology lab reports

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Question description

Looking for a tutor who is willing to write lab reports for the lab manual provided.

Organization of the lab reports:

The lab report documents what you actually did in the lab and is a place for you to include observations, comments and questions. Your lab notebook should be organized by experiment or exercise, and not by week. For each experiment, or connected sets of experiments, you should start a separate entry. Each entry in your lab notebook should include a 1) title, 2) an introduction, 3) a materials and methods section, 4) a results section, 5) a discussion and conclusions section. You should make sure to date (and time as well if it is important) all your entries into your lab notebook. Here is a brief description of what should be included in each section:

Title – this should be the title for the experiments from the lab manual.

Introduction - the introduction should provide some brief background and include a rationale for why the experiment or exercise is being conducted. This section might include a hypothesis if you have formulated one to test. The introduction should not be longer than half a page and it should be clear to anyone who reads it what you are planning to do and why.

Materials and methods – this is the section where you should list the materials needed to carry out the lab and the methods (protocols) you used. This information can often be taken directly from the lab manual. However, remember that the lab notebook should describe what you did, not what you were supposed to do. For example, if the protocol in the lab manual says to incubate a culture of E. coli for 20 minutes at room temperature and instead you actually incubated B. subtilis for 15 minutes at 37 C, then you need to note what you did and that it was a deviation from the protocol. This is critical because when you go to interpret your results it will be important to know exactly what you did. The materials and methods sections should be detailed enough so that someone else could repeat the experiment exactly as you carried it out and obtain similar results.

Results – this section is where you should describe your results and include all the data that you collect. In your lab notebook you can include graphs, charts, tables, drawings, pictures, descriptions or other types of data. Depending on the format of the data you can tape in data to the notebook or create images of the data and include them in your digital version of the lab notebook.

Discussion and conclusions – here is where you should interpret your data and put the results in the context of your original objectives or aims. You can discuss necessary changes or improvements to the materials and methods that would be needed in further experiments. You can address whether or not your hypothesis was confirmed, if you started with a testable hypothesis. In this section you should answer the questions that are posed in the lab manual and you can include other questions you have come up with while carrying out the experiments (and you can answer these or speculate if you’d like).

Microbiology 3004 Lab Manual Fall 2018 Table of Contents Laboratory  Schedule  -­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐  2   Photographic  Atlas  Reading  -­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐  4   Lab  Safety  Instructions  -­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐  5   Laboratory  Practice  -­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐  6   Common  Laboratory  Equipment  -­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐  7   Scientific  Method  of  Inquiry  -­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐  8   The  Spread  of  Contagious  Diseases  -­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐  10   Glo-­‐Germ  Activity  -­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐  10   Questions  -­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐  12   Use  of  the  Microscope  -­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐  13   Parts  of  the  Microscope  and  Magnification  -­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐  15   Questions:  -­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐  15   Culture  Techniques  -­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐  16   Liquid  Culture  Inoculation  -­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐  17   Questions  -­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐  18   Streak  Plate  &  Slant  -­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐  18   Questions  -­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐  19   Spread  Plate  -­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐  21   Questions  -­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐  21   Prokaryotic  survey  -­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐  22   Colony  Morphology  -­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐  23   Cell  Arrangements  -­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐  24   Prokaryotic  Survey  Table  -­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐  25   Survey  of  Prokaryotes  -­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐  26   Cytological  Techniques  -­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐  27   The  Gram-­‐Positive  Bacteria  -­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐  29   The  Gram-­‐Negative  Bacteria  -­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐  30   Common  Staining  Techniques  -­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐  31   Gram  Staining  -­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐  32   Questions  -­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐  33   Micro  Pipettor  Usage  -­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐  34   Enumerating  Microorganisms  -­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐  35   Viable  Count  -­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐  36   Hemocytometer  Direct  Count  -­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐  37   Questions  -­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐  39   Wet  Mount  Technique  -­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐  40   Urban  Microbiome  -­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐  41   Experiment  -­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐  43   Questions  -­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐  43   Part  1:  Experiment  Setup  -­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐  43   Part  2:  Bacteria  Initial  Viability  -­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐  43   Part  3:  DNA  Extraction  -­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐  43   Questions  -­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐  45   Part  4:  DNA  quantification  by  NanoDrop  -­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐  46   Part  5:  Sample  Sequencing  -­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐  46   Part  6:  PCR  amplification  -­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐  46   Part  7:  Gel  electrophoresis  -­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐  48   Part  8:  DNA  sequence  data  analysis  -­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐  48   i Cytological  Techniques:  Special  Cell  Stains  -­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐  49   Acid-­‐fast  Stain  for  Mycobacteria  -­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐  49   Endospore  Stain  -­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐  50   Questions  -­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐  50   Cytological  Techniques:  Bacteria  Motility  -­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐  51   Flagella  stain  -­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐  51   Motility  Stab  and  Oxygen  Requirement  -­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐  52   Questions  -­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐  52   Eukaryotic  Survey  -­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐  53   Question  -­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐  53   Fungi  -­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐  54   Zygomycota  -­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐  54   Ascomycota  -­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐  55   Basidiomycota  -­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐  55   Chytridiomycota  -­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐  56   Deuteromycotes  (abstract  group  not  used  for  taxonomic  identities)  -­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐  56   Protozoa  -­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐  58   Sarcodina  -­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐  58   Actinopodia  -­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐  59   Ciliophora  -­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐  59   Mastigophora  -­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐  59   Apicomplexa  -­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐  60   Euglena  -­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐  60   Streptomyces  and  Antibiosis  -­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐  61   Questions  -­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐  62   Antibiotic  Sensitivity  Testing  -­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐  63   Questions  -­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐  64   Zones  of  Clearing  for  Various  Antibiotics  -­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐  65   Bacterial  Growth  Curve  -­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐  66   Questions  -­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐  69   Optimum  Growth  Temperature  -­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐  70   Questions  -­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐  71   Fermentative  Patterns  of  Gram-­‐Negative  Bacteria  -­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐  72   Questions  -­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐  74   Further  Information:  Biochemical  Tests  -­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐  75   Carbohydrate  Fermentation  and  Gas  Production  -­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐  75   Glucose  Degradation  -­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐  78   Motility,  H2S  Production  and  Indole  Production  -­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐  80   Nitrate  Reduction  -­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐  81   Starch  Utilization/Hydrolysis  -­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐  82   Citrate  Utilization  Test  -­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐  83   Nitrogen-­‐Fixing  Bacteria  -­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐  85   Questions  -­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐  87   Enzyme  Induction  in  E.  coli  -­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐  88   Questions  -­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐  91   Human  Microbiome  -­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐  92   Questions  -­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐  95   Microbes  and  Food  Science  -­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐  96   Lactic  Acid  Bacteria  -­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐  96   Questions  -­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐  97   Assessing  the  -­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐  98   Prevalence  of  Antibiotic-­‐Resistance  in  the  Environment  -­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐  98   Photosynthetic  Bacteria  and  Algae  -­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐  103   ii Questions  -­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐  104   Further  Information  -­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐  105   Cyanobacteria  -­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐  105   Algae  -­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐  107   Bacteriophage:  T3  -­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐  108   Questions  -­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐  110   Water  Quality  Analysis  -­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐  111   Questions  -­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐  113   APPENDICES  -­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐  114   List  of  Reagents  -­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐  115   Biochemical  Table  -­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐  116   Glossary  of  Important  Terms  -­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐  117   iii Laboratory Schedule Lab Session Week 1 Week 2 Week 3 Week 4 Week 5 Week 6 Week 7 Exercises for that Session Lab Safety The Spread of Contagious Diseases: Glo-Germ Activity Routine Techniques Used in Microbiology (aseptic technique, broth culture, streak plate, spread plate) Use of the Microscope Cytological Techniques: Gram Staining Discuss Scientific Method of Inquiry Continued Lesson: Gram Staining Streak for Colony Isolation & Spread Plating (practice) New Lessons: Enumerating Microorganisms and serial dilutions – Part A and B Urban Microbiome – Introduction Continued Lessons: Enumerating Microorganisms – Part C Practice cell enumeration Urban Microbiome – Step 1 (Optional collection – outside of class) New Lessons: Micropipettor Usage Cytological Techniques: Special Cell Stains – Endospore Stain and Acid Fast Stain Wet Mount Technique (optional) QUIZ #1 Continued Lessons: Urban Microbiome – Step 1- 3 New Lessons: Survey of Eukaryotic Microbes Continued Lessons: Urban Microbiome – Step 4 and 5 Survey of Eukaryotic Microbes New Lessons: Cytological Techniques: Bacteria Motility – Flagella stain and Motility stab Streptomyces and Antibiosis – Part A Continued Lessons: Streptomyces and Antibiosis – Part B Urban Microbiome – Step 6 New Lessons: Bacterial Growth Curve – Part A Midterm Practical Exam Continued Lessons: Streptomyces and Antibiosis – Part C Bacterial Growth Curve – Part B Urban Microbiome – Step 7 2 Week 8 Week 9 Week 10 Week 11 Week 12 Week 13 Week 14 New Lessons: Temperature and Growth of Microbes – Part A Fermentative Patterns of Gram (-) Bacteria – Part A Continued Lessons: Temperature and Growth of Microbes – Part B Fermentative Patterns of Gram (-) Bacteria – Part B New Lesson: Enzyme Induction in E. coli Isolation of Nitrogen-Fixing Bacteria – Part A QUIZ #2 Continued Lessons: Isolation of Nitrogen-Fixing Bacteria – Part B New Lessons: Urban Microbiome – Step 8 Human Microbiome – Part A Food Microbiology: Lactic Acid Bacteria – Part A Continued Lessons: Human Microbiome – Part B Food Microbiology: Lactic Acid Bacteria – Part B Urban Microbiome – Step 8 con’t New Lesson: Prevalence of Antibiotic-Resistance in the Environment (PARE) – Part A Photosynthetic Bacteria and Algae Continued Lessons: PARE – Part B Food Microbiology: Lactic Acid Bacteria – Part C New Lessons: Bacteriophage: T3 – Part A Water Quality Analysis – Part A Continued Lessons: PARE – Part C Bacteriophage: T3 – Part B Water Quality Analysis – Part B Final Practical Exam Clean-up 3 Photographic Atlas Reading Book: A Photographic Atlas for the Microbiology Laboratory, Leboffe and Pierce 4th edition. Week 1: Section 1, pages 1 - 4 Section 2, pages 5 - 6 Section 4, pages 31 - 36 Section 6, pages 45 - 48 Section 5, pages 37 - 44 Section 3, pages 19 - 26 Week 2: Section 6, pages 45 - 56 Section 18, pages 217 - 220 Week 3: Section 9, pages 109 - 113 Section 11, pages 123 - 132 Week 4: Section 6, pages 49 - 54 Section 14, pages 169 - 182 Week 5: Section 6, pages 54 - 55 Section 7, pages 82 - 83 Week 6: Section 3, pages 27 - 30 Section 19, pages 223 - 225 Week 8 Section 2, pages 11 - 13 Section 7, pages 57 - 98 Appendix, pages 243 - 250 Week 9 Section 12, pages 133 - 144 Section 19, pages 231 - 234 Section 7, page 86 Week 10 Section 12, pages 133 - 148 Week 11 Section 12, pages 148 - 164 Week 12 Section 10, pages 117 - 122 Section 19, pages 225 - 231 4 Lab Safety Instructions 1) No eating, drinking, or smoking at any time. Do not bring food or drink items into the lab. Avoid all finger/hand-to-mouth contact. 2) No cell phones, smart phones, iPods, etc. These devices can be easily contaminated and transmit microorganisms outside of the lab. Your instructor may confiscate any of these devices if you are seen using them in the laboratory. 3) A lab coat must be worn at all times in the microbiology laboratory. Your lab coat must be stored in the lab between lab sessions. 4) Follow all directions given by the instructor. Bring any safety concerns to the attention of the instructor. 5) Come to lab on time and prepared for that day's experiments. 6) Wash hands, and wipe down bench area with disinfectant prior to working. Before you leave the lab for the day, wipe down your bench area with disinfectant and then wash your hands. Wash your hands at any time during the lab if you think you may have contaminated them. Wipe any surfaces or equipment with disinfectant immediately if you suspect contamination with living cultures. 7) Never pipette by mouth. 8) You must wear gloves when working with living cultures or potentially toxic compounds. 9) You must wear safety glasses when working with an open flame or liquid live microbial cultures. 10) Loose clothing and long hair should be tied back while working to avoid burning with open flames or inadvertent contamination. 11) Open-toed shoes (sandals, Tevas, flip-flops etc.) must not be worn in lab. 12) Use care with the Bunsen burners. Keep paper, alcohol and other flammable items away from the open flame. 13) Treat all living cultures of microorganisms (bacteria, yeast, etc.) as potential pathogens. Avoid spilling or spreading the microorganisms. Place all used materials in the appropriate waste containers designated for cultures (to be autoclaved). Use the techniques specified by the instructor for handling microorganisms. 14) Use care with glassware. Notify the instructor if any culture tubes are broken or spilled. 15) Know where fire extinguishers and safety equipment is located in the lab. 16) If you are immunocompromised for any reason you must consult a physician before taking this lab course. If you are pregnant you must consult a physician before taking this lab course. Microbiology Laboratory Safety Agreement I have read completely and understand all the above safety instructions. I agree to follow all the above safety instructions when working in the microbiology lab. Signature: _______________________________ Date: _____________ 5 Laboratory Practice Correct use and care of the laboratory equipment is considered a fundamental part of good laboratory technique. All students working in the microbiology laboratory are responsible for maintaining equipment and materials in proper working condition. Please read over and follow the instructions listed below: 1. Personal Property – Coats, bags, and books not used in the laboratory are to be stored in the laboratory anteroom and not in the laboratory itself. Do not bring valuables to lab. Do not place books, bags, or coats in your lab drawer, on your seat, on the floor, or on any of the windowsills within the laboratory. Do not bring any food or beverage (including water) into the laboratory. You may not eat, drink, chew gum, or smoke in the laboratory. 2. Microscopes – Always carry the microscope with two hands. At the end of each laboratory period all traces of immersion oil are to be wiped from the 100X immersion lens using lens tissue. You may need to use lens cleaner to clean the lens. Any oil, water, or other residue must be wiped from the microscope stage with tissue paper. When using the microscope always begin focusing at the lowest magnification and work your way up to higher magnification. Never use the course focus when under 40x or 100x magnification. The ocular lenses (eye piece) can be cleaned if necessary using lens cleaner and lens tissue. When returning the microscope to the cabinet make sure the objective is in the lowest magnification position, and that the stage is adjusted down to a low position. Wrap the cord in place around the cord cleat. 3. Pipettes – Used Pasteur and volumetric measuring pipettes are to be discarded in your Roccal waste container at your workstation. Never dispose of any pipettes or pipette tips in the waste cans! 4. Common supplies – Items such as hemocytometers and hand-counters used in several of the lab sessions should be returned clean to the demonstration table following use. Do not keep these materials in your personal laboratory drawer. 5. Prepared slides – Prepared slides that are used during the semester must be returned clean to the trays from which they were taken. 6. Microscope slides – Any disposable glass slides should be discarded in the red sharps container. Do not discard glass slides in the waste cans! 7. Petri dishes and test tubes – All materials used for handling or culturing microorganisms are to be disposed of as follows: test tubes placed in racks on the discard cart, Petri dishes placed in the hazardous waste bin beside the discard cart. 8. Spillage – Any living culture material that is spilled, either on tables or on the floor, is to be treated immediately with disinfectant (Roccal) and cleaned up with paper towels. Notify the instructor of any spills. 9. Cleanliness of the room – Any papers around your bench area and on the floor at the end of the laboratory period are to be picked up and discarded in the wastebasket. Microbiology Laboratory Care Agreement I have read completely and understand all the above instructions. I agree to follow all the above instructions when working in the microbiology lab. Signature: _______________________________ Date: _____________ 6 Microbiology Lab 3004 Common Laboratory Equipment Important Lab Items Serological Pipettes Pipette Aid Sharps Container Pasteur Pipette w/ Pipette Bulb For our lab: These bottles are filled with disinfectant and are benchtop waste containers for all used pipettes Utility Cart 7 Scientific Method of Inquiry Introduction: The scientific method of inquiry is used by researchers to advance our understanding in all fields of science. The process starts with a question that is developed into a testable hypothesis. Experiments are designed to test the hypothesis. In the case that the hypothesis is false or only partially true, the process can circle back to modify the original hypothesis based on what was learned in the experiments. The strength of results from experiments depends on the inclusion of the proper control experiments and the reproducibility of the results by the experimenter and others. Steps of the Scientific Method Detailed Help for Each Step (adapted from www.sciencebuddies.org) Ask a Question: The scientific method starts when you ask a question about something that you observe: How, What, When, Who, Which, Why, How Many or Where? And, in order for the scientific method to answer the question it must be about something that you can measure, preferably with a number. 8 Do Background Research: Rather than starting from scratch in putting together a plan for answering your question, you want to be a savvy scientist using library and internet research to help you find the best way to do things and insure that you don't repeat mistakes from the past. Construct a Hypothesis: A hypothesis is an educated guess about how things work: "If _____[I do this] _____, then _____[this]_____ will happen." A testable hypothesis must be falsifiable, that is it must be possible through experimentation and investigation to prove false. For example, the hypothesis, “because I hear noises at night in the attic I hypothesize that ghosts exist in the attic” is not falsifiable because a supposed attribute of ghosts is that they may go undetected. Because ghosts may go undetected it is not possible to disprove the hypothesis by not detecting ghosts. You must state your hypothesis in a way that you can easily measure, and of course, your hypothesis should be constructed in a way to help you answer your original question. Test Your Hypothesis by Doing an Experiment: Your experiment tests whether your hypothesis is true or false. It is important for your experiment to be a fair test and include the appropriate controls. You conduct a fair test by making sure that you change only one factor at a time while keeping all other conditions the same. You should repeat your experiments several times to make sure that the results are consistent. Analyze Your Data and Draw a Conclusion: Once your experiment is complete, you will collect your measurements and analyze them to see if your hypothesis is true or false. Scientists often find that their hypothesis was false, and in such cases they will construct a new hypothesis starting the entire process of the scientific method over again. Even if they find that their hypothesis was true, they may want to test it again in a new way. Communicate Your Results: To complete your science project you will communicate your results to others in a final publication and/or a poster presentation. Professional scientists do almost exactly the same thing by publishing their final report in a scientific journal or by presenting their results on a poster at a scientific meeting. 9 The Spread of Contagious Diseases Glo-Germ Activity Introduction: Contagious diseases are those that can be easily spread from an infected person to an uninfected person. Some diseases may be spread through airborne droplets released when an infected individual coughs or sneezes (such as cold and flu viruses) while other diseases may be spread by direct contact with an object, such as a faucet or door handle, that has been contaminated by an infected individual (such as strains of methicillin-resistant Staphylococcus aureus; MRSA). A number of human pathogens can be spread via physical contact with an infected individual or through the contamination of food or water as a result of inadequate hygienic measures. In some instances it is necessary to quarantine infected individuals (and those suspected of being infected because of their close contact with infected individuals) to prevent the spread of the disease. For an example of quarantine measures you can see the recent attempts to contain the spread of the Ebola virus in West Africa (see this article from the BBC: http://www.bbc.com/news/worldafrica-28862591). In instances such as these public health officials may advise members of the community against attending events (sports games, concerts and conferences for example) or places (schools, places of worship, airports and public transportation) where close contact among individuals could exacerbate the spread of the disease. Epidemiology is the area of health and medical sciences that study the incidence, distribution and control and containment of disease in a population. Epidemiologists are the scientists that work in this field. Diseases that epidemiologists study include infections diseases caused by microorganisms as well as noninfectious diseases like cancer and asthma. Historical note: The Hungarian-born physician, Ignaz Semmelweis, while working in Vienna in the 1840s, was the first to show that washing hands with a disinfectant could dramatically reduce the incidence of childbed fever (puerperal fever), an infection of mothers following childbirth. However, his ideas of proper hygiene for physicians and healthcare workers did not gain wide acceptance until the late 1800s, following the establishment of the germ theory of disease. Strains of Staphylococcus and Streptococcus are the most common causes of puerperal fever. The Glo-Germ exercise you will participate in today is designed to illustrate how easy it is for an “infected” individual to spread the disease to many other people relatively quickly through a simple handshake. Procedure: 1. You will be provided with a glove and a Petri dish containing a harmless white powder, mineral oil and a piece of sterile cotton gauze. 2. Place the glove on your right hand. Make sure the glove fits snuggly without a lot of folds or wrinkles. 3. With your gloved hand, take the piece of gauze and smear it around in the Petri dish so that it becomes covered with the powder and mineral oil. Move the soaked gauze into the palm of your gloved hand and squish it around so that the entire surface of your palm and 10 fingers are covered with the mix of mineral oil and powder. Place the used gauze back into the Petri dish. 4. Follow the table below for your handshaking partners. You should refer to your bench number and shake hands with the students at the bench number indicated in the order indicated. Your instructor will tell you when to begin each round of handshaking. Introduce yourself to your classmate when you are shaking hands. Be sure to give a firm handshake that lasts for at least two seconds. 5. After the final round of handshaking your instructor will come around with a light source and scan your gloved hand to determine if you were “infected” during one of the handshakes. 6. You should record the results of who in the class is infected and who is not. For 18 Students: Handshaking Partners (your bench number) Round 1 Round 2 Round 3 1 with 18 1 with 10 1 with 3 2 with 17 2 with 18 2 with 4 3 with 16 3 with 9 5 with 7 4 with 15 4 with 17 6 with 8 5 with 14 5 with 8 9 with 11 6 with 13 6 with 16 10 with 12 7 with 12 7 with 11 13 with 15 8 with 11 12 with 15 14 with 16 9 with 10 13 with 14 17 with 18 Round 4 13 with 16 2 with 10 3 with 12 4 with 9 5 with 6 7 with 1 8 with 18 11 with 17 14 with 15 For 20 Students: Handshaking Partners (your bench number) Round 1 Round 2 Round 3 1 with 20 1 with 11 1 with 5 2 with 19 2 with 12 2 with 6 3 with 18 3 with 13 3 with 7 4 with 17 4 with 14 4 with 8 5 with 16 5 with 15 9 with 20 6 with 15 6 with 16 11 with 19 7 with 14 7 with 17 12 with 10 8 with 13 8 with 18 13 with 17 9 with 12 9 with 19 14 with 18 10 with 11 10 with 20 15 with 16 Round 4 1 with 15 2 with 18 3 with 12 4 with 9 5 with 6 7 with 20 8 with 10 11 with 17 14 with 16 13 with 19 Round 1 2 3 4 Who did you shake hands with? Bench # Name 11 Questions 1) Did you become “infected”? Can you determine who was the initial source of the infection (the index case)? Explain how. 2) Does the Glo-Germ activity provide a good model of how a disease such as seasonal flu (influenza) spreads in a community? Explain why and/or why not. 3) In the Glo-Germ activity we assume that each exposure event, a handshake between an “infected” student and an uninfected student, will result in the uninfected student becoming infected. We also assume that each “infected” individual will be detected with the light source. Are these reasonable assumptions to apply to a real instance of the spread of an infectious disease? Explain why or why not. 4) Outbreaks of infectious diseases such as Ebola virus make news headlines and generate a lot of fear. However, when compared to the flu virus or HIV, a disease such as Ebola is less likely to cause as many infections (see figure below). What characteristics of the diseases contribute to this difference? How do some cultural practices facilitate the spread of viruses? 12 Use of the Microscope Procedure: 1. Begin by placing your slide on the microscope stage with sample/smear facing up. 2. When using the microscope always begin focusing at the lowest magnification and work your way up to higher magnification. - Use the lowest power objective (4x or 5x) to look for your smear of cells. 3. Use the coarse focus knob to bring the smear into focus. - The smear will look a bit colored, but you will not be able to see individual cells at this magnification. - It is important to note at this point that the low power objective lens (4x or 5x) is not close to the slide on the stage. The focal distance at low magnification is relatively large, a few centimeters. As you go up in magnification the focal distance will decrease and the objective lens will come closer to the sample on the slide. - At 10x you will still have more than a centimeter distance between the lens and the slide, but at 40x and 100x, the objective lens will be just a few millimeters or less from the sample on the slide. ****Never use the coarse focus at 40x or 100x because it is too easy to make the stage move too close, which will smash the lens into the slide – and that will dirty or damage the lens. Note: To make sure you are looking at something on the slide and not a piece of dirt on the microscope lens, move the stage slightly – if what you are seeing moves with the stage then it is likely to be something on the slide. If what you are looking at does not move with the stage then it is likely to be something on the microscope lens. 4. You will need to adjust the light using the diaphragm condenser and the intensity knob/dial. - You will need more intense light as you go up in magnification. - Take a few seconds to optimize the lighting at each magnification as you work your way up in magnification. - You should also use the condenser height adjustment to make sure that the condenser is in the right position – generally you will want to bring it up as close to the bottom of the stage as it will go. 5. Rotate the objective lens turret to bring the 10x objective in line. 6. Re-focus and adjust the light under the 10x objective. - You should still see your smear but you will not be able to make out individual bacteria (although you should be able to see individual cells if they are larger, like yeast and algae). 7. Rotate the objective lens turret to bring the 40X objective in line. 8. Re-focus and adjust the light under the 40x objective. - At this point you should be able to see individual bacterial cells. 13 - The bacterial cells will be small and of uniform shape and size, without any jagged edges or protrusions. If you are seeing something non-uniform it is probably a speck of dust or dirt. If you are seeing bright perfectly round things that are not stained with dye, you are probably looking at air or water bubbles. Once you have found your cells, record what you see (shape, size, grouping, color, and any other distinguishing characteristic). REMEMBER! Never use the coarse focus at 40x or 100x – use only the fine focus adjustment. Moving to 100x Magnification9. Rotate the objective lens turret so it is between the 40x and 100x objectives 10. Use your lens oil dropper to place a small drop of oil directly on your sample. - You MUST use oil with the 100x objective lens. 11. Rotate in the 100x objective lens. - The lens should be so close to the stage that is touches into the oil drop (but does not touch the slide itself). You can check by leaning down and looking for a little “flash” when the 100x lens touches the oil droplet. 12. Adjust the fine focus knob and the light until you can see your cells clearly. Record what you see if you did not already do so at 40x. You may be able to improve your description when you are looking at 100x magnification. IMPORTANT NOTES: § The 100x objective is the only oil immersion lens – no other objectives should be used with oil or allowed to contact oil on the slide. § You cannot go back to re-focus on 40x if you have oil on your slide, although you can carefully re-focus with the 4x and 10x objectives. § If you get oil on any other lens wipe it off immediately with lens cleaner and lens paper. § Wipe all traces of immersion oil from the 100x immersion lens using lens cleaner and lens tissue. § Oil, once it has dried, is almost impossible to remove from a lens! 1. Storage: o Any oil, water, or other residue must be wiped from the microscope stage. o Put the 4x objective in position and the stage down. o Wrap the cord in place around the cord cleat and return the microscope to its designated spot in the cabinet. Microscope Rules: 1. Always carry microscope with two hands 2. Use the microscope that is numbered according to your station/bench number 3. Always clean lenses with lens paper after use – only use lens cleaner and lens paper DO NOT use bibulous paper or paper of any other kind 4. Store with the stage completely lowered and the lowest objective lens in place 5. Never leave slides on stage when putting away the microscope 14 Note: Here is a link to a couple of YouTube videos on how to use a compound microscope: https://www.youtube.com/watch?v=bGBgABLEV4g https://www.youtube.com/watch?v=9-76svUtJGw Parts of the Microscope and Magnification Fine Focus Knob Coarse Focus Knob Questions: 1. If you are using a 40x objective lens to observe an organism how many times is that organism magnified from its true size? Questions: 1) If you wanted to view an organism 1,000x its true size what objective lens would you use? 2) When working with the 40x or 100x objective lens, which focus knobs can you use safely? 3) I know that my organism is 2um, if I wanted to view my organism so it appears to be 2mm, what objective lens would I want to use? 15 Culture Techniques Summary: In the first few lab sessions you will learn how to work safely with bacteria and other microorganisms. You will learn a number of techniques used routinely in microbiology labs. During the introductory labs you will: 1. Use sterile/aseptic technique to inoculate liquid media and streak plates for colony isolation 2. Prepare Gram stains and use basic light microscopy to observe bacteria 3. Enumerate microorganisms by direct and indirect methods 4. Prepare serial dilutions of samples 5. Examine colony morphology Introduction and Background: The first consideration when working in the lab with microorganisms is your safety and the safety of others in the laboratory. Although the vast majority of microorganisms are harmless and not pathogenic to humans, it is often the case that you may be working with unknown samples that could potentially contain pathogens. As a guiding principle, it is always good practice to treat any microbiological sample as if it were potentially pathogenic. In cases where known potential pathogens are being handled there may be additional precautions taken to avoid contamination or release of the microorganisms. Please make sure you have read and understand the detailed safety instructions provided above. Biosafety Level Definitions (1) BIOSAFETY LEVEL 1 is suitable for work involving well-characterized agents not known to cause disease in healthy adult humans, and of minimal potential hazard to laboratory personnel and the environment. (2) BIOSAFETY LEVEL 2 is similar to Level 1 and is suitable for work involving agents of moderate potential hazard to personnel and the environment. (3) BIOSAFETY LEVEL 3 is applicable to clinical, diagnostic, teaching, research, or production facilities in which work is done with indigenous or exotic agents that may cause serious or potentially lethal disease as a result of exposure by the inhalation route. (4) BIOSAFETY LEVEL 4 is required for work with dangerous and exotic agents that pose a high individual risk of aerosol-transmitted laboratory infections and life-threatening disease. When working in the microbiology lab it is important to make sure that your stock cultures, reagents and instruments remain uncontaminated by microorganisms present in the environment. Sterile technique (or aseptic technique) refers to the set of methods that microbiologists use to minimize the possibility of contamination when handling cultures, growth medias and other reagents (see appendix for more details). The contamination of your stock culture or reagents with foreign microorganisms can make it difficult, if not impossible, to generate reliable and reproducible data. In this lab we will employ basic sterile technique, but in other situations, such as clinical and diagnostic labs, it may be necessary to use more stringent practices to further reduce the chance of contamination. 16 Typically microorganisms are grown in liquid media (batch culture or continuous cultures, sometimes referred to as bioreactors in the case of large scale growth) or on solid media (supplemented with agar to form a solid gel) in plastic disposable dishes (Petri plates). There are numerous media formulations, often developed to support the growth of fastidious microorganisms. Medias, liquid or solid, may be classified as complex or defined in terms of their contents, and selective or differential in regards to their intended use (see the Glossary of terms for definitions). In this lab module you will learn several important techniques that you will use throughout the semester. You will practice your sterile technique as you inoculate liquid media cultures, streak plates for colony isolation and prepare serial dilutions. You will prepare Gram stains from a number of your samples and examine the cell shape and organization by light microscopy. You will prepare a set of serial dilutions and use direct and indirect methods to enumerate the microorganisms in your cultures. Liquid Culture Inoculation In general, you would like to minimize the time that any culture or media is exposed to the air, as many microorganisms float around on dust particles in the air and can find their way into your tubes, bottles, plates, etc. It is often best to tilt your plate, bottle, test tube, when adding or taking out sample as this reduces the open area exposed to dust falling in a downward direction. It is also important to be relatively quick in adding or removing sample because less time spent exposed to the air will reduce the chances of contaminants entering your sample or media. Here is a link to a YouTube video of how to use aseptic technique: https://www.youtube.com/watch?v=bRadiLXkqoU Note: The purpose for all of the links to YouTube throughout the manual is for you to get a general idea of the technique being used. The videos may not be identical to the way that your instructor will demonstrate the technique in the classroom setting. 1. Read through the protocol and make sure you are prepared to begin. 2. Assemble the necessary reagents at your bench and light your Bunsen burner 3. Loosen the bottle/tube cap and then with one hand remove the cap and flame the glass lip of the bottle to destroy any contaminants that may have collected at the lip of the bottle. If you are using plastic then skip the flaming step. Carefully insert a sterile pipette into the media making sure not to touch the lip of the bottle or anything outside the bottle. Remove the desired volume (5 ml) and place it into a sterile test tube (have the test tube in your other hand, removing the cap with your index finger and thumb and flaming the lip just prior to adding the media with your pipette). Recap the bottle and the test tube. 4. If you need to add antibiotics to the media for selection purposes then do it at this point (your instructor will let you know if this is necessary). 17 5. Flame an inoculating loop (or use a sterile disposable inoculating loop) and pick-up a bit of a colony from a plate, or a loop full of media from a liquid culture. Insert the loop into your test tube, flaming as described above. Recap the test tube. 6. Sterilize or dispose of your inoculating loop and place the inoculated test tube in an incubator for growth. 7. Prepare an un-inoculated control tube with just 5 ml of media. Questions 1) What is a pure culture? 2) How is a liquid culture used in a microbiology laboratory? Streak Plate & Slant The objective of streaking a plate for colony isolation is to have isolated colonies to pick from after the bacteria have had a chance to grow. If the streaking on the plate is too dense and the colonies are packed together it becomes difficult, or impossible, to pick a single colony for observation or subsequent inoculation. When you are examining a sample by microscopy, or especially in biochemical/metabolic assays, you want to be certain you are only examining cells from one colony. If you accidentally pick two different colonies because they are growing so closely, or one on top of another, and they happen to be from two different species then your results will be seriously compromised because they represent these two types of bacteria, not a single species. Streaking an agar slant is often to preserve a culture line of bacteria or for a biochemical reaction to observe the bacteria’s metabolism. Here are links to a couple of YouTube videos of how to streak a plate: https://www.youtube.com/watch?v=_1KP9zOtjXk https://www.youtube.com/watch?v=0heifCiMbfY 1. Read through the protocol and make sure you are prepared to begin. 2. Label the plate (the bottom dish, not the lid!) with what you will be streaking with the sample name, dilution, date, your name, the type of media, and any other important information (antibiotics used, for example). It is best to keep your labeling near the edge of the plate (but not on the sidewall) so that the writing does not obscure your view of colonies growing on the agar after inoculation and incubation. 18 3. Light your Bunsen burner and flame an inoculating loop. Then with the loop pick-up a bit of a colony, or a loop full from a liquid culture. 4. Make a thick streak along one edge of the agar plate. (See the diagram for details.) Do not use too much force and avoid making gouges in the agar. 5. Flame the loop for several seconds then let cool. Make a second streak starting from one edge of the original streak. 6. Flame the loop for several seconds then let cool. Make a third streak starting from one edge of the second streak. 7. Flame the loop for several seconds then let cool. Now streak for a fourth time broadly across the center of the plate going in and out of the third streak (see diagram). 8. Label the tube (the glass part, not the lid!) with what you will be streaking with the sample name, dilution, date, your name, the type of media, and any other important information (antibiotics used, for example). It is best to keep your labeling neat and small so that the writing does not obscure your view of colonies growing on the agar after inoculation and incubation. 9. Flame the loop for several seconds then let cool. Again with the loop pick-up a bit of a colony, or a loop full from a liquid culture. 10. Pick up the agar slant. Start a light streak starting from the bottom edge of the agar and move upwards in a broad zigzag to the top edge of the agar. (See diagram for details.) 11. Store the inoculated test tube and plate at room temperature in your drawer for growth over the week. Questions 1) What is the purpose of repeating streaking motions on a single agar plate with sterile loops between each streak? 2) Is this a qualitative or quantitative method? 19                            Streaking  Methods Streaking  on  agar  slant   ng   la u c Ino p Loo Streaking  on  agar  an  agar  plate Steps of Streaking a plate Streaking is used to determine whether a culture is pure (containing only one species of type of or organism) or contaminated (with different types of microorganisms). Besides it also is a test for determining whether a culture is viable or not, that is if growth is possible for a stock culture. 20 Spread Plate The objective of a spread plate is to allow bacteria enough room to grow that the diversity and quantity of bacteria present can be assessed. Often the original sample is diluted and a series of plates at increasing dilutions are made to be able to have an accurate quantity of bacteria in the starting culture 1. Read through the protocol and make sure you are prepared to begin. 2. Label the plate (the bottom dish, not the lid!) you will be streaking with the sample name, dilution, date, your name, the type of media, and any other important information (antibiotics used, for example). It is best to keep your labeling near the edge of the plate (but not on the sidewall) so that the writing does not obscure your view of colonies growing on the plate after inoculation and incubation. 3. Light your Bunsen burner. Flame sterilize a glass/plastic spreader (aka ‘hockey stick’). To flame sterilize, first dip the spreader into 70-90% ethanol and tap off excess liquid. Then quickly place the spreader in the Bunsen burner flame to burn off all the alcohol. BE CAREFUL! DO NOT shake the hockey stick to dry off excess liquid, which can throw the alcohol flame to another surface. DO NOT place the freshly flamed spreader back into the ethanol. If the ethanol container does catch fire, do not panic! Use another dish, preferably larger, and place over the flames to suffocate the fire. 4. Add sample (typically 100 µl) to the center of the agar plate. Use the sterile spreader to smear the sample over the plate. Move the spreader in a circle with the sample in middle and gradually going towards the outside edge (making larger circles or intersecting paths) until the entire sample is distributed. 5. Store the plate(s) at room temperature for growth over the week. Questions 1) What is the purpose of a spread plate? 2) What are the materials needed to prepare and complete a spread plate? 3) Is this a qualitative or quantitative method? 21 Prokaryotic survey Introduction: This exercise is designed to familiarize you with different types of bacteria. You will examine several important genera of bacteria both in lab and by library and/or Internet survey. Procedure: Examine the plates provided and note the colony morphology of the bacteria. Prepare a Gram stain of each bacterium provided. Record the results in your Prokaryotic Survey table below. 1. Using oil immersion and 100x objective lens observe 4 bacterial Gram stains you prepared of organisms from the list below. Fill out the Prokaryotic survey table by drawing what you see in the microscope field and recording the cell shape and arrangement. Staphylococcus epidermidis: Staphylococcus spp. are Gram-positive cocci arranged in irregular, often grape-like, clusters. Measure the diameter of a single coccus. Escherichia coli: Escherichia coli is a Gram-negative bacillus. Measure the length and width of a typical rod. Micrococcus luteus (Sarcina lutea): Micrococcus luteus are Gram-positive cocci that usually exhibit a tetrad or a sarcina arrangement. Note the shape and arrangement of you preparation. Examine other species if provided and time allows. 2. When finished, remove the oil from the prepared slides using paper towel and return them to their proper tray. Remove oil from the objective with lens paper and lens cleaner. 3. Using the Internet or other reference source each team member should investigate and report on one Gram-positive genus not included in the above group. Your lab notebook should include a complete description of all Gram-positive bacteria researched by you and your teammates. 22 Colony Morphology 23 Cell Arrangements Spirilla Spirochete 24 Prokaryotic Survey Table Bacterial species 1 Bacterial species 2 Shape = Arrangement = Bacterial species 3 Shape = Arrangement = Bacterial species 4 Shape = Arrangement = Shape = Arrangement = 25 Survey of Prokaryotes Enter the information in the chart below for each organism you stain and examine under the microscope. This chart will be used throughout the semester. Retain it and keep it legible. Print and add extra pages as needed. Gram Microscopic Organism Morphology Arrangement Macroscopic Size Shape Edge pattern Color Characteristics 26 Cytological Techniques It can be difficult using a bright-field microscope to observe living microorganisms. Although some microorganisms may be pigmented most are nearly transparent and often move rapidly about the slide. Consequently, microbial cells are frequently killed and immobilized, a process called fixation, and stained with dyes. The dyes color the cells, or their background, allowing for better visualization. The staining of the cells helps to provide information about cell size, shape, and cell clustering arrangements. Some stains are able to indicate biochemical or structural properties of the cells in the sample being examined. To stain microorganisms, typically a drop of a liquid containing the microbe is placed on a glass microscope slide and allowed to air dry. The resulting specimen forms a film referred to as a smear. The microorganisms are then killed and fixed to the slide by passing the slide over a flame or in some cases by chemical treatment. A dye is then applied that stains certain components of the cell and the excess unincorporated dye is rinsed off with water. Stains are classified as basic if they carry a positive charge and acidic if they carry a negative charge. Basic dyes stain the negatively charged components of cells, including nucleic acid and many proteins. Common basic dyes include methylene blue, crystal violet, safranin, and malachite green. Common acidic dyes include Congo red, nigrosin, and India ink. A negative stain uses an acidic dye to color the background surrounding negatively charged bacterial cells. The simplest staining methods use a single dye to color bacterial cells so that their size, shape, and arrangement can be observed. A differential stain uses dyes to distinguish one type of bacteria from another. It takes advantage of the fact that bacterial species often have distinct biochemical and structural properties. This leads to differences in their staining properties. Examples of two frequently used differential staining techniques are the Gram stain and the acid-fast stain. These differentiate bacteria based on the biochemical composition and structure of their cell wall. The first dye in a differential stain is called a primary stain, and the second is called a counterstain. A decolorizing step may be used between application of the primary stain and the counterstain. Depending on the composition of the cell wall, bacteria will either retain the primary stain during decolorization or lose the primary stain and take up the counterstain. The Gram stain is designed to differentiate between bacteria that have a single cellular membrane and thick outer peptidoglycan cell wall, the Gram-positive bacteria, and those bacteria that have an inner and outer membrane with a thin peptidoglycan cell wall located in the periplasmic space between the inner and outer membrane layers, the Gram-negative bacteria. After staining Gram-positive bacteria appear purple as a result of the crystal violet dye being trapped within the cells by the thick peptidoglycan cell wall. The thinner peptidoglycan cell wall of Gram-negative bacteria allows the crystal violet dye to wash out during the decolorizing step. Gram-negative bacteria appear pink as a result of the counter staining with safranin. Identifying an unknown bacterial sample as Gram-negative or Gram-positive tells the investigator a good deal about the structure of the bacterial cell envelope. In clinical settings, knowing whether a patient is suffering from a Gram-negative or Gram-positive infection will influence the selection of antibiotics used to treat the infection. Most bacteria will stain as either Gram-positive or Gram-negative, however a few important genera of bacteria lack standard cell walls and do not stain with the Gram technique, such as the Mycoplasmas. 27 Gram Staining: A Historical Perspective One of the most important staining techniques in microbiology was developed in 1882 (published in 1884) by Danish bacteriologist, Hans Christian Joachim Gram (1853-1938). After earning his degree in medicine from University of Copenhagen in 1878 he became a resident physician at the Municipal Hospital. His early works concerned the study of erythrocytes (red blood cells), and his findings were very important to the advancement of the field. The work that earned him international recognition, however, was his method for staining bacteria. While examining lung tissue from patients who had died of pneumonia, Gram discovered that certain dyes were preferentially taken up and retained by bacterial cells. In the first step, he dried a fluid smear on a glass slide over a burner flame and poured Gentian violet solution over it. After a water rinse, he added Lugol’s iodine solution, which acted as a mordant to retain the dye in the cell if possible. Then he poured ethanol over the slide to wash away any of the iodine-dye complex that could easily diffuse out of the bacterial cell. Certain bacteria (pneumococci, for example) retained the color (Gram-positive), while other species did not (Gram-negative). A few years later, German pathologist Carl Weigert (1845-1904), added a final step of staining with safranin. Gram himself never used counterstaining for Gram-negative microbes. The process of staining, as mentioned above, colors the bacteria making them visible by standard light microscopy. The shape of bacterial cells, and the organization or grouping of the cells, can be very useful in identifying an unknown bacterial sample. The basic shapes bacteria take can be rods (bacilli), spheres (cocci), curved rods (vibrios), stubby rods (coccobacilli), spiral shaped (flexible spiral-shaped is considered spirochete and stiff spiral-shaped is referred to as spirilla), or variable in shape lacking any consistent form (pleomorphic). Some types of bacteria remain dispersed and their cells appear singular and unattached, however, other species have a characteristic cell organization. Chains of cocci are called streptococci (such a Streptococcus pyogenes the causative agent of Strep throat and other human infections), whereas grape-like clusters of cocci are called staphylococci (such as Staphylococcus aureus, a common member of the human microflora that may cause a range of infections depending on the specific strain). Bacteria such as Neisseria gonorrhoeae are seen as pairs of cocci, called diplococci. Although staining an unknown bacterial sample and observing by light microscopy the Gram properties, cell shape, and cell organization generally will not give you enough information to identify the exact species of the bacteria, it will help to narrow down the possibilities, and in combination with other information, such as growth and metabolic characteristics, may permit you to make a fairly certain identification. Colony morphology can provide additional information towards identifying a bacterial sample. 28 The Gram-Positive Bacteria - Characteristics of the genus Lactobacillus. Gram-positive, rod-shaped organisms that grow as single cells or loosely associated chains. They vary from short slender rods to short coccobacilli. They are present in decomposing plant material, milk, and other dairy products, microflora of the mouth and the healthy human vagina during childbearing years. - Characteristics of the genus Streptomyces. Branching filamentous bacterial genus of the order Actinomycetales, members of which resemble fungi. Streptomyces are chiefly saprophytic (feed off decaying matter), and produce most of the world’s antibiotics. Great producers of secondary metabolites: antimicrobial, antiviral or anti-tumor activities - Characteristic of the genus Streptococcus. Gram-positive, spherical-shaped organisms that grow as single cells or loosely associated chains. Streptococcus can be found on mucous membranes of the human throat. Their cellular arrangement is in long chains of 6 or more cells. On solid media Streptococcus species form small colonies. These colonies typically appear round, smooth, glistening, flattened, and translucent. Hemolysis may or may not be present on blood agar. Important members 1. Streptococcus pneumoniae: A major human pathogen causing pneumotitis. 2. Streptococcus pyogenes: causes strep throat and pharyingitis (a form of streptococcal infection that affects the pharynx). 3. Enterococcus faecalis: A streptococcus-like organism acclimated to living in the human intestines. E. faecalis is a leading cause of nosocomial (hospital-associated) infections. Hospital-adapted strains are strongly pathogenic due to intrinsic and acquired resistance to conventional antimicrobial drugs. - Characteristic of the genus Staphylococcus Location: Skin (external surfaces and in hair follicles) Microscopic Appearance and Gram Stain Characteristics: Gram-positive coccus. Cellular arrangement: generally occur in grape-like clusters Appearance of colonies on solid media: Staphylococcus spp. form medium-sized round, smooth and glistening colonies that have a soft buttery consistency. The colonies are opaque and usually pearly white, but may have yellow-gold pigmentation. Hemolysis may or may not be present. 1. Staphylococcus aureus: produces large round golden yellow colonies and is hemolytic on blood agar 2. Staphylococcus epidermis: small white or beige colony, non-hemolytic Important members 1. S. epidermidis: is a part of the normal flora of skin 2. S. aureus: This organism is responsible for variety of diseases including skin and wound infections and Toxic Shock Syndrome (TSS). The yellow pigment excreted by this organism is a major factor for virulence. 3. S. saprophyticus: causes urinary tract infections and is resistant to Novobiocin. 29 The Gram-Negative Bacteria The world is full of Gram-negative rods, many of which are members of family Enterobacteriaceae. Members of this family are found in the gastrointestinal tract of animals, but many are also free living in soil and water. Since all of the family members look alike after Gram stain, other tests must be used to differentiate them. Leboffe MJ and Pierce BE. Microbiology: Laboratory Theory and Application. 2nd edition. Englewood, Colorado: Morton Publishing Company. 2006 Characteristics Shared by the Family Enterobacteriaceae Microscopic Morphology: They are short Gram-negative rods with rounded edges and typically 4-5 microns in length. They do not produce spores. They have peritrichous flagella (exception: Klebsiella and Shigella are nonmotile (no flagella)). Macroscopic Morphology: Colonies are usually dome shaped, gray, and smooth. Oxygen Requirement: They are facultative organisms. They can ferment (convert glucose to simpler organic compounds) or respire (oxidize glucose with O2 to produce CO2& H2O) depending upon environmental conditions. Glucose Fermentation: all members can ferment glucose to pyruvate (glycolysis), which is then converted to different end products depending upon the species. These end products can be used in identifying the species. Nitrate reduction: they reduce nitrate to nitrite; some members can convert it to nitrogen. Important members - Escherichia coli: Gram-negative rods, lactose fermenter, normally found in intestines and feces as a normal member of the intestinal flora. Can be an opportunistic pathogen. Shiga toxin producing E. coli (the toxin is produced by a plasmid similar to the toxin producing plasmid of Shigella dysenteriae) has resulted in highly septic variants such as H7:O157. - Klebsiella pneumoniae: Gram-negative rod, ferment lactose, normally found in human intestines, but is also an opportunistic pathogen and can cause pneumonia and dysentery. Forms mucoid (slimy) colonies. - Salmonella typhimurium: Gram-negative rods, non-lactose fermenter. Causative agent of typhoid fever and food poisoning; forms flat, wide colonies. - Proteus mirabilis: Gram-negative rods, lactose negative, widely found in human and animal intestines; opportunistic pathogen. - Shigella sonnei: Gram-negative rods, slow lactose fermenter, not usually found in human digestive tract, all species are human pathogens. Shigella sonnei, also known as Group D, is the leading cause of Shigella-caused dysentery in the United States. - Enterobacter cloacae: Gram-negative rods, lactose fermenter, normally found in human intestines and feces, but can become an opportunistic pathogen causing various infections. - Serratia marcescens: Gram-negative rods, non-lactose fermenter, usually found in feces, prominent opportunistic pathogen. Colonies are red. 30 Common Staining Techniques Simple Stains Basic stains: Methylene blue, crystal violet, Safranin Negative Stain Acidic Stains: Nigrosin and Congo red. Gram Stain Primary stain – crystal violet; Mordant – iodine; Counterstain – Safranin Purpose: Since cytoplasm is transparent, cells are usually stained with a colored dye to make them visible under the microscope. Allows determining cell morphology, size, and arrangement. Basic stains are applied to bacterial smears that have been heat fixed. Purpose: To determine morphology and cellular arrangement in bacteria that cannot withstand heat fixing. Acidic stains are applied to bacterial smear without heat fixing. Cells remain unstained against a colored background. Purpose: The most important differential stain. It is used to distinguish between Gram-positive and Gram-negative cells and determine cell morphology, size, and arrangement. Acid-Fast Stains Primary stain – Carbolfuchsin; Decolorizer – acid alcohol; Counterstain – Methylene blue Purpose: Used as a differential stain to detect cells capable of retaining a primary stain when treated with an acid alcohol. For instance, bacteria in the genus Mycobacterium, some of which are pathogens causing tuberculosis (M. tuberculosis) and leprosy (M. leprae), are identified with this stain. Endospore Stain Primary stain – Malachite green; Counterstain –safranin Purpose: To detect the presence and location of spores in bacterial cells. Among genera that produce spores are Bacillus and Clostridium. Capsule Stain Solution 1 - acidic stain: Congo red or Nigrosin; Solution 2 basic stains – crystal violet, Safranin Purpose: to detect cells capable of producing an extracellular capsule, such as Bacillus anthracis and Streptococcus pneumonia. 31 Gram Staining You will be supplied with cultures of bacteria for Gram staining: Escherichia coli, Staphylococcus epidermidis and an unknown (although these laboratory strains of E. coli and S. epidermidis are non-pathogenic they should be treated as potential pathogens - mutations can occur that alter the properties of these strains). The dyes may also stain your hands and may have toxic effects with repeated or prolonged exposure. YouTube video of the Gram Stain technique: https://www.youtube.com/watch?v=8zd3HPRxx1U Dyes: - Crystal violet - Gram's iodine - 0.5% safranin - Alcohol Cultures: E. coli, S. epidermidis, & unknown Procedure: 1. Start by Gram staining known Gram-positive and Gram-negative samples. a. From a colony on a plate or from an agar slant, first use a sterilized loop to transfer 1-3 loops of sterile DI water onto the slide. Sterilize the loop again and pick up a portion of the colony from the plate or slant (avoid picking up any agar as this will burn on the slide) and smear the bacteria in the drop of water on your slide into a small circle, about the size if a dime. b. From a liquid culture, use a sterile loop to pick up a loop-full of the liquid culture and spread it out as above. You may need 2-3 loops of the liquid culture. Note – if the liquid culture is very dilute then it will be difficult to see any bacteria. 2. Let the samples air dry on the slide for 2-3 minutes or until most of the water has evaporated. 3. Heat-fix the bacteria by passing the slide (with the bacteria on the upper side) rapidly through the full flame of a Bunsen burner several times. The smear is now stable and non-infectious (the heat kills the bacteria). If you do not see a grey smudge on the slide then there are too few bacteria on the slide and they may be difficult to find. 4. Stain by applying the following to the slide, using dropper bottles. a. Add a few drops of crystal violet to the smear and let it sit for 60 seconds. Tip off the dye in to a collection bowl and rinse the slide gently with tap water. Gently blot the slide dry with bibulous paper. Be careful not to wipe off the bacteria. b. Add a few drops of Gram's iodine (mordant) to the smear and let it sit for 60 seconds. Tip off the dye in to a collection bowl and rinse the slide gently with tap water. Gently blot the slide dry. c. Alcohol decolorization: Let the alcohol run over surface of slide until no more crystal violet color comes out of the smear (5-10 seconds). Gently rinse with tap water then blot the slide dry. d. Add a few drops of safranin (counter stain) and let it sit for 30 seconds. Tip off the dye in to a collection bowl and rinse the slide gently with tap water. Gently blot the slide dry with bibulous paper. Be careful not to wipe off the bacteria. 5. Observe your slide under the microscope. 32 Questions 1) What are the staining properties of your unknown microorganism? Can you identify your unknown microorganism? 2) What might cause a Gram-positive cell to appear Gram-negative? 3) What is the purpose of staining a known bacterial sample in parallel with an unknown sample? 4) Can all cells be identified with gram staining? Explain your answer. 33 Micro Pipettor Usage Introduction: The pipettes used in today’s lab are ‘micropipettes’ designed to accurately and reproducibly deliver small volumes (P20 range 2-20 µl; P200 range 20-200 µl; µl = microliter, and 1,000 µl = 1ml). You will be using these pipettes in the lab and you may need to use them in any number of positions in basic research, clinical research, biotech labs or in pharmaceutical labs. There are a number of brands of pipettes, but they all function largely the same. It is important to take the time to learn how to use these pipettes correctly. This exercise is designed to give you practice using the pipettes and checking your consistency. Procedure: 1. Your instructor will demonstrate the correct usage of the pipettes. You can also consult the manufacturer’s instructions on how to use the pipette. 2. Cut a small section of Parafilm (~5 cm) and pick-up a microcentrifuge tube containing the dyed water solution. 3. Using the correct technique and a P20 pipette, make a series of five 5 µl drops in a row on the Parafilm. Make another series of five 15 µl drops in a row. 4. Next, using the correct technique and a P200 pipette, make a series of five 30 µl drops in a row on the Parafilm. Make another series of five 100 µl drops in a row. 5. After you have spotted out your four sets (5 µl, 15 µl, 30 µl and 100 µl) call the instructor over to examine your drops. Each set of five drops should look essentially identical. 6. Repeat this practice if your instructor finds that you first attempt was lacking. Note: Here is a link to a couple of YouTube videos on how to use micropipettes. Keep in mind that the micropipettes and tips shown in the video are not identical to the ones used in class. You should make sure to follow the instructions from your instructor. https://www.youtube.com/watch?v=NgosWmRjjAo https://www.youtube.com/watch?v=waZfBR7fk_8 https://www.youtube.com/watch?v=uEy_NGDfo_8 34 Enumerating Microorganisms When working with a sample of bacteria, or other microorganism, it is often extremely useful to know how many bacteria are present. The cells can be counted directly using a special microscope slide known as a counting chamber or hemocytometer (also used for counting blood cells) that has a grid etched into the surface. The grid allows the number of bacteria in a small area of a known size, to be counted, and from that information the number of cells per milliliter can be calculated. This is referred to as a direct count. Alternatively, the sample of bacteria can be diluted in media then plated onto a solid agar plate to form colonies. Each individual living cell plated can grow to form a visible colony (known as colony forming units, or cfu); counting the colonies gives the number of individual living cells that were originally plated. From the number of colonies and the dilutions made, the cfu/ml of the original sample can be calculated. Since only living cells grow to form colonies, this indirect method, or viable count, gives the number of living cells and does account for dead cells were present in the sample. The direct microscopic count includes the dead bacterial cells since they appear the same as the living cells, unless a special staining method has been used to differentiate the living cells from the dead cells. The direct count and viable count represent classical methods of enumeration. However, there are other options available to quantify the number of microorganisms in a sample, including the use of spectroscopy to measure the optical density of a sample (the amount of light at 600 nm absorbed by the cells in a sample; the OD600 value). To use the optical density approach to determine actual numbers of cells, it is necessary to create a standard curve. The standard curve is created by measuring the OD600 of samples through a range of cell concentrations determined by a direct or viable count. Once the standard curve has been prepared it can be used to translate subsequent OD600 values into cell concentrations. It is necessary to create separate standard curves for each microorganism being quantified because the absorbance properties can differ between species. Measuring the OD600 value of many samples can be done quickly, much more rapidly than a direct count or viable count. There are also laser detection systems that can be used to count individual cells in fine stream, a method know as flow cytometry. In laboratory settings, bacteria are typically grown in favorable conditions that they grow to high densities. Therefore, it is often necessary to dilute the bacteria to a lower concentration for other applications using a set of serial dilutions to create a range of cell concentrations. Serial dilutions are usually made in 1:10 steps, although other increments can be used. Very simply, a 1:10 dilution is made from the original ‘stock’ culture then a second 1:10 dilution is made from the first 1:10 dilution, making it a 1:100 dilution relative to the starting concentration. Then another 1:10 dilution can be made from the second 1:10 dilution (a 1:1000 dilution relative to the starting concentration). This dilution process continues until the desired estimated concentration is reached. 35 Viable Count Procedure: Part A – 1. Label the tubes and any plates you intend to use. In this exercise you will prepare dilutions down to 10-7. 2. Use sterile technique to prepare seven tubes each containing 4.5 ml of water. Continue to use sterile technique throughout the preparation of your serial dilutions. 3. From the yeast (baker’s yeast – Saccharomyces cerevisiae) culture provided, use a sterile one ml pipette to add 0.5 ml of the original culture to the 4.5 ml of water in the tube labeled 10-1. This is a 1:10 dilution. Mix the dilution by flicking the tube or tapping it against your palm. 4. Using a sterile 1 ml pipette, transfer 0.5 ml from the 10-1 tube (NOT the original culture) to the 4.5 ml in the tube labeled 10-2. Mix the new dilution. This is another 1:10 dilution and represents a combined 1:100 dilution from the original stock. 5. Repeat for the remaining tubes, always transferring 0.5 ml from the previously prepared dilution to the subsequent dilution tube and homogenizing directly before the transfer. Use a new sterile pipette at each transfer step and remember to mix well after each addition. Your final dilution, 10-7, represents a 1:10,000,000 dilution from the original culture. If the starting culture was at a density of ten million cells per ml, then the final dilution tube should contain ~1 cell per ml. 6. Set dilutions aside and complete the Direct Count experiment 0.5mL 0.5mL 4.5mL 0.5mL 0.5mL 4.5mL 4.5mL 0.5mL 4.5mL 0.5mL 4.5mL 0.5mL 4.5mL 4.5mL 10x 10-1 10-2 10-3 10-4 10-5 10-6 10-7 1:10 1:10000 1:10000000 Serial Dilution Illustration Note: Make sure you understand the math underlying the dilutions. You should be able to make these calculations relatively easily, if not, practice more until you can. Here are links to a couple YouTube videos of how to prepare a serial dilution: https://www.youtube.com/watch?v=ZqdU3VfQ_Tc https://www.youtube.com/watch?v=tdXSM0EUxDQ 36 Hemocytometer Direct Count Procedure: Part B – 1. Wipe hemocytometer and cover slip gently with water or lens cleaner before starting. 2. Use the dilutions of yeast (Saccharomyces cerevisiae) you prepared in the serial dilution exercise. You will probably want to start by counting the yeast in your 1:10 (10-1) or the 1:100 (10-2) dilution. 3. Place the coverslip on the counting chamber. 4. Mix the tube of yeast immediately before adding them to the counting chamber. a. The yeast settles quickly, if they are not mixed evenly this will bias your count. 5. Load the a chamber by touching a drop of your diluted yeast at the edge of the cover slip using a Pasteur pipette and let the capillary action draw the cells into the chamber b. Avoid getting air bubbles under the coverslip. If you overfill the chamber or get liquid onto the surface of the coverslip, rinse off the counting chamber and reload. 6. Place the counting chamber on your microscope stage and locate the grid under lowpower magnification (4x or 10x objective). You may need to adjust the diaphragm condenser on the microscope to set the light level so that you can see the grid. Too much light will prevent you from seeing the grid. 7. Count cells using the 10x or 40x objective lens. DO NOT USE 100X OIL IMMERSION LENS. Use clicking counter to count the cells. 8. Count at least three of 0.04mm2 boxes and at least a total of 100 cells. Record the number of cells in each box you counted. 9. Calculate the concentration in the number of cells per mL for the starting culture. (total # cells) ÷ [(total # of 0.04mm2 boxes counted) x (4*10-6ml) x (dilution)] = cells/ml 10. Each group member should make a spread plate of the same serial dilution. Choose a serial dilution tube that would give you a viable count between 500-1000 cells/ml. 11. Make a spread plate with 100 µl of the chosen dilution. (See page 21 for instructions) Theodore Deleted 37 Note: Enumerating microorganisms using a hemocytometer takes practice so you should repeat this process a number of times. The more cells you count the more accurate your calculation of the cells per ml. Verify your numbers, you should get close to the same value each time you make the count. If the numbers you are coming up with from multiple counts are off by more than 10%-20% then there is likely a problem with the counts. If the counts are off, then restart the process by reloading the sampling to the hemocytometer. Here is a link to a couple YouTube videos of how to use a hemocytometer: https://www.youtube.com/watch?v=89SB1tAa6kg https://www.youtube.com/watch?v=vBJIy5TRQ2E Calculating stock concentration (cells/ml) using the hemocytometer: (average # cells per box) ÷ [(vol. of one of the boxes counted) x (dilution)] = cells/ml 1/25mm2 (0.04mm2) Hemocytometer box/square volume: Cover slip sits 0.1mm above the surface of the hemocytometer. 1cm = 10mm 1cm3 = 1mL Volume of each 0.0625 mm2 square (1/16): 0.0625 mm2 x 0.1 mm = 0.00625 mm3 0.00625 mm3 = 0.00000625 cm3 = 6.25x10-6 mL Volume of each 0.04 mm2 square (1/25): 0.04 mm2 x 0.1 mm = 0.004 mm3 0.004 mm3 = 0.000004 cm3 = 4x10-6 mL 1/16mm2 Volume of each 0.0025 mm2 square (1/400): 0.0025 mm2 x 0.1mm = 0.00025 mm3 0.00025 mm3 = 0.00000025 cm3 = 2.5x10-7 mL Notes: • Cells might be on the border of a square: count cells on two borders only, and on the same two sides for each square. Usually on the upper and left border (red) • The most accurate count will be obtained when between 100 and 200 cells are counted. Dilute the culture sample if counts for each box exceed 100. • Use a homogenous (completely mixed and vortexed) suspension of cells • Avoid trapped air-bubbles. • The rectangular boxes on the grid are not used for counting. 38 • To differentiate between live or dead cells, a number of stains can be used. Live cells possess an intact cellular membrane that can actively exclude certain dyes (like Trypan blue), whereas dead cells do not have such integrity and so are stained in a blue color. Review resource for using a hemocytometer: http://mathbench.umd.edu/modules/measurement_hemocytometers/page01.htm Part C – Examine the spread plates prepared in the previous period. Count the number of yeast colonies (colony forming units; cfu). From this number, and from the total dilution of the original suspension, calculate the number of yeast cells/ml (or cfu/ml) in the original culture, using the formula: (average # colonies) ÷ [(vol mLs plated) x (dilution)] = viable count (CFU) For example, if you plated 0.1 ml of the 1:105 (or 10-5) dilution and counted and average of 95 colonies, the viable count would be 95 ÷ (10-5 x 0.1), or 95 ÷ (1/100,000 x 1/10), which comes out to 9.50 x107 cells/ml, or cfu/ml. Questions 1) What is the accuracy of the direct and viable cell counts of the suspension? Is one method more precise enumerating cells in the starting cell suspension? 2) You dilute an original sample 1:30. You then count the number of yeast in the 1:30 dilution. You count an average of 7 cells in the 1/25 mm2 sized boxes. What is the concentration, in cells/ml, of the original sample? 3) You count the number of bacteria in 5 of the 1/400 mm2 small boxes of the central grid on the hemocytometer. Your results are: 12 cells in box #1, 17 cells in box #2, 17 cells in box #3, 14 cells in box #4, and 16 cells in box #5. You are counting a 1:10 dilution of the original sample. What is the concentration of the original culture (in cells/ml)? 4) You count 47 cfu on a spread plate. The plate was prepared by spreading 0.2 ml of a 1:10000 dilution of the original sample. What is was the concentration of the original culture (in cells/ml)? 5) You have a culture of yeast that is at a concentration of 6.74 x 106 cells/ml. You dilute the sample 1:100, and then 1:100 again, and finally you dilute the sample an additional 1:3. You add 0.1 ml of the final dilution to a spread plate. Assuming that most of the cells in the original culture were living, how many CFUs do you expect to count on your spread plate the next day? 6) What is the average size of a yeast cell? 39 Wet Mount Technique Yeasts are unicellular fungi that are spherical, ellipsoidal or oval in shape and usually do not form hyphae (fungal filaments) except in case of Candida. Baker’s yeast or Saccharomyces cerevisiae is the first microbe domesticated for human use and is a member of the group of Ascomycetes or sac fungi. Yeasts commonly reproduce asexually by budding, a process in which new cell (called a daughter cell) is formed by the parent cell as a bud. During sexual reproduction yeasts produce sexual spores (e.g. ascospores). The alcohol beverage (beers, wines, etc.) and bread-making industries are entirely dependent on fermentation by S. cerevisiae. Most commonly found yeast in healthy individual mouth and vaginal tract is Candida albicans. When normal flora of an individual is compromised, this can act as an opportunistic pathogen. Wet Mount Some samples can be placed directly under the microscope. However, many samples look better when placed in a drop of water on the microscope slide. This is known as a "wet mount" (Fig. 4). The water helps support the sample and it fills the space between the cover slip and the slide allowing light to pass easily through the slide, the sample, and the cover slip. No elaborate fixation or staining of sample is required. However, to aid visualization of cellular morphology simple stains like methylene blue, trypan blue or nigrosin are often used. Figure 4: Procedure: 1. Mark two circles on a clean slide with wax pencil. To one add few drops of water-iodine solution and to another methylene blue solution. 2. Add one or two drops of the original yeast culture provided to the slide. 3. Mount the coverslip and view under 10X and 40X objective lenses. 4. Note the shape, size and presence or absence of budding. Look for the small nucleus and large vacuole. Record and draw observations. 5. Calculate the average dimensions of a yeast cell. Measurement of yeast dimensions using ocular micrometer Prepare a wet mount as described above from any dilutions you have prepared to be able to view isolated cells under the microscope. Measure the length and width of at least 10 yeast cells (under the high-dry objective-40X) using the ocular micrometer in the eyepiece. In the micrometer, the distance between any two lines is 1 micrometer (mm) or 1 micron (1 µm) Observations: (Look also for the small nucleus, large vacuole and glycogen granules.) 40 Urban Microbiome Introduction: Microorganisms are present in almost every natural environment and they are often the most numerous type of organism. Until recently, studies of microbial diversity were limited to methods where only microorganisms whose growth could be detected in the lab in nutrient media could be identified, quantified and studied. It is estimated that only <10% of the microbial diversity in any given sample can be cultivated in the lab and detected in this way, therefore much of the microbial diversity has gone undetected. Metagenomic approaches have been introduced that detect a much greater portion of the actual diversity in a sample. Metagenomic approaches are culture-independent, which means that it is not necessary to cultivate the microorganisms to detect their presence. Instead, genomic DNA (or RNA in the case of some viruses) is isolated from the environmental sample being studied and using “signature” DNA sequences as a proxy to quantify and characterize the microorganisms present in the sample. The isolated DNA contains genomes representing, in principle, all of the microorganisms that were present in the sample (hence the prefix “meta”, in metagenomics). Identifying the Urban Microbiome Environmental samples Genomic DNA extraction PCR amplification of 16S rRNA gene Next generation sequencing Sequence data analysis Workflow for the analysis of bacterial communities. Adapted from Tringe and Rubin, 2005. Metagenomic approaches have now been used to analyze numerous natural environments across the globe as well as the microbiomes of humans and a variety of other organisms. More research is focused on microbial communities in the ocean and distant regions of the earth than the urban microbial community – despite the fact that a majority of the world’s population now resides in cities (Yergeau et al., 2010; Tringe et al., 2005). The metagenomic approach can be used to analyze any set of organisms from the environment, however, for the work in this lab we will begin by focusing our investigation on the bacterial members of the urban microbial community and leave viruses, fungi and other groups microorganisms to future studies. A specific region of each microbial genome is examined to focus on a subset of organisms– in the case of bacteria this is most often the gene coding for the 16S rRNA. The 16S rRNA gene is used because it is present in all bacteria and it has regions of sequence that are shared among all bacteria as well as variable regions in the gene that differ from species to species. 41 To investigate the urban bacterial communities of Brooklyn you will collect samples from the environment and prepare and analyze these samples. Each of your samples contains the genomic DNA from all of the organisms present. After collecting the environmental sample the DNA from the bacteria needs to be purified away from the bacterial cells and all the other unwanted dirt and debris that is collected. We will use universal PCR primers to amplify a variable region within the bacterial 16S rRNA gene. The universal primers are designed to be variable in several positions (that is, a given position in the sequence of the primer could be A, C, G or T for example, and the resulting primers will not all be identical in sequence) for the amplification of 16S rRNA gene from as broad a range as possible of the bacterial species represented in the sample. Each PCR sample will be visually examined by gel electrophoresis to confirm the correct band size and each sample will be quantified using a NanoDrop 2000 spectrophotometer. The company that sequences our DNA samples uses the Illumina MiSeq technology. The sequence data from the 16S rRNA genes can them be compared to a database to determine the bacterial diversity of the sample. Required background reading: 1. N. Pace review article: Mapping the Tree of Life: Progress and Prospects; Microbiology and Molecular Biology Reviews, Dec. 2009, p. 565–576 2. J. Handelsman review article: Metagenomics: Application of Genomics to Uncultured Microorganisms; Microbiology and Molecular Biology Reviews, Dec. 2004, p. 669–685 3. Microbiology; A Human Perspective, 6th edition, Nester et al., chapter 1 and chapter 11 (or equivalent chapters from other introductory microbiology texts). Recommended background reading: * These reading cover next generation sequencing technologies and metagenomics data analysis. They can be technical, but reading them to get the general sense of things can be very useful. 1. Logares, R., Haverkamp, T. H. A., Kumar, S. et. al.. (2012). Environmental microbiology through the lens of high-throughput DNA sequencing: Synopsis of current platforms and bioinformatics approaches. Journal of Microbiological Methods, 91(1), 106-113. 2. Metzker, M. L. (2010). Sequencing technologies the next generation. Nature Reviews Genetics, 11(1), 31-46. 3. Scholz, M. B., Lo, C., & Chain, P. S. G. (2012). Next generation sequencing and bioinformatic bottlenecks: The current state of metagenomic data analysis. Current Opinion in Biotechnology, 23(1), 9-15. 4. Shokralla, S., Spall, J. L., Gibson, J. F., & Hajibabaei, M. (2012). Next-generation sequencing technologies for environmental DNA research. Molecular Ecology, 21(8), 1794-1805. 42 Experiment A study of the bacterial diversity from urban soil samples collected from around Brooklyn. Procedure - Discuss as a class an overall research question to guide sampling locations. Collect soil samples from Brooklyn. Extract microbiome community DNA and sequence with Illumina MiSeq. *Students work in a group of 3 students (each bench island forms a group; 6 groups per Micro 3004 lab section) Questions 1) What is your group’s hypothesis for this experiment? 2) What are the controls for this experiment? What are key environmental data to collect that cannot be controlled between samples? Part 1: Experiment Setup 1. Collect exposed soil or sand avoiding roots, twigs, stones, and other debris. Using sterile tube or plastic bag. Label the containers with your sample location, group number/identifier, date, and lab instructor’s name. 2. Store at 4°C (refrigerator) until Part 2 & 3. Part 2: Bacteria Initial Viability 1. Using the lid of a sterile 1.7 ml microcentrifuge tube, transfer 2 lid full of soil from the container into a test tube with 9mL of sterile water & appropriately labeled. Flick tube vigorously to mix. The soil will settle to the bottom. Perform a 1:10 dilution: Transfer 1 ml of “dirty” water from the first test tube to a second test tube of 9 ml of sterile water (labeled 1:10). Flick tube vigorously to mix. 2. Spread plate 100 µl (0.1 ml) from your 2 tubes (undiluted and 1:10) on to a labeled Nutrient agar plate. Part 3: DNA Extraction 1. Using the lid of a sterile 1.7 ml microcentrifuge tube, transfer 2 lid full of soil from container into a labeled Power Soil powerbead tube. Gently tighten cap on the powerbead tube. This is step 1 of the DNA extraction protocol. 43 Follow the Qiagen PowerSoil kit (#12888) protocol for isolation of genomic DNA form your sample. The steps are copied here, essentially identical to those written in the Qiagen PowerSoil protocol. 1. Already completed: Transfer a cap full of soil to a Power Soil PowerBead tube. After your sample has been loaded into the PowerBead tube the next step is homogenization and lysis. The PowerBead tube contains a buffer that will (a) help disperse the debris particles, (b) begin to dissolve any humic acids that may be present and (c) protect the nucleic acids (genomic DNA) from degradation. 2. Gently vortex or invert PowerBead tubes to mix. 3. Add 60 µl of solution C1 to your PowerBead tube and invert several times to mix or vortex briefly. 4. Secure PowerBead tube horizontally using a vortex adapter. Vortex for 10 minutes at maximum speed This vortexing step is critical for complete homogenization and cell lysis. A combination of chemical agents from steps 1-4 and mechanical shaking lyse the cells. By randomly shaking the beads in the presence of disruption agents, collisions of the beads with the microbial cells will cause the cells to break open. 5. Place your PowerBead tube in the centrifuge; make sure your tube is balanced against another PowerBead tube (if there is not another group’s tube to balance with, then use a microcentrifuge tube containing water that weighs the same as your PowerBead tube). Centrifuge your tube at 10,000 x g for 30 seconds at room temperature. 6. Transfer the supernatant to a clean, labeled, 2 ml collection tube. Discard the PowerBead tube. Expect between 400-500 µl of supernatant at this step. The exact recovered volume depends on the absorbency of your starting material and is not critical for the procedure to be effective.. 7. Add 250 µl of solution C2 to the supernatant and vortex for 5 seconds. Incubate at 4°C for 5 minutes. 8. Centrifuge the tubes at room temperature for 1 minute at 10,000 x g. Make sure to face the hinge of the tube to the outside (away from the center of the rotor – this is so any pellet will collect at the bottom or side of the tube underneath the hinge). 9. Avoiding the pellet (there may not be a visible pellet), transfer up to 600 µl of the supernatant (liquid) to a clean 2 ml collection tube. 10. Add 200 µl of solution C3 and vortex briefly. Incubate at 4°C for 5 minutes. 11. Centrifuge the tubes at room temperature for 1 minute at 10,000 x g. 12. Transfer up to 750 µl of supernatant to a clean 2 ml collection tube. The pellet at this point contains non-DNA organic and inorganic material including cell debris and protein. For the best DNA yields and quality, avoid transferring any of the pellet to the collection tube. 44 13. Add 1.2 ml (2x 600 µl) of solution C4 to the supernatant. Be careful not to overflow the tube by keeping the pipette too much inside the tube, the whole volume will fit. Close tube and vortex for 5 seconds. 14. Load 675 µl of your DNA containing solution into a spin filter tube and centrifuge at 10,000 x g for 1 minute at room temperature. Remove the spin filter column and discard flow through from the collection tube – dump into a waste container or in the sink. Replace the spin filter column into the collection tube. A total of three loads for each sample are required. The DNA is selectively bound to the silica membrane in the spin filter device in the high salt solution. 15. Add 500 µl of solution C5 to the spin filter and centrifuge at 10,000 x g for 30 seconds at room temperature. 16. Discard the flow through from the collection tube as described above. 17. Centrifuge the spin filter at 10,000 x g for 1 minute at room temperature to remove any residual liquid from the filter or the sides of the filter. 18. Carefully place the dry spin filter in a clean, labeled, 2 ml collection tube. Avoid getting any residual C5 solution on the filter insert. 19. Add 100 µl of solution C6 to the center of the white filter membrane. 20. Centrifuge at room temperature for 30 seconds at 10,000 x g. 21. Discard the spin filter. The DNA is in the collection tube is now ready for downstream applications. 22. Store your DNA in the collection tube at 4°C. Questions 1) What type of conclusions can be made from initially culturing on nutrient agar (e.g., qualitative assessment, quantitative assessment, preliminary, estimate, descriptive, or bacterial diversity)? 2) What is the purpose of the ethanol during the DNA extraction? 3) Our primers target the 16S rRNA gene, are there other genes used for metagenomic sequencing? 45 Part 4: DNA quantification by NanoDrop Once you have eluted your final purified metagenomic DNA sample, quantify the sample using the NanoDrop and/or by running your sample on a 1% agarose gel. 1. In order to use the NanoDrop instrument you will need a P2 or P10 pipette and tips, a small volume of elution buffer and control lambda DNA of known concentration, in addition to your PowerSoil DNA sample. 2. Your instructor will demonstrate the use of the NanoDrop instrument. 3. Add 1µl of sterile water to the pedestal, move the arm into place, and the open again and use a Kim wipe to gently wipe away the drop of water. 4. Place 1µl of the appropriate blank (the same solution you eluted your DNA with – most likely C6 buffer from the PowerSoil kit) on the pedestal and close the arm. Blank the instrument. Then open the arm and gently wipe away the blank with a Kim wipe. 5. Place 1µl of a known concentration of lambda DNA control DNA or your sample on the pedestal and close the arm. Type in the control or sample name into the sample field on the computer. Now click on the “measure” icon to measure your sample. After measuring, lift the arm and wipe away your sample. 6. Repeat step 5 with any additional samples. Part 5: Sample Sequencing Sample DNA is sent for sequencing. Ideal quantity of DNA is at least 10ng/µL and quality of 1.8 (measured by the wavelength ratio 260/280, which indicates the absorbance of light from non-DNA elements such as: proteins, ethanol, and salts). Sequencing still requires a few weeks (mostly the lab facility sample que and about a day on the actual sequencing machine) to complete, therefore we will continue with our own validation procedures of PCR and gel electrophoresis. Part 6: PCR amplification Using universal primers that recognize conserved regions of the bacterial 16S rRNA gene you will amplify a variable region from the 16S rRNA gene. The DNA samples contain 16S rRNA genes from all the different bacteria present – therefore the PCR product will represent a mix of 16S rRNA sequences. The following list shows and the set-up for a 25 µl reaction (Qiagen HotStart kit (#203445)): Single 25 µL reaction Forward primer 1 µl Reverse primer 1 µl 2X master mix 12.5 µl dH2O 8.5 µl DNA Template 2 µl Prepare your template DNA for two PCR reactions**. 46 Reaction A – 1. 23 µl Master mix (HotStart 2x mix, water, and primers (Forward & Reverse)) 2. Use 2 µl of your undiluted sample as template Reaction B – Need to make a 1:10 dilution of sample DNA 1. Pipette 18 µl of sterile, molecular grade water into a clean microcentrifuge tube. 2. Add 2 µl of your isolated metagenomic DNA sample to the tube to make a 1:10 dilution of your template. Make sure your tube containing the 1:10 dilution is clearly labeled. 3. With diluted DNA set up second PCR reaction: a. 23 µl Master mix b. 2 µl of the 1:10 dilution of your DNA sample as template. *Remember that one of your PCR tubes will receive 2 µl of undiluted template DNA and the other tube will receive 2 µl of the 1:10 dilution of the template DNA that you prepared above. **Your instructor will work with you to prepare a positive and negative control PCR reaction to be used by the lab as a group. Your instructor will introduce you to the working of the thermal cycler. The thermal-cycler should be set as follows (according to the HotStart basic protocol): Step 1 Step 2 Step 3 Step 4 Step 5 Step 6 Step 7 Initial heat activation Denaturation/melting Annealing Extension Cycle to step 2 for 34 times Final extension Hold/Pause 15 min 30 sec 45 sec 45 sec 95°C 95°C 55°C 72°C 10 min Indefinitely 72°C 10°C On the graphical display you can watch the how the instruments runs through the cycles and an estimate of when your samples will have finished their run. After the PCR run, make sure your instructor has your samples and that they have been places in a box or rack for storage at -20C. 47 Part 7: Gel electrophoresis In order to check whether or not a PCR product of the expected size was amplified you will run a fraction of your PCR reactions on a 1% agarose gel to visualize the DNA bands that are present in your two PCR reactions. A 1% agarose gel was made as follows: 1. Make sure you have gloves and safety glasses on. 2. In a ~250 ml Erlenmeyer flask measure out 100 ml of 1x TAE buffer. 3. Weigh out 1 gram of agarose powder and add it to the Erlenmeyer flask with the buffer. 4. Carefully microwave the agarose in the buffer at ~70% power in a microwave for 2-4 minutes. Do this in 30 second increments to avoid having the liquid boil over. All the agarose powder should go into solution and the buffer should be clear with no agarose floating. 5. Allow the melted agarose to cool for 5 min. Then carefully add 2 drops of ethidium bromide (this is a carcinogen, so nitrile gloves must be worn). Gently swirl to mix. 6. Pour the melted agarose into the casting cassette (make sure it is balanced) and insert the appropriate comb to create the wells. 7. Let the agarose cool and gel for ~30 minutes. **The gel box and gel are arranged with the wells of the gel closest to the side with the negative electrode (black). DNA is negatively charged and will move towards the positive electrode (red). Procedure: Loading the Gel with PCR product – 1. In each 25 µL PCR reaction tube add 5 µL of loading dye. 2. Flick the tubes to mix the sample with the loading dye. 3. Load 10 µL of sample into assigned gel lane. The gel should be run at ~80-100 volts for 45-75 minutes. Make sure not to run the gel so long that the dye front runs off the gel or at such a high voltage that the gel box heats up. The higher the voltage will cause the DNA to move faster so the run time should be shorter. Smaller DNA sequences will move farther through the gel than larger sequences. When the gel has finished running, DNA bands can be seen using a UV light box (transilluminator). Check for clear ladder bands, no band in the negative lane and a band in the positive lane. Know that a reasonable amount of primer dimer (extra primers and when primers bind to themselves) will show up as a band at the smallest end of the ladder. Compare the size of any bands in your sample lanes with the size standard and record your results. Part 8: DNA sequence data analysis Your instructor will provide more information and details related to the data analysis component of the experiment on soil microbiome diversity 48 Cytological Techniques: Special Cell Stains Introduction: The following procedures are designed to illustrate various cytological structures and/or characteristics of microorganisms. Many of these techniques have diagnostic value in identifying bacteria from clinical samples. Your laboratory instructor may ask you to demonstrate one or more of these slides for grading. Keep your special staining slides in your drawer! Acid-fast Stain for Mycobacteria The cell wall structure of the mycobacteria is unique and distinct from both the typical Grampositive or Gram-negative cell wall. Mycobacteria have a cell wall similar to that of the thick peptidoglycan cell walls of Gram-positive bacteria, however, the outer most cell wall layer of mycobacteria is comprised of waxy mycolic acids. This outer later protects bacteria from drying out and inhibits the entry of many antimicrobial drugs. These bacteria do not stain well using the Gram stain method, but they can be stained with a fat-soluble red dye, basic fuchsin. The sample is decolorized using acidified alcohol, and the surrounding tissue is stained with water-soluble methylene blue. Acid-fast organisms appear bright red in color; Dyes: other bacteria and occasional epithelial cells in the smear appear - Basic fuchsin blue. Using the acid-fast staining technique allows these - Acidified alcohol pathogens to be detected, whereas other methods, such as Gram - Methylene blue staining, leave these bacteria uncolored. Mycobacterium tuberculosis (the cause of tuberculosis) and Mycobacterium leprae Cultures: Mycobacterium sp. (the cause of Hansen’s disease, also known as leprosy) are clinically important pathogenic species. Acid-fast Stain (Kinyoun method) 1. Obtain a heat-fixed smear of mycobacteria (this will be prepared for you). 2. Stain 5-10 minutes (without heating) in basic fuchsin solution 3. Tip the dye into the collection bowl. Rinse in warm tap water 4. Agitate your slide for 30-60 seconds in acidified alcohol 5. Tip the alcohol into the collection bowl. Rinse in cold water. 6. Counter stain 30-60 seconds in the methylene blue solution 7. Tip dye into the collection bowl. Rinse with water and carefully blot dry. 8. Examine your slide with 100x oil immersion objective -­‐ Remember to start at the 4x and move your way in magnification to the 100x objective Notes: Acid-fast organisms appear a bright red color; other bacteria and occasional epithelial cells in the sputum smear appear blue. 49 Endospore Stain Endospores are formed by some species of bacteria in response to harsh or adverse environmental conditions not suitable for growth. Endospores are dormant and undergo little or no metabolic activity until stimulated to germinate. The protective endospore is of diagnostic value in distinguishing members of the genera Bacillus and Clostridium from other non-endospore forming bacteria. Cultures of Bacillus sp. (Bacillus cereus, Bacillus brevis, or Bacillus lichenformis) will be available for spore staining, using the technique outlined in the next section. Dyes: - 5% aqueous malachite green - 0.5% Safranin Cultures: Bacillus sp. Procedure (Schaeffer-Fulton method) 1. Prepare a heat-fixed slide of the bacteria and place the slide over a boiling water bath. 2. Add a few drops of 5% aqueous malachite green to the smear, and cover with a piece of bibulous paper to reduce evaporation. 3. Leave the slide on top of the water bath for 10-15 minutes, adding more malachite green to the bibulous paper as needed. Do not allow the paper to dry. 4. Following heating, discard the paper form the slide surface and gently wash the slide with running water 5. Counter-stain the smear with 0.5% aqueous Safranin for 1 minute. 6. Tip the dye into the collection bowl. Rinse with water and carefully blot dry. 7. Examine your slide with the 100x oil immersion objective a. Remember to start at 4x and move you way up in magnification to the 100x objective. Notes: Vegetative cells stain pink/red in this procedure, whereas endospores stain green/blue. Some endospores may be seen within the former vegetative portion of the cell. ***Also prepare Gram stains of the endospore forming and the non-spore forming bacteria from the cultures provided for this exercise. Questions 1) Can everything on your slides be identified by Gram stain? Explain your answer. 2) Why was a basic solution then an acidic solution used for the Acid-fast stain? 3) What environmental conditions might trigger endospore formation? 4) Why do the stained endospores appear different from the vegetative cells? 50 Cytological Techniques: Bacteria Motility Flagella stain The flagella stain highlights the delicate fibers that provide force for the motility of bacteria. This procedure is actually not a stain, but rather a pattern of silver deposition on the bacterial surface and its flagella. Differences in distribution of flagella are of diagnostic value in identification of bacterial species. Washed cultures of several different bacteria (Staphylococcus, Escherichia, Proteus, and Pseudomonas) will be provided. Silver Nitrate Flagella Stain: Preparation of culture: A fresh agar slant having an appreciable Dyes: amount of condensation water at its base is inoculated with the test - 20% tannic acid bacteria and incubated at the optimal temperature for 18-24 hours. - 5% ammoniacal Afterwards, a new slant is inoculated with a loop of the condensation silver nitrate water. After another 18-24 hours of incubation, condensation water Cultures: with heavy growth is removed with a Pasteur pipette into a tube Proteus sp., containing sterile distilled water. The aqueous suspension of bacteria Pseudomonas sp., E. coli is incubated for 2-4 hours at the optimum temperature. After the incubation period, a loop of the bacterial suspension is transferred to one end of a clean microscope slide. Do not heat-fix or wash the preparation. It is recommended that you outline the area of the smear on the underside of the slide with your wax marking pencil to make it easier to locate the bacteria following staining. Flagella Stain Procedure 1. Add a loop of bacterial sample to one end of a clean slide and then tilt it to allow the drop to run down the slide. Allow the sample to air dry. Do not heat-fix. 2. Flood the slide with mordant (20% tannic acid), and place on a steaming water bath for 24 minutes. 3. Add more mordant as necessary to prevent drying of the slide. 4. Gently rinse the slide in tap water. Wipe excess mordant from the edges of the slide. 5. Flood with 5% ammoniacal silver nitrate solution and steam on the water bath for approximately 1 minute or until a "silver mirror" appears on the slide surface. Warning: the silver film should be only slightly reflective (if it is too dense then you will not see bacteria). 6. Rinse with tap water and blot dry. Notes: Scan the slide with the high-dry objective (40x) to find an area where the bacteria are clearly visible. Change to the oil immersion 100x objective and look for the flagella extending from the cell surface. ** You should also inoculate a tube of motility agar with a sample of each of the bacteria you are staining for flagella (see next page). 51 Motility Stab and Oxygen Requirement Motility test medium is a semisolid medium designed to detect bacterial motility. It is inoculated by stabbing with a needle. TTC (2,3,5, triphenyltetrazolium chloride) is added to the medium to make interpretation easier. Bacteria use TTC as an electron acceptor; in its oxidized form, TTC is colorless and soluble when reduced to formazin, it is red and insoluble. Growth of microorganisms capable of reducing TTC will appear as a red color along the stab line as well as in areas into which the cells have migrated. Cultures of Staphylococcus, Escherichia, Proteus, and/or Pseudomonas will be available. Motility Stab Procedure 1. Use a sterile needle to pick a well-isolated colony. 2. Stab the medium to within 1 cm of the bottom of the tube. a. Be sure to keep the needle in the same line it entered as it is removed from the medium. 3. Incubate at room temperature for 18hrs or until growth is evident. Notes: A red turbid area extending away from the line of inoculation indicates a positive motility test. A negative test is indicated by red growth along the inoculation line, but no further. Questions 1) Did you observe any flagellated bacteria on your slides? As accurately as you can, draw the organisms you observed. 2) Explain how the flagella stain works. 52 Eukaryotic Survey Introduction: In 1760, Carolus Linnaeus grouped all living organisms into plants (non-motile) and animals (motile). It took almost two centuries to develop the more appropriate five-kingdom classification proposed by Whittaker and Lynn Margulis in 1968. The kingdoms proposed were Monera (unicellular prokaryotes including bacteria and cyanobacteria), Protista (unicellular eukaryotes including protozoa and unicellular algae), Plantae (autotrophs), Fungi (saprotrophs) and Animalia (heterotrophs). With modern DNA-based technology such as PCR and rDNA (ribosomal DNA) sequence analyses the living world is currently classified as a three-domain system with Bacteria, Archaea and Eukarya; described by Carl Woese in 1990. In this exercise, we will examine representative types of eukaryotic microorganisms: fungi and protozoa. Algae will be studied later under photosynthetic organisms. Although some of the fungi-like, protozoa-like and plant-like organisms now have a different eukaryotic kingdom based on the rDNA genetic analyses and may be grouped into different super-kingdoms, we will classify them under broader class of fungi and protozoa based on their morphological and physiological types. Essentially, eukaryotes have a nuclear membrane and membrane-bound organelles which prokaryotes lack. Eukaryotes are also relatively larger and do not have a peptidoglycan cell wall. Procedure: Each student will make all the wet-mounts to visualize the given set of samples. Also the corresponding commercial or prepared slides for each sample will be available for comparison. (Refer to page 40 in the manual) 1. Suspend the sample in water if it is not already provided in a liquid culture. 2. Use a depression slide and coverslip as demonstrated by your instructor. 3. Place a drop of the sample in the center of the well of the depression slide. Handle the sample gently (no shaking or pipetting) to avoid disruption of any cellular structures. The structures are important to identify the organisms. 4. Take a clean coverslip and place one edge of the slide next to the droplet of sample. Slowly lower the coverslip. You may want to use a pencil or loop to lower the coverslip slowly. Avoid trapping air bubbles under the coverslip. If many air-bubbles are trapped it will obscure the viewing so prepare the slide again. 5. Observe the sample using your microscope under 10x or 40x objective lenses. 6. Prepare detailed drawings in your notebook of the specimens you observe. Question 1) What type of morphology, cellular structures, or arrangement did you observe in your samples? 53 Fungi Fungi are the heterotrophs that live on decomposing organic matter and are called saprophytes. Most are aerobic or facultative aerobes, grow terrestrial in slight moisture, with high humidity and acidic environment. Except Chytrids with flagellated gametes that have adapted to live in water, all fungi are non-motile and found in terrestrial habitat. Fungi are economically important as they are used in production of beer, wine, alcohol, bread (Saccharomyces cerevisiae), Sake (Aspergillus), cheese (for adding flavor), and antibiotics (Penicillium- antibacterial penicillin). There is also a symbiotic role for fungi as lichens (fungi and photosynthetic organisms) or mycorrhizae (symbiosis with plants). Morphologically fungi are grouped as filamentous or yeasts. Filamentous fungi (mold) have long branching cellular structure called hyphae that connects the two or more dividing cells and shares the cytoplasm. Several hyphae may form a mat called a mycelium. Yeasts are unicellular fungi with oval or spherical shape that replicate either by uneven (budding) or even (binary fission) cell division. Many pathogenic fungi are dimorphic and can grow generally either as yeast or mold. Most fungi replicate both sexually and asexually. Asexual reproduction can occur by fragmentation where a small portion of a colony is distributed (budding) or by asexual spore formation. Sexual reproduction occurs when two cells of opposite mating orientation fuse and form a diploid fruiting structure that differentiates to form sexual spores. Different types of sexual spores are the basis for classifying fungal groups: Divisions (for this course we focus on only 4 divisions) 1. Zygomycota form zygospores 2. Ascomycota form ascospores 3. Basidiomycota form basidiospores 4. Chytridiomycota form zoospores And the none taxonomic group of: Deuteromycota do not form any sexual spores. Zygomycota Zygomycota produce the sexual spores, zygospores from the fusion of hyphae of different mating types. A mature zygospore of Rhizopus is shown with harden cell wall representing the sexual stage. The vegetative or asexual stage is recognized by the asexual spores (sporangiospores) formed in the sporangium (dark-colored structures located at the tip) of the stem-like structure known as sporangiophore. NOTE: Examine prepared slides to observe both forms of reproduction Some Rhizopus spp. are opportunistic agents of human zygomycosis. They may cause serious (and often fatal) infections in humans and animals because of their rapid growth rate and growth at relatively high temperatures. Some species are plant pathogens (R. stolonifer, R. microspores); two are used in food fermentation (R. oligosporus, is used in www.olympusmicro.com www.allergy-details.com 54 the production of tempeh, a fermented food derived from soybeans; R. oryzae and S. cerevisiae are used in the production of sake, as one fungus reduces starch in rice to glucose, while the other ferments the glucose to alcohol). R. stolonifer, commonly called bread mold, appears as a white mycellial-growth with black sporangia and produces black spores. Ascomycota Saccharomyces cerevisiae, baker’s yeast, is a member of this group of fungi that is widely used in food industry. Record and draw observations. Lichens are also ascomycetes forming symbiotic relation with photosynthetic algae. These are sac fungi forming sexual ascospores (4 meiotic cells) in a pod-like structure called an ascus. Basidiomycota The Basidiomycetes include fungi that form fruiting bodies large enough to be visible to the naked eye. These fruiting structures are termed mushrooms (when edible) or toadstools (when poisonous). However the bulk of the living fungus resides as tiny mycelial threads spread through several cubic yards of soil. Fruiting organs of the basidiomycetes that are found near trees are probably closely associated with tree roots as ecto- or endo- mycorhizae (ex. morels & truffles). Other species are either associated with the roots of smaller plants such as grasses and bushes and therefore the fruiting bodies appear in outcroppings in open areas such as meadows (mushrooms such as Coprinus, Agaricus, Amanita, etc.). These species are likely to be degrading organic material in the soil, such as dead tree roots. In short, the basidiomycetes (and other fungi) play a large role in recovering organic matter locked in the tissues of dead organisms, and in increasing the fertility of soils overall. Coprinus is a basidiomycete, notice the stalk (or stipe), which supports the cap at the top of the mushroom. Beneath the cap are numerous gills which give rise to sexual basidiospore. The gills are seen in a cross-section through the fruiting body, under low magnification and enlarged. The fruiting structure is diploid, produced by the mating of haploid mycelia in the soil. Each basidiospore (sexual spore) is supported by a knoblike structure called basidium. NOTE: Examine prepared slides to observe gills, basidia, and basidiospores http://www.fungi4schools.org/Reprints/Photoset01/Coprinus_comatus_Shaggy_inkcap.jpg Coprinus: a) a large species of Coprinus, b) a cross-section of the fruiting body, c) higher magnification and differential staining shows the immature basidiospores. 55 Chytridiomycota Saprobic – degrade chitin or keratin Act as parasites *aquatic fungi Deuteromycotes (abstract group not used for taxonomic identities) The fungi of this group reproduce asexually, but they do not form sexual spores and hence often called fungi imperfecti. Aspergillus (Division Ascomycota) and Penicillium (Division Ascomycota) are common fungi representative of the group Deuteromycetes. Candida albicans (Division Ascomycota), the yeast is also a member of this group that causes vaginal infections. These organisms do not show cell-cell fusion and hence do not produce sexual zygospores. Instead they produce asexual spores by sporangia. http://fungalgenomes.org/blog/2008/03/riping-in-an-asexual-fungus/ http://faculty.ivytech.edu/~bsipe/MICRO/penicillium1.htm In Penicillium and Aspergillus, asexual reproductions is by formation of conidia (conidiospores) that derive from the specialized stalks known as conidiophores that remain attached via slimy projections called sterigma. The flask shaped fruiting body thus gets projected from conidiophores is known as phialide. Conidia are also called mitospores due to the way they are generated through the cellular process of mitosis. They are haploid cells genetically identical to the haploid parent, can develop into a new organism if conditions are favorable, and serve in biological dispersal. The spores are non-motile but the air currents typically carry the conidia to new colonization sites. The distinctive feature is that Penicillium display a brush-shaped, elongate and open phialide while Aspergillus display more like a single bunch or dens-fanshaped phialide attached via vesicle to the conidiophores. Penicillium is a fungus that is widely distributed in nature. Several Penicillium molds, including P. chrysogenum, P. roquefortti, and P. glaucum have blue or blue-green conidia. P. chrysogenum is the first species used to produce the antibiotic penicillin, while P. roquefortti is used to flavor Roquefort and Stilton cheeses, and P. glaucum is used to flavor Blue and Gorgonzola cheeses. Other Penicilliums have white conidia including P. camemberti and P. candida and are used in making of camembert and brie cheeses. P. marneffei, a mold with yellow conidia, is a human pathogen cause penicilliosis in HIV patients and prevalent in Southeast Asia. (http://www.wikipedia.org/penicilium) 56 Aspergillus is a genus of around 200 molds found throughout much of the nature worldwide and is common contaminants of starchy foods (such as bread and potatoes), as well as grows in or on many plants and trees. A. niger can be found growing on damp walls, as a major component of mildew. In Asian countries, alcoholic beverages such as Japanese sake are made from rice. First, A. oryzae (koji mold) converts the starch in the rice to sugars (saccharification), which then subsequently fermented by other microorganisms such as yeast (Saccharomyces) and/or lactic acid bacteria. A. niger ferments glucose to citric acid, and represents the main source of citric acid production as well as many commercial enzymes including those used in laundry detergents. A. terreus secretes a compound, and was the initial source of lovastatin (Lipitor), a cholesterolreducing drug. A. fumigatus, A. flavus, and sometimes, A. niger are human pathogens that cause lung and systemic infections called aspergillosis. A. parasiticus is a food-contaminating organism that produces the toxin and carcinogen, aflatoxin. In addition to the Deuteromycota, the Ascomycota, the Zygomycota, the Basidiomycota, there is also a close relative of the fungi, the Oomycetes. Saprolegnia is a member of the Oomycetes group referred to as water molds. They were once included under the fungi due to the similar morphology such as white threads emerging through decaying matter. Under current classification scheme they are grouped under slime molds and water molds. During its life cycle, it undergoes both asexual and sexual reproduction, the former by means of biflagellated zoospores. Members of Oomycestes are responsible for many plant diseases and rapidly get infected via motile zoospores (e.g. potato blight, mildew of grapes). NOTE: Examine prepared slides to observe the sexual and asexual reproductive stages. Examine living cultures for general growth characteristics, DO NOT OPEN PLATES. 57 Protozoa In keeping with the old terminology, the protozoa are a diverse group of unicellular eukaryotes found free-living in all kinds of habitats. They are consumers of living and decaying matter; some are parasites, others live symbiotically. Most protozoa possess a single nucleus, but some species may have two or more. They usually reproduce by splitting in two (binary fission), but sexual reproductive processes are also known to occur. Protozoa are further classified into groups based on the type of structures they used for locomotion (motility). Amoeboid protozoa use cytoplasmic projections called pseudopodia; flagellates use flagella; ciliates use cilia, or sporozoans if they lack any motility structures. Protozoa often form a dormant stage (e.g. cyst) resistant to adverse environmental conditions and many pathogenic protozoa have the ability to form cyst, allowing them to infect effectively. Some species of microorganisms are not grouped under Protozoa but have achieved similar modes of movement through convergent evolution (e.g. zoospores of Oomycetes-and the following). Physarum This organism is a slime mold. It has a complex life-cycle including a small amoeboid stage, large plasmodial stage, fruiting bodies containing spores and, under conditions of drying, a desiccation-resistant sclerotial stage. NOTE: Examine prepared slide for the detailed morphology of the plasmodium Sarcodina This group consists of amoeboid protozoa that use pseudopodia for locomotion. Examples include Amoeba (Entamoeba histolytica is the cause of amoebiasis) that do not have outer covering on the pseudopodia (naked) or have protein or mineral coating over the pseudopodia. The foraminiferans and radiolarians are amoeboid protozoa widespread in marine environment and contribute to the ocean sediments significantly from their external shells made up of either CaCO3 or silica, respectively. (Figure: http://spd.fotolog.com/photo/29/26/28/thechoice/1114406626_f.jpg) 58 Actinopodia The members of this phylum also use slender pseudopodia for locomotion. These organisms differ from the Foraminifera in the composition of the shells that surround them. In the Actinopodia, the shells are made of silica, the same material in glass. (Figure: http://io.uwinnipeg.ca/~simmons/16cm05/1116/16protis.html) Ciliophora Include ciliated protozoan that possess numerous cilia for locomotion. Paramecium is a unicellular organism found in freshwater throughout the world. The paramecium has a stiff outer covering that gives it a permanent slipper shape. It swims rapidly by coordinated wavelike beats of its many cilia: short, hair-like projections of the cell. The paramecium has an external oral groove lined with cilia and leading to a mouth pore and gullet; food is digested in food vacuoles. Paramecium can divide asexually by cell division called fission and can also undergo conjugation exchanging nuclear material between two cells. Tetrahymena A ciliated protozoan with an oral apparatus used for feeding on bacteria. The organism swims by means of rows of cilia arranged longitudinally over the surface of the organism. The organism has two nuclei in the cell that perform different functions. Several discoveries in cellular physiology were established by studying this organism. NOTE: Add a small drop of nigrosin to your wet-mount preparation to observe uptake of particles. Mastigophora This group includes the flagellated protozoa. Trichomonas vaginalis is an example causing vaginal infection and more potent trichomoniasis. Trypanosoma are another example of flagellates. Trypanosomes are microscopic, one-celled protozoans of the genus Trypanosoma, of which hundreds of species are known. A trypanosome is long, pointed and possesses a flagellum. The flagellum arises at the front, or anterior end of the parasite and curves back to form the edge of a long undulating membrane used in locomotion. T. gambiense and T. rhodesiense cause African sleeping sickness and both are transmitted by tsetse flies. (Figure: http://io.uwinnipeg.ca/~simmons/16cm05/1116/16protis.html, http://www.britannica.com/eb/art-55545) 59 Apicomplexa Members are referred to as sporozoans as they lack locomotion structures. Example is Plasmodium is the genus of responsible for malaria (caused by P. falciparum transmitted via by a female Anopheles mosquito vector) in humans and other animals. In humans, the parasite is found intracellularly in red blood cells and is used as diagnosis of malaria. NOTE: Look for the parasite within the red blood cells present in the smear in the prepared slide available for examination. Euglena Common flagellated protozoan and are found in nutrient-rich freshwater, except for a few marine species. The cells vary in length from around 20 to 300 µm, and are typically cylindrical, oval, pear or spindle-shaped with a single emergent flagellum for movement. There are usually many bright green chloroplasts, although some species are colorless. If sunlight is not available, it can absorb nutrients from decayed organic material. Euglena is also found in sewage systems. Thus euglena is unique like a plant carry out photosynthesis and like an animal has whippy flagellum to move through the water and survive on decaying matter. (Figure: http://water.me.vccs.edu/courses/env108/lesson2_3.htm) NOTE: Examine the prepared slide available and identify cell characteristics. 60 Streptomyces and Antibiosis Streptomyces is a bacterial genus of the Order Actinomycetales, members of which resemble fungi in their branching filamentous structure and are chiefly saprophytic (feed off decaying matter). A number of Streptomyces sp. produce antibiotics, and also play an important role as degraders of biopolymers, such as starch. Part A – Students will work in groups. In this exercise, you will attempt to isolate an antibioticproducing microorganism from soil and demonstrate its capacity to inhibit the growth of other microorganisms. Bacteria from the Gram-positive genus Streptomyces are commonly isolated from soil samples, and several species produce anti-microbial compounds. Procedure: 1. Add 1 gram of soil to 99 ml of water in a bottle for use as a stock suspension (~10-2). 2. Prepare two subsequent 1:100 serial dilutions (1:10,000 (10-4), and 1:1,000,000 (10-6)) by transferring 1 ml amounts to 99 ml water bottles. 3. Plate 0.1 ml of the 10-2, 10-4, and the 10-6 dilutions to starch agar plates (a total of 3 plates per group). 4. Incubate the plates at room temperature in your drawer. Part B – Procedure: 1. Examine the plates and look for colonies fitting the description of the genus Streptomyces. a. Look for leathery, raised, colonies that may be pigmented. b. The surface of the colony may look powdery, rough, or velvety. 2. Use your loop to suspend a bit of the colony in sterile water and Gram stain 2-3 loops of potential Streptomyces colonies (or take a sample from a prepared plate for comparison). Streptomyces should appear as Gram-positive cells growing in chains. 3. Obtain 2 NA plates and 2 tubes of molten agar. 4. Inoculate 3-4 loops (or 2-3 drops with Pasteur pipette) of Escherichia coli and Staphylococcus epidermidis into two separate 10 ml tubes of molten agar. 5. Pour into sterile NA plates, swirl until evenly dispersed and allow them to solidify on the bench while you select your putative Streptomyces colonies. a. **These are pour plates. Your instructor will demonstrate how to do this: inoculate, pour, tilt or swirl to evenly distribute agar. 61 6. Label the plates with the identity of the bacteria you added to the molten agar and with a crayon or Sharpie, divide each plate into two sections. 7. Select colonies typical of Streptomyces from the plates prepared last period. With your loop, cut out small disks of agar containing a colony and invert them on one section of each plate. You may use a scalpel to do this if you are unable to extract the sample with a loop. Remember to place the agar disk upside down on the plate of bacteria. Mark on the bottom of the plates (Unknown Streptomyces) where you placed your putative Streptomyces colony or colonies. 8. **Additionally, a Streptomyces plate will be provided as a control. 9. With your loop, cut out two small squares of agar containing a control Streptomyces colony and invert them on the remaining section of each plate. Label the bottom of this section, “Known Streptomyces”. 10. Place the plates in the container provided by your instructor. Part C – Examine the plates for evidence of antibiotic activity. Record your observations in a table. Questions 1) Why did bacteria such as Streptomyces species evolve toxins against other bacteria? 2) Describe the colonies you isolated and tested in the table you prepared (see above). Turn this table in with your questions. What evidence did you use to conclude that they might be Streptomyces species? 3) Did the colonies you isolated exhibit antibiosis or antimicrobial activity? Describe the results of your antibiosis tests. 62 Antibiotic Sensitivity Testing Antibiotics are compounds that are produced as secondary metabolites by certain groups of microorganisms, especially Streptomyces, Bacillus, and a few molds (Penicillium and Cephalosporium) that are inhabitants of soils. Antibiotics may have a bactericidal (killing) effect or a bacteriostatic (growth inhibitory) effect on a range of microbes. The range of bacteria or other microorganisms that are affected by a certain antibiotic is expressed as its spectrum of activity. Antibiotics that kill or inhibit a wide range of Gram-positive and/or Gram-negative bacteria are said to be broad spectrum. If effective mainly against a few Gram-positive or a few Gram-negative bacteria, they are narrow spectrum. Part A – Several techniques are available for testing antibiotic sensitivity of a particular microbial isolate. One of the easiest to perform is the Kirby-Bauer disk diffusion technique. This technique is carried out in microbiology laboratories in order to determine optimal antibiotic (or antibiotic concentration) therapy in treating a bacterial infection. It involves paper disks impregnated with known concentrations of a number of antibiotics and placed on top of bacterial plates. The plates are incubated to allow growth of the bacteria and time for the agent to diffuse into the agar. In order to determine if an antibiotic will be effective in treating the bacterial infection, the zone of inhibition must be measured and compared to a standard. If the zone of inhibition is smaller than the predetermined zone for that compound, the organism is considered resistant to that antibiotic. If it is within the range or larger, it is considered sensitive to the antibiotic being tested. Here is a link to a YouTube video: (http://www.youtube.com/watch?v=O5NwOGazOAA) Students will work in groups. 1. Prepare suspensions of 0.1 ml Escherichia coli and Staphylococcus epidermidis cultures in two separate 9.0 ml tubes of sterile water. Mix thoroughly by vortexing. Use sterile technique in obtaining bacteria from the stock culture test tube in which they are provided. 2. Using a sterilized cotton swab, wet the swab in the suspended bacteria and swab it across the entire surface of a Mueller-Hinton agar plate as demonstrated by the laboratory instructor. Cover in a uniform carpet, rotating 90 degrees until entire plate is inoculated. Divide each plate into four sections, or five if a blank disk is given. 3. Use alcohol-sterilized forceps to place the disks on the plates, and push them gently onto the surface to keep them from dropping off when the plate is inverted. Push them down into the agar with the forceps. a. You must use penicillin and ampicillin impregnated discs in your experiment, the other two antibiotics may be of your choice. b. You must use the same disks for the two different bacteria in order to compare the sensitivity of the organisms to the different types of antibiotics. c. Make sure the disk is in center of quadrant 4. Place the plates in the container provided by your instructor. 63 Part B – 1. Examine the plates and look for zones of inhibition around each of the disks. The presence of a zone indicates sensitivity to the antibiotic. 2. To define sensitivity, measure the diameter of the zone of inhibition and compare it to known diameters using the same organism and antibiotic to determine sensitivity or resistance (refer to the appendix). 3. Record the results in a table and turn it in to your instructor with the answers to the questions at the end of the exercise. Example: Ampicillin (A10): E. Coli - 16-22 mm S. aureus - 27-35 mm Questions 1) What is the difference between a bactericidal antibiotic and an antibiotic that is bacteriostatic? 2) Examine the zone of inhibition around each disk for the presence of micro colonies within the zone. What do these micro colonies represent? 3) Why didn’t plants evolve anti-microbial agents against all the microbes in their environment? 64 Zones of Clearing for Various Antibiotics Inhibition Zone - Diameter to Nearest mm Resistant Intermediate Susceptible Antibiotic (Antimicrobial Agent) Disc Code Amoxicillin (other) AMC Amoxicillin (Staph) AMC Ampicillin **Gram(-) rods and enterococci AM 11 12 - 13 14 Ampicillin ***Staphylococci and highly penicillin-sensitive organisms AM 20 21 - 28 29 Bacitracin B 8 9 - 12 13 Carbenicillin (other) CB 17 18 - 22 23 Carbenicillin (Pseudomonas) CB 13 14 - 16 17 Cephalothin CF 14 15 - 17 18 Cefoxitin FOX 14 15 - 17 18 Chloramphenicol C 12 13 - 17 18 Clindamycin CC-2 14 15 - 20 21 Ciprofloxacin CIP-5 15 16 - 20 21 Colistin CL 8 9 – 10 11 Doxycycline D 12 13 – 15 16 Enoxacin ENX 14 15 - 17 18 Erythromycin E 13 14 – 17 18 Gentamicin GM 12 13 – 14 18 Kanamycin K-30 13 14 - 17 18 Methicillin (Staph) M or DP 9 10 – 13 14 Oxacillin (Staph) OX 10 11 - 12 13 Oxacillin OX 17 18 – 24 25 Nalidixic Acid NA 13 14 – 18 19 Penicillin G (Enterococcus) P 14 15 Penicillin G (Staph) P 28 29 Streptomycin Sulfamethoxazoletrimethoprim Tetracyclin S-10 14 15 - 20 21 SXT 10 11 - 15 16 Te-30 14 15 - 18 19 Tobramycin NN-10 12 13 - 14 15 Vancomycin Va-30 9 10 - 11 12 http://delrio.dcccd.edu/jreynolds/microbiology/2421/lab_manual/KB_antibiotic.pdf 65 Bacterial Growth Curve Cellular respiration: Heterotrophic bacteria obtain their energy for cell growth and division by means of either respiration or fermentation. Both catabolic systems convert the chemical energy of organic molecules to high-energy bonds in adenosine triphosphate (ATP). In respiration, glucose is converted to ATP in three distinct phases: 1) glycolysis, 2) the tricarboxylic acid cycle (Krebs cycle), and 3) oxidative phosphorylation (sometimes called the electron transport chain, or ETC). Glycolysis splits the six-carbon glucose molecule into two pyruvate molecules, composed of 3 carbon molecules, with the production of ATP and reduced coenzymes. Krebs cycle is the complex pathway in which acetyl-CoA (from the conversion of pyruvate) is oxidized to CO2 and more coenzymes are reduced. ATP is also a product. Electron Transport Chain (ETC) is a series of oxidation-reduction reactions that receives electrons from the reduced coenzymes produced during glycolysis and the Krebs cycle. At the end of the ETC is an inorganic molecule called the terminal electron acceptor. When oxygen is the final electron acceptor, the respiration is aerobic. If the terminal electron acceptor is an inorganic molecule other than oxygen (e.g., sulfate or nitrate) the respiration is anaerobic. As cells grow and divide a population cycle develops. Cell division is by binary fission and at a constant rate depending upon the composition of the growth medium and the conditions of incubation. There are four characteristic phases of this growth cycle Cells/mL (Log Scale) Stationary Phase Logarithmic Phase Death Phase Lag Phase Time 66 Lag Phase: Period of little or no cell division. 1. Cells do not immediately reproduce in new medium. 2. The cell density remains temporarily unchanged. 3. Cells may be growing in volume or mass, synthesizing enzymes, proteins, RNA, etc., and increasing in metabolic activity. The length of the lag phase is dependent on a wide variety of factors including: • the size of the inoculum • time necessary to recover from physical damage or shock in the transfer • time required for synthesis of essential coenzymes or division factors • time required for synthesis of new (inducible) enzymes that are necessary to metabolize the substrates present in the medium. Log/exponential growth Phase: cells dividing à period of growth à increase logarithmically • Phase when cells are most active metabolically. • The rate of exponential growth of a bacterial culture is expressed as generation time, also the doubling time of the bacterial population. Generation time (G): defined as the duration time (t) of growth divided by the number of generations (n) G = t/n The number of generations (n) is calculated using the formula below where Nt is the number of cells at time (t) and No is the number of cells at the start of growth. Nt = No x 2n For example if the number of cells at time t is 256 and the number of cells at No is 8, then: 256 = 8 x 2n! 2n = 32 ! n = 5 If the time at Nt is 100 minutes, then G = 100/5, or a generation/doubling time of 20 minutes. Stationary Phase: When the number of deaths is equivalent to the number of new cells • The growth rate slows and there is no net change in cell density. • Population growth is limited by one of three factors: 1. Exhaustion of available nutrients 2. Accumulation of inhibitory metabolites or end products 3. Exhaustion of space: a closed system such as a test tube or flask. • Bacteria that produce secondary metabolites, such as antibiotics, do so during the stationary phase of the growth cycle. • During this stationary phase, spore-forming bacteria have to induce or unmask the activity of dozens of genes that may be involved in the sporulation process. Death Phase–When the number of deaths exceeds number of new cells formed 1. If incubation continues after the population reaches stationary phase, a death phase follows, in which the viable cell population declines. 2. During the death phase, the number of viable cells decreases geometrically (exponentially), essentially the reverse of growth during the log phase. 67 Procedure: You will follow the growth of E. coli over the course of the period (3 hrs) by making direct counts of the bacterial suspension by measuring the turbidity of a sample at a given time with a spectrophotometer. The data obtained from the direct counts will be used to plot a partial growth curve. Summary: Turbidity Counts with the Spectrophotometer to measure absorbance at 600. Direct Counts with a Hemocytometer and cells stained with crystal violet. 1. Turn on the spectrophotometer at least 15 minutes prior to use. Set the unit to “absorbance” and the wavelength to OD600. 2. The class is provided a 250 ml Erlenmeyer flask of with 190 ml nutrient media. Add 10ml of 40% glucose for a final concentration of 2% glucose and final volume of 200 ml. 3. Remove 10 ml of the growth media-glucose mixture from the flask and place into a separate spectrophotometer tube to use as a blank for the spectrophotometer (no bacteria). 4. Insert the media blank into the spectrophotometer and adjust the spectrophotometer dial to zero. **Do not adjust the dials on the spectrophotometer after you blank it or your future readings will be incorrect. 5. Add 10 ml of E. coli stock culture provided to the 190 ml media-glucose mixture for a 1:20 dilution and homogenize. 6. Remove 7 ml of the E. coli growth mixture from the flask, place in a spectrophotometer tube and immediately measure the OD600. This is the T=0 absorbance. 7. Add one drop of crystal violet to the spectrophotometer tube. (At about T3 first dilute the culture 1:100) Mix well and let sit for 5 min. Fill a hemocytometer counting chamber and let sit for 5 min. Count at least 100 cells and 3 of the smallest boxes. Record current direct count concentration = (Ave. cell count x 2.5*10-7)/Dilution. 8. Keep the flask containing the E. coli growth media-glucose mixture in the 37ºC orbital shaker. 9. Lab groups will trade off taking readings every 30 minutes for at least 3 hours. a. Each group should take a minimum of 1 reading. 10. Once the OD600 reading reaches 1.00, you must dilute the culture as follows to obtain accurate readings: 1 ml culture into 6 ml of media + glucose. Read the OD600 of the dilution and multiply the value by 7 to obtain an accurate OD600 reading. 68 Questions 1) Plot the OD600 readings and cell concentration for each time point on a scatterplot with a line connecting the points. Have the measured data (OD600 and cell concentration) on the y-axis against the time on the x-axis using Excel (or other computer based data/graphing program). It is helpful to set the y-axis on a log scale. 2) Can you recognize the different phases of the growth curve in your plot of the data? Label them on the graph. If not, what are possible reasons for difference in the growth curve you observed vs. what is expected? 3) Use the logarithmic phase of the growth curve to estimate the generation time (g units are hours/generation; also known as the doubling time) of E. coli. How does the generation time that you estimated compare to the published generation times for E. coli growing under optimal conditions? What are some of the possible reasons for differences in the generation times? What are possible sources of error in this exercise? 4) Plot the cell concentration (x-axis) vs. OD600 (y-axis). Determine the standard curve of your culture by fitting a regression line to the points or calculating the average slope between points. What is the future benefit of knowing the standard curve for this culture and being able to predict the cell concentration for any OD600? 69 Optimum Growth Temperature Microorganisms differ in the temperatures at which they grow optimally. Many microorganisms grow well at “normal” temperatures that are tolerated by humans, higher plants and animals. However, certain bacteria grow at temperatures (extreme heat or cold) at which few higher organisms can survive. Depending on their preferred temperature range, bacteria are divided into psychrophiles, mesophiles, thermophiles and extreme thermophiles. Part A – Cultures of the following organisms will be available. a. Pseudomonas fluorescens b. Bacillus stearothermophilus c. Serratia marcescens d. Saccharomyces cervisiae 1. Prepare 4 tubes of tryptic soy broth (TSB) for each of the bacteria (a through c) and 4 yeast extract-peptone-dextrose broth (YEPD) tubes of the yeast (d). 2. Incubate one tube of each set at each of the following temperatures: 4°C - 6°C (refrigerator) 20°C - 25°C (room temperature) 37°C (incubator) 55°C (incubator) 70 Part B – Record the relative amounts of growth in each culture, using the symbols, 0 = no growth through ++++ = heavy growth. Classify each organism according to its temperature range and optimum temperature for growth. Note any additional effects of temperature on the organism. Chart of Temperature Effect on Growth 4°C -6°C 20°C -25°C 37°C 55°C Pseudomonas fluorescens Bacillus stearothermophilus Serratia marcescens Saccharomyces cerevisiae Questions 1) What potential contamination hazards do psychrophiles pose? 2) In what ways might a microorganism's ability to grow at low or high temperatures be reflected in the structure or expression of various proteins? 3) How might microorganisms growing in the soil or water adapt to seasonal changes in temperature? 4) Analyze your data. What are your observations for each species? Write a summary of your data and submit it with the table to your instructor. 71 Fermentative Patterns of Gram-Negative Bacteria Introduction and Background: Members of the Enterobacteriaceae family are found in the gastrointestinal tract of animals, but many are also free living in soil and water. There are a number of Enterobacteriaceae that are important human pathogens, typically causing gastrointestinal illness (e.g., salmonellosis and shigellosis/bacterial dysentery). Since all of the family members look alike after Gram stain, other tests must be used to differentiate them. Characteristics of the Enterobacteriaceae Microscopic morphology: • They are Gram-negative rods and typically 1-5 microns in length. • They do not produce spores. • They have peritrichous flagella (exception: Klebsiella and Shigella are non-motile and have no flagella). Macroscopic colony morphology: □ Colonies are usually dome shaped, gray, and smooth. Oxygen requirement: □ They are facultative anaerobic bacteria. They can ferment or respire depending upon the level of oxygen available. Catalase activity: □ All members are catalase positive with exception of Shigella dysenteriae. Glucose fermentation: □ All members can ferment glucose to pyruvate (pyruvic acid), which is then converted to different end products depending upon the species. These end products can be used in identifying the species. Nitrate reduction: □ Most members reduce nitrate to nitrite. Some members can further reduce nitrite to nitrogen. Bacteria Fermentation: In contrast to respiration, fermentation is the metabolic process by which glucose acts as an electron donor and one or more of its organic products act as the final electron acceptor. Reduced carbon compounds in the form of acids and organic solvents, as well as CO2, are the typical end products of fermentation. The lactose fermentation ability of the members of Enterobacteriaceae is one of the key characteristics used in identification. Testing for lactose fermentation can distinguish between a lactose negative pathogen and the lactose positive Enterobacteriaceae. 72 Procedure: Part A – Inoculations Each group will perform the tests on the known bacteria, but each student will obtain one unknown for testing as an individual. Bacteria that will be provided as ‘known controls’: Enterobacteriaceae: Shigella sp., Salmonella sp., E. coli, Proteus spp. (choice between 2 species, most likely P. vulgaris and P. mirabilis), Enterobacter sp., & Klebsiella sp. Others (used as controls for some reactions in addition to Enterobacteriaceae): Pseudomonas putida & Bacillus sp. Experiment set-up: For each group: Label and inoculate the tubes or plates according to the procedures listed below. Unknowns: For each assay, divide a shared plate for the unknowns into three sectors and additionally label each sector with the group member’s initials and unknown’s number. Media - Tubes Glucose Lactose Clarks & Lubbs SIM tubes Nitrate Broth Citrate Slant # of tubes 5 5 5 6 5 5 Control Bacteria # Unknowns E. coli, Shigella 3 E. coli, Shigella 3 E. coli, Enterobacter 3 E. coli, Klebsiella, Proteus sp. 3 E. coli, Pseudomonas putida 3 E. coli, Klebsiella 3 Control Bacteria – # Unknowns # of Media - Plates divided equally onto divided equally onto plates 2 plates 1 plate All 6 Enterobacteria EMB 4 (Proteus alone 3 on center of 1 plate) HEK 3 All 6 Enterobacteria 3 MAC 3 All 6 Enterobacteria 3 E.coli, Bacillus Starch 2 None (on a single plate) EMB = Eosin Methylene Blue Agar; HE = Hektoen Enteric Agar; MAC = MacConkey Agar Inoculating tubes of liquid media 1. Label tube wall with group number and organism name. 2. Place a drop of culture into each tube using a Pasteur pipette or inoculating loop. Inoculating differential agar media plates 1. Label the plates with group number and organism name. 2. For each new organism, flame the loop and complete a single ‘isolation’ streak. Incubate all media at 37°C for 24 hours then at 4°C until the next lab. 73 Part B – Interpretation of Biochemical Assays Perform following tests on the cultures inoculated in the previous period. Consult Chapter 5 of your Photographic Atlas (Alexander and Strete) for details on the biochemical tests. Prepare a table in your notebook detailing the results of each biochemical assay. Procedure: MEDIA SIM Reagent ***Perform the motility and H2S observations first • Nitrate Broth 1. Clark & Lubb’s Broth ** perform using the same broth tube. Add Kovac’s Reagent and shake Indole Production 1. Add Nitrate Reagent 1 & 2 2. If no pink color, add powdered zinc Nitrate Reduction § Starch Agar 2. 3. 4. TEST NAME Cover surface of the entire plate with Lugol’s iodine Add a few drops of methyl red to culture Add 3 ml naphthol reagent Add 1 ml KOH-creatin Shake well Starch Utilization Methyl Red Vogues-Proskauer Check list of tests to be interpreted: - Carbohydrate fermentation (lactose) - EMB agar - MacConkey agar - Hektoen agar - Starch agar plates à Starch utilization - Glucose broth = Phenol red à Carbohydrate fermentation (glucose) and gas production - Lactose broth = Phenol red à Carbohydrate fermentation (lactose) and gas production - Clark & Lubb’s = Methyl Red Test à Acid end products from glucose fermentation - Clark & Lubb’s = Voges-Proskauer Test à Acetoin production from glucose degradation - SIM tubes à Motility, H2S production and Indole production - Nitrate broth tubes (see detailed description) à Nitrate reduction - Simmon’s citrate agar à Citrate utilization Questions 1) What type of bacteria are you looking for with these tests? 2) Where are the majority of these bacterial species found? 3) Would you be able to identify these species from each other by gram stain? Explain your answer. 74 Further Information: Biochemical Tests Carbohydrate Fermentation and Gas Production Phenol Red & Acid Fuchsin Broth Fermentation Tests are used to differentiate members of Enterobacteriaceae and to distinguish them from other Gram-negative rods. The medium is a basal recipe in which a fermentable carbohydrate is added (i.e., glucose, lactose etc.). Fermentation of glucose begins with the production of pyruvate, accomplished by glycolysis in most organisms. Pyruvate is then fermented further and results in the production of a variety of acids, alcohols and H2 or CO2 gases, depending on what organism is doing the fermenting. Phenol Red Broth includes peptone and the pH indicator phenol red (PR) in the basal media. PR indicates whether or not fermentation is occurring. PR is yellow below pH 6.8, pink above pH 7.4 and red in between. By-products of fermentation are acids; therefore, if the PR Broth remains yellow fermentation of a sugar is taking place. To test for gas production it is necessary to invert a Durham tube in the PR Broth. This tube captures any gas being produced by organisms in the broth, which is indicated by a bubble or pocket in the Durham tube. + Gas Production + Sugar Fermentation - Gas Production + Sugar Fermentation Result yellow broth bubble in tube yellow broth no bubble in tube red broth no bubble in tube pink broth no bubble in tube - Gas Production - Sugar Fermentation http://lactosesintolerances.blogspot.com/2013/04/h ome-test-for-lactose-intolerance.html Phenol Red Interpretation Symbol (A= acid or B = basic/G= gas) Fermentation with acid and gas end products A/G Fermentation with acid end products; no gas produced A/- No fermentation -/- Degradation of peptone; alkaline end products B/- Several selective and/or differential agars can be used to determine whether an organism is able to ferment lactose. The following text provides information on the agars used in this laboratory. (Plate images from: http://www.mc.maricopa.edu/~johnson/labtools/Dbiochem/3emb.jpg; http://www2.austin.cc.tx.us/microbugz/assets/images/HESalm.jpg) 75 Eosin Methylene Blue (EMB) Agar: Contains peptone, lactose, sucrose, and the dyes eosin Y and Methylene blue. It is selective because the dyes inhibit growth of Gram-positive bacteria. It is a differential media for bacteria that have enzymes to allow lactose fermentation; acid is produced and the change in pH will cause the colony to be colored. EMB agar is used for the isolation of fecal coliforms. Coliforms are members of the Enterobacteriaceae that produce acid (and gas) from lactose fermentation. Non coliforms are members of the Enterobacteriaceae that do not ferment lactose 1. Strong lactose fermenters are dark purple/green metallic (E. coli). 2. Slower lactose fermenters produce smaller amounts of acid (Enterobacter aerogenes: slow lactose fermenters) that results in a pink coloration of growth. Nonfermenters are colorless—they either retain their normal color or take on the coloration of the medium. Result Poor growth or no growth (P) EMB agar Interpretation Organism is inhibited by eosin and Methylene blue Good growth (G) Organism is not inhibited by eosin and Methylene blue Gram-negative Growth is pink (P) Organism ferments lactose with little acid production (A) Possible coliform Ex: Klebsiella (mucoid) Enterobacter Organism ferments lactose and/ or sucrose with much acid production (A) Probable coliform Ex: E. coli Organism does not ferment lactose or sucrose. No reaction (NR) Non-coliform Ex: Shigella Growth is “dark” (purple to black, with or without green metallic sheen) (D) Growth is “colorless” (no pink, purple, or metallic sheen) (C) Presumptive ID Gram-positive Further information about EMB: https://catalog.hardydiagnostics.com/cp_prod/Content/hugo/EMBAgar.htm Hektoen Enteric (HE) Agar: Hektoen Enteric agar (HE) is another selective/differential agar used for isolation and identification of Gram-negative bacteria that are found in feces (called enteric and/or coliform bacteria). HE agar is designed to specifically detect and differentiate between Salmonella and Shigella based on 1) the ability to utilize lactose, sucrose or salicin, and 2) the ability to reduce sodium thiosulfate to hydrogen sulfide gas (H2S). Ferric ammonium citrate, present in the media, reacts with H2S to form a black precipitate. Bromothymol blue and acid fuchsin dyes are added as colored pH indicators that detect acid production from the fermentation of sugars (differential characteristic). Bile salts are included to inhibit most Gram-positive bacteria (selective characteristic). 76 E. coli produces a yellow color because acid is an end product of its fermentation of lactose (It’s growth moderately inhibited). Shigella is also a lactose non-fermenter and is blue-green; it is not a sulfur reducer. Salmonella enterica does not ferment lactose but does produce a black precipitate from the reaction between ferric ammonium citrate in the medium and H2S from sulfur reduction. E. faecalis is inhibited by the bile salts. Further information about HE: https://catalog.hardydiagnostics.com/cp_prod/Content/hugo/HektoenEntericAgar.htm Escherichia coli Salmonella enterica Enterococcus faecalis Shigella flexneri Result HE agar Interpretation Yellow/orange colonies + Fermentation of Lactose Greenish blue colonies with black centers Greenish blue colony color + Reduction of Sodium Thiosulfate to H2S - Fermentation of Lactose - Fermentation of Lactose - Reduction of Sodium Thiosulfate to H2S MacConkey (MAC) Agar: MacConkey agar is a culture medium designed to grow Gram-negative bacteria and stain them for lactose fermentation. It contains bile salts, crystal violet dye (inhibits Gram-positive bacteria, selective characteristic), neutral red dye (stains microbes fermenting lactose), lactose and peptone. Neutral red dye is an indicator that is colorless above a pH of 6.8 and red at a pH below 6.8. Acid accumulation from lactose fermentation turns the dye red. By utilizing the lactose available in the medium, Lac+ bacteria such as E. coli and Klebsiella will produce acid, which lowers the pH of the agar below 6.8 and results in the appearance of red/pink colonies. (Note the precipitated bile salts around the E. coli.) Non-Lactose fermenting bacteria such as Salmonella and Shigella use peptone and excrete ammonia, which raises the pH of the agar. Non-lactose fermenting bacteria appear as white/colorless colonies or retain the color of the media. Result Pink Colonies Non-pink Colonies MAC agar Interpretation + Fermentation of Lactose - Fermentation of Lactose 77 Further information about MAC: https://catalog.hardydiagnostics.com/cp_prod/Content/hugo/MacConkeyAgar.htm Escherichia coli Enterobacter aerogenes Proteus mirabilis Shigella sonnei Glucose Degradation Clark & Lubb’s Broth: Clark and Lubb’s, also known as MR-VP Broth, is the combination medium used for both Methyl Red (MR) and Voges-Proskauer (VP) tests. It is a simple solution containing only peptone, glucose, and a phosphate buffer. The peptone and glucose provide protein and a fermentable carbohydrate while the potassium phosphate is included to resist pH changes in the medium. This media is designed to distinguish what pathway an organism uses to breakdown the main product of glycolysis, pyruvate. The Enterobacteriaceae can be further differentiated based upon other metabolic characteristics, including the end products of glucose fermentation. All members of Enterobacteriaceae can break down glucose into pyruvate. The two major pathways for further break down of the pyruvate are called the Mixed Acid (MR test) and the 2, 3-Butanediol pathways (VP test). Determination of these two kinds of metabolic patterns, as well as other tests described, have been put to practical use in the sanitary analysis of water supplies in which it is necessary to test for indicators of fecal contamination. For example, tests that detect low acid and acetoin will distinguish between non-fecal enteric bacteria (2, 3-butanediol fermenters, such as Enterobacter, Erwinia, and Serratia) from fecal enterics (mixed acid fermenters, such as E. coli, Salmonella and Shigella). **Klebsiella can use both pathways. 1) The Mixed Acid Pathway: Products are a mixture of lactic acid, acetic acid, formic acid, succinate and ethanol. If the bacterium possesses the enzyme formate dehydrogenase, which cleaves formate to generate the gases (CO2 and H2). Glucoseà Phosphoenolpyruvate (PEP)à Pyruvateà Lactate, CO2, H2, Ethanol, acetate, formate, succinate Note: End products are listed in order of abundance. Most formate is converted to H2 and CO2 gases. The amount of succinate is between acetate and formate, but is derived from PEP, not pyruvate. 78 Methyl Red Test The test is designed to detect the few enterics capable of overcoming the potassium phosphate buffer and lowering the pH through the mixed acid fermentation. The acids produced by these organisms tend to be stable, whereas acids produced by other organisms tend to be quickly converted to more neutral products. It is for this reason that media inoculated for this test is incubated for 5 days. After incubation, an aliquot of broth is removed and Methyl Red indicator is added. Methyl red is red at pH 4.4 and yellow at pH 6.2. Between these two pH values it is various shades of orange. Red color is the only true indication of a positive result; orange broth is considered negative or inconclusive. Yellow is negative. TIP: Use no more than five drops of MR (3 is fine). Result Methyl Red Interpretation Symbol Red Mixed acid fermentation + Shades of Orange Inconclusive - Yellow No mixed acid fermentation - No Color change No mixed acid fermentation - 2) The 2, 3-Butanediol Pathway: 2, 3-butanediol and ethanol are the main end products, but a small amount of acid is produced. Alpha-D-Glucoseà Pyruvate àAcetaldehydeà alpha-AcetolactateàAcetoinà 2, 3-butanediol Enough acid is released to detect a color change on a lactose fermentation test plate. This indicates lactose positive E. coli and Klebsiella pneumonia, additionally E. coli makes so much acid using the mixed acid pathway that the colonies are very dark and produce a green sheen. Voges-Proskauer Test This test identifies organisms able to produce acetoin from the degradation of glucose during a 2, 3 butanediol fermentation (non-fecal enterics). The Voges-Proskauer test was designed for organisms that are able to ferment glucose, but quickly convert their acid products to acetoin and 2, 3- butanediol. Addition of VP reagents to the medium oxidizes the acetoin to diacetyl, which in turn reacts with guanidine nuclei from peptone to produce a red color. A positive VP result, therefore, is red. No color change is negative. A copper color is a result of interactions between the reagents and should not be confused with the true red color of a positive result. Use of positive and negative controls for comparison is usually recommended. Result Voges-Proskauer Interpretation Symbol Red Copper No Color change 2,3 butanediol fermentation (acetoin produced) Reactions occurred between reagents No mixed acid fermentation (acetoin is not produced) + - 79 Motility, H2S Production and Indole Production SIM (Sulfide-Indole-Motility) Medium: SIM medium is used for determination of three bacterial activities: sulfur reduction, indole production (as opposed to tryptophan production), and motility. The semisolid medium includes casein and animal tissue as sources of amino acids, an iron-containing compound, and sulfur in the form of thiosulfate. SIM medium is used to identify bacteria that are capable of producing indole, using the enzyme tryptophanase. The Indole Test is one component of the IMViC battery of tests (Indole, Methyl red, Voges-Proskauer, and Citrate) used to differentiate the Enterobacteriaceae. SIM medium also is used to differentiate sulfur-reducing members of Enterobacteriaceae, especially members of the genera Salmonella, Francisella, and Proteus from the negative Morganella morganii and Providencia rettgeri. In addition to the first two functions of SIM, motility is an important differential characteristic of Enterobacteriaceae. Further information about SIM: https://catalog.hardydiagnostics.com/cp_prod/Content/hugo/SIMMedium.htm Note: When reading your test results, make the motility and H2S determinations before adding the indole reagent. Motility Motility determination in SIM medium is made possible by the reduced agar concentration and the method of inoculation. The medium is inoculated with a single stab from an inoculating needle. Motile organisms are able to move about in the semi-solid medium and can be detected by the radiating growth pattern extending outward in all directions from the central line. Growth that radiates in all directions and appears slightly fuzzy is an indication of motility. This should not be confused with the (seemingly) spreading growth produced by lateral movement of the inoculating needle when stabbing. Result Growth radiating outward from the stab line No radiating growth Motility Interpretation Motility Non-motile Symbol + - Sulfur Reduction Bacteria can accomplish sulfur reduction to H2S in two different ways depending on the enzymes present. • The enzyme cysteine desulfurase catalyzes the putrefaction of the amino acid cysteine to pyruvate. • The enzyme thiosulfate reductase catalyzes the reduction of sulfur (in the form of sulfate) at the end of the anaerobic respiratory electron transport chain. Both systems produce hydrogen sulfide (H2S) gas. When either reaction occurs in SIM medium, the H2S produced combines with iron, in the form of ferrous ammonium sulfate, to form ferric sulfide (FeS), a black precipitate. Any blackening of the medium is an indication of sulfur reduction and a positive test. No blackening of the medium indicates no sulfur reduction and a negative reaction. Result Black in medium No black in the medium Sulfur reduction Interpretation Sulfur reduction (H2S production) Sulfur is not reduced Symbol + 80 Indole Production Indole production in the medium is made possible by the presence of tryptophan (contained in casein and animal protein). Bacteria possessing the enzyme tryptophanase can hydrolyze tryptophan to pyruvate, ammonia (by deamination) and indole. The hydrolysis of tryptophan in SIM medium can be detected by the addition of Kovac’s reagent after a period of incubation. Kovac’s reagent contains dimethylaminobenzaldehyde (DMABA) and HCl dissolved in amyl alcohol. When a few drops of Kovac’s reagent are added to the tube, DMABA reacts with any indole present and produces a quinoidal compound that turns the reagent layer red. The formation of red color in the reagent layer indicates a positive reaction and the presence of tryptophanase. No red color is indole-negative. Result Indole Production Interpretation Symbol Red in the alcohol layer of Kovac’s reagent Tryptophan is broken down into indole & pyruvate + Reagent color is unchanged Tryptophan is not broken down into indole & pyruvate _ Nitrate Reduction Most nitrate-reducing bacteria (especially the enterics) contain the enzyme nitrate reductase and perform a single step reduction of nitrate (NO3) converting it to nitrite (NO2). Other bacteria, in a process known as de-nitrification, are believed to contain several other enzymes capable of reducing nitrate and its products all the way to molecular nitrogen (N2). Some other products of nitrate reduction include ammonia (NH3), nitric oxide (NO) and nitrous oxide (N2O). ammonium nitrate nitrite nitric oxide nitrous oxide nitrogen Nitrate Broth contains beef extract, peptone, and potassium nitrate (KNO3). In contrast to many differential media, no color indicators are included. The color reactions obtained in nitrate broth take place as a result of reactions between metabolic products and reagents added after incubation. Sulfanilic acid and alpha-naphthylamine (reagents A and B) are added to the medium to test for nitrate reduction to nitrite (NO-3 à NO2). • Nitrite, if present, will form nitrous acid (HNO2) in the aqueous medium. • Nitrous acid reacts with the added reagents to produce a red, water-soluble compound. Red color formation after the addition of reagents indicates that the organism reduced nitrate to nitrite. 81 If no color change takes place with the addition of reagents, the nitrate either was not reduced or was reduced to one of the other nitrogenous compounds (NO3 à N2 or NH4). Because it is visually impossible to tell the difference between these two occurrences, another test must be performed. The test needed to determine if nitrate (NO3) was not reduced or it was reduced to either nitrogen (N2) or ammonium (NH4) involves adding a small amount of powdered zinc to the broth. This addition of zinc will catalyze the reduction of any nitrate (NO3) present to nitrite (NO2). If nitrate (NO3) is present at the time of zinc addition, the above-described reaction between nitrous acid and reagents will follow and turn the medium red. Red color after the addition of zinc powder indicates that nitrate was not reduced by the organism. No color change after the addition of zinc indicates that the organism reduced the nitrate to NH3, NO, N2O, or some other non-gaseous nitrogenous compound. ***For figures showing the different results of this test, refer to A Photographic Atlas for the Microbiology Laboratory 4th Edition by Leboffe and Pierce pg. 84-85 Result Red color (after addition of reagents A and B) No color (after the addition of reagents) No color change (after addition of Zinc) Red color (after addition of zinc powder) Nitrate Reduction Interpretation Symbol Nitrate reduction to nitrite (NO3àNO2) + Incomplete test; requires the addition of zinc powder Nitrate reduction to non-gaseous nitrogenous compounds (NO3àNO2ànon gaseous nitrogenous products) No nitrate reduction + _ Starch Utilization/Hydrolysis Starch agar - Starch is a polysaccharide composed of repeating alpha-D-glucose subunits. Bacteria that produce the extracellular enzyme amylase break down starch into single subunits of alpha-D-glucose. These single subunits are transported into the cell, where they are broken down in cell respiration. Starch agar is used to test for the breakdown of starch by amylase. The medium contains beef extract and peptone to support growth, soluble starch and agar. An isolate is inoculated onto a plate with a sterile transfer loop. The plate is flooded with Gram’s iodine, which reacts with starch to produce a purple-blue color throughout the agar medium. Result Starch Utilization Interpretation Symbol Clearing in the medium The organism hydrolyzed the starch in the media providing a clearing + No Clearing in the medium None of the starch in the medium was hydrolyzed - 82 Bacillus subtilis Before Gram's Iodine Treatment After Gram's Iodine Treatment Escherichia coli Clearing (http://homepages.wmich.edu/~rossbach/bios312/LabProcedures/Starchpos.jpg) Citrate Utilization Test The citrate utilization test is used to determine the ability of an organism to use citrate as its sole carbon source. In many bacteria, citrate (citric acid) is produced by acetyl coenzyme A (from oxidation of pyruvic acid or the b-oxidation of fatty acids) that reacts with oxaloacetate at the entry to the Krebs cycle. Citrate is then converted through a complex series of reactions back to oxaloacetate, which begins the cycle anew. In a medium where citrate is the only available carbon source, bacteria that possess citratepermease can transport the molecules into the cell and produce pyruvate (pyruvic acid) by a reversal of the above-described reaction. Pyruvate can then be fermented to a variety of products depending on the pH environment. Simmon’s Citrate Agar This agar contains sodium citrate as the sole carbon source and ammonium phosphate as the sole nitrogen source. Bromothymol blue dye is the indicator used in this citrate test. Bromothymol blue is green at pH 6.9 and blue at pH 7.6. 83 Bacteria that survive in the medium and utilize the citrate also convert the ammonium phosphate ((NH4)2HPO4) to ammonia (NH3) and ammonium hydroxide (NH4OH). Both products tend to alkalinize the agar. As the pH goes up, the medium changes from green to blue. Thus, the conversion of the medium to blue is a positive citrate test result. Note: 1. Citrate utilization is an aerobic process. As a result, agar slants are used to increase the surface area exposed to air. 2. A green color can also occur with heavy growth, so be careful not to over inoculate. Result Blue (even a small amount) No color change; growth No color change; no growth Citrate Utilization Interpretation Citrate is utilized Citrate is not utilized Citrate is not utilized Symbol + - http://www.chemistrylearning.com/krebs-cycle/ 84 Nitrogen-Fixing Bacteria This process converts N2 in the atmosphere into NH3 (ammonia), which is assimilated into amino acids and proteins. It occurs in some free-living bacteria (Azotobacter, Clostridium, and cyanobacteria) in the soil and also in symbiotic bacteria within the roots of leguminous plants, the rhizobia bacteria (Rhizobium and Frankia), within characteristic nodules. These microorganisms are important in the nitrogen cycle, returning fixed nitrogen to the soil. In Azotobacter the nitrogenase enzyme responsible for nitrogen fixation is anaerobic, but the exceedingly high respiratory rate of the Azotobacter species consumes O2 so rapidly that an anaerobic environment is maintained inside the cell. Rhizobium live endosymbiotically with leguminous plants. These plants include clover, alfalfa, peas, peanuts and soybeans. The plant synthesizes the protein leghemogloben when infected with Rhizobium, which binds to O2 and depletes the levels of O2 in the nodule allowing the Rhizobium to fix nitrogen. Root nodules of Rhizobium http://biology.unm.edu/ccouncil/Biology_203/Images/Monera/rhizobium.jpg Rhizobium trifoli (micrograph: Frank Dazzo; photo: www.msu.edu/.../size_machine/rhizobium.gif) 1000 Isolated sample from white clover this summer microscopy, gram stained crushed root nodule. Rhizobia colonies are often slimy, due to synthesis of exopolysaccharide, and pigmented. Note the large size of the cells in the wet mount of an isolated colony. Both cysts (phase bright ovals) and vegetative cells (phase dark bacilli) are visible. 85 Procedure: Part A – 1. Select a large, firm nodule from a clover plant root, leaving a short piece of root on either side. Rinse root with tap water to remove soil. 2. Place the root nodule in a microfuge tube and immerse the nodule in 1 ml of 10% solution of sodium hypochlorite (bleach, a disinfectant). Close the cap and invert several times. Tap or shake the tube to make sure the entire root is washed. 3. Drain the bleach from the tube and replace it with sterile water. Do this 4 times. Do not dump out the root nodule! It is important to make sure that the bleach is completely removed from the root nodule. 4. Remove the root with sterile forceps and place on a glass slide. (Clean slide with ethanol first.) Crush the nodule between two clean glass slides. Do this on the bench-top to avoid breaking the slides. 5. With a small amount of the organisms extracted from the root nodule, prepare a streak plate on yeast extract-mannitol agar (YEM) and incubate until next week in your drawer. 6. Prepare a Gram stain of the organisms from the crushed root nodule. Draw the organisms in your notebook. Part B – 1. Select a single colony from the organisms derived from the crushed root nodule that grew on the YEM plate. 2. Prepare a Gram stain. 3. Draw Gram stain in your notebook. Record characteristic observations. 4. Compare the appearance of the microorganisms from the plate with those obtained last week directly from he crushed nodule. Are there any notable differences? Record your results. NOTE: The Rhizobia from the nodule are pleomorphic (may have multiple shapes or forms with an inconsistent Gram stain). The Rhizobia grown on a plate and stained will have the typical form of Gram-negative rods. Ø Further reading: Van Rensburg HJ, Hahn JS, Strijdom BW (1973) Morphological Development of Rizobium bacteroids in nodules of Arachis hypogaea L. Phytophylactica 5, 119-122 86 Questions 1) Describe the general appearance of the colonies on the YEM plates. 2) What are the microscopic and macroscopic morphological characteristics of Rhizobium? 3) Is there evidence of pigmentation in the colonies? 4) Identify and describe another beneficial plant-microbe alliance. 5) What benefit does the plant derive from the Rhizobium-clover alliance? 6) What benefit does the bacterium derive? 7) Describe your observations of the wet mount slides for Rhizobium. 87 Enzyme Induction in E. coli Introduction: Cells do not constantly synthesize all the proteins coded for in their DNA. In some cases a bacterium will synthesize an enzyme only if the substrate for that enzyme is present. This is called an inducible enzyme. A set of genes whose expression is coordinated by an operator is defined as an operon. The genes within an operon often work together to complete a complex task. Operons are frequent in prokaryotes and often are inducible or repressible (or both). In this exercise, you will study conditions affecting the induction of the Lac operon in E. coli by monitoring the activity of the enzyme β-galactosidase. The Lac operon codes for proteins required to transport lactose into the cell and break it down to glucose and galactose. The operon is activated in the presence of lactose (and low levels of glucose) and the β-galactosidase enzyme is synthesized following the induction of the lac operon. In order for bacteria to ferment lactose, they must possess two enzymes: lactose permease, a membrane- bound transport protein, and β-galactosidase, an intracellular enzyme that hydrolyzes the disaccharide lactose into the monosaccharides glucose and galactose. Bacteria that can synthesize both enzymes are active lactose fermenters. The compound o-nitrophenyl-β-D-galactopyranoside (ONPG) is a substrate analog of lactose and can be used to measure the induction of the lac operon. Because of its similarity to lactose, ONPG can become the substrate for any β-galactosidase enzyme present. In the reaction that occurs ONPG is hydrolyzed to galactose and o-nitrophenol (ONP), which is yellow. In this experiment ONP is the indicator used to show the presence of B -galactosidase. 88 In order for E. coli to start taking up and using lactose as its main energy supply, the Lac operon must be activated. Two types of transcriptional controls regulate the Lac operon: Positive and Negative. Positive Regulation - Occurs when the DNA-binding form of a protein works to turn a gene on. These proteins aid RNA polymerase in binding to the promoter region. CAP (Catabolite Activator Protein) is the protein responsible for turning the Lac operon on, leading to gene expression. CAP is used in bacteria to enable the use of alternative carbon sources in the absence of glucose. CAP is able to bind to the Lac operon when cyclic-adenosine monophosphate (cAMP) is present. cAMP binds to CAP and allows the protein to bind to the Lac operon. The levels of cAMP are dependent on whether or not glucose is present. If glucose is abundant cAMP levels are low; therefore, CAP is not in a DNA-binding form because cAMP is not bound to it. Catabolite Control of the Lac Operon The Lac operon is inducible by lactose to the highest levels when cAMP and CAP form a complex. 1. Under conditions of high glucose, a product of glucose breakdown inhibits the enzyme adenylate cyclase, preventing the conversion of ATP into cAMP. 2. Under conditions of low glucose, there is no product of glucose break down, and therefore adenylate cyclase is active and cAMP is formed. 3. When cAMP is present, it acts as an allosteric effector, complexing with CAP. 4. The cAMP CAP complex acts as an activator of lac operon transcription by binding to a region within the lac promoter. (Griffiths AJ, Gelbart WM, Miller JH, Lewontin RC. Modern Genetic Analysis. 2nd edition. New York. W.H. Freeman & Co., 2002) If glucose is abundant for the bacteria's use, it would be a waste of cellular energy for CAP to activate the Lac operon. That being the case, the Lac operon is not just controlled by CAP. Even if CAP is present, the negative control could still repress the gene. Negative Regulation - Occurs when the DNA-binding form of a protein works to turn a gene off. These proteins work to inhibit the binding of RNA polymerase to the operon. The Lac repressor protein is the protein responsible for inhibiting the expression of the Lac operon. The Lac repressor protein is able to bind to the Lac operon when lactose is absent. Allolactose is an isomer of lactose that binds to the Lac repressor protein and removes it. Removing the repressor protein is one of two necessary steps for the activation/transcription of the Lac operon. The other being the binding of CAP. The use of both the CAP and the Lac repressor protein allows for the Lac operon to be highly expressed when two conditions are met: lactose must be present and glucose must be absent. Carbon Source + Glucose + Lactose + Glucose - Lactose - Glucose - Lactose - Glucose + Lactose Activator Protein - CAP Repressor Protein - Lac Repressor Operon Expression Not Bound to Operon Not Bound to Operon OFF Not Bound to Operon Bound to Operon OFF Bound to Operon Bound to Operon OFF Bound to Operon Not Bound to Operon ON 89 Diagram of the of Dual of the LacofOperon: Diagram theControl dual control the Lac Operon: This Lac operon system results in a typical diauxic growth curve, a result of two different exponential growth phases, separated by a time when the culture does not grow. Escherichia coli grown in a medium containing a mixture of glucose and lactose will produce this type of growth curve. During the first few hours the bacteria divide exponentially, using the glucose as the carbon and energy source. When the glucose is used up, there is a brief lag period while the lac genes are switched on before the bacteria return to exponential growth, now using up the lactose. Example of a Diauxic Growth Curve http://rsif.royalsocietypublishing.org/content/5/Suppl_1/S29/F1.large.jpg 90 Procedure: In this exercise you will study conditions affecting the synthesis of the enzyme β-galactosidase in Escherichia coli. 1. Obtain four tubes, each containing 1 ml of a suspension of E. coli that has been grown in a glucose-containing medium, washed, and resuspended in non-nutrient buffer. 2. To the first tube add 0.1 ml of 1% yeast extract and 0.1 ml of 5% lactose. 3. To the second tube add 0.1 ml of 1% yeast extract and 0.1 ml of 5% glucose. 4. To the third tube add 0.1 ml of 1% yeast extract, 0.1 ml of 5% glucose, and 0.1 ml of 5% lactose. 5. To the fourth tube add 0.1 ml of 1% yeast extract, 0.1 ml of 1% glucose, and 0.1 ml of 5% lactose. 6. Mix, label the tubes appropriately, and incubate in a water bath at 37°C for 1 - 1.5 hours. 7. Add 3 drops of toluene to each tube and vortex vigorously. It will help to make the cells permeable to the ONPG substrate. Allow the tubes to stand at room temperature for 10 minutes. 8. Add 1 ml of 0.05% O-nitro-phenyl-β-galactoside (ONPG) and observe for 5-10 minutes. Record your results. Questions 1) What are the advantages to a microbe of having inducible enzymes? 2) What is the function of toluene in this experiment? 3) What are the advantages to a microbe as a consequence of such an arrangement of genes, and what is this arrangement called? 91 Human Microbiome Introduction: A healthy adult is colonized by trillions of microorganisms (bacteria and also human viruses, bacteriophage, and fungi). There are actually three microbial cells for every one human cell, and all these microorganisms add up to ~1.5-2.7 kilos in a healthy adult. These microorganisms constitute the normal, healthy human microflora, also referred to as the human microbiome. ***For more details visit the Human Microbiome Project website: http://www.hmpdacc.org/ ***For an interactive overview: explore-human-microbiome Bacteria are most numerous in the large intestine (the colon), but they can be found anywhere that is exposed to the outside environment. Bacteria heavily colonize the skin, oral cavity, scalp and nostrils. The human body contains many environmental niches that can vary in regards to the availability of water, salinity, temperature, pH and the presence of fats and oils. Microorganisms are generally well adapted to the niche they occupy on/in the human body. There is a great diversity of bacteria associated with a healthy human (~1,000 different species) although a smaller number of abundant types make up the majority of the bacterial population while much of the diversity is represented by relatively rare species. Background Reading: 1. The Human Microbiome Project Consortium. Structure, Function and Diversity of the Healthy Human Microbiome. Nature, 2012, June; 486:207-214. (http://www.nature.com/nature/journal/v486/n7402/full/nature11234.html) 2. Grice EA and Segre JA. The skin microbiome. Nat Rev Microbiol. 2011 Apr;9(4):244-53. (http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3535073/) Isolation of Commensal Bacteria The genus Staphylococcus is found on normal skin (external surfaces and in hair follicles). The bacteria are facultative anaerobes and chemoorganotrophic, requiring nutritionally rich media. The colonies are opaque and usually pearly white, but may have yellow-gold pigmentation. Clinical tests are used to differentiate the pathogenic Staphylococcus spp. from other Grampositive cocci. The pathogen most often encountered is S. aureus that causes various infections (e.g., carbuncles, osteomyelitis, bacterial pneumonia, wound infection, food poisoning, toxic shock syndrome). Additionally, S. epidermidis is a common commensal of the healthy skin, but can be an opportunistic pathogen, while S. saprophyticus can be isolated from the vagina and may be responsible for urinary tract infections in females. Each group of three students will be provided with: 3 nutrient agar (NA) plates and 1 Mannitol Salt Agar (MSA) plate Make sure you: Ø Invert your plates for incubation. The plates are generally moist and condensation can form if the lid is up (above the media), droplets may drip down onto the surface of the media. Ø Discard all used swabs in the red (biohazard) plastic waste containers provided in the work areas. 92 Part A – Procedure: With 2 NA plates perform the following steps. Forearm 1. Moisten a sterile cotton swab with dH2O (or swab buffer provided) and rub on the skin of your forearm. 2. Streak the swab across the entirety of a labeled NA plate. 3. Invert the plate and place it in a basket for incubation at 37 °C. Forehead 1. Moisten a sterile cotton swab with dH2O (or swab buffer provided) and rub vigorously the skin on your forehead. 2. Streak the swab across the entirety of a labeled NA plate and place it in a basket for incubation at 37 °C. With 1 NA plate and 1 MSA plate perform the following steps Nostril 1. Use a sterile cotton swab to sample from the nostrils of one member of the group. 2. Streak the swab across the last labeled NA plate and incubate with the other plates. 3. Repeat the procedure with a fresh swab and inoculate a MSA plate. 4. If possible, incubate plates at 37°C in an anaerobic environment (consult your lab instructor). To Gram stain bacteria collected from the biofilm on your teeth: 1. Using a sterile toothpick, carefully scrape the surface of your teeth to remove bacteria and plaque. 2. Spread the material from the toothpick onto a slide and let it air dry for several minutes. 3. Proceed with the Gram stain protocol as described previously. Notes: on the teeth the most common Gram-positive cells are streptococci (usually S. mutans) and the most common Gram-negative bacteria are from the genera Neisseria. You may also see cells from your cheek and gums on the slide. To Gram stain bacteria collected from skin: 1. Take a moistened sterile swab and swab a region of your skin thoroughly – for at least 30 seconds. 2. Place the swab end into a microcentrifuge tube containing ~250 µl (0.25 ml) of sterile water and twist and squeeze it around to release any bacteria into the water in the microcentrifuge tube. 3. Remove the swab and discard it in the biohazard waste. Now place the microcentrifuge tube into the centrifuge and spin at high speed (~13,000 rpm) for one minute. Make sure to balance the tube in the microcentrifuge tube in the rotor (ask your instructor). 93 4. Remove your tube and carefully dump the liquid into the sink. You should have a tiny pellet remaining in the bottom of the tube with a little bit of water. Flick your tube with your finger, or vortex, to resuspend the pellet in the small volume of water remaining. 5. Use a sterile loop or pipette to transfer the resuspended pellet material to a clean slide. Let the sample air dry on the slide. 6. Now continue with a standard Gram stain protocol. Examine your two slides under the microscope. Record what you see. Make sure you note the Gram property, the shape, size, grouping and relative abundance of the various bacterial types. Part B – Catalase Test: Bacterial cells produce hydrogen peroxide (H2O2) during aerobic respiration. If H2O2 accumulates in the cell, it becomes toxic. Therefore, most aerobic and facultative anaerobic bacteria possess an enzyme called catalase, which breaks down H2O2 to water and oxygen. Catalase positive bacteria, such as species of Staphylococcus and Micrococcus, can be identified with bubbly formation (O2) from catalase-negative species of Streptococcus and Enterococcus, which lack this enzyme. Procedure: 1. Select a colony from one of the NA plates. Record the colony description: morphology & color, relative abundance on plate. 2. Prepare a gram stain with half of the colony. 3. Clean a glass slide and dived it into 3 sections and label each section as ‘positive’, ‘negative’, and ‘unknown’. 4. Place a small drop of water on to each section of the slide. 5. With the other half of the selected colony place a small, thick smear onto the ‘unknown’ section of the slide. 6. Use a small portion of the positive and negative control cultures for the remaining sections on the slide. 7. Allow some of the liquid to air dry on the slide. 8. Place a drop of fresh 3% H2O2 on each of the smears. 9. Look for the production of gas. Record your results Ex. of catalase test slide: S. epid = positive culture, Strep. = negative culture, then unknown with positive reaction 94 Questions 1) Do your results support your hypothesis? Explain why or why not. 2) Can you tell how many different species of bacteria you are looking at in each sample? 3) What can you say about the teeth and skin (or wherever your second sample was taken from) in regards to the bacteria they host? 4) In what other ways could you address your hypothesis, assuming you were not limited by your skills and had access to whatever equipment and reagents you might need? 95 Microbes and Food Science Lactic Acid Bacteria Introduction: Microorganisms have been used for centuries in the preparation and preservation of various foods. The lactic acid formed from sugars in the food by lactic acid bacteria inhibits the growth of less tolerant microorganisms. The metabolic action of the bacteria on vegetable and dairy products results in such food preparations such as buttermilk, sour cream, pickles and sauerkraut. Important genera of lactic acid bacteria include: Streptococcus, Lactobacillus, Enterococcus, Lactococcus, and Leuconostoc. Some species are part of the normal flora of the human body and are found in the oral cavity, GI tract and vagina. Certain oral lactic acid bacteria are responsible for the formation of dental plaque and the initiation of dental caries (cavities). The initiation of cavities is caused by the extra acidity produced by these bacteria; it dissolves the calcium phosphate in tooth enamel leading to the start of a cavity. A major part of the lactic acid bacteria group is the previously mentioned Lactobacilli. They are gram positive, rod shaped organisms that grow as single cells or loosely associated chains. Lactobacillus varies from short slender rods to short coccobacilli. They are present in decomposing plant material, milk, and other dairy products, and are found in the microflora of the mouth and the healthy human vagina during childbearing years. Lactic acid fermentation is an anaerobic fermentation reaction, which can be categorized in two ways: The first being homolactic fermentation, in this reaction two molecules of lactic acid are produced from the conversion of one glucose molecule. The second being heterolactic fermentation, in this reaction carbon dioxide and ethanol as well as lactic acid are produced from glucose. The presence of lactic acid bacteria can be determined by their ability to form large amounts of acid. In addition, the formation of vitamins and other substances often increases the nutritional value of the food. The agar used in this lab is called Yeast Dextrose Calcium carbonate agar (YDC). This is a differential agar that determines whether or not the plated organism produces lactic acid. The CaCO3 in the YDC plates can be broken down to CO2 gas by lactic acid, producing a clearing. This clearing is the indicator for a lactic acid positive bacteria. The Durham Fermentation Tubes, used in Part B of this lab, are used to determine whether an organism is a homolactic fermenter or a heterolactic fermenter. The media is a basal media with a high glucose concentration and a pH indicator. The Durham tube is included in order to identify whether or not gas is produced from the metabolic activities of the organism in the media. 96 Part A – Students will work in groups. Fermented vegetable and milk products (eg. buttermilk) will be available for examination. Media: YDC plates (yeast, dextrose, calcium carbonate) 1. Using a marking pencil, divide the bottom of a YDC plate into four parts 2. Choose 4 fermented products 3. Streak the plate with the fermented products selected by your group. Demonstration: On a separate YDC plate, the lab instructor will place a drop of dilute HCl. Part B – Media: Glucose fermentation tubes with Durham tubes 1. Examine the YDC streak plates for the presence of colonies of lactic acid bacteria. 2. Select colonies from each fermented product and Gram stain. Note: Pay particular attention to those colonies that are surrounded by a clear area. After you Gram stain and test for catalase... 3. Obtain Durham Fermentation Tubes from the class instructor 4. Using aseptic technique, inoculate each tube with bacteria from an isolated colony from each of the fermented products on the YDC plate. 5. Incubate tubes at room temperature until next week Part C – 1. Examine the fermentation tubes inoculated last period and classify the organisms according to whether they are homolactic fermenters or heterolactic fermenters. 2. Record your observations of the bacteria examined by Gram staining. Give positive or negative results for each on the YDC plates and catalase test and describe the reactions observed in the fermentation tubes. Be sure to note the food source from which you isolated each organism. Product Organism Originated In Homofermentor Heterofermentor Questions 1) What is the function of the calcium carbonate added to the agar? What accounts for the clear area surrounding some of the colonies? 2) What is the predominant type of organism present in each of the foods? 97 Assessing the Prevalence of Antibiotic-Resistance in the Environment (PARE) Introduction: A team of four students will collect a soil sample. Each class or collection team will discuss an interesting site for sample collection. Is the site in an urban area, rural, near a factory, or near a waterway? Or would your class like to track changes at the same location over time (each team collects samples from the same general location each year)? The few published studies monitoring antibiotic-resistant bacteria in soil show vastly different frequencies, depending on collection location (zero detectable resistant colonies at some sites and up to 80% of total colonies resistant at other sites). Any information students obtain will be valuable to help understand the dynamics of antibiotic-resistance. Procedure: Part A – Sample Collection 1. Each collection team will collect a soil sample from their chosen collection site. These will be labeled with the team name; all data pertaining to the soil sample will later be linked to the team name in the database. Team member and team name should be recorded on the data sheet. 2. Review the Soil Collection Data Sheet at the end of this document (or provided by your instructor) prior to sample collection so that you will know what characteristics must be recorded. *Obtain permission for collection from private property. 3. Use a stick or rock at the sample site location (or the plastic collection tube itself, if using one) to loosen a sample of dirt about the size of an ice cream scoop and transfer it into the collection vessel (tube or bag) without touching the dirt (to avoid contaminating with bacteria on your hands). 98 4. Use a smartphone to capture latitude and longitude coordinates, preferably in decimal degrees format. (for example: lat 41.1509; lon. -73.1415). If latitude and longitude information cannot be captured, record the location information as accurately as possible. 5. Enter information for each indicated data field into the Soil Collection Data sheet while at the collection site. Do not lose this form; this information must be entered into the electronic database at a later date. 6. Label the outside of the tube or bag with the your team name and collection date. 7. Bring your sample to lab/class. Part B – Serial Dilution and Plating Our goal is to determine the percent tetracyclineresistant bacterial cells in each soil sample. To do this, we need to know the total number of cells present and, of those, how many are resistant to tetracycline. One standard method used for measuring microbes in soil is to assess the number of colony forming units (CFUs) per gram of soil. Counting the number of colonies that grow provides an estimate of the number of cells plated. The values obtained are estimates because cells that were dead or that could not grow under our particular growth conditions will not be detected. Mixing the soil in water separates the cells and allows us to perform incremental dilutions. Because there is a high, but unknown concentration of microbes in each soil sample, plating several different dilutions results in at least one plate with colonies separated enough for accurate counting. Each soil sample will be subjected to one round of serial dilution and at least 2 rounds of plating. Making at least 2 sets of plates from the same soil sample and serial dilution is called a technical replicate. Measuring the number of CFUs from a soil sample at least twice helps makes sure that the data you generate is accurate and helps you identify possible mistakes. If the two colony count results are not similar, we can assume that an error took place at some stage in the methods. 1. Complete a serial dilution for 10-1 to 10-6 a. Label each of the sterile tubes: 1/101 ;1/102 ; 1/103 ; 1/104 ; 1/105 ; 1/106 b. Use a sterile pipet to transfer 9 ml sterile water into each of the tubes. Measure 1g soil, preferably without rocks or any large debris. If a spatula is used to scoop soil, it should be sterilized with ethanol prior to each use. 99 c. Add the 1 g of soil to the 9 ml sterile water in the 1/10 dilution tube and seal the cap. This is the 1/10 dilution. (The dilution factor is 10.) Vortex for 1 minute. d. Use a sterile transfer pipet to transfer exactly 1ml of the 1/10 dilution into the tube labeled 1/102. Pipet up and down several times to mix well and without setting the pipet down, transfer 1ml of this dilution to the next tube to create the 1/103 dilution. 2. Plate each dilution No antibiotic plates Note: On the NA (no antibiotic) plates, you will plate only the 1/102-1/106 dilutions a. For each plating series, label 5 MacConkey agar plates (or other “no antibiotic” medium) with your team name, the plate type, the series code (U1, U2, H1, or H2) and the dilution for dilutions 1/102 through 1/106 . Experience indicates that the 1/10 dilution will likely have too many colonies to count. b. Use a sterile pipet to transfer 0.2 ml from the 1/106 dilution onto the 1/106 plate. Spread the liquid around evenly on the plate using a sterile spreader or sterile glass beads. Repeat for the other dilutions. Take care not to touch or contaminate the sterile items prior to use. Antibiotic plates Note: On the tetracycline plates, you will plate only the 1/10 – 1/103 dilutions a. For each plating series, label the three Tet3 (3 µg/ml tetracycline) plates with your team name, the plate type, series code (U1, U2, H1, or H2) and the dilution (1/10 through 1/103 ). Repeat for the three Tet30 (30 µg/ml tetracycline) plates. b. Spread 0.2 ml of each dilution onto the corresponding plate as directed in step 2. c. Wrap all plates with parafilm and incubate lid side down at 28°C for 72 hours. Incubating lid side down will prevent condensation from forming on your agar surface. d. Remove plates and record the incubation temperature and the duration of incubation. This information is required for the PARE database. Part C – Calculate the CFU per gram soil Soil contains an enormous diversity of species resulting in many different colony morphologies on the chosen medium. Determining the total number of colonies per plate will be challenging— even the tiniest colonies should be counted. Other colonies will be visible “under” larger colonies. Since each colony is derived from a cell that landed on the plate, each (regardless of size) must be counted to the best of your ability. The challenge of determining an accurate count is one reason that each sample was diluted and plated at least twice. Each plate series for your soil sample will be counted by two team members. Our goal is to determine what proportion of the total bacterial cells is resistant to tetracycline. We are using CFUs as a measure of total cells present, so determining the ratio of tetracycline resistant CFUs per gram of soil to the total CFUs per gram soil results in the frequency of tetracycline-resistant cells per gram soil. Some bacterial cells can grow in the presence of low levels of tetracycline but are inhibited at a higher concentration. In addition, a concentration that inhibits growth of one species may not have any detrimental effect on another. We tested, and 100 will report, the frequency of resistance at two different concentrations of tetracycline (3 µg/ml and 30 µg/ml). 1. For each plating series, refer to the number of colonies on the countable NA (without antibiotic) plate (Data Worksheet Table 1). Transfer this information to the first row of Table 2. 2. In the second row of Table 2, indicate the volume of cells plated onto each countable plate. If you followed the methods in this handout, you plated 0.2ml onto each plate. 3. Determine the dilution factor for the countable plates. The 1/10 dilution was diluted by a factor of 10, so the dilution factor is 10. The 1/102 dilution was diluted by a factor of 100, so the dilution factor is 100 and so on. If the most countable plate for a series resulted from the 1/100 dilution, the dilution factor for that plate would be 100. Enter this information in Table 2. 4. Use the formula below to calculate the total number of colonies per gram of soil for each series and enter into Table 2. Note, 1g = 1ml. A volume of 0.2 ml was plated, so we need to multiply by 5 (5 x 0.2ml = 1ml) to arrive at the number of cells per ml. The volume plated was also diluted relative to the original soil sample, so we also need to multiply by the dilution factor. CFUs on plate x 5 x dilution factor For example, if there are 210 colonies on the 1/103 dilution plate: 210 x 5 x 103 = 1,050,000 = 1.05 x 106 CFUs per gram soil Note: The database does not allow entry of exponential numbers; 1.05 x 106 should be entered as 1,050,000 or 1.05E6 Part D - Calculate Frequency of Tetracycline-Resistant Colonies 1. Fill in the appropriate values Table 3 (transfer values for the Tet3 and Tet30 plates in Table 1 to row 1 of Table 3). If no colonies appear on any of these plates, we can say that there are no detectable tetracycline-resistant colonies under our testing conditions. Tetracycline-resistant bacteria may be present, but perhaps we didn’t plate enough cells to detect them (they are present at a relatively low frequency) or they were unable to grow under our chosen growth conditions (perhaps a critical nutrient was missing). 2. For each concentration of tetracycline, calculate the tetR CFUs per gram of soil: CFUs on plate x 5 x dilution factor For example, if there are 160 colonies on the 1:102 dilution of the Tet3 plate (3 µg/ml tetracycline), that equates to 8 x 104 tetR cells per gram of soil. 160 x 5x 100 = 80,000 = 8.0 x 104 tet3R cells/gram soil 101 In Table 3, record the results for each concentration of tetracycline for all plating series. 3. Calculate the relative frequency (percent) of resistant cells as a function of the total number of CFUs/gram soil calculated in Section 6. Divide the total number of tetR CFUs per gram of soil by the total CFUs per gram of soil (on the non-antibiotic plate) to arrive at the frequency of tetR colonies. Multiplying by 100 results in the percent resistant cells. For example: 4. Record percent values in Table 3 of the Data Worksheet. Part E – Record Soil Data and Colony Count Results Into National Database 102 Photosynthetic Bacteria and Algae Introduction: Current classification defines five major groups of photosynthetic bacteria, four of which are anoxygenic phototrophs (green sulfur, green non-sulfur, purple sulfur, and purple non-sulfur). These organisms oxidize H2S or organic material in order to generate reducing power using sunlight. They do not produce O2 and are either obligate or facultative anaerobes and can grow in the presence of O2. Sulfur bacteria preferentially use H2S while non-sulfur bacteria utilize organic matter as their source of electrons for generating reducing power through NADH. The names green or purple reflect the kind of photosynthetic pigments they possess imparting color to the bacteria. There are also certain structural differences. Green-sulfur bacteria do not have flagella but have gas vacuoles that allow them to float at the surface, they also have extra-cellular S granules. Some purple-sulfur bacteria have flagella as well as gas vacuoles and the S-granules are stored intracellularly. The fifth group is of the cyanobacteria that are oxygenic phototrophs, they generate O2 and use water as their photosynthetic electron donor. They are considered as the primary producers that made the atmosphere change from anoxic (or reducing) to oxygenic. Thus, the current world is more facilitative for aerobic organisms. Members within cyanobacteria include: Merismopedia, Gloeocapsa, Anabaena, Nostoc, Oscillatoria and Lyngbya. Photosynthetic eukaryotic microorganisms include a large group of algae, specifically the green algae from which higher plants have emerged. The microscopic green algae include unicellular as well as various colonial, coccoid, and filamentous forms of flagellates (usually with two flagella per cell), that all contain chloroplasts. Genera within the green algae include: Euglena, Chlamydomonas, Volvox, Ulothrix, and Spirogyra. In this exercise, you will examine several representative types of cyanobacteria and the green algae for structural and functional organization. Individual students will utilize wet mount techniques learned earlier in this course to examine given set of samples. When making slides from cultures, be careful not to mix up the pipettes for taking samples from culture tubes. Wet-Mount Preparation Procedure: (Refer to page 40) The usual wet mount can be performed by putting a small drop of sample onto the clean slide in the center of a waxed circle using Pasteur pipette. Spread the drop evenly and place the cover slip gently over the drop avoiding any air-bubbles. 1. Use 40x or low-power 10x to visualize the samples. 2. Draw the cyanobacteria in large scale in your notebook. Pay attention to fine details. Be careful not to mix up the pipettes in taking samples from culture tubes. 3. After studying the living material, add a drop of nigrosin (negative stain) to the edge of the coverslip and allow it to diffuse across the wet-mount. This will accentuate appearance of certain features of the living cells. 103 Note: The diffusion can take 15-30 min to be able to visualize. If the wet-mount is already dried out during the observation prepare a fresh one and add nigrosin from the edges and allow it to diffuse. Do not put too much nigrosin, which will obscure all the visibility. Look for the difference between the cells that make up the filaments (called trichomes). Can you see nuclei and chloroplasts in any of the cells? Are all cells in the filaments identical to one another? Look for the presence of specialized nitrogen fixing cells, such as heterocysts, in some of the filaments. Questions 1) How are heterocysts adapted for nitrogen fixation? 2) Observe the filaments for an unusual type of movement termed gliding motility. What is the mechanism of this type of movement? 3) Make simple line drawings of the different bacteria and algae showing some of the basic features referred to in this description of the group. 4) For Gloeocapsa, what features can be seen in the nigrosin-stained preparation that was not visible in the unstained sample? 5) Name two microbial algae that are used as food. Name two macroscopic algae that are used as food. 104 Further Information Cyanobacteria The cyanobacteria deserve special emphasis because of their great ecological importance in the global carbon, oxygen and nitrogen cycles, as well as their evolutionary significance in relationship to plants. Chloroplasts (plastids) are formed by permanent enslavement of cyanobacteria in plants: land plants, green algae (e.g. chlamydomonas) and red algae. Of the 5 major groups of photosynthetic bacteria, only cyanobacteria are oxygenic during photosynthesis. They are often called blue-green algae, even though not all members are blue-green color and they are definitely not algae. Pigments Most cyanobacteria have a slimy sheath, or coating, which is often deeply pigmented, particularly in species that occur in terrestrial habitats. These pigments impart color to individual cells and colonies as well as to nuisance blooms of cyanobacteria that rise to the surface affecting the other aquatic life. The colors of different species include light gold, yellow, brown, red, green, blue, violet, and blue-black. Cyanobacteria utilize chlorophyll a, which is responsible for their green coloration. In addition, they have unusual accessory pigments called phycobilins, which absorb wavelengths of light for photosynthesis that are missed by chlorophyll and the carotenoids, thus giving bluegreen or reddish color to cyanobacteria. Morphology Most cyanobacteria have a Gram-negative type cell wall that consists of an outer membrane component, even though they may show a distant phylogenetic relationship with certain Gram-positive bacteria. The cyanobacteria are morphologically a heterogeneous mixture of bacteria. Some are unicellular, with typical prokaryotic shapes such as cocci rods, and spirals. Such species come in two forms depending on whether they divide by binary fission (unicellular) or multiple fission (colonial). In the latter, a mucilaginous sheath holds individual cells together to produce the colonial forms. Gloeocapsa, for example, secrete individual gelatinous sheaths which can often be seen as sheaths around a recently divided cell, two daughter cells, that remained together temporarily by outer sheaths or they generate mucilaginous sheaths around several cells (colonial). For Merismopedia, cell division is restricted to two directions resulting in a row-like arrangement of cells in flat colonies. The cells are held together by mucilage. In some species, cytokinesis (division of cytoplasm) is incomplete; thus the dividing cells remained attached through a filament. Such 105 filamentous multi-cellular associations are called trichomes and may or may not be enclosed within a sheath. Cell wall pores allow cells in a filament to communicate with each other, so neighboring cells are not completely separated. As in other filamentous or colonial bacteria, the cells of cyanobacteria may be joined by their walls or by slimy sheaths, but each cell is an independent unit of life. Further the filamentous forms are either branching or non-branching. In the former, a single cell divides in two directions forming a connected branch (Ex: Stigonematales). The nonbranching types are divided into two groups depending on the presence or absence of special nitrogen fixing heterocysts. Anabaena and Nostoc have heterocysts. Oscillatoria and Lyngbya do not. Motility Some of the filamentous cyanobacteria are motile by means of gliding or rotating around a longitudinal axis. The mechanism for this movement is unexplained but may be connected to the extrusion of slime through small pores in their cell wall, together with contractile waves in one of the surface layers of the wall. Habitat Cyanobacteria are found in most aerobic environments where water and light are available for growth. They inhabit a wide range of environments, including freshwater and marine habitats, soils, and the surfaces of rocks. They can also be found in the desert where they remain dormant most of the time, taking advantage of the occasional rains. In aquatic environments they often form thick mats. Like many bacteria they have a higher tolerance for heat and low pH than the green plants, so they are often the main autotrophs in hot springs. Cyanobacteria inhabiting the surface layers of water are part of a complex microbial community called plankton. They adjust their buoyancy by inflating or deflating gas vacuoles, enabling them to adjust their position in the water column, floating near the surface during the day for photosynthesis and sinking deeper at night to harvest nutrients. When numerous cyanobacteria become unable to regulate their gas vesicles properly (for example, because of extreme fluctuations of temperature or oxygen supply), they may float to the surface of a body of water—where they die and decay— forming nuisance blooms. Some cyanobacteria that form these blooms secrete poisonous substances that are toxic for animals that ingest large amounts of the contaminated water. Nitrogen Fixation Some cyanobacteria can fix nitrogen. We learned earlier this semester, that nitrogen fixers must avoid oxygen. Cyanobacteria are aerobic and regulate oxygen levels with specialized cells called heterocysts, which appear as enlarged segments in filamentous cyanobacteria. They are surrounded in a thickened, specialized glycolipid cell wall that slows the rate of diffusion of O2 into the cell. Any O2 that diffuses into the heterocyst is rapidly reduced by hydrogen, or is expelled through the cell wall. Heterocysts have intercellular connections to adjacent vegetative cells, and there is continuous movement of the products of nitrogen fixation moving from heterocysts to vegetative cells, and the products of photosynthesis moving from vegetative cells to heterocysts. 106 Algae There are about 6000 species of green algae; many species live most of their lives as single-cells, other species form colonies or long filaments. Algae employ simple reproductive structures and lack the extensive vascular structures characteristic of higher plants. Eukaryote organisms are capable of oxygenic photosynthesis. They are classified into different groups on the basis of morphology, types of chlorophylls, carbon reserve storage materials, cell wall composition, and habitat. Although many algae, like the higher plants, are non-motile, they may have motile reproductive cells. Pigments Although eukaryotic algae all have chlorophyll a pigments, giving them a bright green color. The algae are differentiated by their accessory pigments (such as chlorophylls b, c1, c2, or d, as well as a variety of carotenoid pigments) that catch photons of wavelengths to which chlorophyll a is not sensitive. The specific secondary pigments depend on the color of the light available in the organism’s local environment. Green algae that live at the surface of oceans and lakes use carotene (a yellow molecule) to trap purple and UV radiation. Farther down from the surface of the ocean, red (using phycobilins), brown (using fucoxanthin), and golden algae (using xanthophill) trap the blue-green light that penetrates to deeper levels of the oceans. All land plants appear to have evolved from green algae, perhaps because the pigment combination of green algae is better able to tolerate high light conditions and UV radiation and resist photo bleaching (the destruction of photosynthetic pigments by excess light). Diatoms Diatoms belong to a large group called the heterokonts, including both autotrophs (e.g. golden algae, kelp) and heterotrophs (e.g. water moulds). There are more than 200 genera of living diatoms, and it is estimated that there are approximately 100,000 extant species. Diatoms have two hard cell walls (called frustules) composed of silicon oxide. Their yellowish-brown chloroplasts contain pigments such as fucoxanthin. Diatoms are a widespread group and can be found in the oceans, in freshwater, in soils and on damp surfaces. Most live in open water, although some live as surface films at the water-sediment interface, or even under damp atmospheric conditions. They are especially important in oceans, where they are estimated to contribute up to 45% of the total oceanic primary production. Euglena Euglena is a flagellated cell with the same chlorophylls (a and b) as found in higher plants. Euglena is unusual for an alga in that it can lose its chloroplasts when kept in the dark or treated with the antibiotic streptomycin, but it is able to survive as a heterotroph. Thus, this organism has affinity to both algae and protozoa. For this reason, Euglena is claimed by botanists as an alga, but as a protozoan by zoologists. 107 Bacteriophage: T3 Introduction: Viruses that infect bacteria are called bacteriophages. These can interact with the host cell in two main ways: Lytic and Lysogenic. Lytic phage- Take over the metabolism of the host cell completely, resulting lysis of the cell. Steps of Infection: a. Attachment to the bacterial cell wall b. Injection of viral DNA into the bacterial cytoplasm. c. The viral genome then commands the cell to produce viral proteins, which are used for assembly of phages. d. Once assembly is complete, the cell lyses and releases the phages, which then attack other bacterial cells and begin the replication cycle anew. Example: T3 (infects E. coli) Temperate/Latent phage (Lysogenic cycle) - Live in harmony with the host cell and multiply as the host cell multiplies. Steps of Infection: 1. Attachment to the bacterial cell wall 2. Injection of viral DNA into the bacterial cytoplasm. 3. Viral nucleic acid becomes integrated into the host cell's chromosome - Takeover of cellular processes is not immediate 4. Replication of phage is delayed - This process is called lysogeny, which results in a prophage that can remain dormant for long periods of time 5. Under environmental stressful conditions the lysogenic prophage switches to the lytic mode to try and escape from the cell before cell death. Example: Lambda (infects E. coli) Life cycle of the typical temperate phage coliphage -λ Campbell, A., Nature Reviews Genetics 4, 471-477 (June 2003) Note: Stimuli that induce the switch from a lysogenic to a lytic phage include exposure to environmental stress factors like radiation and toxic chemicals. Thus, in the lytic cycle, infection by viral DNA leads directly to the multiplication of the virus and lysis of the host bacterial cell. In the lysogenic cycle, a prophage is replicated as part of the host's chromosome. 108 Lytic bacteriophage: Counting Plaques Lysis of bacterial cells growing on an agar plate produces a clearing that can be viewed with the naked eye. These clearings are called plaques. A plaque assay uses this phenomenon as a means of calculating the phage concentration in a given sample. When a sample of bacteriophage is added to a plate inoculated with enough bacterial host to produce a lawn of growth, the number of plaques formed can be used to calculate the original phage titer or density. The plaque assay technique is similar to the standard plate count, in that it employs a serial dilution to produce countable plates needed for later calculations. Diluted phage is added directly to a small amount of broth culture. Then this phage-host mixture is added to a tube of soft agar, mixed and poured onto prepared nutrient agar plates as an agar overlay. The consistency of the solidified soft agar is sufficient to immobilize the bacteria while allowing the smaller bacteriophages to diffuse short distances and infect surrounding cells. During incubation, the phage host produces a lawn of growth on the plate in which plaques appear where contiguous cells have been lysed by the virus. At suitable dilutions of phage particles, each plaque that forms represents the point at which a single phage particle was deposited. Each student will determine the number of phage particles/ml (or plaque forming units; pfu) of the suspension provided. Procedure: Part A – Each group will obtain the following materials: • 3 small tubes containing 3 ml of 0.7% nutrient agar (melted and kept at 50°C in a water bath) • 3 nutrient agar plates • 3x 9 ml sterile nutrient broth dilution blanks • 1ml pipettes • Suspension of phage T3 • E. coli suspended in nutrient broth 1. Prepare 1:10, 1:100, and 1:1,000 serial dilutions of the phage suspension in broth. 2. Add 1.0 ml of each dilution to separate tubes of 0.7% agar. 3. Add 0.1 ml of an E. coli suspension to each of the tubes of 0.7% agar. 4. Mix the contents of the tubes by swirling, and pour as an overlay onto the nutrient agar plates. 5. Allow the overlay to harden before moving the plates 6. Incubate them at room temperature in box provided by your instructor top facing up to avoid dislodging the soft agar overlay. 109 Part B – Enumeration of plaques: Bacteriophage counts are made by counting the number of plaques formed on host bacteria grown on agar. At suitable dilutions of phage particles, each plaque that forms represents the point at which a single phage particle was deposited. This is analogous to the plate count of bacteria where each colony formed represents a single bacterium in the initial inoculum. Each group will determine the number of phage particles/ml (or plaque forming units; pfu) of the suspension provided. Examine the plates prepared last period and determine the number of phage particles/ml of the original suspension, using the formula: Pfu/ml = (# of plaques) x (1/ml plated) x (1/dilution) Examples: If 162 PFUs were counted after inoculating a plate with 0.1 mL of phage suspension, diluted 1:109, the equation would be set up as follows. 162 pfu / 0.1ml x (1/10-9) = 1.62 x 1012 If 347 plaques were obtained when 0.1 mL of a 10-6 diluted phage suspension was plated, the equation would be set up as follows 347 pfu / 0.1ml x (1/10-6) = 3.47 x 109 Questions 1) Determine the number of phage particles/ml (or plaque forming units; pfu) of the suspension provided. 2) Can we grow bacteriophage on NA plates? Why or why not? 3) What causes the clear zones on the lawn of bacteria? 110 Water Quality Analysis Introduction: In New York City, the first public well was dug near Bowling Green in 1677. Almost a 100 years later (1776) the population had grown to 22,000 and the first reservoir was needed and built on the East side of Broadway between Pearl and White streets. As the demand on the water system grew the number of wells increased, eventually the supply was not enough and the water was getting polluted. The solution was to broaden the sources of water through drawing from the Catskill and Delaware watersheds. Currently, water for NYC is drawn from three upstate reservoir systems, which include 19 reservoirs and 3 controlled lakes that store 580 billion gallons of water. In 1989, the Total Coliform Rule was passed and set a both a health goal and legal limits for the presence of total coliforms in drinking water. This rule mandated regular monitoring (by the local government and the Environmental Protection Agency, EPA) of drinking water systems; the frequency of monitoring was set by the size of the population served (<1000 people = once/month, >2.5 million = ~420x/month) less than 5% of the samples could be positive. Most water-borne diseases result from transmission through fecal contamination. There are various pathogens, which may be present in low numbers compared to other bacteria making it difficult to specifically detect the pathogens during regular water quality monitoring. Therefore, indicator organisms are used– a typical coliform or group of coliforms as an indication of fecal contamination. The most commonly used organisms are Escherichia coli and Enterococci spp.. This is because they are 1) live longer than most pathogens, 2) more abundant, and 3) less risky to collect and culture. You will perform simple tests to determine the presence of indicator organisms in water samples provided to check the purity of water. The IDEXX Enterolert system is one detection and monitoring system that is recognized and approved by the EPA for water quality analysis. This method is based on the concept of Most Probable Number as a way to best estimate the amount of bacteria in a culture. Using proprietary media, IDEXX Enterolert selects for Enterococci spp.; when their cells are present they fluoresce blue under UV light. The IDEXX trays are specifically divided into small and large wells, the number and ratio of these cells that fluoresce blue can be used in a calculation to determine the MPN of Enterococci spp. in the original sample. For more information: http://www.nyc.gov/html/dep/html/drinking_water/history.shtml water.epa.gov/type/rsl/monitoring/vms511.cfm https://www.youtube.com/watch?v=LbfMa3BmD18 http://microbiol.org/wp-content/uploads/2010/12/Sutton.jvt_.16.3.pdf Procedure: Part A – Students work in lab groups and have aliquot of each sample Water Samples: Media: Drinking Fountain water 3 Lactose broth tubes with Durham tubes Environmental water 2 EMB agar plates 2 MacConkey agar plates 2 IDEXX media and tray (for the class) 111 1. Using a sterile tube, go to the campus pond (or other local water source) to collect a surface water sample (~ 5 ml). 2. Label lactose broth tubes with the water sample names. Inoculate with 0.1 ml of the water samples. a. Create a negative control with the third tube with 0.1 ml of Distilled water. 3. Label the EMB agar plates with Fountain water and Environmental water. Repeat for and the 2 MacConkey agar plates. 4. Spread plate 100 µl of each sample on their agar plate accordingly and incubate at 37ºC overnight. 5. Dissolve IDEXX media into 90 ml of distilled water in a flask. Repeat to have a flask for each sample. Label the flasks for each sample. 6. Add 10 ml of sample to IDEXX media flask. Mix well by swirling the flask. 7. Pour solution into IDEXX tray. Tap the bottom to remove air bubbles. 8. Align IDEXX tray wells with the rubber gasket. Place through the heat sealer with the larger well going through last. 9. Incubate IDEXX tray at 41ºC overnight. Part B – 1. Examine: a. the lactose broth tubes for fermentation of the sugar lactose. b. the EMB agar plate for the presence of coliform bacteria. c. the MacConkey plates for evidence of lactose fermentation. 2. Observe the IDEXX tray under UV light. Count the number of small wells then large wells that fluoresce. 3. Refer to the IDEXX Most Probable Number (MPN) table to find the number of cells in the tray. Multiply by the dilution factor (10) to determine the number of cells estimated to be in the original sample. 112 Questions 1) What is a coliform and where are they found abundantly? What makes them a good indicator organism? 2) Did you see any of the lactose broth tubes changing into pink or red color with gas in inverted tubes? If so, how would you interpret your results? 3) How would you interpret your observations with EMB and MacConkey agar plates? 4) Is the amount of bacteria detected using the IDEXX system within acceptable drinking or recreational water quality limits? 5) What are the most recent results for biological/bacterial contamination from the water quality monitoring of the New York City drinking water? 113 APPENDICES 114 List of Reagents Reagent Voges-Proskauer test ONPG 0.05% aqueous Dorner’s negative stain – used for capsule stain Crystal Violet for gram stain Silver Nitrate Stain Sudan Black for fat droplets (0.3%) Methylene Blue (1.5%) for Acid Fast Stain Mordant for Flagella Stain Safranin for Gram Stain Safranin for Spore stain and Fat droplet stain Malachite Green 5% for spore stain Methyl Red Basic Fuchsin for Acid Fast stain and Flagella stain Preparation - 40 %KOH and 0.3% Creatine - 5% Alpha Naphtol – 10 gms. Alpha naphtol in 200ml absolute alcohol - o-nitrophenyl-b-D-galactopypanoside dissolved in water - 10 % nigrosin (India ink) stain - 0.5% crystal violet - Solution A § 30 gm crystal violet § 400 ml ethanol (95%) - Solution B § 16gm Ammonium Oxalate o 1600ml water - When both are completely dissolved, add solution A to solution B and allow to age for 24 hours or longer before use. - 5% Silver nitrate - Add aqueous ammonium hydroxide until brown precipitate disappears - 0.3 gm Sudan black B (powdered) - 100ml Ethanol 70% - Let stand overnight - 01.5 gm Methylene blue - 100ml 95% ethanol - Use 30 ml 1.5% Methylene blue with 100ml 0.01% potassium hydroxide - 20% tannic acid – 20gm/100ml water - ferrous sulphate 15.6 gm/100ml water - Safranin O – 2.5% 2.5 gm - Ethanol (95%) 100ml - Dilute Safranin for Gram Stain (2.5%) 1:5 to make 0.5% safranin for spore stain and fat droplet stain. - Malachite green 5gm - Distilled water 100ml - 0.1gm methyl red dye - 300ml ethanol 95% - 200 ml distilled water 1) 3% basic fuchsin in 95% ethanol 2) Stir well and let sit for 1 week 115 Biochemical Table 116 Glossary of Important Terms Acid-fast: bacteria that retain dyes after decolorizing with acid alcohol. Acid-fast staining is typically used to identify Mycobacterium tuberculosis and other species of Mycobacteria. Agar: a complex polysaccharide that is commonly used as a solidifying agent in media plates. It is similar in function to gelatin, but agar’s benefits include remaining solid at temperatures reaching 80 ºC to 90 ºC and not being degraded by most microorganisms. It is purified from red algae (a type of seaweed). Aseptic: free from contamination caused by harmful bacteria, viruses, or other microorganisms. Bacillus (plural: bacilli): a rod-shaped bacterium. Batch culture: a closed culture containing a single batch of medium and inoculated with a microorganism. Coccobacillus (plural: coccobacilli): a stubby rod-shaped bacterium. Coccus (plural: cocci): the spherical shape of some bacterial cells. Colony: a cluster of microorganisms, generally originating from a single cell, that form a visible group on a solid media plate. Complex media: a culture media containing ingredients, such as yeast extract or beef extract, of unknown chemical composition. Continuous culture systems (bioreactors and chemostats): a culture system for growing microorganisms where environmental conditions are maintained, fresh media is added regularly and wastes are removed. It is generally used for large cultures grown for extended period of time. Defined media: a culture media with precisely know chemical composition. Differential media: media used in plates that distinguished between different microorganisms based of their growth and/or metabolic properties. Direct count: method for enumerating microorganisms by direct counting using a counting chamber (hemocytometer) and microscope. Enumerate: to count or listing off items one by one. In microbiology, it is often refer to a method that allows the quantity of bacteria to be measured. Fastidious bacteria: microorganisms that require special nutrient additions in media in order to grow. Fermentation: A metabolic process of bacteria and yeast that converts sugar to acids, gases, and/or alcohol. 117 Frank pathogen: A frank pathogen is a microorganism capable of producing disease in either normal healthy persons or immunocompromised persons. Frank pathogens – enteric viruses, Salmonella, Shigella, E. coli 0157:H7, Yersinia, Campylobacter, Vibrio, Helicobacter, Cryptosporidium, Giardia, Entamoeba histolytica, Cyclospora, Toxoplasma gondii, helminthes. Gram stain: a staining technique used to differentiate between Gram-positive (single membrane with thick outer peptidoglycan cell wall) and Gram-negative (inner and outer membrane layers with a thin peptidoglycan cell wall located in the periplasmic space between the inner and outer membranes) based on the differences in their cell envelope structure. Homogenous: A completely uniform mixture/solution. In the laboratory when using a solution of any kind, it is briefly vortexed to ensure it is completely mixed. Indicator organisms: are a basic monitoring tool used to measure both changes in environmental water quality or conditions, and the potential presence of hard-to-detect pathogenic organisms. Inoculate: to initiate the growth of a microbial culture by adding a small amount (inoculum) of the desired microorganism to be grown. Media: the liquid or solid nutrient source used for the growth of microorganisms. Niche: Smallest unit of a habitat that is occupied by an organism. A habitat niche is the physical space occupied by the organism; an ecological niche is the role the organism plays in the community of organisms found in the habitat. Nosocomial: Originating in a hospital. Often referring to hospital acquired infections. Opportunistic pathogen: a pathogen that takes advantage of certain situations when an individual has a compromised immune system—such as bacterial, viral, fungal or protozoan infections that usually do not cause disease in a healthy host, one with a healthy immune system. Pleomorphic: bacteria that vary in shape and lack any single distinct form. Saprophytic: to live on dead or decomposing matter. Secondary metabolite: Organic compounds that are not directly involved in the normal growth, development, or reproduction of an organism. Sometimes they are used as toxins to prevent predation or overcome competition. Selective media: a media that promotes the growth of the desired type, or types, of bacteria while inhibiting the growth of most other types of bacteria. Serial dilution: a set of dilutions increasing by regular steps (usually ten-fold) used to create a range of concentrations of the desired component (which may be living microorganisms from a culture). Smear: a sample of bacteria added to a slide and dispersed to aid in subsequent observation by microscopy. 118 Spirillum: a rigid, spiral-shaped, bacterium. Spirochete: a flexible, spiral-shaped, bacterium with periplasmic flagella. Spore: a minute, typically one-celled, reproductive unit capable of giving rise to a new individual without sexual fusion, characteristic of lower plants, fungi, and protozoans. In microbiology often is referring to a rounded resistant form adopted by a bacterial cell in adverse conditions. Staphylococcus: cocci-shaped bacteria organized in grape-like clusters. Sterile technique: A procedure is performed under sterile conditions to reduce the chances of contaminating samples or reagents with microorganisms present in the environment. Often this includes techniques and procedures, such as, flame sterilization, autoclaving materials/media, and working in a laminar airflow environment. Streptococcus: cocci-shaped bacteria growing in long attached chains. Symbiosis: Interaction between organisms; can be mutually beneficial (mutualism), one benefits at the other’s expense (parasitism/predation), or neither benefit (commensalism). Vegetative cell: a cell of a bacterium or unicellular alga that is actively growing rather than forming spores. Viable count: method for enumerating microorganisms by plating a sample for a living culture and then counting the colonies that grow from the individual microorganisms originally plated. This method enumerates only living microorganisms. Vibrio: slightly curved rod-shaped bacteria. 119

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