Carbohydrate Fermentation Microbiology Lab Exercise #7 Dr. Carol G. Leggett 2 De Enathan Name Carbohydrates are a group of organic macromolecules that can be utilized for energy by living organisms. This group includes sugars, starches, and cellulose. Bacteria can be biochemically categorized according to which sugars they are capable of utilizing or fermenting. This laboratory exercise will require the inoculation of three different sugars; dextrose (glucose), sucrose, and lactose. We will also inoculate a starch agar plate to determine if your unknown bacterium is capable of producing amylase, an enzyme that can breakdown the starch molecule into simple sugar. Carbohydrate Fermentation - Lactose (+) (+) E, aerogenes Uninoculated Ps. aeruginosa P. vulgaris E. coli Photo courtesy of www.biosci.usc.edu This photograph demonstrates the color variation seen in carbohydrate fermentation tubes following inoculation. The first tube on the left is uninoculated and has an orange-red color. Inside the test tube is a smaller inverted tube known as a Durham tube. The Durham tube collects gas that is released during the fermentation process. These carbohydrate tubes are read according to their color and whether or not gas has been collected in the Durham tube. 1 OBSERVATION #1- Inoculation of Carbohydrate Tubes to Test for Fermentation and Gas Production Obtain 1 tube of lactose, sucrose and dextrose media. Each tube is a reddish color due to the addition of phenol red, which is a pH indicator. Below pH 6.6, phenol red is a bright yellow color. Between pH 6.6-8.0, phenol red is a red-orange color, and above pH 8.1, it is a bright the sugar is broken down, and sometimes gas is released. Inoculate each tube using your loop. Label each tube with your initials and place in a rack in the 37°C incubator. Volstodolat .con o mulyatoso 1 gmie on OBSERVATION #2 - Inoculation of a Starch Agar Plate Obtain a starch agar plate and label the bottom of the petri dish with your initials and the date. Using your loop, inoculate the center of the agar plate. Do not do a streak plate, just place the bacteria in a circle (about the size of a nickel) in the center of the plate, SEE DIAGRAM. Once inoculated, invert the agar plate and place in the incubator. Next lab, iodine will be poured onto the plate, if a clear halo is evident around the bacterial growth, then amylase was produced and broke down the starch surrounding the colonies. Place Bacteria Staphylococcus epidermidis Here Bacillus subnlis Starch Agar Plate oto courtesy of www.academic.pgcc.edu NAME: DATE: POST LAB QUESTIONS FOR MICROBIOLOGY EXERCISE #7 After viewing the carbohydrate tubes, indicate if your unknown fermented each sugar and whether or not gas was released in the process. acid 993 ACID GAS 1. Glucose 2. Sucrose || 111 3. Lactose Photo courtesy of http://student.ccbcmd.edu/courses/bio141/labmanua/lab8/prgas.html 4. What was the name of the pH indicator used in the tubes, and what color did it turn in an acid environment? 5. Did your unknown bacteria produce amylase? PRE LAB QUESTIONS FOR MICROBIOLOGY EXERCISE #8 1. What three sugars are in the TSI media? 2. What three tests are performed using SIM media? 3. A positive citrate test turns what color? 4. If hydrogen sulfide is produced by bacteria in a SIM tube, what color is seen in the media? 5. What are the two tests performed with MRVP media? 3
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