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Enumeration Of Bacteria Using Turbidity And Standard Plate Method

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Biology
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George Mason University
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Enumeration of Bacteria using Turbidity and Standard Plate Methods
Introduction
Investigative Purpose
This lab was conducted to find the number of colonies forming unit of the bacterial
sample in a given culture.
Technical Purpose
The technical goal of this lab was to practice the turbidity and standard plate methods of
enumerating bacteria, compare them, make serial dilutions, pour plate and make a standard
curve.
Hypothesis
It can be hypothesized that there will be a strong correlation between the absorbance of
the cultures and the number of bacteria CFUs in respective cultures.
Theoretical Background
In a culture, growth of microbes requires favorable conditions. For sustained growth of
organisms, additives or nutrients should be added. The most widely used nutrient-rich medium
for supporting bacteria growth is the Luria-Bertani. It exists both as ready-to-use liquid and
powder. Agar is usually added to LB to form a gel that forms sites for the growth of bacteria.
The bacteria are not in a position to digest agar but instead obtain their nutrition of LB.
The rate of population growth of bacteria is exponential. This means that every fission
cycles the population increase is by a factor 2. The time required for completion of a fission
cycle is known as doubling time or generation. In a normal growth curve, there are four phases.
These include the lag phase, exponential growth phase, stationary phase, and death phase.

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Several methods are used in the measurement of growth of microbes. These methods include
standard plate count (SPC), filtration, most probable number method, direct microscopic count,
flow-cytometry and indirect methods of estimation e.g. the turbidity method. The focus of this
lab is on the turbidity method and standard plate count (SPC).
In SPC, the counts of bacteria or microbes are reported as colony-forming units (CFUs).
A CFU is an entity that can form a colony on an agar plate. As opposed to a single bacterium, it
forms from short segments of a chain or bacterial clump. As a result of uncertainty determining
the actual number of cells forming a colony, this makes it necessary to report the counts as CFU.
To obtain the right number of CFUs, serial dilution is employed to dilute the original inoculum.
The bacteria are spread on a plate surface and then counts are performed.
In the turbidity method, instead of directly counting cell numbers, the change in turbidity
that occurs as the number of bacteria increases is easily used to monitor the growth of cultures.
The advantage of this method is that it is a simple and most rapid way of checking bacteria
growth. A standard curve is made for each bacterial species is made by comparing actual cell
numbers to optical density readings. The cell number of an unknown culture can be estimated by
determining its turbidity. Turbidity is a measure of cloudiness done using a spectrophotometer.
Materials and Methods
Materials and Equipment
Nutrient Agar
Erlenmeyer Flasks
Bacteria culture
Nutrient broth

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Enumeration of Bacteria using Turbidity and Standard Plate Methods Introduction Investigative Purpose This lab was conducted to find the number of colonies forming unit of the bacterial sample in a given culture. Technical Purpose The technical goal of this lab was to practice the turbidity and standard plate methods of enumerating bacteria, compare them, make serial dilutions, pour plate and make a standard curve. Hypothesis It can be hypothesized that there will be a strong correlation between the absorbance of the cultures and the number of bacteria CFUs in respective cultures. Theoretical Background In a culture, growth of microbes requires favorable conditions. For sustained growth of organisms, additives or nutrients should be added. The most widely used nutrient-rich medium for supporting bacteria growth is the Luria-Bertani. It exists both as ready-to-use liquid and powder. Agar is usually added to LB to form a gel that forms sites for the growth of bacteria. The bacteria are not in a position to digest agar but instead obtain their nutrition of LB. The rate of population growth of bacteria is exponential. This means that every fission cycles the population increase is by a factor 2. The time required for completion of a fission cycle is known as doubling time or generation. In a normal growth curve, there are four phases. These include the lag phase, exponential growth phase, stationary phase, and death phase. Several methods are used in the measurement of growth o ...
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