Introduction to Lab Ex. 19: Enumeration of Bacteria

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Introduction to Lab Ex. 19 Enumeration of
Bacteria - Pour Plate Technique
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Lab Ex. 19 Enumeration of Bacteria Pour Plate
Technique Bacterial growth may be determined
based on increase in the number of cells.
Reproduction and thus growth leads to increase
in cell number and thus the population of the
bacterial culture. Bacterial growth follows
specific patterns as seen in the growth curve
patterns for bacteria. The time it takes for a
population of bacterial cells to double in
number is called the generation
time. Bacterial growth may be determined by
using either Cell Mass methods or Cell Number
methods.
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Cell Mass methods Turbidity measurements by
Spectrophotometry Total Protein Total
DNA Cell Number methods Direct Microscopic
Counts (hemocytometer) Pour Plate Most
Probable Number Membrane Filter Enumeration
methods may yield either total counts or viable
counts. Total count is a count of cells including
dead and live cells. Viable count is a count of
only those cells that are alive in the sample.
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Pour Plate method This is the most commonly
used method for enumeration of bacteria in a
wide variety of samples including milk, food,
meat, soil etc. Pour plate methods yield a
count of only the living cells in the sample
and thus are a viable count. There are two
steps to the process dilution of the sample so
that various dilutions of the sample may be
inoculated onto plates and a count of the
colonies that grow made the second step is the
plating of the dilutions so that each cell in
the diluted sample may then grow and form
colonies that will in turn become visible to
the naked eye and can be counted.
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Dilution is important since the colonies will
have to be counted and with a concentrated
sample there may be too many colonies than can
be accurately counted. Plating is important
since a count of only the living cells is
required in this procedure (only living cells
will be able to multiply and form colonies)
Samples of milk, meat and soil will be used in
the exercise. Rules to keep in mind - only
living cells are counted. Why? - only plates
with colony numbers between 30-300 are
useable (lt30 or gt300 are not statistically
valid) - use aseptic technique - dilutions to
be done accurately
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Procedure - aseptically dilute sample with
sterile water blanks - make 110 dilutions as
instructed, based on sample - aseptically
transfer 1ml volume of dilutions that are to
be plated onto sterile Petri plates - pour
molten agar onto the sample in Petri dish and
mix thoroughly - incubate as instructed for
growth of colonies - during next lab period,
count colonies on plates - use the formula
cfu/ml of colonies x 1
dilution