Introduction to Lab Ex. 20: Enumeration of Bacteria -

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Introduction to Lab Ex. 20 Enumeration of
Bacteria - Most Probable Number
method Membrane Filter method
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Water Quality

• Microbes are filtered from water that percolates
  into groundwater.
• Some pathogens are transmitted to human in
  drinking and recreational water.
• Resistant chemicals may be concentrated in the
  aquatic food chain.
• Mercury is metabolized by certain bacteria into a
  soluble compound, concentrated in animals

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Waterborne Diseases
Table 27.2
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Most Probable Number Membrane Filter
methods These are methods used to enumerate the
numbers of bacteria in water samples. The
Most Probable Number method is used to check
potability (if water is safe enough to be
drinking water) of water. The MPN method looks
for the presence of potential pathogenic
bacteria that may be in the water due to fecal
contamination of the water supply. Water
supplies are generally derived from ground
sources and have to be checked for safety
levels of bacterial contamination.
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MPN method enumerates the enteric bacteria called
coliforms, specifically fecal coliforms (E.
coli) Coliforms are Gram negative bacilli that
have the ability to ferment lactose with the
production of acid and gas. Fecal coliforms are
those coliforms that are normally found in the
feces of warm blooded animals (including
humans) MPN method thus enumerates the fecal
coliforms in water samples. E. coli is thus
used as an indicator organism.
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MPN test includes 3 levels of testing
Presumptive, Confirmed, Completed. The
presumptive test looks for presence of fecal
coliforms in the water sample by inoculating
lactose broths with the water sample. Those
tubes that show presence of acid and gas are
scored and those with no acid/gas as -.
Three sets of lactose broths are inoculated
with varying dilutions of the sample first
set of 3 or 5 tubes inoculated with 10ml of
sample second set of tubes inoculated with
1ml of sample third set of tubes inoculated
with 0.1ml of sample.
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The combination of positives in the 3 sets is
used to figure out the MPN /100ml of water
using the table provided. The tubes that show
positive in the presumptive test should be
confirmed to contain E.coli. This done in the
confirmed test using the selective/differential
medium EMB (that uniquely highlights E.coli
growth on it). The completed test is done only
where legal issues are involved wherein the
bacterial culture is then identified by a full
complement of tests including gram stain to
show that it is indeed E.coli
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Bacteriological analysis of water Most
Probable Number (MPN) technique
coliform acid and gas from lactose lt24
hours/370C indicator organism E. coli
  Water double strength single strength
single strength sample 10
ml sample 1.0 ml sample 0.1 ml
sample   Normally 5 Durham tubes are inoculated
but this exercise is modified to three tubes in
the interest of economy.
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Multiple-Tube Method
Figure 6.18a
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Multiple-Tube Method
Figure 6.18b
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Bacteriological analysis of water Membrane
filter technique
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