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CE 170: Environmental Engineering
Background Notes on Measuring Microbes (E. Dammel and J. Johnston)
Total Coliform Measurement
Total coliform bacteria are defined as bacteria capable of fermenting lactose in the
presence of lauryl sulfate (a mix often referred to as lauryl triptose). The fermentation of
lactose is a common biochemical process, that takes place in the intestines of warmblooded animals. The lauryl sulfate serves to mimic the presence of bile salts found in
the intestinal tract of all warm-blooded animals. Thus, if a bacteria can ferment lactose in
the presence of lauryl sulfate, it can ferment lactose in the intestine of a warm-blooded
animal. The presence of such an organism in a water sample was thought to be an
indicator of fecal pollution. In spite of the many problems associated with this test, it
remains the regulatory standard for assessing the microbiological quality of recreational
waters such as the American River.
Counting Bacteria Using an MPN Scheme
In nature, bacteria occur in huge numbers and with many different species present at the
same time. Because a bacterium is a discrete entity, it is possible to dilute a solution to
the point were the number of bacteria present approaches zero. This is the principle you
used in inoculating groups of tubes with successive 10:1 dilutions. With a dilute enough
solution, it is considered probable that no organisms will be introduced to one or more of
the 5 tubes in that dilution. This is a random process where we are attempting to
statistically estimate the number of organisms present per a given volume.
The French mathematician, Siméon Denis Poisson, 1781-1840, developed a distribution
used to describe the occurrence of unlikely events in a large number of independent
repeated trials. Finding a bacterium in a dilute bacterial suspension is an unlikely
occurrence and the five tubes in each dilution is a repeated trial. For the MPN test, the
Poisson distribution is:
y

1
1 - e -n1
a
 e   1 - e  e 
p1
- n1 q1
-n 2 p 2
-n 2 q 2
1 - e

- n 3 p 3
e  
- n 3 q 3
where:
y
a
n1,n2,n3

p1,p2,p3
q1,q2,q3
=
=
=
=
=
=
probability of occurrence of a given result
constant for a given set of conditions
sample size in each dilution, mL
coliform density, number / mL
number of positive tubes in each sample dilution
number of negative tubes in each sample dilution
The solution to this problem is iterative for each dilution and probability, with a selected
y and solution for . Solutions for combinations with reasonable probabilities using three
successive 10:1 dilutions are listed in the table from Standard Methods that is appended
to your procedures.
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Diluting Samples
The standard MPN test involves using three successive 10:1 dilutions. This procedure
can count up to 1600 coliform per 100 mL. If the sample has more than this, we need to
dilute it until we get it into the test range. Domestic sewage, for instance, contains
millions of coliform per 100 mL. Unfortunately, we don't know the coliform count
before doing the test. So, like in the BOD test, we may have to prepare a series of
dilutions and look for valid test results.
The Presumptive Coliform Test
Determining the nummber of bacteria isn't always the goal in engineering practice. For
drinking water regulation, we often want to simply detect the presence of bacteria. In
domestic water supply, if you get a positive result, you presume that coliform are present
and take appropriate actions to protect public health. Simultaneously, you take steps to
determine if the positive test result is really caused by coliform bacteria, including the
start of a confirmed test. The confirmed test is only the first of several sequential steps
needed to determine if the lactose-fermenting bacteria found in the presumptive test
belong to the coliform group.
The Heterotrophic Plate Count (HPC) Test
Another measure of the microbial content of a water sample is the Heterotrophic Plate
Count (HPC) test. In this test, a sample of water is mixed with warm R2A agar (a
nutrient substrate) that is solidifying in a petri dish. After the agar completely solidifies,
the petri dish is incubated for 48 hours. Then the colonies that grew in the agar are
counted. The assumption here is that one bacterium results in one colony.
This test differs from the MPN test in two ways. First, it is a direct count, rather than a
probabilistic estimate. Second, it is a less selective test. In other words, the R2A agar
can be used by many (if not most) heterotrophic bacteria. Therefore, HPC's should
always be higher than MPN's because a wider variety of bacteria are detected in the HPC
test than the MPN test.
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