Microbiology:
Testing for Bacteria
Linda Wolf
Glencoe High School
SWRP Teacher for 12 years
Pathogens
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Pathogens are organisms capable of
causing disease
The following are some of the “bad guys”:
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Protozoa: Giardia, Cryptosporidium
Bacteria: Salmonella typhi, Legionella,
Shigella, Vibrio cholerae, Vibrio vulnificus
Virus: Hepatitis, Polio
Sizes
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Bacteria are 2 - 4 µm
Viruses are 0.02 - 0.09 µm
For reference:
 106 microns (or micrometer, µm) = 1 meter
 1000 µm = 1 mm
Testing for Pathogens
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Direct testing for pathogens is impractical
Pathogens are usually found in low
numbers
Can’t survive for very long outside the
warm confines of a human or animal body
Too many methods are too sophisticated
and expensive
Indicator Bacteria
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Some bacteria can be good indicators of
human pollution – the source for most
pathogens
Bacteria present in sewage pollution
Survive longer than pathogens
Easily detectable
Common Indicator Bacteria
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Total Coliforms
Fecal Coliforms
E. coli
Enterococci
Total
Coliforms
Fecal
Coliforms
E. coli
Total coliforms
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Rod-shaped, gram negative bacteria
Ferment lactose at 35°C
Found in intestinal tracts of cold and warmblooded animals
Group members: Escherichia, Klebsiella,
Enterobacter, Serratia, Citrobactera,
Edwardsiella
Fecal coliforms
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Subset of Total coliform group
Present in sewage and indicate possibility
of human pathogens
Distinguished from Total coliform by
ability to ferment lactose at 44.5°C
Group members: E. coli and Klebsiella (not
always fecal often associated with paper,
textile & pulp waste)
Fecal coliforms
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Common in the intestines of both warm
and cold-blooded animals
If fecal coliforms are present it is presumed
that human or animal excrement is present
Diseases such as typhoid fever, hepatitis,
gastroenteritis, dysentery and ear infections
can be contracted in water with high Fecal
coliform levels
E. coli
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Escherichia coli is a specific species within
the Fecal coliform group
Specific to intestines of mammals and
other warm blooded animals
Only specific strains (i.e. O157:H7) are
pathogenic
According to EPA, is the best indicator of
health risks from water contact recreation
Enterococci
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Survives in salt water
More human specific
Found primarily in the intestinal tract of
warm-blooded animals
Used in some states as indicator organism
in estuarine and marine waters
Bacterial Measurement
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Membrane Filtration Methods
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Quantify bacteria numbers by filtering water,
growing bacteria, and counting
Most Probable Number Methods
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Estimate bacterial numbers based upon a
color change or amount of gas produced
through a specific bacterial metabolic process
Membrane Filtration
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Known volume of water is filtered through
a filter (0.45 µm) that is capable of trapping
all bacteria
Filter transferred to Petri dish containing
growth media
Individual bacterial cells will grow on the
filter into visible colonies in 24 hours
Membrane Filtration
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m-ColiBlue24 broth
Due to the metabolism of the bacteria on
the media:
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Blue colonies indicate E. coli
Red colonies indicate other Total coliform
bacteria
E. coli turn blue from the action of βglucuronidase enzyme on 5-bromo-4-chloro3-indolyl-Beta-B-glucuronide
Procedure
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Collect water in a sterile container
Filter water within 6 hours*
Place sample in cooler if taking to lab
*6 hours is standard holding time, but samples
should definitely be filtered within 24
hours
Prepare plates
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Determine amount of water to filter
5 plates for each site
For each site label one plate 0 mL for a
“blank”
# plates
Label
Volume
1
0 mL
10 mL sterile
2
10 mL
10 mL sample
2
30 mL
30 mL sample
Prepare Plates
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Label bottom of plate with:
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Date and time
Sampling site
Volume to be filtered
Use sterile forceps to place sterile absorbent pad
in each plate, if plates don’t already have them
Place about 2 mL of broth on each pad, using
either a sterile pipette or by shaking and pouring
ampule
Filter Samples
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Sterilize forceps, place membrane filter on
filter holder
Use sterile water for small samples of
water (1 mL) to wet the filter
Pump until most of water is through filter
Release pressure
Sterilize forceps and place filter grid-sideup on the absorbent pad
Plates
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Put cover on plate
Leave upright until all plates are filtered
Incubate upside down for 24 hours in an
incubator at 35° C
Calculating Results
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Count the blue and red colonies on each
plate
Blue colonies are E. coli
Red + Blue = Total Coliforms
If there are greater than 200 colonies report
that plate as TNTC (Too numerous to
count)
Most accurate plates
The best are when the colony counts are in the
range of:
 20 – 80 colonies per plate for E. coli, and
 50 – 200 for Total coliforms
Calculating Results
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Standard Units = CFU/100 mL (Colony Forming
Units)
Average colony counts x 100 = CFU/100 ml
Volume Filtered (mL)
If fewer than 20, estimate CFU/ 100 ml using all
plates.
Add total number of colonies and total volume
Total colony counts x 100 = CFU/100 ml
Total mL filtered
Other problems
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If over 200, but colonies are clearly
countable, use the same general formula.
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Conflicting colony counts:
go with the smaller sample size
0 mL filtered
0 colonies
counted
1 mL filtered
6 colonies
counted
10 mL filtered
The 10 mL plate
would be used for
calculating CFU/100
mL:
30 mL filtered
24 / 10 x 100 =
240 CFU/100 mL
24 colonies
counted
67 colonies
counted
0 mL filtered
1 mL filtered
1 mL plate has more
colonies that 10 mL
plate.
Possible problem(s):
• mislabeled plate
• contaminated
apparatus
0 colonies
counted
23 colonies
counted
The 1 mL plate
would be used for
calculating CFU/100
mL:
10 mL filtered
30 mL filtered
23 / 1 x 100 =
2300 CFU/100 mL
18 colonies
counted
52 colonies
counted
0 mL filtered
0 mL filtered
Possible
problem:
finger on filter
or
contaminated
forceps
6 colonies
counted
7 colonies
counted
The sterile water
“blank” or 0 mL
plate is a quality
control measure –
bacterial growth on
the blank makes the
other plate counts
suspect.
Possible
problem:
“sterile” water
not sterilized
1 mL filtered
23 colonies
counted
Possible problem: filter not wetted with
sterilized water before filtering low
volume sample – sample concentrated in
one area of filter.
30 mL filtered
Estimate or
report as Too
Numerous to
Count (TNTC)
Example of plate with more than 200
colonies. Colonies could be counted
or estimated, and results flagged as
“estimate”.
Water Quality Standards
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In Oregon, based upon contact recreation
126 CFU/100 mL for 5 samples within a 30
day period
406 CFU/100 mL for a single sample