Microbiology: Testing for Bacteria

1
MicrobiologyTesting for Bacteria

• Linda Wolf
• Glencoe High School
• SWRP Teacher for 12 years

2
Pathogens

• Pathogens are organisms capable of causing
  disease
• The following are some of the bad guys
• Protozoa Giardia, Cryptosporidium
• Bacteria Salmonella typhi, Legionella, Shigella,
  Vibrio cholerae, Vibrio vulnificus
• Virus Hepatitis, Polio

3
Sizes

• 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

4
Testing for Pathogens

• Direct testing for pathogens is impractical
• Pathogens are usually found in low numbers
• Cant survive for very long outside the warm
  confines of a human or animal body
• Too many methods are too sophisticated and
  expensive

5
Indicator Bacteria

• 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

6
Common Indicator Bacteria

• Total Coliforms
• Fecal Coliforms
• E. coli
• Enterococci

7
Total coliforms

• Rod-shaped, gram negative bacteria
• Ferment lactose at 35C
• Found in intestinal tracts of cold and
  warm-blooded animals
• Group members Escherichia, Klebsiella,
  Enterobacter, Serratia, Citrobactera, Edwardsiella

8
Fecal coliforms

• Subset of Total coliform group
• Present in sewage and indicate possibility of
  human pathogens
• Distinguished from Total coliform by ability to
  ferment lactose at 44.5C
• Group members E. coli and Klebsiella (not always
  fecal often associated with paper, textile pulp
  waste)

9
Fecal coliforms

• 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

10
E. coli

• 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. O157H7) are
  pathogenic
• According to EPA, is the best indicator of health
  risks from water contact recreation

11
Enterococci

• 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

12
Bacterial Measurement

• Membrane Filtration Methods
• Quantify bacteria numbers by filtering water,
  growing bacteria, and counting
• Most Probable Number Methods
• Estimate bacterial numbers based upon a color
  change or amount of gas produced through a
  specific bacterial metabolic process

13
Membrane Filtration

• 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

14
Membrane Filtration

• m-ColiBlue24 broth
• Due to the metabolism of the bacteria on the
  media
• Blue colonies indicate E. coli
• Red colonies indicate other Total coliform
  bacteria
• E. coli turn blue from the action of
  ß-glucuronidase enzyme on 5-bromo-4-chloro-3-indol
  yl-Beta-B-glucuronide

15
Procedure

• 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

16
Prepare plates

• 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
17
Prepare Plates

• Label bottom of plate with
• Date and time
• Sampling site
• Volume to be filtered
• Use sterile forceps to place sterile absorbent
  pad in each plate, if plates dont already have
  them
• Place about 2 mL of broth on each pad, using
  either a sterile pipette or by shaking and
  pouring ampule

18
Filter Samples

• 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-side-up
  on the absorbent pad

19
Plates

• Put cover on plate
• Leave upright until all plates are filtered
• Incubate upside down for 24 hours in an incubator
  at 35 C

20
Calculating Results

• 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)

21
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

22
Calculating Results

• 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

23
Other problems

• If over 200, but colonies are clearly countable,
  use the same general formula.
• Conflicting colony counts
• go with the smaller sample size

24
0 mL filtered
1 mL filtered
6 colonies counted
0 colonies counted
10 mL filtered
30 mL filtered
The 10 mL plate would be used for calculating
CFU/100 mL 24 / 10 x 100 240 CFU/100 mL
24 colonies counted
67 colonies counted
25
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
30 mL filtered
10 mL filtered
The 1 mL plate would be used for calculating
CFU/100 mL 23 / 1 x 100 2300 CFU/100 mL
52 colonies counted
18 colonies counted
26
0 mL filtered
0 mL filtered
Possible problem finger on filter or
contaminated forceps
Possible problem sterile water not sterilized
6 colonies counted
7 colonies counted
1 mL filtered
30 mL filtered
The sterile water blank or 0 mL plate is a
quality control measure bacterial growth on the
blank makes the other plate counts suspect.
Estimate or report as Too Numerous to Count (TNTC)
23 colonies counted
Possible problem filter not wetted with
sterilized water before filtering low volume
sample sample concentrated in one area of
filter.
Example of plate with more than 200 colonies.
Colonies could be counted or estimated, and
results flagged as estimate.
27
Water Quality Standards

• 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