BI 218 Food Microbiology
COLIFORM TESTING
THEORY:
Purpose and Background
This method is applicable to the enumeration of viable coliforms and/or E. coli, in foods, food ingredients,
and environmental samples (swabs). Coliforms in food or food processing environments serve as an indicator of
inadequate sanitation. If unexpected levels of coliforms are detected in a food sample, the possibility of a
processing error or post-processing contamination should be considered.
Coliform bacteria are defined as Gram negative, nonsporulating, rod-shaped bacteria that produce acid and
gas by lactose fermentation at 35°C in 48 hours. Coliform bacteria are not a traditional taxonomic group, like
Salmonella, Escherichia coli, or Listeria. Instead, coliform bacteria are a collection of strains in the
Enterobacteriaceae family. E. coli, Enterobacter spp., Klebsiella spp., and Citrobacter spp. are the most common
coliforms; however, all isolates of these groups are not coliforms.
Petrifilms:
E. coli and other coliforms can be enumerated on ready-to-use 3M Petrifilm E. coli count plates, or if only
coliforms are required, the 3M Petrifilm Coliform count plates can be used. This method uses bacterial culture
plates containing dry medium and a cold water-soluble gelling agent. 1mL samples are added directly to the plates.
Pressure, when applied to the plastic spreader placed on the overlay film, spreads the sample over 20 cm2. The
gelling agent is allowed to solidify, and the plates are then incubated and counted.
The plates contain violet-red bile, lactose, nutrients, a cold water-soluble gelling agent, and 2,3,5-triphenyl
tetrazolium chloride (TTC) indicator dye. Reduction of the indicator by the bacteria causes the colonies to become
red and facilitates enumeration. When coliforms ferment lactose, gas is produced. The film traps the gas around the
coliform colonies and thus differentiates them from colonies of other gram-negative organisms. The blue colonies
are formed when the indicator 5-bromo-4-chloro-3-indolyl-D-glucuronide (BCIG) is cleaved, an indicator of
glucuronidase activity. E. coli and coliforms form gas from the fermentation of lactose.
Similar to most other E. coli and coliform media, 3M Petrifilm plates will not specifically indicate whether
any E. coli O157 strains are present. This is because most E. coli O157 strains are atypical E. coli, so they will
appear as non-E. coli coliforms (red with gas).
Most Probable Number:
The MPN method is accomplished by performing a multi-tube lactose fermentation test. Turbidity and
gas production that surpasses 10% of the volume of a Durham tube after incubation at 32C for 24-48 hours
constitutes a (+) result. Several optional MPN methods are available. MPN method measures only viable
organisms. MPN results are to be considered only as quantitative estimates of coliform density, since the results
are based on the frequency of positive fermentation reactions occurring at random in a series of tubes obtained
from a multiple-inoculation procedure. Since one or more coliform organisms may be responsible for (+)
lactose fermentation, the most probable number of coliforms then depends on the laws of probability and
random sampling. MPN is derived by applying the theory of probability of the determination of population
densities of microorganisms in a substrate (sample). To apply the theory of probability, certain basic
assumptions must be made. It is assumed that the organisms are distributed randomly throughout the sample,
that there is equal likelihood of finding an organism in one portion as in another, that the organisms exist as
simple entities (not as groups or cluster), and that they do not repel one another. It is further assumed that each
portion of the sample, when incubated in the culture medium, is certain to exhibit growth if the inoculum
contains one or more organisms, and that the analyst always selects the proper nutrients and physical conditions
to assure growth of the organisms in question.
Originated by: Allan Richardson Revised by: Yvonne Henry & Samiha Mohammad Sharif 1
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The selection of the number of dilutions prepared from a sample is generally based on the expected
population of the sample. The most reliable results are obtained when a series of tubes yields all positives at the
lower dilution and all negatives at the higher dilution. The greater the population density in a sample, the higher
serial dilutions will be required. Thus, if an initial sample population is completely unknown, it is best to make
a range of dilutions that would permit MPN determination. The most common technique is to inoculate either 3
or 5 tubes with portions of the sample that form a series of 10-fold dilution. The confidence limits for an MPN
are narrowed as the number of tubes inoculated with each dilution increases.
The MPN technique has some limitations. For example, it is not as precise as the plate count for high-
density populations. Also, it must be recognized that an MPN value is an estimate of the population, and not a
precise enumeration of viable organisms. On the other hand, it can be used to advantage in several instances.
For instance, it is particularly useful for low levels of organisms (<10 per g) in milk, water, and foods where
particulate matter interferes with accurate colony counts. Proper choice of media can give early results for some
of the biochemical characteristics also, and estimates of mixed populations can be obtained by using selective
media. Frequently, more samples can be analyzed with greater facility.
The tables generally recommended for use contain only those combinations that occur often enough to have
statistical significance. Those combinations that are highly improbable are omitted from the tables because they
are not acceptable for important decisions. In addition, most MPN tables include the 95% confidence limits and
some even include 99% confidence limits. If the combination of positive tubes is not listed in the table, it is
advisable to do the test again with the original sample if possible. If there is no residual sample, and an MPN is
imperative, the analyst should either consult another complete table or calculate the MPN based on the observed
results. For coliform testing, the lactose fermentation test is considered the first of three MPN testing steps:
Presumptive Test, Confirmatory Test, and Completed Test.
Membrane Filtration:
The MF method is a valuable procedure for the detection and enumeration of coliform bacteria in large
volumes of water and food samples with LOW coliform densities. This method has a very high degree of sensitivity,
as it can concentrate low numbers of microbes in large volume samples (e.g. 100 mL) and demonstrate growth of
colonies within an 18-24 hour period on an appropriate medium. It gives a direct count with better reproducibility of
results but is not useful when sample is turbid with particulate matter (ex. milk with fat content, algae, sediments,
etc.).
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BI 218 Food Microbiology
COLIFORM TESTING
FLOW DIAGRAMS:
Petrifilms MPN
Sample Preparation
3-tube by 3 dilution Transfer stick
MPN series in LST of broth to BGB Turbidity
broth. Incubate Incubate 48 h and gas? Yes
48 ±4 hr at 35 ±1oC at 35C ± 1oC
Report coliform
Yes Count
Turbidity No
and gas?
Yes
Sample Preparation Transfer stick
of broth to EC
Incubate 48+/-4 h
at 44.50C ± 0.2oC
Faecal Coliform
Pipet sample onto Petrifilm™ plates. not present
Incubate face up in stacks of 20 or less. Turbidity No
Incubate at 35 ± 1C for 24 ± 2/ 48 ± 4h and gas?
Yes
Yes
Faecal Coliform
Streak to EMB Present
No Blue colonies Red colonies No incubate 18-24h at 35 ±1oC
with gas? with gas?
E. coli Coliforms
not Yes Yes not
present present Typical No
colonies?
E. coli present
Yes
Coliforms present
Calculate Streak to MAC, NA or
Plate EMB
Count
IMViC tests or API
Report Results ID as No E. coli
[Link]? not present
Yes
* Blue colonies with gas plus red colonies with gas provide total coliform count. Look up
E. coli present MPN/g index
Report results
EQUIPMENT AND SUPPLIES:
• 1 – 200mL PO4 for Water sample (Boston bottle)
• 1 – 50mL Rinse water (in 500mL Boston bottles)
• 3 – Double strength LSTB + Durham tubes (In large tubes containing 10mL)
• 6 – Single strength LSTB + Durham tubes (In small tubes containing 10mL)
• 1mL pipettes
• 3 – 9.9 mL PO4 buffer
• 1 – 9 mL PO4 buffer
• 1 – m-FC plate
• 1 – m-Endo plate
• 1 – Membrane filtration unit
• 2 – Membrane filters of pore size: 0.45 m
• 1 – Sterile 100mL graduated cylinder
• 1 – E. coli 3M Petrifilm
• Up to 3 – BGLB broth tubes
Originated by: Allan Richardson Revised by: Yvonne Henry & Samiha Mohammad Sharif 3
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• 1 – EMB plate
• MPN tables: two versions of the tables are supplied (MPN / mL and MPN / 100 mL )
• CULTURES: You will CREATE a spiked water sample using a mixed culture of:
Escherichia coli ATCC #: _____________ & Klebsiella pneumoniae ATCC #: ___________
OBJECTIVE:
• To create a spiked water sample to be tested
• To isolate and enumerate coliform bacteria following adapted protocols of Health Canada Method MFHPB-
19 for MPN testing, MFHPB-26 for Membrane Filtration testing, and MFHPB-34 for Petrifilm testing
• To observe colony morphology and enumerate the number of total and fecal coliform bacteria via MPN
testing, MF, and a Petrifilm to compare the merits and limitations of each method
METHODS:
Day 1
PROCEDURE A: SAMPLE PREPARATION
Note: This is NOT part of the Coliform Testing method; since our tap water is relatively sterile, we are going to
add a known concentration of mixed culture of Escherichia coli (faecal coliform) & Klebsiella pneumoniae
(coliform) to create a “spiked water sample” from which the bacteria will be recovered using MPN, MF, and
Petrifilm methods.
1. The initial concentration of the 1:1 mixed culture of Escherichia coli & Klebsiella pneumoniae is
~1.0x109cells/mL
2. Dilute the culture using 9.9mL PO4 buffer tubes until the last tube is to 10-6. The final bacterial cell
concentration should be ~1.0x103 cells/mL in this tube. This will be used to prepare the spiked water sample.
3. Transfer 0.2 mL from the last test tube (10-6 dilution tube) to 200 mL bottle of sterile water. This results in
your final concentration of bacteria being ~1 cell/1mL
Additional step:
4. Using one 9 mL PO4 buffer, make a 10-7 dilution tube from the 10-6 tube (made in Step 2 above). The final
bacterial cell concentration should be ~1.0x102 cells/mL in this tube. This will be used for the Petrifilm.
PROCEDURE B: MPN METHOD
Presumptive test:
1. Inoculate 3 tubes of double strength LSTB (in large tubes) with 10 mL of spiked water sample
2. Inoculate 3 tubes of single strength LSTB with 1 mL of spiked water sample
3. Inoculate 3 tubes of single strength LSTB with 0.1 mL of the water sample
4. Incubate tubes for 35°C for 48 hours
PROCEDURE C: MF METHOD
1. Sterilize forceps in flame quickly; ensure they do not get too hot and are completely cool before using
2. Place sterile filter (0.45 m) into sterile membrane filtration apparatus with the grid up
3. Attach aspirator (at the staining sink) to the filtration unit and turn on water to begin suction
4. Measure and filter 50 mL of “spiked” water sample using a graduated cylinder
a. Pour sample in small aliquots and let drain completely to avoid tearing the filter
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COLIFORM TESTING
5. Once sample has passed through the filter, pour ~20 mL of sterile Rinse water/buffer in the same graduated
cylinder and filter this also
a. This step ensures all bacteria from the sides of cylinder and filtration apparatus are collected
6. Following filtration and rinsing, aseptically transfer membrane filter to m-ENDO plate
a. Ensure that the filter is placed on the surface of the agar with the grid up
7. Repeat filtration procedure (steps 4-6) and transfer membrane filter to m-FC agar plate
8. Incubate m-ENDO plates at 35oC and FC plates at 45oC for 24 hours.
PROCEDURE D: COLIFORM/ E. coli PETRIFILM
1. Lift the top film and transfer 1.0 mL of the required dilution in the centre of dry media. Carefully roll top film
down to avoid entrapping air bubbles
2. Distribute sample evenly using a downward pressure on the centre of the plastic spreader
a. Do not slide the spreader across the film
b. Leave plate undisturbed for at least 1 minute to permit the gel to solidify
3. Incubate plates at 35°C for 24 hours
Day 2
Procedure A: MPN – Presumptive Test Results, Performance of Confirmed and Completed tests
1. Examine LSTB tubes for gas formation and turbidity and record results. This is your MPN-code
a. Be careful not to shake or invert the tubes as they are slide-caps
b. These are the 'positive tubes'
Confirmed test:
2. Transfer a sterile loopful from each series (10mL, 1mL, and 0.1mL) if there is a positive LSTB tube present
to one BGLB broth tube per series
a. You should have a maximum of 3 BGLB tubes
b. ex. If my positives are: + + + for 10mL, + - + for 1mL, and - - - for 0.1mL, then I would require only
2 BGLB tubes to confirm the presence of coliforms in my 10mL and 1mL series
3. Incubate BGLB tubes for 48 hours at 45oC
Completed test:
Note: This is a cheat. Technically the completed test should be performed USING the BGLB results
4. Streak one EMB plate using a loopful of broth from the HIGHEST DILUTION that has a positive
a. In the example provided in step 2. One of the positive 1mL LSTB tubes would be used
5. Incubate plates 24 hours at 35oC
Procedure B: MF - Results
1. Examine the m-ENDO plates and count both the dark red/purple colonies with a metallic sheen ([Link] /
faecal coliforms) and the pink juicy colonies (Klebsiella/ non-faecal coliforms). The combined count is
the “total coliform count”
2. Examine the m-FC plates and count only the dark BLUE colonies ([Link] / faecal coliforms)
Calculation:
(Colony Count) x 100 = CFU/100 mL
(Volume of sample used in mL)
Originated by: Allan Richardson Revised by: Yvonne Henry & Samiha Mohammad Sharif 5
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Procedure C: Petrifilms
1. Count plates promptly after incubation period. A total coliform count is a count that includes the red colonies
with gas and the blue colonies with gas
a. Non-faecal coliform colonies are red and have a gas bubble
b. Confirmed E. coli colonies are blue and have gas bubbles
i. The size or the intensity of the blue does not matter
ii. Blue colonies that are not associated with gas are not counted as [Link]
c. Gas bubbles may be located in various arrangements and still be associated with the colony (colonies
with gas bubbles that have a distance greater than one colony diameter between the colony and gas
bubble are not counted as coliforms)
Note: Do not count colonies on the white foam dam since they are removed from the selective influence of
the medium.
d. Some [Link] and other coliforms, when present in large numbers, may cause one or more of the
following: excessive gas formation, small indistinct colonies, or a dark red or blue growth area. E.
coli will cause the growth area to turn blue, while high concentrations of other coliforms (non-E. coli)
will cause the growth area to turn a dark red. When any of these occurs, this is TNTC. Technically
the sample should be diluted further to obtain a more accurate count. Make estimates on plates
containing more than 150 colonies by counting the average number of colonies in one or more
representative squares and determining the average number per square. Multiply the average number
by 20 to determine total count per plate.
2. Compare results obtained by the MPN and the MF methods. Compare results for coliforms and faecal
coliforms.
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COLIFORM TESTING
Day 3
1. Examine BGLB tubes for gas production in the Durham tubes. Record tubes with gas formation as positive
tubes
2. Examine EMB plates and MAC plates for expected results.
REFERENCES:
Compendium of Methods for the Microbiological Examination of Foods, 4th ed., Chapters 2, 4. American
Public Health Association (APHA), 2001.
Bacteriological Analytical Manual, online ed., Chapters 3, January 2001.
Jay, James M. Modern Food Microbiology 6th Edition, Aspen Publishers, Gaithersburg Maryland. 2001
ISBN-0-8342-1671-X
MFHPB-18. Determination of the Aerobic Colony Count in Foods. July 2015. Volume 2. Compendium of
Analytical Methods, Health Canada, Ottawa.
MFHPB-19. Enumeration of Coliforms, Faecal Coliforms and E. coli in Foods Using the MPN Method. April
2002. Compendium of Analytical Methods, Volume 2, Health Canada, Ottawa.
MFHPB-26. Enumeration of Escherichia Coli in Foods by the Hydrophobic Grid Membrane Filter (Hgmf)
Method, March 2001. Volume 2, Compendium Of Analytical Methods, Health Canada, Ottawa.
MFHPB 33. Enumeration of Total Aerobic Bacteria in Food Products and Food Ingredients Using 3M Petrifilm
Aerobic Count Plates. April 2015. Volume 2, Compendium of Analytical Methods, Health Canada,
Ottawa.
MFHPB 34. Enumeration of E. coli and coliforms in food products and food ingredients using 3M Petrifilm
E. coli count plates. July 2013. Volume 2, Compendium of Analytical Methods, Health Canada,
Ottawa.
MFHPB-35 Enumeration of Coliforms in Food Products and Food Ingredients using 3M Petrifilm Coliform
Count Plates. February 2001. Volume 2, Compendium of Analytical Methods, Health Canada, Ottawa.
Petrifilm Aerobic Count Plate Interpretation Guide – Brochure. 3M Canada, London ON Canada, 2005.
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MPN TABLES:
Modified from: Standard Methods for the Examination of Water and Wastewater. 14th Edition, APHA-AWWA.
MPN Table 3 Tubes per Dilution 5 Tubes per Dilution
Per 1.0 g 95% Confidence Limits Per 1.0 g 95% Confidence Limits
Combination MPN Index Lower Upper MPN Index Lower Upper
of Positives per mL per mL
(0-0-0) <0.03 <0.02
(0-0-1) 0.03 <0.005 0.09 0.02 <0.005 0.07
(0-1-0) 0.03 <0.005 0.13 0.02 <0.005 0.07
(0-2-0) -- 0.04 <0.005 0.11
(1-0-0) 0.04 <0.005 0.2 0.02 <0.005 0.07
(1-0-1) 0.07 0.01 0.21 0.04 <0.005 0.11
(1-1-0) 0.07 0.01 0.23 0.04 <0.005 0.11
(1-1-1) 0.11 0.03 0.36 0.06 <0.005 0.15
(1-2-0) 0.11 0.03 0.36 0.06 <0.005 0.15
(2-0-0) 0.09 0.01 0.36 0.05 <0.005 0.13
(2-0-1) 0.14 0.03 0.37 0.07 0.01 0.17
(2-1-0) 0.15 0.03 0.44 0.07 0.01 0.17
(2-1-1) 0.2 0.07 0.89 0.09 0.02 0.21
(2-2-0) 0.21 0.04 0.47 0.09 0.02 0.21
(2-2-1) 0.28 0.1 1.5 -- -- --
(2-3-0) -- 0.12 0.03 0.28
(3-0-0) 0.23 0.04 1.2 0.08 0.01 0.19
(3-0-1) 0.39 0.07 1.3 0.11 0.02 0.25
(3-0-2) 0.64 0.15 3.8 -- -- --
(3-1-0) 0.43 0.07 2.1 0.11 0.02 0.25
(3-1-1) 0.75 0.14 2.3 0.14 0.04 0.34
(3-1-2) 1.2 0.3 3.8 --
(3-2-0) 0.93 0.15 3.8 0.14 0.04 0.34
(3-2-1) 1.5 0.3 4.4 0.17 0.05 0.46
(3-2-2) 2.1 0.35 4.7 -- -- --
(3-3-0) 2.4 0.36 13 -- -- --
(3-3-1) 4.6 0.71 24 -- -- --
(3-3-2) 11 1.5 48 -- -- --
(3-3-3) >24 -- -- --
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COLIFORM TESTING
MPN Table 3 Tubes per Dilution 5 Tubes per Dilution
per 100 mL 95% Confidence Limits per 100 mL 95% Confidence Limits
Combination MPN Index Lower Upper MPN Index Lower Upper
of Positives per 100 mL per 100 mL
(0-0-0) <3 <2
(0-0-1) 3 <.5 9 2 <0.5 7
(0-1-0) 3 <.5 13 2 <0.5 7
(0-2-0) -- 4 <0.5 11
(1-0-0) 4 <.5 20 2 <0.5 7
(1-0-1) 7 1 21 4 <0.5 11
(1-1-0) 7 1 23 4 <0.5 11
(1-1-1) 11 3 36 6 <0.5 15
(1-2-0) 11 3 36 6 <0.5 15
(2-0-0) 9.0 1 36 5 <0.5 13
(2-0-1) 14 3 37 7 1 17
(2-1-0) 15 3 44 7 1 17
(2-1-1) 20 7 89 9 2 21
(2-2-0) 21 4 47 9 2 21
(2-2-1) 28 10 150 -- -- --
(2-3-0) -- 12 3 28
(3-0-0) 23 4 120 8 1 19
(3-0-1) 39 7 130 11 2 25
(3-0-2) 64 15 380 -- -- --
(3-1-0) 43 7 210 11 2 25
(3-1-1) 75 14 230 14 4 34
(3-1-2) 120 30 380 --
(3-2-0) 93 15 380 14 4 34
(3-2-1) 150 30 440 17 5 46
(3-2-2) 210 35 470 -- -- --
(3-3-0) 240 36 1300 -- -- --
(3-3-1) 460 71 2400 -- -- --
(3-3-2) 1100 150 4800 -- -- --
(3-3-3) >2400 -- -- --
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