1. Lab 7: Enumeration of coliforms, fecal coliforms and E. coli in foods

2. Enumeration of coliforms fecal coliforms and E
Enumeration of coliforms fecal coliforms and E. coli in foods using the MPN method
Members of two bacteria groups, coliforms and fecal coliform, are used as indicators of possible sewage contamination because they are commonly found in human and animal feces.
an indicator organism or group of organisms is one whose numbers in a product reflect the success or failure of GMP "Good Manufacturing Practices".
The most commonly tested fecal bacteria indicators are
Total coliforms
Fecal coliforms
Escherichia coli
Enterococci.

3. Total Coliforms: Aerobic or facultative anaerobic Gram negative
non-spore-forming
rod-shaped
Most commonly used indicator for: drinking water, wastewater treatment.
gas production during lactose fermentation within 48 hours at 37°C
Total coliforms include a wide variety of bacteria including Escherichia coli and a broad spectrum of other bacteria.
Escherichia coli is species found in lower gastrointestinal tract of many vertebrates and is indicative of FECAL CONTAMINATION.
Examples: Escherichia, Citrobacter, Klebsiella and Enterobacter

4. Fecal Coliforms:
Fecal coliforms, a subset of total coliform bacteria, are more fecal-specific in origin.
Able to ferment lactose and produce both acid and gas at 44.5°C in 24 hours
Include Escherichia and Klebsiella, which are exclusively fecal in origin (perhaps. . .)
Fecal coliforms are still being used in many states as the indicator bacteria.

5. E. coli
E. coli is a type of fecal coliform bacteria commonly found in the intestines of warm blooded animals and humans.
The presence of E. coli in water is a strong indication of recent sewage or animal waste contamination.

6. Enterococci
Enterococci are a subgroup within the fecal streptococcus group.
Enterococci are distinguished by their ability to survive in salt water, and in this respect they more closely mimic many pathogens than do the other indicators.
Enterococci are typically more human-specific than the larger fecal streptococcus group.

8. Sources of Coliform Bacteria
Soil and Vegetation
Intestines of warm blooded animals
On site sewage system and sewer line leaks
Agricultural waste runoff
Surface water and Flood water
Ground water

9. Principle: 1. inadequate processing and/or post-processing
The terms “coliform” and “faecal coliform” have no taxonomic validity and, therefore, are only meaningful when expressed in terms of the analytical test parameters of medium, time and temperature of incubation.
The presence of “indicator organisms” in foods processed for safety may indicate one of the following possibilities:
1. inadequate processing and/or post-processing
contamination; and/or
2. microbial growth.
The presence of coliforms, faecal coliforms and
aerogenic E. coli in food and water may be determined
by means of the MPN procedure.

10. Gas production is used as an indication of ability to ferment lactose from Lauryl Sulfate Tryptose (LST) broth (presumptive coliform test).
Gas production from Brilliant Green Lactose 2% Bile (BGLB) broth is considered confirmation of coliform presence.
Gas production at 44.5 or 45o C from EC broth is used as confirmation of faecal coliform presence; and appearance of typical nucleated, dark-centred colonies with or without metallic sheen when positive Escherichia coli (EC) broths are streaked onto Levine's Eosin Methylene Blue (L-EMB) agar are indicative of E. coli.
The typical colonies on L-EMB agar must be confirmed by further biochemical tests to prove the presence of E. coli.

11. Basic Steps Collecting Samples Transporting Samples Delivering Samples
Lab Preparation
Preparing Sample
Incubating Sample
Reading Sample
Disposing Sample
Data Management
Data Interpretation

12. Materials and special equipment:
1. Peptone Water (0.1% and 0.5%)
2. Aqueous Sodium Citrate (2.0%), tempered to 40-45oC
3. Lauryl Sulfate Tryptose (LST) broth
4. Brilliant Green Lactose 2% Bile (BGLB) broth
5. Escherichia coli (EC) broth or EC broth with MUG (4-methylumbelliferyl-ß-D-glucuronide)
6. Levine's Eosin Methylene Blue (L-EMB) agar or Endo agar
7. MacConkey agar
8. Nutrient Agar (NA) or other non-selective agar
9. Covered water baths, with circulating system to maintain
temperature of 44.5°C and 45 ° C.
Water level should be above the medium in immersed tubes.

13. Materials and special equipment:
10. Thermometer, calibrated and traceable
11. Incubator, 37 °C.
12. Stomacher, blender or equivalent.
13. Control cultures (use ATCC cultures or equivalent):
positive control(s): E. coli that is known to produce gas at 44.5 / 45°C and is capable of fermenting lactose to produce typical reactions on L-EMB agar.
if using EC-MUG, a strain that is known to produce ß-glucuronidase EMB / IMViC (E. coli ++--)
negative control: Enterobacter aerogenes or an equivalent gram negative rod that does not produce “positive” reactions on EMB and is
indole-negative, methyl red negative, Voges- Proskauer-positive,
and citrate positive.
MPN broths negative control: Salmonella berta or an
equivalent gram negative rod that is gas-negative in MPN
broths and in the secondary EC broth.

14. NOTE: Some strains of Enterobacter aerogenes will give false-positive reactions in the MPN broths (LST, BGLB and EC broths) by producing a small gas bubble.
14. pH meter capable of distinguishing to 0.1 pH units within the range of pH 5.0 to 8.0 or pH paper
capable of distinguishing from 0.3 to 0.5 pH units,
within the same range.

15. 15. Supplies needed for confirmation (commercially available):
A. IMViC media and reagents:
Tryptone (or tryptophane) broth Indole reagents (available commercially).
ii. Buffered Glucose broth Voges-Proskauer test reagents (available commercially) Methyl red solution.
iii. Simmon's Citrate (SC) agar
B. Rapid Identification Kits or Systems (such as API,
Vitek or equivalent).

16. Procedures Handling of Sample Units
Each sample unit may be analyzed individually or the analytical units may be combined where requirements of the applicable sampling plan can be met.
Carry out the test in accordance with the following instructions:
Handling of Sample Units
In the laboratory prior to analysis, except for shelf-stable foods, keep sample units refrigerated (0-5°C) or frozen, depending on the nature of the product.
Thaw frozen samples in a refrigerator, or under time and temperature conditions which prevent microbial growth or death.
2. Analyze sample units as soon as possible after
their receipt in the laboratory. Shellfish must be
analyzed within 24 hours of collection.

17. Procedures Preparation for Analysis
1. Have ready sterile peptone water.
2. Clean the surface of the working area with a suitable disinfectant.
3. Arrange LST broth tubes in rows of five and mark them identifying the sample unit and the dilution to be inoculated.

18. Typical growth results observed for brilliant green lactose bile broth
Typical growth results observed for brilliant green lactose bile broth. Use these tubes to determine an MPN

19. Typical growth results observed for EC broth.
Use these tubes to determine an MPN

20. Levine's Eosin-methylene Blue Agar
(EMB) is primarily a differential medium. However, it does inhibit the growth of some Gram positive bacteria.
EMB is used to differentiate between enteric lactose fermenters (coliforms) and non-lactose fermenters as well as specifically identifying E. coli.
Eosin Methylene Blue agar contains peptone, lactose, and the dyes eosin Y and methylene blue. The sugars provide fermentable substrates to encourage growth of fecal coliforms. The dyes inhibit growth of Gram-positive organisms and, under acidic conditions, also produce a dark purple.

21. The eosin and methylene blue dyes cause lactose fermenters to have pink colonies. E. coli incorporates so much of the dye that the dyes precipitate in the cells and give the colonies a metallic green sheen.
Non-lactose fermenters will remain colorless or take on the color of the medium such as Salmonella (one of the causative agents of food poisoning).
Note: This media is used to confirm the presence of E. coli in water samples contaminated with sewage or fecal material.
You will use this agar again with in the water sampling experiment to differentiate between E. coli and Enterobacter.

22. Colony types observed on EMB agar
Colony types observed on EMB agar. Both fish-eye-type and coli-type colonies are shown.
Coliform colonies will show a relatively dark color die to the acid produced from lactose fermentation. There can be a variety of colony types, but the two "classical" types of coliform colonies are often noted: Click on images for alternate and/or larger views in separate window.
Coli-type colonies are very dark, almost black, when observed directly against the light. By reflected light a green sheen can be seen which is due to the precipitation of methylene blue in the medium from the very high amount of acid produced from fermentation. Those which form this type of colony are methyl red-positive lactose-fermenters such as most strains of E. coli and some strains of Citrobacter.
Aerogenes-type colonies are less dark. Often a dark center is seen surrounded by a wide, light-colored, mucoid rim – resulting in a "fish-eye" type of colony. Those which form this type of colony are methyl red-negative lactose-fermenters which include most strains of Klebsiella and Enterobacter.
Non-lactose-fermenting colonies produce no acid from fermentation, so the lighter-colored alkaline reaction is seen. Colonies of Pseudomonas (a strictly-aerobic non-fermenter) are shown at right

23. Colony types observed on EMB agar
Colony types observed on EMB agar. Both fish-eye-type and coli-type colonies are shown.

24. The dark colonies produced on EMB agar is a result of the acid produced during lactose fermentation precipitating the dyes in the media.

26. Lactose fermentation broth
Typical reactions in LFB. For coliforms, all tubes should be positive. Why?

27. Simmon citrate medium
Indole reaction
Methyl red reactions

28. Rapid Identification Kits.
Rapid identification kits may be used to identify E. coli.
Follow manufacturer’s instructions.

30. Membrane Filter Test
The membrane filtration method involves filtering several different-sized portions of the sample using filters with a standard diameter and pore size, placing each filter on a selective nutrient medium in a petri plate, incubating the plates at a specified temperature for a specified time period, and then counting the colonies that have grown on the filter.
This method varies for different bacteria types (variations might include, for example, the nutrient medium type, the number and types of incubations, etc.).

31. Membrane Filter Test Used to detect coliforms
Filter 100 mL water through a 0.45 m filter
Incubate filter on pad soaked with a differential medium (Endo medium; contains lactose and Basic Fuchsin dye) at 35°C for hours
Count colonies that grow on filter
coliforms will be dark red with metallic gold sheen
To enumerate Fecal Streptococci, grow on Streptococcus agar at 37°C for 24 hours. Fecal streptococci reduce 2,4,5-triphenyltetrazolium chloride to formazan, which makes colonies appear red
Much quicker and easier than MPN method

32. Colilert Test Used to detect total coliforms and E. coli
Add packet of salts and nutrients to water sample and incubate 24 hours
MUG
ONPG
Total coliforms can convert o-nitrophenyl--D-galactopyranoside (ONPG) to yellow nitrophenol with -galactosidase
E. coli can metabolize 4-methylumbelliferone glucuronide (MUG) to a molecule that fluoresces under UV light with glucuronidase
Colilert Quanti-Tray
Enumeration Method Lab Procedures
1) Different types of water samples require different types of preparation as follows:
a) For sterile (blank) water or relatively clean fresh water pour 100 ml of sterile water or sample directly
into the sterile 100 ml mixing bottle (by filling to the 100 ml line) and add one package of the reagent.
Cap and shake until dissolved.
b) For fresh water that is suspected to contain contamination, pour 50 ml of sterile distilled water into
the mixing bottle and add one package of the reagent. Cap and shake until dissolved. Then, after the
foam subsides, using a sterile pipette add 10 ml of sample and top off with 40 ml (to the 100 ml line).
Cap and shake again. This is a 1:10 dilution.
c) For all marine or estuarine water samples (salinity greater than 5 ppt), pour 50 ml of sterile distilled
water into the mixing bottle and add one package of the reagent. Cap and shake until dissolved. Then,
using a sterile pipette add 10 ml of sample and top off with 40 ml (to the 100 ml line). Cap and shake
again. This is a 1:10 dilution.
2) Make sure there is little or no foam left in the headspace of the mixing bottle prior to moving on to
the next step.
3) Pour sample/reagent mixture from the mixing bottle into a quanti-tray and seal in the IDEXX Sealer.
4) Place the sealed tray in a 35±0.5°C incubator for a minimum of 18 hours and a maximum of 22 hours
(includes warming time). This is the incubation period.
May not detect up to 1/3 of E. coli strains (including pathogenic ones!)
Broth and agar plate techniques involving ONPG and MUG also exist

33. Colilert: measures coliform bacteria for freshwater
Colilert-18 Measures coliform bacteria for marine waters
Analyte
Total coliform and E. coli
Time to result: 24 h
Results:
Colorless: Negative
Yellow: total coliform
Yellow fluorescent:
E. coli

34. As viewed under UV light
Enterolert Test
Enterolert™ reagent is used for the detection of enterococcus bacteria(enterococci) such as E. faecium and E. faecalis in fresh and marine water.
This product is based on Defined Substrate Technology® (DST™) and utilizes a nutrient indicator that fluoresces when metabolized by enterococci.
Analyte:
Enterococcus
Time to result:
24 h
Result:
Nonfluorescent: Negative
Fluorescent: Enterococcus
As viewed under UV light
Enterolert Quanti-Tray
Enumeration Method Lab Procedures
1) Different types of water samples require different types of preparation as
follows:
a) For sterile (blank) water or relatively clean fresh water pour 100 ml of sterile
water or sample directly into the sterile 100 ml mixing bottle (by filling to the 100
ml line) and add one package of the reagent. Cap and shake until dissolved.
b) For fresh water that is suspected to contain contamination, pour 50 ml of sterile
distilled water into the mixing bottle and add one package of the reagent. Cap and
shake until dissolved. Then, after the foam subsides, using a sterile pipette add 10
ml of sample and top off with 40 ml (to the 100 ml line). Cap and shake again.
This is a 1:10 dilution.
c) For all marine or estuarine water samples (salinity greater than 5 ppt), pour 50
ml of sterile distilled water into the mixing bottle and add one package of the
reagent. Cap and shake until dissolved. Then, using a sterile pipette add 10 ml of
sample and top off with 40 ml (to the 100 ml line). Cap and shake again. This is a
1:10 dilution.
2) Make sure there is little or no foam left in the headspace of the mixing bottle
prior to moving on to the next step.
3) Pour sample/reagent mixture from the mixing bottle into a quanti-tray and seal
in the IDEXX Sealer.
Place the sealed tray in a 41° ± 0.5° C incubator for a minimum of 24 hours and a
maximum of 28 hours (includes warming time). This is the incubation period
When the reagent is added to the sample and incubated, bacteria down to one MPN (most probable number) in a 100ml sample can be detected within 24 hours.

35. Colilert ™ Enterolert ™

36. Reading the Sample
Count the yellow cells that are positive and mark it.
Use a 6-watt 365nm UV light within 5 inches of the sample in a dark environment and count the positive cells.
Wear anti-UV glasses/goggles.
Filling the Quanti-Tray
1-Begin with sterile Quanti-Trays
2-Use one hand to hold the
Quanti-Tray upright with the
well side facing the palm
(bubble side).
3-Squeeze the upper part of the Quanti-Tray so that tray bends towards the palm.
4-Open the Quanti-Tray by Pulling the foil tab away from the well side. (Do not touch inside of the foil
or tray.)
5-Pour the reagent/sample mixture directly into the Quanti-Tray avoiding contact with the foil tab.
6-Tapping the Quantitray can help remove bubble. Allow the foam to settle.
Sealing the Quanti-Tray
1 Set the Quanti-Tray
within the proper
rubber insert.
2 Place the Quanti-
Tray/Inset onto the input
shelf and feed it into the
sealer.
2a Note:If the tray/insert will
not feed and it was feed into the
sealer correctly, gently lift up on
the input shelf’s outside lip
during feeding.
3 Remove Quanti-Tray and
insert. If they were inserted
crooked you may stop and
reverse the insertion and reinsert
them.
4 Sealed Quanti-
Tray removed
from the rubber
insert.

37. Most Probable Number
(MPN) Tables

39. END OF LECTURE