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

2. 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

9. Principle:  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.  The typical colonies on L-EMB agar must be confirmed by further biochemical tests to prove the presence of E. coli.  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.

11.  The typical colonies on L-EMB agar must be confirmed by further biochemical tests to prove the presence of E. coli.  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.

12. 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.

13. Membrane Filter Test This method varies for different bacteria types (variations might include, for example, the nutrient medium type, the number and types of incubations, etc.).

14. 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 18-24 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

15. Colilert Test  Qualitative NOT quantitative  Used to detect total coliforms and E. coli  Add packet of salts and nutrients to water sample and incubate 24 hours  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  E. coli can metabolize 4-methylumbelliferone glucuronide (MUG) to a molecule that fluoresces under UV light with glucuronidase  Total coliforms can convert o-nitrophenyl-  -D-galactopyranoside (ONPG) to yellow nitrophenol with  -galactosidase ONPG MUG

16. 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

17. 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. 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. Analyte: Enterococcus Time to result : 24 h Result: Nonfluorescent: Negative Fluorescent: Enterococcus As viewed under UV light

18. Reading the Sample  Count the yellow cells that are positive, and mark the cell with a “Sharpie”.  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.

19. Most Probable Number (MPN) Tables

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

22. 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 44.5oC and 45oC. Water level should be above the medium in immersed tubes.

23. Materials and special equipment: 10. Thermometer, calibrated and traceable 11. Incubator, 35oC. 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 / 45o 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 negative control: Enterobacter aerogenes or an equivalent gram negative rod that does not produce “positive” reactions on EMB and is indole-negative, methyl rednegative, 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.

24.  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.

25. 15. Supplies needed for confirmation (commercially available): A. IMViC media and reagents: i.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).

26. Procedures  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 1.In the laboratory prior to analysis, except for shelf-stable foods, keep sample units refrigerated (0-5oC) 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.

27. 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.

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

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

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

31. 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.

32.  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.  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.

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

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

36. Methyl red reactions Simmon citrate medium Indole reaction

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

39. END OF LECTURE