Transcript Lab7-Enumeration of total coliform, fecal colifrom and E.coli in foods

Lab 7:
Enumeration of coliforms, fecal
coliforms and E. coli in
foods
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
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Total coliforms
Fecal coliforms
Escherichia coli
Enterococci.
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
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 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
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.
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.
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 humanspecific than the larger fecal streptococcus
group.
Sources of Coliform Bacteria
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.
 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.
 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.
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.
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.).
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
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
MUG
 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
 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
ONPG
• 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
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
• 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.
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.
Most Probable Number
(MPN) Tables
Basic Steps
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Collecting Samples
Transporting Samples
Delivering Samples
Lab Preparation
Preparing Sample
Incubating Sample
Reading Sample
Disposing Sample
Data Management
Data Interpretation
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-Dglucuronide)
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.
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.
 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. 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).
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.
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.
Typical growth results observed for brilliant green lactose bile
broth. Use these tubes to determine an MPN
Typical growth results observed for EC broth.
Use these tubes to determine an MPN
Colony types observed on EMB agar. Both fish-eye-type and
coli-type colonies are shown.
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.
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.
 The dark colonies produced on EMB agar is a result of the
acid produced during lactose fermentation precipitating the
dyes in the media.
Lactose fermentation broth
Typical reactions in
LFB. For coliforms, all
tubes should be
positive. Why?
Indole reaction
Methyl red reactions
Simmon citrate
medium
 Rapid Identification Kits.
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Rapid identification kits may be used to identify E. coli.
• Follow manufacturer’s instructions.
END OF LECTURE