AN

ASSIGNMENT
ON

DETECTION OF COLIFORM FROM ANIMAL ORIGIN

FOOD
Submitted To: Submitted By:
Dr. C. V. Savalia Alpesh P.Suthar
VPH-602
Professor & Head M.V.Sc Scholar
(Practcle Assign.)
Dept. of Vet. Public Health & Dept. of Vet. Public Health &
Epidemiology Epidemiology

Department of Veterinary Public Health and Epidemiology

Vanbandhu College of Veterinary Science and Animal Husbandry
Navsari Agricultural University
Navsari – 396 450
 INTRODUCTION
• Coliform bacteria are a commonly used indicator of sanitary
quality of foods and water.
• They are defined as rod shaped Gram-negative non-spore
forming and motile or non-motile bacteria which
can ferment lactose with the production of acid and gas when
incubated at 35–37°C. 
• Coliforms can be found in the aquatic environment, in soil and on
vegetation; they are universally present in large numbers in the
feces of warm-blooded animals.
• While coliforms themselves are not normally causes of serious
illness, they are easy to culture, and their presence is used to
indicate that other pathogenic organisms of fecal origin may be
present.
 DETECTION OF
COLIFORM FROM
ANIMAL ORIGIN FOOD
(MILK )
 Materials and methods
• All the samples were collected in sterilized container
and were brought in ice box to the Laboratory, for the
isolation of E. coli.
• Microbiological methods
• Preparation of serial dilutions:
• Ten ml from each sample of unpasteurized raw milk
were transferred to 90 ml sterile peptone water (0.1%)
and thoroughly mixed to give 1:10 dilution 'first
dilution'; serial dilutions were prepared by transferring
one ml from first dilution to 9ml peptone water and so
on as described by Harrigan and McCance (1976).
• Coliform bacteria, E. coli:
• The coliform test was done according to Harrigan by plating
one ml sample onto Mac Conkey agar media.
• The plates were incubated at 37ºC for 48 h and the counts
were presented as colony forming unites per gram (cfu/ml).
• Plates showing positive coliform were subjected to the
confirmatory test using Brilliant green bile lactose broth in
test tubes with inverted Durham tubes and incubated at 44ºC
for 48 h.
• Each positive tube was sub cultured into E.C. broth medium
and then incubated at 44.5ºC for 24 h. Tubes showing gas
productions were considered E. coli positive.
• All the samples positive for E. coli contamination were
confirmed using Gram's staining, cultural and biochemical
examinations.
• For the isolation and identification of E. coli, the enriched
sample was cultured on selective medium Levine Eosin
Methylene Blue (EMB) Agar and incubated at 37ºC for 24 h.
• Morphologically typical colonies (at least 4/plate) producing
metallic sheen were taken into nutrient broth for further
identification. Biochemical tests were performed to confirm E.
coli using Gram staining, Catalase test, Indole, Methyl red,
Voges- Proskauer test, Nitrate reduction, Urease production,
Simmon's citrate agar, and various sugar fermentation tests.
Biochemical characterization of E. coli
 DETECTION OF
COLIFORM FROM
ANIMAL ORIGIN FOOD
(MEAT)
 Presumptive test for coliform bacteria
• Sample preparation:
• Weigh 50 g meat sample into sterile high-speed blender jar.
• Add 450 ml Butterfield's phosphate-buffered dilution water
and blend 2 min.
• Frozen sample can be softened by refrigerating 25 g
portion for 18 h at 2-5°C.
• Preparation of serial dilutions:
• Prepare decimal dilutions with 90 ml sterile dilution water
plus 10 ml from previous dilution.
• Number of dilutions to be prepared depends on anticipated
coliform density.
• Shake all suspensions 25 times in 30 cm arc for 7 s. Do
not use pipets to deliver <10% of their total volume.
• Transfer 1 ml portions to 3 LST tubes for each dilution
for 3 consecutive dilutions.
• Hold pipet at angle so that its lower edge rests against
tube. Let pipet drain 2-3 s.
• Not more than 15 min should elapse from time sample is
blended until all dilutions are in appropriate media.
• Incubate tubes 48 +/- 2 h at 35°C. Examine tubes at
24 +/- 2 h for gas, i.e., displacement of medium in
fermentation vial or effervescence when tubes are
gently agitated.
• Reincubate negative tubes for additional 24 h.
Examine a second time for gas. Perform a confirmed
test on all presumptive positive (gassing) tubes.
 Confirmed test for coliforms
• Gently agitate each gassing LST tube and transfer
loopful of suspension to tube of BGLB broth.
• Hold LST tube at angle and insert loop to avoid
transfer of pellicle (if present).
• Incubate BGLB tubes 48 +/- 2 h at 35°C. Examine for
gas production and record.
• Calculate most probable number (MPN) of coliforms
based on proportion of confirmed gassing LST tubes
for 3 consecutive dilutions.
 Test for Faecal Coliforms
• Most Probable Number (MPN) method was used to
• determine faecal coliforms in the sample.
• Serial dilutions of 10-1 to 10-4 were prepared by
picking 1 ml of the sample into 9 ml sterile distilled
water.
• 1 ml aliquot from each of the dilution were inoculated
with 5 ml of Mac Conkey broth and inoculated into at
44C for faecal coliforms for 18-24 hours.
• Tubes showing changes from purple to yellow were
identified as positive for faecal coliforms. Counts per
100 ml were calculated from Most Probable Number
(MPN) tables.
• Test for Total Viable Count (TVC)
• Microorganisms were isolated and enumerated by the pour plate
method and growth on plate count agar (PCA).
• Serial dilutions of 10-1 to 10-4 were prepared by diluting 1g of
the sample (meat or fish) into 10ml of sterilized distilled water.
• One millimeter aliquots from each of the dilutions were
inoculated into petri dishes with already prepared PCA.
• The plates were then incubated at 35oC for 24hrs. After
incubation all white spots or spread were counted and recorded
as total viable counts using the colony counter. The counts for
each plate were expressed as colon forming unit of the
suspension (cfu/g).
• Test for the presence of Escherichia coli (Thermo
tolerant Coliforms)
• From the positive tubes that were identified, a drop was
transferred into a 5 ml test tube of trypton water and incubated at
44C for 24 hours.
• A drop of Kovacs reagent was then added to the tube of trypton
water.
• All tubes showing a red colour development after gentle agitation
denoted th presence of indole and were recorded as presumptive
for thermo tolerant coliforms (E. coli).
• Counts per 100 ml were calculated from Most Probable Number
(MPN) tables.
• A drop of culture from TVC plate was placed on slide, spread with
a flamed sterile loop, allowed to dry and fixed the bacteria by
passing the slide two times through a Bunsen flame.
 REFERANCES

• Harrigan, W. F. (1998). Laboratory methods in food

microbiology. Gulf Professional Publishing.

• Harrigan, W.F. and MacCance, M.E. (1976). Laboratory

Methods in Food and Dairy Microbiology.1st Edn.,
Academic Press, London.