Isolation and detection of
Coliforms and Fecal coliforms in
Foods
MCB 180 T-4L Exercise 5
Blas, June Vincent Caresosa, Ellaine Joyce
Coliforms
• Gram-negative, rod-shaped bacteria
• Non-spore forming, lactose fermenting
• common in
– soil and surface water
– human and animal waste
• indicator organisms- indicates the potential
presence of pathogens in water
Procedure LSTB
Presumptive Test for the presence of coliforms-
Lauryl Sulfate Tryptose Broth
Reagents:
• Sodium lauryl sulfate- inhibits gram-positive
microorganisms, selective agent for coliforms
• Lactose-detection of lactose fermentation by
coliforms
• Potassium phosphate-acts as buffer
• Sodium chloride-maintain osmotic equilibrium
• Durham tube- detect the production of gas
Presumptive test for presence of coliforms-
esult:
R
sit ive uction
Po prod y
a s it
→G Turbid
→
Result- LSTB
Food Sample 10-1 10-2 10-3 MPN
Isaw 3 3 2 1100
Siomai 3 0 0 23
Palamig 3 3 3 >1100
Procedure BGLBB
Confirmatory test for the presence of coliforms-
Brilliant Green Lactose Bile Broth
Reagents:
• Peptone- contains bile that inhibits gram-positive
microorganism, source of nitrogen
• Brilliant green dye- inhibits selected gram-negative
bacili
• Durham tube- detection of lactose-fermenting
organisms by gas production
Confirmatory test for the presence of coliforms-
esult:
R
sit ive uction
Po prod y
a s it
→G Turbid
→
Result-BGLB
Food
10-1 10-2 10-3 MPN
Sample
Isaw 3 3 3 >1100
Siomai 2 0 0 9
Palamig 3 3 3 >1100
Procedure EMBA
Test for Escherichia coli- Eosin
Methylene Blue Agar
Reagents:
• Eosin Y and methylene blue- inhibits gram-
positive bacteria
• Peptone- contains bile that inhibits gram-
positive microorganism, source of nitrogen
• Lactose-detection of lactose fermentation by
coliforms
• Potassium phosphate-acts as buffer
Gram negative bacteria
that ferment the
lactose produce acid
which turns the
colonies dark purple
as the acid acts upon
the dyes.
Lactose non-fermenters
are either colorless or
light lavender
• Certain lactose-
fermenting bacteria
produce flat, dark
colonies with a green
metallic sheen.
• [Link] colonies have
a characteristic green
sheen.
Test for Escherichia coli
e sult: n
it ive R shee
Pos etallic
r een m
G
Result- EMBA
Food Sample Positive result- Greenish metallic sheen
Isaw + -
Siomai + +
Palamig - -
Standard Methods Agar- for culture
growth
Reagents:
• Glucose- energy source
• Yeast extract- B-complex vitamins
• Tryptone- bacterial growth nutrients, amino
acid source
Procedure- IMViC
IMViC Test
Indole test
• to determine the
ability of an organism
to split amino acid
tryptophan to form
the compound indole
Identification of [Link] by IMViC test-
Indole
Reagents:
• 1% Tryptone Broth- contains tryptophan that
is hydrolysed by tryptophanase to produce
three possible end products – one end
product; indole
• Kovac's Reagent-contains 4 (p)-
dimethylamino benzaldehyde, reacts with
indole (red compound)
Identification of [Link] by IMViC test-
Indole
lt: dark
R esu rface
o sitive on su
P ring
re d
Methyl Red (MR) test determines whether
the microbe performs mixed acids fermentation
when supplied glucose.
• Color Red- indicated by large amonts of acid,
pH<4.4, positive result
• Color yellow- pH>5.1
Identification of [Link] by IMViC test-
Methyl red test
Reagents:
• MRVP Medium- for Methyl Red (MR) test and
Voges-Proskauer (VP) test
• Methyl red test- contains methyl red (p-
dimethylaminoaeobenzene-O-carboxylic acid)
that serves as pH indicator
Identification of [Link] by IMViC
test- Methyl Red
e sult: n
R
sit ive solutio
Po lored
co
red
Voges-Proskauer test
• determines whether the
microorganisms produces
2,3-butanediol, a
fermentation product from
glucose
• 2,3-butanediol- detects
acetoin that reacts with
reagents to turn red
Identification of [Link] by IMViC test-
Voges-Proskauer test
Reagents:
• MRVP Medium- for Methyl Red (MR) test and
Voges-Proskauer (VP) test
• Solution A: Alpha-naphthol 5%- color intensifier
• Solution B: Potassium hydroxide 40%- oxidizing
agent
Identification of [Link] by IMViC test-
Voges-Proskauer test
e sult:
R
sit ive olor
Po ink c
p
Citrate Utilization-
• to determine the ability of bacteria
to utilize sodium citrate as its only
carbon source and inorganic
(NH4H2PO4) is the sole fixed
nitrogen source.
• Growth usually results
in the bromothymol blue
indicator, turning from
green to blue.
• agar color is deep forest
green at neutral pH.
• pH> 7.6 bromothymol
blue changes from
green to blue
Identification of [Link] by IMViC test-
Citrate utilization
Reagents: Simmons Citrate Agar
• Potassium phosphate-acts as buffer
• Sodium chloride-maintain osmotic equilibrium
• Magnesium sulfate- cofactor for metabolic
reaction
• Bromthymol blue- pH indicator
• Sodium citrate- carbon and energy source
Identification of [Link] by IMViC test- Citrate
utilization
e sult:
it ive R lue
Pos en to b
gre
IMViC reaction of colonies results
IMViC test Isaw Siomai Palamig (b,p)
Indole test + + - - + -
Methyl red test + + - - + +
Voges-Proskauer
- - - - - -
test
Citrate utilization
- + + + + +
test
[Link]
Possible identity [Link] [Link] [Link]
dii
Escherichia coli
• gram-negative, facultatively
anaerobic, rod-shaped non-spore-forming
rod
• constitute about 0.1% of gut flora
• can cause serious food poisoning
Citrobacter freundii
• facultative anaerobic, gram-negative, long
bacterial rods
• Most C. freundii cells generally have several
flagella
• Slow lactose fermenter & urea hydrolyzer
soil organism but can also be found in:
intestinal tracts of animals and humans.
water
sewage
food
• responsible for reducing nitrate to nitrite in
the environment
• Pathogen to humans:
nosocomial infections of the respiratory
tract, urinary tract, blood, and many other
normally sterile sites in patients
Citrobacter koseri
• Gram-negative, nonspore-forming bacillus
• facultative anaerobe capable of aerobic
respiration
• motile via peritrichous flagella
• part of the normal flora of human and
animal digestive tracts
LST-MUG test for coliforms and [Link]
Reagents
• Tryptose- provides the nitrogen, carbon
compounds, vitamins and amino acids.
• Lactose- the fermentable sugar.
• Sodium lauryl sulphate- inhibits organisms other
than coliforms.
• Potassium phosphates- controI the pH during
fermentation of lactose.
• βD-glucoronidase (produced by E. coli)
cleaves 4-methylumbelliferyl-β-D-
glucuronide to 4-methylumbelliferone and
glucuronide.
• The fluorogen 4-methylumbelliferone can be
detected under a long wavelength UV lamp.
Colilert test for coliforms and [Link]
Colilert -
• commercially available enzyme-substrate
liquid-broth medium that allows the
simultaneous detection of total coliforms and
Escherichia coli (E. coli).
• a chromogen that reacts with the enzyme
found in coliforms (galactosidase), and a
fluorogen that reacts with an enzyme
found in E. coli (glucuronidase).
• Positive reaction-
coliform turns the
medium yellow;
• E. coli-positive
reaction causes the
medium to fluoresce
under a long-wave
ultraviolet light (366
nm).
Petrifilm-
• common method of
analyzing water for
coliforms
• uses membrane filter to
capture bacteria
• filter then placed on petri
dish and colonies formed
were counted