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.  

• E.coli 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 E.coli 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 E.coli 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 E.coli 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 E.coli 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 E.coli 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 E.coli 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 E.coli 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 E.coli 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

C.freun
Possible identity E.coli C.koseri C.koseri
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 E.coli
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 E.coli

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