Detection of E.

coli from food sample

For B.Sc Biotechnology 6th Semester Practical

By
Dr Shally Sultana Choudhury
Asst. Professor
Department of Biotechnology
Pandu College: Maligaon: Assam
 Introduction
• Coliform bacteria are rod-shaped, gram-negative, non-
spore forming bacteria which can ferment lactose with the
production of acid and gas when incubated at 37° C.
• E. coli are gram-negative, facultative anaerobic, rod-shaped
coliform bacteria, produce indole from tryptophan within
24 hours of incubation.
• Detection of E. coli is a very important Microbiological
testing parameter for Food, Feed, Water and Environmental
sample analysis. This presentation represents a detailed
procedure for the detection of E. coli in a sample.
 Principle
 Chromocult® Coliform Agar
• Chromocult® Coliform Agar ES is intended for use in Microbiology
laboratories analyzing food and animal feeds. This chromogenic
culture medium is selective and differential and enables E. coli and
coliforms from food matrices, such as raw ground beef, raw ground
chicken and raw milk, to be detected, differentiated and
enumerated within 24 hours.
• Fresh food usually contains a high microbial load and generally does
not contain stressed or injured bacteria. The high level of
accompanying flora requires higher selectivity of the culture
medium to ensure inhibition of unwanted bacteria and allow the
target organisms to grow well.
• The combination of suitable peptones and the buffering using MOPS allow
rapid growth of coliforms and an optimal transformation of the
chromogenic substrates. The amount of bile salts and propionate largely
inhibit growth of Gram-positive and Gram-negative accompanying flora.

• The simultaneous detection of total coliforms and E. coli is achieved using

the combination of two chromogenic substrates. The substrate Salmon™-β-
D-GAL is cleaved by β-D-galactosidase, characteristic for coliforms, resulting
in a salmon to red colouration of coliform bacteria colonies. The substrate X-
β-D- glucuronide is cleaved by β-D-glucuronidase, characteristic for E. coli,
causing positive colonies to turn blue in colour.
• As E. coli cleaves Salmon™-β-D-GAL as well as X-β-D-
glucuronide, its colonies turn to a dark violet colour and are
easily differentiated from the other coliforms having a salmon-
red colour.
 Eosin Methylene Blue agar(EMB agar)
• EMB agar is characterized by the presence of a combination of
the two dyes - eosin and methylene blue in the ratio of 6:1.
• Gram-negative bacteria that ferment the lactose produce acid
which lowers the pH. This encourages dye absorption by the
colonies and turns the colonies dark purple as the acid acts
upon the dyes.
• In EMB agar, most of the strains of E. coli colonies have a
characteristic green sheen. Rapid fermentation of lactose &
production of strong acids, thus a rapid reduction in the pH of
the EMB agar, the critical factor in the formation of the green
metallic sheen observed with E. coli.
 Sorbitol MacConkey Agar (sMAC)
• This is only slightly selective, since the concentration of
bile salts, which inhibits gram-positive microorganisms, is
low in comparison with other enteric plating media.
• Peptone and proteose peptone supply necessary
nutrients like nitrogenous and carbonaceous
compounds, long-chain amino acids, minerals, vitamins
and trace ingredients for the growth of organisms.
• Sodium chloride maintains osmotic equilibrium. Neutral
red is an indicator. D-Sorbitol is the fermentable
carbohydrate.
• Crystal violet present in the medium inhibits the growth
of gram-positive bacteria, especially enterococci and
staphylococci.

• Differentiation of enteric microorganisms is achieved by

the combination of sorbitol and the neutral red indicator.
The growth of E.coli on MacConkey Agar with Sorbitol
shows colorless colonies. Colorless or pink to red
colonies are produced depending upon the ability of the
isolate to ferment the carbohydrate sorbitol.
 Requirements
 Sample: Raw ground chicken/mutton
 Apparatus and Equipments:
 Electronic balance
 Autoclave
 Laminar air flow cabinet
 Inubator
 Petridish
 Micropipette and tips
 Mixer grinder
 Inoculating loop
 Spatula
 Chemicals:
 Chromocult coliform agar
 EMB agar
 Sorbitol MacConkey agar
 EC broth
 Peptone
 Sodium chloride
 Procedure
 Step 1- Media and diluent preparation
 Step 2- Sample preparation
 Step 3- Selective enrichment
 Step 4- Streaking on CCA plate
 Step 5- Confirmatory test
 Step 6- Result observation
 Step 7- Result interpretation
 Step 1
 Preparation of Chromocult coliform agar:
 Suspend 34.5 g in 1000 ml of purified water and heat to
boiling at 100°C with frequent agitation until completely
dissolved (approximately 4- 5 minutes).
 Do not autoclave, do not overheat.
 Immediately cool the medium in a water bath at 45-50 °C (a
precipitate appears if a period of 2 hours is exceeded).
 pH: 7.0 ± 0.2
 Pour the medium in sterile Petriplates under Laminar air
flow and allow them to solidify.
 Preparation of EC broth:
 Suspend 1.85g of EC broth in a conical flask and add 50 ml of
distilled water.
 Shake to dissolve the medium completely.
 Tranfer 10 ml to test tubes and autoclave at 121° C and 15 lbs
pressure for 15 minutes.

 Preparation of EMB agar:

 Suspend 3.6 g of EMB agar in 100 ml distilled water.
 Autoclave the medium at 121° C and 15 lbs pressure for 15
minutes.
 Distribute 15ml into sterile petriplates. Cool and store in dark
condition.
 Preparation of Sorbitol MacConkey Agar:
 Suspend 5g in 100 ml distilled water. Heat if necessary to dissolve
the medium completely.
 Autoclave the medium at 121° C and 15 lbs pressure for 15
minutes.
 Distribute 15ml into sterile petriplates and allow to cool.
 Preparation of diluent/Peptone salt:
 Suspend 0.5g peptone into a large conical flask.
 Add 4.25g NaCl into the same conical flask.
 Dissolve the content into 500ml distilled water.
 Autoclave the diluent at 121° C and 15 lbs pressure for 15
minutes.
 Step 2
 Sample Preparation:
 Weigh 10g of raw chicken/mutton and put it in a sterile
blender.
 Add about 50ml of sterile diluent to it and blend completely.
 Take the sample to the Laminar flow hood and add another
50 ml to it. Mix thoroughly to make a 10¯¹ dilution.
 Take 1ml of 10¯¹ dilution and add to test tube having 9ml of
diluent. This makes a 10¯² dilution.
 Carry out the serial dilution of the sample till 10¯⁶ dilution.
 Step 3
 Selective enrichment of E.coli:
 Take two EC broth for Trial-1 and Trial-2.
 Shake the sample of 10¯³ dilution and transfer 1ml of it to EC broth (T-1) tube for Trial-
1.
 Discard the tip.
 Plug the mouth of the tube and shake gently to mix the content.
 Attach another sterile tip and transfer 1ml of sample into another EC broth (T-2) tube
for Trial-2.
 Discard the tip.
 Plug the mouth of the tube and shake gently to mix the content.
 Incubate the enrichment broth at 37°C for 24 hours.
 After 24 hours take out the tubes from the incubator.
 Cloudy appearance of the broth indicates that enrichment has been done successfully.
 Step 4
 Streaking on Choromocult coliform agar plate:
 Burn an inoculating loop to red hot and cool for 5 minutes inside the
LAF.
 Shake the T-1 EC broth tube and pick one loopful of EC broth culture.
 Streak the culture on first quadrant of Chromocult coliform agar plate
 Burn to red hot and cool the loop again.
 Now streak on the second, third and fourth quadrant of the plate.
 Similarly, streak from T-2 EC broth tube .
 Incubate the plates at 37°C for 24 hours.
 Take out the plates after 24 hours and observe the colonies.
 Violet and blue colonies appear. These are suspected as E.coli.
 Step 5
 Confirmatory test:
 Confirmatory test # 1: Cultivation of suspected E.coli colony on
Sorbitol MacConkey Agar
 Pick a violet-blue colony from CCA plate and streak on sMAC agar plate.
 Label the plate as ‘Presumptive E. coli’.

 Confirmatory test # 2 : Cultivation of suspected E.coli colony on

EMB Agar plate.
 Pick another colony from CCA plate and streak onto EMB agar plate.
 Label the plate as “Presumptive E. coli’.
 Incubate all the plates at 37°C for 24 hours.
 After 24 hours, take out the plates and observe the colony characteristics.
 Step 6
 Result Observation:
 Confirmatory test result # 1: Colonies on sMAC agar are
pink in colour which confirms the colonies as E.coli.

 Confirmatory test result # 2: Colonies on EMB agar

produce a metallic green sheen which confirms the
presence of E. coli.
 Step 7
 Result interpretation: Violet colonies on CCA plate, Pink
colonies on sMAC agar and Metallic Green sheen on EMB
agar plate are observed.
 All the results confirm that E.coli is detected in the sample.

CCA plate sMAC agar plate EMB agar plate