Aerobic Mesophilic Plate Count

Sample diluted by 1:10 (11ml in 99ml, 11g in 99ml or 50g in 450ml)

Blend the Sample at low speed (app 8000rpm) in the Stomacher for 30-60 seconds
or shake vigorously (50 times through 30cm arc)

If count is more than 2.5x103 per ml or g

Prepare decimal dilutions as follows; shake each dilution 25 times through 30cm arc

Pipette 1ml of food homogenate into a tube containing 9ml of the diluents

From the first dilution transfer 1ml to second dilution tube containing 9ml of the
diluents, repeat until the desired dilution is obtained

Label all the petriplates with sample number, dilution etc

Pipette 1ml of food homogenate from the required dilutions into the petriplates in
duplicate

Pour 10 to 12ml of molten Plate Count Agar (PCA) cooled to 42-450C into the
petridish within 15min of dilution

Mix the media and dilutions by swirling gently clockwise, anti-clockwise, to and fro
thrice and taking care that the content does not touch the lid

Allow to set and incubate the plates at 350C for 48+2 hours

Following incubation count all the colonies on dish containing 30-300 colonies and
record the result as per the dilution
 Determination of Aciduric Flat Spore Former in Foods
(Bacillus coagulans responsible for spoilage of Canned products)

Sample diluted by 1:10 (11ml in 99ml, 11g in 99ml or 50g in 450ml)

Blend the Sample at low speed (app 8000rpm) in the Stomacher for 30-60 seconds or
shake vigorously (50 times through 30cm arc)

If count is more than 2.5x103 per ml or g

Prepare decimal dilutions as follows; shake each dilution 25 times through 30cm arc

Pipette 1ml of food homogenate into a tube containing 9ml of the diluents

From the first dilution transfer 1ml to second dilution tube containing 9ml of the
diluents, repeat until the desired dilution is obtained

Diluted samples in test tubes are heat shocked at 880C for 5 min and cooled
immediately

Label all the petriplates with sample number, dilution etc

Pipette 1ml of food homogenate from the required dilutions into the petriplates in
duplicate

Pour 18 to 20ml of molten Dextrose Tryptone Agar (DTA) with bromocresol

purple cooled to 42-450C into the petridish within 15min of dilution

Mix the media and dilutions by swirling gently clockwise, anti-clockwise, to and fro
thrice and taking care that the content do not touch the lid

Allow to set and incubate the plates at 550C for 48+2 hours

Following incubation count all the colonies on dish containing 30-300 colonies and
record the result as per the dilution
 Determination of Bacillus cereus in Foods and Beverages
(Plate Count Method)

Sample diluted by 1:10 (11ml in 99ml, 11g in 99ml or 50g in 450ml)

Blend the Sample at low speed (app 8000rpm) in the Stomacher for 30-60 seconds
or shake vigorously (50 times through 30cm arc)

If count is more than 1000 per ml or g

Prepare decimal dilutions as follows; shake each dilution 25 times through 30cm arc

Pipette 1ml of food homogenate into a tube containing 9ml of the diluents

From the first dilution transfer 1ml to second dilution tube containing 9ml of the
diluents, repeat until the desired dilution is obtained

Label all the petriplates with sample number, dilution etc

Pipette 0.1ml of food homogenate from the required dilutions into the
Mannitol Egg Yolk Polymyxin (MYP) Agar Plates

Spread the sample (Surface Plating) evenly with sterile bent glass streaking rods
(hockey sticks)

Incubate the plates at 300C for 24 hours

Following incubation count all the Eosin Pink colonies surrounded by lecithinase
zone and record the result as per the dilution

If colonies are not clear incubate plates for another 24 hours and count the plates
containing 15-150 colonies

Five or more colonies of presumptive B.cereus are picked from plates and transferred
to Nutrient Agar Slants for Confirmation
 Incubate the streaked Nutrient Agar Slants at 300C for 24 hours

After incubation Gram Stain the colony and examine under microscope

B.cereus will appear as large Gram Positive bacilli in short to long chains; spores
are ellipsoidal, central to sub-terminal and do not swell sporangium

Transfer 3mm loopful of this culture to a tube containing 0.5ml sterile diluents

Vortex mix, Inoculate or streak into the following media and read the biochemical
reaction

S.No Media Incubation at 350C Typical Reaction

1 Phenol red dextrose broth Incubate Acid produced (colour
anaerobically changes from red to
for 24 hours yellow)
2 Nitrate broth For 24 hours Reduces nitrates to
nitrites
3 Modified VP Medium For 48 hours Positive
4 Nutrient agar with tyrosine For 48 hours Positive
5 Nutrient broth with lysozyme For 24 hours Growth positive

Bacillus cereus = Present/Absent

Bacillus cereus/g
 Determination of Bacillus cereus in Foods and Beverages
(Most Probable Number (MPN) Method)

Sample diluted by 1:10 (11ml in 99ml, 11g in 99ml or 50g in 450ml)

Blend the Sample at low speed (app 8000rpm) in the Stomacher for 30-60 seconds
or shake vigorously (50 times through 30cm arc)

If count is less than 1000 per ml or g

Prepare decimal dilutions as follows; shake each dilution 25 times through 30cm arc

Pipette 1ml of food homogenate into a tube containing 9ml of the diluents

From the first dilution transfer 1ml to second dilution tube containing 9ml of the
diluents, repeat until the desired dilution is obtained

Inoculate 1ml of food homogenate into three Trypticase soy polymyxin broth from
each dilutions

Incubate the plates at 300C for 48 hours

Examine for dense growth typical of B.cereus

Vortex mix, inoculate loopful form each growth positive tube into the
Mannitol Egg Yolk Polymyxin (MYP) Agar Plates and Streak to obtain colonies

Incubate the plates at 300C for 48 hours

Following incubation count all the Eosin Pink (Mannitol fermentation positive)
colonies surrounded by lecithinase zone and record the result as per the dilution

Five or more colonies of presumptive B.cereus are picked from plates and transferred
to Nutrient Agar Slants for Confirmation
 Incubate the streaked Nutrient Agar Slants at 300C for 24 hours

After incubation Gram Stain the colony and examine under microscope

B.cereus will appear as large Gram Positive bacilli in short to long chains; spores
are ellipsoidal, central to sub-terminal and do not swell sporangium

Transfer 3mm loopful of this culture to a tube containing 0.5ml sterile diluents

Vortex mix, Inoculate or streak into the following media and read the biochemical
reaction

S.No Media Incubation at Typical Reaction

350C
1 Phenol red dextrose broth Incubate Acid produced (colour
anaerobically for changes from red to
24 hours yellow)
2 Nitrate broth For 24 hours Reduces nitrates to
nitrites
3 Modified VP Medium For 48 hours Positive
4 Nutrient agar with tyrosine For 48 hours Positive
5 Nutrient broth with lysozyme For 24 hours Growth positive

Bacillus cereus = Present/Absent

The Confirmed Bacillus cereus count is determined using the following table and
reported as MPN of Bacillus cereus/g
MPN per 1g of Sample using 3 tubes
Positive tubes Positive Tubes Positive tubes Positive tubes
0.1 0.01 0.001 MPN 0.1 0.01 0.001 MPN 0.1 0.01 0.001 MPN 0.1 0.01 0.001 MPN
0 0 0 <3 1 0 0 3.6 2 0 0 9.1 3 0 0 23
0 0 1 3 1 0 1 7.2 2 0 1 14 3 0 1 39
0 0 2 6 1 0 2 11 2 0 2 20 3 0 2 64
0 0 3 9 1 0 3 15 2 0 3 26 3 0 3 95
0 1 0 3 1 1 0 7.3 2 1 0 15 3 1 0 43
0 1 1 6.1 1 1 1 11 2 1 1 20 3 1 1 75
0 1 2 9.2 1 1 2 15 2 1 2 27 3 1 2 120
0 1 3 12 1 1 3 19 2 1 3 34 3 1 3 160
0 2 0 6.2 1 2 0 11 2 2 0 21 3 2 0 93
0 2 1 9.3 1 2 1 15 2 2 1 28 3 2 1 150
0 2 2 12 1 2 2 20 2 2 2 35 3 2 2 210
0 2 3 16 1 2 3 24 2 2 3 42 3 2 3 290
0 3 0 9.4 1 3 0 16 2 3 0 29 3 3 0 240
0 3 1 13 1 3 1 20 2 3 1 36 3 3 1 460
0 3 2 16 1 3 2 24 2 3 2 44 3 3 2 1100
0 3 3 19 1 3 3 29 2 3 3 53 3 3 3 >1100
 Determination of Anaerobic Mesophilic Spore Formers
(Clostridium perfringens)

Inoculate 2g or ml of food sample into 15 to 20ml of Cooked Meat Media in

duplicates

Incubate the plates at 350C for 24 hours

Positive tubes showing turbidity and gas production are streaked on to

Tryptone sulfite cycloserine (TSC) Agar

Incubate plates upright, anaerobically at 350C for 18-20 hours

Count all the colonies that are black in color surrounded by a zone of precipitate

Inoculate the black colony from TSC Agar on to Motility Nitrate Agar and Lactose
Gelatin Agar by Stabbing and thioglycolate broth

Incubate the streaked Nutrient Agar Slants at 350C for 24 hours

After incubation Gram Stain from the thioglycolate broth and

examine under microscope

Presence of large Gram Positive rods

Growth only in the stabbed area in Motility Agar (Non motile) and are
positive for Nitrate reduction indicated by red/orange colour in the medium

Growth in Lactose gelatin medium indicates Lactose fermentation indicated by gas

bubbles and change in colour of medium from red to yellow

Clostridium perfringens = Present/Absent

Detection, Determination and Confirmation of Coliforms by Pour Plate Method

Sample diluted by 1:10 (11ml in 99ml, 11g in 99ml or 50g in 450ml)

Blend the Sample at low speed (app 8000rpm) in the Stomacher for 30-60 seconds
or shake vigorously (50 times through 30cm arc)

If count is more than 2.5x103 per ml or g

Prepare decimal dilutions as follows; shake each dilution 25 times through 30cm arc

Pipette 1ml of food homogenate into a tube containing 9ml of the diluents

From the first dilution transfer 1ml to second dilution tube containing 9ml of the
diluents, repeat until the desired dilution is obtained

Label all the petriplates with sample number, dilution etc

Pipette 1ml of food homogenate from the required dilutions into the petriplates in
duplicate

Pour 10 to 12ml of molten Violet Red Bile Agar (VRBA) cooled to 480C into the
petridish within 15min of dilution

Mix the media and dilutions by swirling gently clockwise, anti-clockwise, to and fro
thrice and taking care that the content does not touch the lid

Allow to solidify and overlay 3 -5ml VRBA and solidify and

Incubate the plates at 350C for 18-24 hours

Following incubation count all the colonies that are purple red in colour, 0.5mm in
diameter or larger and surrounded by a zone of precipitated bile acids in the plate
containing 30-300 colonies and record the result as per the dilution.

Number of Coliforms per g/ml

 Detection, Determination and Confirmation of Coliforms by MPN Method
(Used for Low Coliform Density and Large quantity Sampling)

Sample diluted by 1:10 (11ml in 99ml, 11g in 99ml or 50g in 450ml)

Blend the Sample at low speed (app 8000rpm) in the Stomacher for 30-60 seconds
or shake vigorously (50 times through 30cm arc)

Prepare decimal dilutions as follows; shake each dilution 25 times through 30cm arc

Pipette 1ml of food homogenate into a tube containing 9ml of the diluents

From the first dilution transfer 1ml to second dilution tube containing 9ml of the
diluents, repeat until the desired dilution is obtained

Inoculate 1ml of food homogenate into three Lauryl Tryptose Sulphate (LST) broth
(containing inverted Durham tubes) from each dilutions and
Incubate the tubes at 350C for 24-48 hours

Examine for gas production at 24hrs and

reincubate the gas negative tubes and record after 48 hours

Transfer a loopful of each gas positive tubes on to

Brilliant Green Lactose Bile Broth (BGLB) and incubate at 350C for 48 hours

Formation of Gas Confirms Coliform bacteria

The Confirmed Coliform count is reported as MPN of Coliform/g
MPN per 1g of Sample using 3 tubes
Positive tubes Positive Tubes Positive tubes Positive tubes
0.1 0.01 0.001 MPN 0.1 0.01 0.001 MPN 0.1 0.01 0.001 MPN 0.1 0.01 0.001 MPN
0 0 0 <3 1 0 0 3.6 2 0 0 9.1 3 0 0 23
0 0 1 3 1 0 1 7.2 2 0 1 14 3 0 1 39
0 0 2 6 1 0 2 11 2 0 2 20 3 0 2 64
0 0 3 9 1 0 3 15 2 0 3 26 3 0 3 95
0 1 0 3 1 1 0 7.3 2 1 0 15 3 1 0 43
0 1 1 6.1 1 1 1 11 2 1 1 20 3 1 1 75
0 1 2 9.2 1 1 2 15 2 1 2 27 3 1 2 120
0 1 3 12 1 1 3 19 2 1 3 34 3 1 3 160
0 2 0 6.2 1 2 0 11 2 2 0 21 3 2 0 93
0 2 1 9.3 1 2 1 15 2 2 1 28 3 2 1 150
0 2 2 12 1 2 2 20 2 2 2 35 3 2 2 210
0 2 3 16 1 2 3 24 2 2 3 42 3 2 3 290
0 3 0 9.4 1 3 0 16 2 3 0 29 3 3 0 240
0 3 1 13 1 3 1 20 2 3 1 36 3 3 1 460
0 3 2 16 1 3 2 24 2 3 2 44 3 3 2 1100
0 3 3 19 1 3 3 29 2 3 3 53 3 3 3 >1100
 Detection, Determination and Confirmation of E.coli by Pour Plate Method

Sample diluted by 1:10 (11ml in 99ml, 11g in 99ml or 50g in 450ml)

Blend the Sample at low speed (app 8000rpm) in the Stomacher for 30-60 seconds
or shake vigorously (50 times through 30cm arc)

If count is more than 2.5x103 per ml or g

Prepare decimal dilutions as follows; shake each dilution 25 times through 30cm arc

Pipette 1ml of food homogenate into a tube containing 9ml of the diluents

From the first dilution transfer 1ml to second dilution tube containing 9ml of the
diluents, repeat until the desired dilution is obtained

Label all the petriplates with sample number, dilution etc

Pipette 1ml of food homogenate from the required dilutions into the petriplates in
duplicate

Pour 10 to 12ml of molten L-Eosin Methylene Blue Agar (L-EMB) cooled to 480C
into the petridish within 15min of dilution

Mix the media and dilutions by swirling gently clockwise, anti-clockwise, to and fro
thrice and taking care that the content does not touch the lid

Allow to solidify and overlay 3 -5ml L-EMB, solidify and

Incubate the plates at 350C+0.50C for 24+2 hours

Following incubation count all the colonies that are typical nucleated dark centered
colonies with or without sheen.

Typical colonies are transferred to PCA Slant and incubate at 350C+0.50C for
18-24 hours and proceed for biochemical tests
 Transfer growth from PCA Slants to
Tryptone broth and incubate for 24+2 hours at 350C+0.50C,
MR-VP broth for 48+2 hours at 350C+0.50C,
Koser citrate broth for 24+2 hours at 350C+0.50C,
and LST broth for 48+2 hours at 350C+0.50C

Perform Gram stain in a smear prepared from 18 hours PCA Slant,

Presence of Gram negative rods confirms Escherichia coli

S.No Indole MR VP Citrate Type

1 + + - - Typical E.coli
2 - + - - Atypical E.coli
3 + + - + Typical Intermediate
4 - + - + Atypical Intermediate
5 - - + + Typical Enterobacter aerogens
6 + - + + Atypical Enterobacter aerogens
 Detection, Determination and Confirmation of E.coli by MPN Method
(Used for Low E.coli Density and Large quantity Sampling)

Sample diluted by 1:10 (11ml in 99ml, 11g in 99ml or 50g in 450ml)

Blend the Sample at low speed (app 8000rpm) in the Stomacher for 30-60 seconds
or shake vigorously (50 times through 30cm arc)

Prepare decimal dilutions as follows; shake each dilution 25 times through 30cm arc

Pipette 1ml of food homogenate into a tube containing 9ml of the diluents

From the first dilution transfer 1ml to second dilution tube containing 9ml of the
diluents, repeat until the desired dilution is obtained

Inoculate 1ml of food homogenate into three Lauryl Tryptose Sulphate (LST) broth
(containing inverted Durham tubes) from each dilutions and
Incubate the tubes at 350C for 24-48 hours

Examine for gas production at 24hrs and

reincubate the gas negative tubes and record after 48 hours

Transfer a loopful of each gas positive tubes on to

Brilliant Green Lactose Bile Broth (BGLB) and incubate at 350C for 48 hours

Streak one plate L-Eosin Methylene Blue Agar (L-EMB) from each positive BGLB
tube and incubate at 350C for 24+2 hours

Examine the plate for typical nucleated dark centered colonies with or without sheen.

Typical colonies are transferred to PCA Slant and incubate at 350C+0.50C for
18-24 hours and proceed for biochemical tests
Transfer growth from PCA Slants to
Tryptone broth and incubate for 24+2 hours at 350C+0.50C,
MR-VP broth for 48+2 hours at 350C+0.50C,
Koser citrate broth for 24+2 hours at 350C+0.50C,
and LST broth for 48+2 hours at 350C+0.50C
 Perform Gram stain in a smear prepared from 18 hours PCA Slant,

Presence of Gram negative rods confirms Escherichia coli

Compute MPN of E.coli per g or ml considering gram negative, non spore forming
rods producing gas in lactose and classify biochemical types as follows

S.No Indole MR VP Citrate Type

1 + + - - Typical E.coli
2 - + - - Atypical E.coli
3 + + - + Typical Intermediate
4 - + - + Atypical Intermediate
5 - - + + Typical Enterobacter aerogens
6 + - + + Atypical Enterobacter aerogens

The Confirmed E.coli count is reported as MPN of E.coli/g

MPN per 1g of Sample using 3 tubes

Positive tubes Positive Tubes Positive tubes Positive tubes
0.1 0.01 0.001 MPN 0.1 0.01 0.001 MPN 0.1 0.01 0.001 MPN 0.1 0.01 0.001 MPN
0 0 0 <3 1 0 0 3.6 2 0 0 9.1 3 0 0 23
0 0 1 3 1 0 1 7.2 2 0 1 14 3 0 1 39
0 0 2 6 1 0 2 11 2 0 2 20 3 0 2 64
0 0 3 9 1 0 3 15 2 0 3 26 3 0 3 95
0 1 0 3 1 1 0 7.3 2 1 0 15 3 1 0 43
0 1 1 6.1 1 1 1 11 2 1 1 20 3 1 1 75
0 1 2 9.2 1 1 2 15 2 1 2 27 3 1 2 120
0 1 3 12 1 1 3 19 2 1 3 34 3 1 3 160
0 2 0 6.2 1 2 0 11 2 2 0 21 3 2 0 93
0 2 1 9.3 1 2 1 15 2 2 1 28 3 2 1 150
0 2 2 12 1 2 2 20 2 2 2 35 3 2 2 210
0 2 3 16 1 2 3 24 2 2 3 42 3 2 3 290
0 3 0 9.4 1 3 0 16 2 3 0 29 3 3 0 240
0 3 1 13 1 3 1 20 2 3 1 36 3 3 1 460
0 3 2 16 1 3 2 24 2 3 2 44 3 3 2 1100
0 3 3 19 1 3 3 29 2 3 3 53 3 3 3 >1100