AB Mauri India Private Limited WI: CSL M6

MICROBIOLOGY: STANDARD OPERATING PROCEDURE Issue Date: 14/04/2016

Revision Date: 04/01/2019

Method Title: COLIFORMS - MPN METHOD

1. Purpose :
To enumerate Coliforms in spice and spice products by MPN method

2. Apparatus:
2.1 Pipettes-with 1ml tips
2.2 Test tubes (18 X 150 mm)
2.3 Polypropylene bottle (250 ml)
2.4 Incubator 35+/-1°C
2.5 Sterile spoons and spatulas
2.6 Electronic Weighing Balance
2.7 Inoculation loop
2.8 Durham’s tube
2.9 Spirit lamp

3. Reagents :
3.1 Phosphate Buffer (M461 Himedia)
3.2 Lauryl Tryptose Broth (M080 Himedia)
3.3 Brilliant Green Bile Broth (M121 Himedia)

4. Preparation of Media:
4.1.Preparation of Sterile Buffered Dilution blank: Add 1.25ml of stock Butterfield
Phosphate Buffer to 1L deionized water. Mix well and dispense 9ml & 90ml into required
no. of tubes and bottles respectively. Sterilize by autoclaving at 15lbs pressure for 15
minutes.
(Stock Butterfield Phosphate Buffer: 34g Phosphate Buffer per 1L deionized water. pH
7.2+/-0.2).
4.2.Preparation of Lauryl Tryptose Broth: Suspend 35.6 grams of LT Broth in 1L distilled
water. Boil to dissolve the medium completely. Dispense 10ml to test tubes and insert
Durham’s tubes. Sterilize by autoclaving at 15lbs pressure for 15 minutes.
4.3.Preparation of Brilliant Green Bile Broth: Suspend 40.0 gms of BGB in 1000ml distilled
water. Mix well and dispense 10ml to test tubes and insert Durham’s tubes. Sterilize by
autoclaving at 15lbs pressure for 15 minutes.
5. Procedure:
5.1. Sample Preparation:
Aseptically weigh 10g of sample into a 250 ml PP bottle containing 90ml sterile dilution blank.
This dilution is 10-1. Shake at least 25times in a gt.arc.
5.2. Preparation of Dilutions:

ABM Analytical Methods Manual – Coliforms Page 1 of 3

AB Mauri India Private Limited WI: CSL M6
MICROBIOLOGY: STANDARD OPERATING PROCEDURE Issue Date: 14/04/2016
Revision Date: 04/01/2019

5.2.1.To prepare 10-2dilution, transfer 1 ml of 10 -1dilution to 9ml dilution blank and shake
25 times in a 1ft arc.
5.2.2.To prepare 10-3dilution, transfer 1 ml of 10 -2dilution to 9ml dilution blank and shake
25 times in a 1ft arc. (Use separate sterile pipette /tip for each dilution)
5.2.3.Prepare as many decimal dilutions as necessary (at least 3) depending on the
anticipated coliform density.
5.2.4.Shake each dilution before preparing each sub sequential dilution.
5.3. Presumptive test:
5.2.1.Transfer 1ml aliquots of each dilution to 3 LT tubes.
5.2.2.Shake well all suspension 25 times in 1 ft arc.
5.2.3.Do not use pipettes to deliver <10% of their volume.
5.2.4.Do not insert the pipette more than 2.5cm below the surface of the sample.
5.2.5.If the pipette becomes contaminated before completing transfers, replace it with a
sterile microtip.
5.2.6.Complete the preparation of initial dilution and pouring of agar within 15 minutes.
5.2.7.Incubate tubes for 24-48 hrs at 35+/-1°C.
5.2.8.Examine tubes at 24+/-2 hrs for gas formation ie displacement of medium in
Durham’s tubes or effervescence when tubes gently agitated. Re-incubate negative
tubes for an additional 24 hrs. Examine.
5.2.9.Perform confirmed test on all positive tubes.
5.4. Confirmed Test:
5.2.1.From each gassing LT tube, transfer a loopful of suspension to BGB broth tubes.
Avoid pellicle if present.
5.2.2.Incubate 24-48 hrs at 35°C.
5.2.3.Examine the tubes for gas production.
5.2.4.Calculate most probable number (MPN) of coliforms based on proportion of
confirmed gassing LT tubes for 3 consecutive dilutions.

6. Calculation:
6.1. When more than three dilutions are used, select last (highest) dilution in which three
tubes are positive and next two dilutions. If this is not possible, select last three positive
dilutions.
6.2. Record the number of positive tubes in each dilution. Refer MPN table to find MPN
index.

MPN/g = (MPN/g from table/100) X dilution factor of the middle tube

7. Reference:
7.1. FDA Bacteriological Analytical Manual
7.2. Compendium of Methods for Microbiological examinations of Foods.

ABM Analytical Methods Manual – Coliforms Page 2 of 3

AB Mauri India Private Limited WI: CSL M6
MICROBIOLOGY: STANDARD OPERATING PROCEDURE Issue Date: 14/04/2016
Revision Date: 04/01/2019

8. Environmental aspects:
On completion of work all contaminated materials should be sterilized.

Coliform Bacteria Count - MPN Method

Most Probable Number (MPN) per 1g of sample, using 3 tubes with 0.1, 0.01 and 0.001 g portions

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

MPN Table - from ASTA Microbiological methods, Method M-3.1

ABM Analytical Methods Manual – Coliforms Page 3 of 3