Validation of Microbial Limit Test Method

 

Validation of Microbial Limit Test Method

PURPOSE

To lay down the procedure for Validation of Microbial limit Test Method as per USP.

SCOPE:

This protocol is applicable for Verification of Microbial Limit Test Methods for Raw material and non-sterile Products

MATERIAL AND EQUIPMENT

1.  Head cap, Lab coat ,Slippers, Mask
2.   Disposable and Sterilized Hand Gloves
3. Liquid Hand Sanitizer (70% IPA)
4.  Buffered Sodium chloride peptone solution pH 7.0
5.  Soyabean Casein Digest Medium
6.  Soyabean Casein Digest Agar
7.   Sabouraud Dextrose Agar
8.   Cetrimide Agar
9.  Mac Conkey Agar
10.  XLD Agar
11.  Mannitol Salt Agar
12.   Instruments: Incubator, Autoclave, Colony Counter, Required Glasswares
13.  Microbiological Testing Area (Laminar Air Flow/ Microbiological Safety Cabinet)
14. Test sample

Ø  Microbial Culture

a. Bacillus subtilis ATCC 6633

b. Candida albicans ATCC 10231

c. Pseudomonas aeruginosa ATCC 25619

d. Staphylococcus aureus ATCC 6538

e. E. coli ATCC 8739

f. Salmonella typhimurium ATCC 14028

g. Shigella boydii ATCC 8700

 

Preparation of Inoculum

• Ø  Harvest the organism from master culture slant of each organism individually in Buffered sodium chloride peptone solution pH 7.0 and Perform serial dilution up to 10⁸   to bring the microbial count between 10 to 100 cfu/ml.
• Ø  Inoculate 1 ml test solution from 10^5, 10^6, 10⁷ and 10⁸ dilutions in Triplicate and pour approximately 20-25 ml Soyabean casein digest agar (SCDA) for enumeration of bacteria and Sabouraud dextrose agar (SDA) for enumeration of Yeast and Mould. Allow to solidify
• Incubate SCDA plate at 35°C for 24-48 hours and SDA plate at 25°C for 3-5 days

 

Ø  Calculate the number of colonies obtained in plates using formula,

                TAMC/TYMC (cfu/ml) =   No. of colonies per ml X dilution factor

                                                                     Volume of inoculum in ml

Ø  Select the dilution containing 10-100 cfu/ml organism and use it for test.

 

Sample Preparation

Weight accurately 10 gm of sample and dissolve in 100 ml 1. Sterile Buffered Sodium chloride peptone solutions pH 7.0, or 2. Soyabean casein digest medium or 3. Fluid A (Test Sample).

 

Preparation of Fluid A:

Dissolve 1 gm of Peptic digest of animal tissue in water to make 1 litre and filter if necessary. Adjust to a P^H of 7.1±0.2.

Study Scheme

1.Test Group

In this group, product is subjected to the neutralization method and less than 100 cfu of challenge organism is inoculated for recovery.

2.Peptone Control Group

In this group, neutralization method is used with peptone as test solution and less than 100 cfu of challenge organism is inoculated for recovery.

3.Viability Group

In this group, actual Inoculum of less than 100 cfu is used, without exposure to the neutralizer is used for recovery.

Test Procedure

1.      Test Group

Ø  Inoculate 9 ml of test sample as prepared in “sample preparation” in 7 test tube and label each test tube with respective organism as

Test 1:  Bacillus subtilis

Test 2: Candida albicans

Test: Pseudomonas aeruginosa

Test 4: Staphylococcus aureus

Test 5: E.  Coli

Test 6: Salmonella typhimurium.

Test 7: Shigella boydii

• Ø  Add 1 ml inoculum of Bacillus subtilis containing less than 100 cfu in test 1 and mix well to dissolve. Inoculate 1 ml sample from Test 1 in Soyabean casein digest agar in triplicate, mix well and let it be solidified at room temperature. Incubate the plate at 30-35°C and observe the result after 3-5 days.
• Ø  Add 1 ml inoculum of Candida albicans containing less than 100 cfu in test 2 and mix well to dissolve. Inoculate 1 ml sample from Test 2 in Sabouraud dextrose agar in triplicate, mix well and let it be solidified at room temperature. Incubate the plate at 20-25°C and observe the result after 5 -7 days.
• Ø  Add 1 ml inoculum of Pseudomonas aeruginosa containing less than 100 cfu in Test 3 and mix well to dissolve. Inoculate 1 ml sample from Test 3 in Cetrimide agar in triplicate, mix well and let it be solidified at room temperature. Incubate the plate at 30-35°C and observe the result after 3-5 days
• Ø  Add 1 ml inoculum of Staphylococcus aureus containing less than 100 cfu in Test 4 and mix well to dissolve. Inoculate 1 ml sample from Test 4 in Mannitol salt agar in triplicate, mix well and let be solidified at room temperature. Incubate the plate at 30-35°C and observe the result 3-5 days
• Ø  Add 1 ml inoculum of E.  Coli containing less than 100 cfu in Test 5 and mix well to dissolve. Inoculate 1 ml sample from Test 5 in Mac conkey agar in triplicate, mix well and let it be solidified at room temperature. Incubate the plate at 30-35°C and observe the result after 3-5 days
• Ø  Add 1 ml inoculum of Salmonella typhimurium containing less than 100 cfu in Test 6 and mix well to dissolve. Inoculate 1 ml sample from Test 6 in XLD agar in triplicate, mix well and let it be solidified at room temperature. Incubate the plate at 30-35°C and observe the result after 3-5 days.
• Ø  Add 1 ml inoculum of Shigella boydii containing less than 100 cfu in test 7 and mix well to dissolve. Inoculate 1 ml sample from Test 6 in XLD agar in triplicate, mix well and let it be solidified at room temperature. Incubate the plate at 30-35°C and observe the result after 3-5 days.

Ø  Calculate the number of colonies obtained in plates using formula,

            TAMC/TYMC (cfu/ml) =   No. of colonies per ml X dilution factor

                                                        Volume of inoculum in ml

Peptone Group

Ø  Inoculate 9ml of Buffered sodium chloride peptone solutions pH 7.0 or Fluid A in 7 test tube and label each test tube with respective organism as

           Test 1:  Bacillus subtilis

           Test 2: Candida albicans

           Test 3: Pseudomonas aeruginosa

           Test 4: Staphylococcus aureus

           Test 5: E.  Coli

           Test 6: Salmonella typhimurium

           Test 7: Shigella boydii

• Ø  Add 1 ml inoculum of Bacillus subtilis containing less than 100 cfu in Test 1and mix well. Inoculate 1 ml sample from Test 1 in Soyabean casein digest agar in triplicate, mix well and let solidify at room temperature. Incubate the plate at 30-35°C and observe the result after 3-5 days.
• Ø Add 1 ml inoculum of Candida albicans containing less than 100 cfu in Test 2 and mix well. Inoculate 1 ml sample from Test 2 in Sabouraud dextrose agar in triplicate, mix well and let solidify at room temperature. Incubate the plate at 20-25°C and observe the result after 5-7 days.

Ø  Add 1 ml inoculum of Pseudomonas aeruginosa containing less than 100 cfu in Test 3 and mix well to dissolve. Inoculate 1 ml sample from Test 3 in Cetrimide agar in triplicate, mix well and let solidify at room temperature. Incubate the plate at 30-35°C and observe the result after 3-5 days.

Ø  Add 1 ml inoculum of Staphylococcus aureus containing less than 100 cfu in Test 4 and mix well. Inoculate 1 ml sample from Test 4 in Mannitol salt agar in triplicate, mix well and let solidify at room temperature. Incubate the plate at 30-35°C and observe the result after 3-5 days.

• Ø  Add 1 ml inoculum of E.  Coli containing less than 100 cfu in Test 5 and mix well. Inoculate 1 ml sample from Test 5 in Mac conkey agar in triplicate, mix well and let solidify at room temperature. Incubate the plate at 30-35°C and observe the result after 3-5 days.
• Ø  Add 1 ml Inoculum of Salmonella typhimurium containing less than 100 cfu in Test 6 and mix well. Inoculate 1 ml sample from Test 6 in XLD agar in triplicate, mix well and let solidify at room temperature. Incubate the plate at 30-35°C and observe the result after 3-5 days
• Ø  Add 1 ml Inoculum of Shigella boydii containing less than 100 cfu in Test 7 and mix well. Inoculate 1 ml sample from Test 6 in XLD agar in triplicate, mix well and let solidify at room temperature. Incubate the plate at 30-35°C and observe the result after 3-5 days.

Ø  Calculate the number of colonies obtained in plates using formula,

               TAMC/TYMC (cfu/ml) =   No. of colonies per ml X dilution factor

                                                                  Volume of inoculum in ml

 Viability Group

Ø  Inoculate 1 ml, inoculum of Bacillus subtilis containing less than 100 cfu/ ml in Soyabean casein digest agar in triplicate, mix well and let solidify at room temperature. Incubate the plate at 30-35°C and observe the result after 3-5 days.

Ø  Inoculate 1 ml, inoculum of Candida albicans containing less than 100 cfu/ ml in Sabouraud dextrose agar in triplicate, mix well and let solidify at room temperature. Incubate the plate at 20-25°C and observe the result after 5 -7 days.

Ø  Inoculate 1 ml, inoculum of Pseudomonas aeruginosa containing less than 100 cfu/ ml in Cetrimide agar in triplicate, mix well and let solidify at room temperature. Incubate the plate at 30-35°C and observe the result after 3-5 days.

Ø  Inoculate 1 ml, inoculum of Staphylococcus aureus containing less than 100 cfu/ ml in Mannitol salt agar in triplicate, mix well and let solidify at room temperature. Incubate the plate at 30-35°C and observe the result after 3-5 days

Ø  Inoculate 1 ml, inoculum of E.  Coli containing less than 100 cfu/ ml in Mac conkey agar in triplicate, mix well and let solidify at room temperature. Incubate the plate at 30-35°C and observe the result after 3-5 days.

Ø  Inoculate 1 ml, inoculum of Salmonella typhimurium containing less than 100 cfu/ ml in XLD agar in triplicate, mix well and let solidify at room temperature. Incubate the plate at 30-35°C and observe the result after 3-5 days.

Ø  Inoculate 1 ml, inoculum of Shigella boydii less than 100 cfu/ ml in XLD agar in triplicate, mix well and let solidify at room temperature. Incubate the plate at 30-35°C and observe the result after 3-5 days.

Ø   Calculate the number of colonies obtained in plates using formula,

                         T AMC/TYMC (cfu/ml) =   No. of colonies per ml X dilution factor

                                                                               Volume of inoculum in ml

Recovery Comparison

 

 

Ø  Test result must show the recovery of individual organism less than 100 cfu/ ml is not inhibited by the test sample and neutralization method.

Ø  Recovery between the test group and peptone group demonstrate adequate neutralizer efficacy and recovery between peptone group and viability group demonstrate adequate neutralizer toxicity.

Ø  Verification method shall demonstrate neutralizer effect and neutralizer toxicity by comparing recovery among three distinct test groups:

                        a.Test Group (Neutralized product with Inoculum)

                        b.Peptone Group (Challenge Inoculum control in buffer solution)

                        c.Viability Group (Inoculum in absence of product or neutralizer)

Ø  At least 3 replicate of the test should be performed and each test should demonstrate that the average number of cfu recovered from Test Group is not less than 70% of that recovered from Viability Group.

 

REFERENCE:

USP.36 “General chapter (1227) Validation of Microbial Recovery from Pharmacopoeial Articles”.