MLT (Microbial Limit Test) Validation

The validation test is done by following methods –
A. Preparation of culture suspension
B. Recovery of Viable Microorganism

A. Preparation of the Test Suspensions

1. Remove following culture slant from the refrigerator and allow it to attain room temperature.
• Bacillus subtilis
• Candida albicans
2. Inoculate loop full of the culture from each slant separately into 10 ml of sterile saline solution (0.9% sodium chloride solution).
3. Transfer 1.0 ml of the solution into 9.0 ml of sterile saline solution (0.9% sodium chloride solution) to obtain a test dilution of 10^-1.
4. Transfer 1.0 ml of the 10^-1 dilution into 9.0 ml of sterile saline solution to give 10^-2 dilution.
5. Similarly serially dilute the culture suspension to obtain dilution of 10^-3, 10^-4, 10^-5, 10^-6, 10^-7  and 10^-8.
6. Plate 1.0 ml of the culture suspension from dilution 10^-3 to 10^-8 in duplicate into sterile Petri dishes.
7. Pour approximately 15-20 ml of sterile Soybean Casein Digest Agar cooled to about 45°C in each plate. Incubate at 32.5 ± 2.5°C for 24 to 48 hours.
8. Count the number of colonies on each plate and select the dilution, which gives a count of 10 to 100 cfu for recovery of viable microorganisms.

B. Recovery of Viable Microorganisms

For Total Bacterial Count:

Step No	Negative control	Positive control	Product Control	Positive Product control
1	Add 1.0 ml of sterile water into the 100ml of Soyabean Casein digest Medium.	Add 1.0 ml of the culture suspension (containing 10 to 100 cells of B. subtilis) in 100 ml of sterile Soybean Casein digest medium.	Prepare sample by dissolving 10g of product under test in 100 ml of Soybean Casein digest medium.	Prepare sample by dissolving 10g of product under test in 100 ml of Soybean Casein digest medium and add 1 ml of culture suspension (containing 10 to 100 cells of B.Subtilis).
2	Aseptically pipette out 1 ml of the pretreated specimen in duplicate in sterile petridishes.	Aseptically pipette out 1 ml of the pretreated specimen in duplicate in sterile petridishes.	Aseptically pipette out 1 ml of the pretreated specimen in duplicate in sterile petridishes.	Aseptically pipette out 1 ml of the pretreated specimen in duplicate in sterile petridishes.
3	To each of the petridishes add about 20 ml of Soybean Casein Digest Agar (HiMedia Code No. M290), mix and allow to solidify	To each of the petridishes add about 20 ml of Soybean Casein Digest Agar (HiMedia Code No. M290), mix and allow to solidify	To each of the petridishes add about 20 ml of Soybean Casein Digest Agar (HiMedia Code No. M290), mix and allow to solidify.	To each of the petridishes add about 20 ml of Soybean Casein Digest Agar (HiMedia Code No. M290), mix and allow to solidify.
4	Invert the petridishes and incubate at 30°c to 35°c for five days.	Invert the petridishes and incubate at 30°c to 35°c for five days.	Invert the petridishes and incubate at 30°c to 35°c for five days.	Invert the petridishes and incubate at 30°c to 35°c for five days.
5	Count the number of Colony Forming Units developed at the end of incubation and   record the results.	Count the number of Colony Forming Units developed at the end of incubation and record the results.	Count the number of Colony Forming Units developed at the end of incubation and report that highest number of Colony Forming Units obtained in the plates. Multiply the highest number of Colony Forming Units by 10 and report this value as Total bacterial Count per 1 gm of the sample.	Count the number of Colony Forming Units developed at the end of incubation and report that highest number of Colony Forming Units obtained in the plates. Multiply the highest number of Colony Forming Units by 10 and report this value as Total bacterial Count per 1 gm of the sample.

For Total Fungal Count:  

Step No	Negative control	Positive control	Product Control	Positive Product control
1	Add 1.0 ml of sterile water into the 100ml of Soybean Casein digest Medium.	Add 1.0 ml of the culture suspension (containing 10 to 100 cells of C. albicans) in 100 ml of sterile Soybean Casein digest medium.	Prepare sample by dissolving 10g of product under test in 100 ml of Soybean Casein digest medium.	Prepare sample by dissolving 10g of product under test in 100 ml of Soybean Casein digest medium and add 1 ml of culture suspension (containing 10 to 100 cells of C. albicans).
2	Aseptically pipette out 1 ml of the pretreated specimen in duplicate in sterile petridishes.	Aseptically pipette out 1 ml of the pretreated specimen in duplicate in sterile petridishes.	Aseptically pipette out 1 ml of the pretreated specimen in duplicate in sterile petridishes.	Aseptically pipette out 1 ml of the pretreated specimen in duplicate in sterile petridishes.
3	To each of the petridishes add about 20 ml of Sabouraud Dextrose  Agar (HiMedia Code No. M063), mix and allow to solidify	To each of the petridishes add about 20 ml of Sabouraud Dextrose  Agar (HiMedia Code No. M063), mix and allow to solidify	To each of the petridishes add about 20 ml of Sabouraud Dextrose  Agar (HiMedia Code No. M063), mix and allow to solidify.	To each of the petridishes add about 20 ml of Sabouraud Dextrose  Agar (HiMedia  Code No. M063), mix and allow to solidify  .
4	Invert the petridishes and incubate at 20°c to 25°c for five days.	Invert the petridishes and incubate at 20°c to 25°c for five days.	Invert the petridishes and incubate at 20°c to 25°c for five days.	Invert the petridishes and incubate at 20°c to 25°c for five days.
5	Count the number of Colony Forming Units developed at the end of incubation and record the results.	Count the number of Colony Forming Units developed at the end of incubation and record the results.	Count the number of Colony Forming Units developed at the end of incubation and report that highest number of Colony Forming Units obtained in the plates. Multiply the highest number of Colony Forming Units by 10 and report this value as Total Fungal Count per 1 gm of the sample.	Count the number of Colony Forming Units developed at the end of incubation and report that highest number of Colony Forming Units obtained in the plates. Multiply the highest number of Colony Forming Units by 10 and report this value as Total Fungal Count per 1 gm of the sample.

Acceptance Criteria

The product under test is considered non-inhibitory to microorganism under the defined test condition if the following condition is met.

• The test is valid, i.e. negative control shows no growth. 
• The recovery of the organism from positive product control is not less than 75% when compared with the recovery of organisms from positive control.

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