Total Microbial Count test refers to the counting of viable microorganisms in any dosage form or raw material etc. There are several official methods for conducting total microbial count test. In this article, I will give you a step by step guide on how to perform total microbial count test.
Now let us talk about the procedure of conducting this test. There are two official tests for conducting total microbial test which I think are easy to perform and are cost-effective.
- Pour Plate Method
- Spread Plate Method
Pour Plate Method
Prepare all the require culture media (Tryptic Soy Agar, Tryptic Soy Broth and Sabouraud Dextrose Agar) as per supplier instructions and autoclave it.
Take solid dosage form equivalent to 10 grams (tablets, capsules, powder) and 10 ml of the liquid dosage form (syrups, suspension) suspended/dissolved in 90 ml of Trypticase Soy Broth aseptically.
The result of the addition of 10 g/ml in 90 ml of broth is 100 ml. Now take aseptically 1 ml from the 100 ml broth in two pairs of Petri plates (previously sterilized by dry heat sterilization). Incubated the remaining broth at 30-35 °C for 24-48 hours.
After completing the above step pour about 15 to 20 ml of molten Trypticase Soy Agar in the plates and the plates swirled gently for even mixing.
About 15 to 20 ml of molten Sabouraud Dextrose Agar (SDA) poured in the remaining two plates. These plates swirled gently for even mixing.
The next step is to allow poured culture media to solidify.
Incubate the plates at 30-35°C for 3-5 days.
The Sabouraud Dextrose Agar (SDA) plates incubated for 5-7 days at 20-25 °C.
Spread Plate Method
Take 10 grams of the solid dosage form (tablets, capsules, powder) and 10 ml of the liquid dosage form (syrups, suspension) suspended/dissolved in 90 ml of Trypticase Soy Broth aseptically.
Aseptically add 1 ml of inoculated broth on Tryptic Soy Agar plate. The remaining broth incubated at 30-35 °C for 24-48 hours.
The inoculated sample distributed uniformly on the TSA plate with the help of a glass spreader.
The plates incubated at 30-35°C for 3-5 days.
Follow the same procedure for SDA plates but incubated for 5-7 days at 20-25 °C.
After completion of incubation, the results expressed in the form of colony-forming units. If no growth of microbial colonies observed than the results expressed as less than 10 microorganisms per gram or per ml.
Documentation and Reporting of Results
Record the number of counts (CFU) on the microbiological testing record. Where,
Bacterial (Aerobic) Count = B
Mold & Yeast Count = M
Pathogen = P
The number of cfu found on TSA is equal to the:
The Total Aerobic Microbial Count (TAMC)
If colonies of fungi seen on TSA then count as a part of TAMC.
The number of cfu found on SDA is equal to:
The Total Combined Yeast &Mold (TCYM)
You should count colonies of bacteria as a part of TYCM if found on SDA plates.
Results reported by filling Microbiological Testing Report.
Results calculated by multiplying with factor 10.
Table Acceptance Criteria for Microbiological Quality of Non-Sterile Dosage Forms
|
Total Microbial Count |
Acceptance Criteria |
||
|
TAMC (cfu/g or cfu/ml) |
TCYM (cfu/g) |
Specified microorganisms |
|
|
Non-aqueous preparation for oral use |
103 |
102 |
Absence of E.coli in 1g or 1ml. |
|
Aqueous preparation for oral use |
102 |
101 |
Absence of E.coli in 1g or 1ml. |
|
Oromucosal use |
102 |
101 |
Absence of staphylococcus aureus in 1g or 1ml Absence of Pseudomonas aeruginosa in 1g or 1ml. |
|
Vaginal use |
102 |
101 |
Absence of Candida albicans in 1g or 1ml Absence of Pseudomonas aeruginosa in 1g or 1ml Absence of Staphylococcus aureus in 1g or 1ml. |