In the intricate world of pharmaceuticals, maintaining the highest standards of quality and safety is non-negotiable. Microbiological analysis, particularly Total Aerobic Microbial Count (TAMC) and Total Yeast and Mold Counts (TYMC), emerges as a crucial player in guaranteeing the microbial purity of pharmaceutical products. Let's explore the intricate processes involved in these tests, unveiling the meticulous measures undertaken to ensure the integrity of medicines.
Understanding TAMC and TYMC:
TAMC and TYMC tests are quantitative assessments that enumerate mesophilic bacteria, total aerobic microbial count, and fungus count, including total combined yeasts and mold counts. These microorganisms thrive under aerobic conditions, making their enumeration imperative to guarantee pharmaceutical safety.
Sample Preparation:
The journey begins with meticulous sample preparation. A 1:10 concentration in Buffered Peptone Water (1401) at pH 7.0 ensures an optimal environment for subsequent analysis. This stock solution can be further fortified with neutralizers and surfactants to eliminate any interfering substances. The Most Likely Number Method (TAMPC) as mentioned in Part C, has extra steps unique to this test.
A- Membrane Filtration Method
1. Transfer solutions
- TAMC and TYMC Totals: Transfer 10 ml of the solution to a filter membrane (pore size 0.45 μm).
- Wash each filter membrane 3x100ml of Buffered Peptone Water (1401) at pH 7.0
2. Transfer plates and incubate
TAMC: Transfer the membrane to a Trypticasein Soy Agar (1068) and incubate at 30-35 °C //5 days
TYMC: Transfer the membrane to a Sabouraud Dextrose Agar plates (1024) and incubate at 20-25 °c//5-7 days
3. Calculate
Calculate CFU per gram or milliliter of the product.
B1- Plate Count Method (Pour Plate Method)
1. Transfer solutions and incubate
TAMC and TAYC Totals: Transfer 1ml of the stock solution into at least on empty and sterilized petri dish.
TAMC: Add 15-20 ml of Trypticasein Soy Agar (TSA), incubation at 30-35 °C//5 days
TYMC: Add 15-20 ml of Sabouraud Dextrose Agar, incubation at 20-25 °C//5-days
2. Calculate
Select the plates corresponding to the given dilution and present the highest number of colonies lower than 250 for TAMC and 50 for TYMC.
Calculate the number of CFY per g or ml of product.
B2- Plate Count Method (Surface Method)
1. Transfer solutions and incubate
TAMC: Transfer at least 0.1 ml of the stock solution to the Trypticasein Soy Agar (TSA), incubation at 30-35 °C//120 hours
TYMC: Transfer at least 0.1 ml of the stock solution to a Sabouraud Dextrose Agar plate, incubation at 20-25 °C//5-7 days
2. Calculate
Calculate the number of CFU per g or ml of product
C- Most Likely Number Method (TAMPC)
1. Sample Preparation
Besides the sample being prepared in Buffered Peptone Water, there are few additional steps unique to this test.
Make two serial dilutions from the stock solution of 1ml/ dilution in two tubes in 9ml each of the Trypitcasein Soy Broth (1224)
Using the initial three dilutions, carry out three further dilutions with at least 3 tubes/dilution in a concentration in a 1:10 in Trypticasein Soy Broth (TSB). Incubation at 30-35 °C/120 hours
Using the initial three dilutions, carry out three further dilutions with at least 3 tubes/dilution in a concentration of 1:10 in Trypticasein Soy Broth (TSB). Incubation at 30-35°C/max 3 days
2A: Confirmation with Turbidity
Perform a subculture in the same broth or on a plate or TSA.
Determine the most probable number of microorganisms by g or ml of product.
2B: Confirmation without Turbidity
Determine the most probable number of microorganisms by g or ml of product.
To conclude, microbiological analysis, specifically TAMC and TYMC, stands as an unwavering guardian in the pharmaceutical industry. Following standardized protocols outlined in references such as the 11th edition of "Standard Methods for the Examination of Dairy Products" and the "European Pharmacopoeia 9.3," these tests ensure that pharmaceuticals meet stringent microbial purity standards. As technology advances, these methods will continue to evolve, fostering a future where pharmaceuticals remain synonymous with quality and safety.
Reference:
Brewer. JAMA, 115. 1940. Vera. J. Bact. 47:59, 1944.
Pittman. J. Bact. 51:19, 1946.
European Pharmocopoeia 9.3.