1.0 ) OBJECTIVE:
To lay down the procedure for carrying out microbiological analysis of filled capsules.
2.0 ) PROCEDURE:
2.1 ) MEDIA PREPARATION:
Preparation of Culture media:
- Weigh the required quantity of dehydrated Soyabean Casein Digest Medium (Tryptone Soya Broth), Soyabean Casein Digest Agar (for bacteria) and Sabouraud Dextrose agar (for fungus) and add required quantity of freshly collected distilled water in each sterilized flasks.
- Mix well and heat the media to dissolve properly and check the pH of media before sterilization.
- Autoclave the media for sterilization at 121ºC temperature and 15 psi pressure for 15 minutes.
2.2 ) MICROBIAL ANALYSIS OF FILLED CAPSULE
A.) Total microbial count B.) Pathogen detection
A.) TOTAL MICROBIAL COUNT
- Pour plate method by serial dilution:
- Take 2 test tubes and label them A & B and 6 petriplates (3 for SCDA & 3 for SDA) and label the petriplates as 10ˉ¹, 10ˉ², 10ˉ³.
- Pour 9ml buffer solution (Nacl / Peptone water) in each test tube.
- Take 99ml of Soyabean Casein Digest Medium (Tryptone Soya Broth) in borosilicate glass bottle and add 1 gm of capsule sample to make 1:100 dilution and mix well.
- Pipette out 1ml of this dilution with the help of sterile micropipette and open the lids of empty sterile petriplate labeled 10ˉ¹ and dispense into the middle of each petriplate aseptically and then close the lid of petriplate.
- Transfer 1ml of capsule sample to the test tube labeled A to make 1:10 dilution and mix well.
- Take 1ml of sample from test tube A and dispense aseptically into the petriplate labeled 10ˉ².
- Transfer 1ml of sample from test tube A to test tube B and mix well.
- Then take 1ml of sample from test tube B and dispense the sample into the petriplate labeled 10ˉ³.
- Remove the cotton plug from the flask of molten agar (SCDA & SDA) and pass the rim of opened flask through the flame of Bunsen burner.
- Open the lid of petriplate containing the sample and pour 20-25ml sterile Soyabean Casein Digest Agar (for TAMC) and Sabouraud Dextrose Agar (for TYMC) cooled at 45ºC into the petriplates.
- Close the lid of petriplates and mix thoroughly by gently swirling the plates & allow solidifying the media at room temperature.
- Use aseptic technique throughout the procedure.
- Invert and incubate the SCDA plates at 30- 35ºC for 3-5 days (for TAMC) and SDA plates at 20-25ºC for 5-7 days (for TYMC).
- Also incubate the Soyabean Casein Digest Medium containing capsule sample for 24 hours at 30- 35ºC temperature for pathogen detection test.
- After incubation examine the SCDA plates for total bacterial count and SDA plates for total yeast and mold count.
- Take maximum from the two plates and calculate the no. of CFU/ml of water sample.
- PATHOGEN DETECTION
Detection of Escherichia coli:
- Streak a loopful culture of enriched pre-incubated Soyabean Casein Digest Medium containing capsule sample on the surface of sterile MacConkey Aagr plate.
- Incubate the plates at 30-35°C for 18-72 hours.
- After incubation presence of brick red colonies on MacConkey agar indicate the presence of Coli.
- Further confirmation is done by gram’s staining.
Detection of Salmonella:
- Transfer 0.1 ml of enriched Soyabean Casein Digest medium to 10 ml of Rappaport Vassiliadis Salmonella enrichment broth and incubate at 30-35°C for 24-48 hours.
- If subcultured on plate Xylose-Lysine Deoxycholate Agar and incubate at 30-35°C for 24-48 hours. Well developed red colonies with or without black centre indicates the possibility of presence of
- Further confirmation is done by gram’s staining.
Detection of Pseudomonas aeruginosa:
- Streak a loopful culture of pre-incubated Soyabean Casein Digest Medium (containing capsule sample) on the surface of Cetrimide Agar medium and incubate at 30-35°C for 18-72 hours.
- A greenish colored colony indicates the possibility of presence of aeruginosa.
- Carry out the further confirmation by gram’s staining.
- Further confirmation done by oxidase test.
Detection of Staphylococcus aureus:
- Streak a loopful culture of pre-incubated Soyabean Casein Digest medium (containing capsule sample) on the surface of Mannitol Salt Agar plates. Incubate the plates at 30-35°C for 18-72 hours.
- Examine the plates after incubation.
- If upon examination of incubated plates none of them contains yellow colonies with yellow zones the sample meets the requirements for the absence of Staphylococcus aureus.
- If growth occurs further confirm by streak the colonies on the surface of Baired Parker agar plates black, shinny colonies surrounded by clear zones indicates the presence of Staphylococcus aureus.
- Further confirm by gram’s staining.
Detection of Aspergillus brasiliensis:
- Streak a loopful culture of enriched fluid Soyabean Casein Digest medium on the surface of Sabouraud Dextrose Agar plates. Incubate the plates at 30-35for 18-72 hours.
- Growth of black color colonies may indicate the presence of Aspergillus brasiliensis.
- Further confirm by microscopic examination.
Test for Candida albicans:
- Streak a loopful culture of enriched fluid Soyabean Casein Digest medium on the surface of Sabouraud Dextrose Agar plates. Incubate the plates at 30-35for 18-72 hours.
- Growth of white colonies may indicate the presence of Candida albicans.
- Further confirmation was done by microscopic examination.
TESTING PARAMETERS
| Sr. No. | PARAMETERS | LIMITS |
| 1. | Description | As per sample observation |
| 2. | Identification test | Should be comply |
| 3. | Weight of 20 capsules | Informative |
| 4. | Average weight | Informative |
| 5. | Average fill | informative |
| 6. | Uniformity of wt. | Should be comply (±7.5% w/w) |
| 7. | Disintegration time | NMT 30 Minutes |
| 8. | Microbiological limits | |
| Total bacterial count | Not more than 1000 CFU/ml | |
| Total Fungal count | Not more than 100 CFU/ml | |
| Escherichia coli | Should be absent | |
| Salmonella | Should be absent | |
| Staphylococcus aureus | Should be absent | |
| Pseudomonas aeruginosa | Should be absent | |
