SOP Title: Serial Dilution Preparation of Microbial Cultures
- Objective:
To lay down the Procedure for Serial Microbial Cultures Dilution.
- Scope
This procedure is applicable for Serial Microbial Cultures Dilution in the Microbiology Section of Quality Control Department.
- Responsibility:
3.1 Quality Control: To prepare and review the SOP. To follow the procedures for Serial Dilution Preparation of Microbial Cultures as per this SOP.
- Quality Assurance Department: To review and approve the SOP and Annexure.
- Accountability:
Head Quality Control Department, Head Quality Assurance Department
- Procedure:
| 5.1 | Safety Precautions: | ||
| 5.1.1 | Wear personal protective equipments such as protective hand gloves, nose masks and over gown during operation. | ||
| 5.1.2 | Microbiologist should disinfect the hand gloves with 70% v/v IPA before carrying out operation. | ||
| 5.2 | Procedure for Serial Dilution: | ||
| 5.2.1 | Remove the slants of the Working Cultures from the refrigerator and allow to stand for at least 2-3 hrs to attain room temperature. | ||
| 5.2.2 | Refer Procedure for Media Preparation and Sterilisation as per SOP for Media preparation. | ||
| 5.2.3 | Inoculate a loopful of the bacterial cultures from working culture slants in test tubes containing 10 ml of Soyabean Casein Digest Medium and fungal cultures in Saborauds Dextrose Broth. Both SCDM and SDB are considered as stock cultures for dilution (100). Refer Annexure for List of cultures. Refer SOP for maintainance of cultures. | ||
| 5.2.4 | Incubate the bacterial cultures at 30-35°C for 18-24 hours, fungal cultures at 20-25°C, Candida albicans for 48hrs and Aspergillus brasilliensis for 72 to 120 hours or until good sporulation. | ||
| 5.2.5 | Use Buffered Sodium Chloride Peptone Solution to make the culture dilutions. | ||
| 5.2.6 | 0.05% polysorbate 80 may be added to the buffered solution to suspend spores of Aspergillus brasilliensis | ||
| 5.2.7 | Switch ‘ON’ the LAF and proceed for operation. | ||
| 5.3 | A) Small Volume Serial Dilutions: | ||
| 5.3.1 | Label four screw-capped tubes as 1:100, 1:10,000, 1:1,00,000 and 1:1,000,000. | ||
| 5.3.2 | In scientific notation this would be 10–2, 10–4, 10–5 and 10–6. For details, refer Annexure. | ||
| 5.3.3 | Using a sterile 10 ml pipette, add 9.9 ml of sterile BSCP aliquot into first, second and fourth tubes and add 9.0 ml in third tube. | ||
| 5.3.4 | Using a 100μL micropipette and sterile tip, transfer 0.1 ml of broth culture into the 10-2 tube and Cap the tube. Glass pipette of 0.1 ml can also be used. | ||
| 5.3.5 | Mix it for a few seconds vigorously flick the tube to adequately disperse the bacteria evenly throughout the tube and break up bacterial clumps.
Note: Do not shake the test tube. |
||
| 5.3.6 | With new sterile tip, transfer 0.1ml from first tube (1:100 or 10-2) and add it to the tube labeled as 1:10,000 or 10-4. Repeat the tube mixing procedure. | ||
| 5.3.7 | With new sterile tip, transfer 1.0 ml broth culture from the second tube (1:10,000 or 10-4) and add it to third tube labeled as 1:100,000 or 10-5
Repeat the tube mixing procedure. |
||
| 5.3.8 | With new sterile tip, transfer 0.1 ml from the second tube (1:10,000 or 10-4) and add it to fourth tube labeled 1:1000,000 or 10-6. Repeat the tube mixing procedure. | ||
| 5.3.9 | Plate out 0.1 ml and 1.0 ml of each dilution on sterile Petri plates in duplicate.
Also plate out stock cultures (100) in duplicates. |
||
| 5.3.10 | Pour about 20 ml of Soyabean Casein Digest Agar cooled to 45°C in to petri plates for bacterial culture and Saborauds Dextrose Agar with or without antibiotic for fungal cultures. | ||
| 5.3.11 | Rotate clockwise and anticlockwise for uniform mixing. Repeat this procedure for all the cultures. | ||
| 5.3.12 | Incubate the plates at 30-35°C for 48 hrs except plate containing dilutions of Candida albicans which is incubated at 20-25°C for 48 hours and Aspergillus Brasilliensis for 72 to 120 hours. | ||
| 5.3.13 | After completion of incubation period observe the plates and note down the colonies and select the dilutions which show colonies NMT 100 CFU(approximately 10–100 CFU). If the dilutions are showing more than 100 CFU then further dilution to be done. | ||
| 5.3.14 | Record the results of Serial Culture Dilution in Annexure. | ||
| 5.4 | B) Large Volume Serial Dilutions: | ||
| 5.4.1 | Select four sterile bottles and labeled as 1:100, 1:10,000,100,000 and 1:1,000,000. | ||
| 5.4.2 | In scientific notation this would be 10-2, 10-4, 10-5 and 10-6. For details refer Annexure No. 01. | ||
| 5.4.3 | Add measure volume of sterile buffer solution in to the bottle as follows.
Labeled Bottle as. 1:100(10-2), 1:10,000 (10-4) and 1:1,000,000.(10-6) add 99 mL and for 100,000 (10-5) add 49 ml sterile buffer solution respectively. |
||
| 5.4.4 | Using a sterile 1mL pipette, transfer 1mL of broth culture into the 10-2 bottle Repeat the tube mixing procedure as per 5.3.5. | ||
| 5.4.5 | Using a new sterile 1ml pipette, transfer 1ml from the (1:100 or 10-2) and add it to the bottle labeled 1:10,000 (10-4). Repeat the bottle shaking procedure. | ||
| 5.4.6 | Using a new sterile 1 ml pipette, transfer 1 ml from the 1:10,000 (10-4) bottle and add it to the 1:1,00,000 (10-5) bottle. Repeat the shaking procedure. | ||
| 5.4.7 | Using a new sterile 1 ml pipette, transfer 0.1 ml from the 1:10,000 (10-4) bottle and add it to the 1:1,000,000 (10-6) bottle. Repeat the shaking procedure. | ||
| 5.4.8 | Follow the procedure as per 5.3.9 to 5.3.15. | ||
| 5.4.9 | After completion of incubation and result recording, the petriplates and test tubes shall be decontaminated as per define SOP. | ||
| 5.4.10 | Store the test tubes or bottles containing 100 CFU culture at 2-8°C in refrigerator for its further use. Discard the rest of the dilution tubes. | ||
| 5.4.11 | If the Serial dilution results are not proper, than the microbiologist should investigate the cause. The Serial Dilution has to be performed again to get count within range. | ||
| 5.5 | Serial Dilutions Preparations | ||
| 5.5.1 | Frequency : Monthly | ||
| 5.5.2 | Serial dilutions to be performed for all micro organisms. | ||
- Definitions / Abbreviations:
- Definitions:
- Abbreviations:
| Abbreviation | Expansion |
| LAF | Laminar Air Flow |
| °C | Degree centigrade |
| ml | mililiter |
| µl | microlitre |
| % | percentage |
| v/v | Volume by volume |
| SCDA | Soyabean Casein Digest Agar |
| SDA | Saborauds Dextrose Agar |
| SDB | Saborauds Dextrose Broth |
| BSCP | Buffered Sodium Chloride Peptone Solution |
| SCDM | Soyabean Casein Digest Medium |
List of Cultures for Serial Dilutions
| Culture Name | Abbreviation |
| Bacillus subtilis | B. subtilis |
| Candida albicans | C. albicans |
| Escherichia coli | E. coli |
| Pseudomonas aeruginosa | P. aeruginosa |
| Salmonella abony | S. abony |
| Staphylococcus aureus | S. aureus |
| Shigella boydii | S. boydii |
| Aspergillus brassiliensis | A. brasilliensis |
*Note – Ready to use SOP available in “DOWNLOAD” Section.