SOP for Growth Promotion Test | growth Promotion Test Procedure | Media Growth Promotion Testing |
- Growth Promotion Test SOP covers below points :
- General precautions
- Preparation of media
- Growth promotion test of microbiological media/medium procedure
- growth promotion test of the Agar medium
- growth promotion test of the Broth medium
- growth promotion test of selective media (Indicative reactions)
- growth promotion test acceptance criteria
- Growth Promoting, Inhibitory and Indicative properties of media
1.0 OBJECTIVE :
- To provide a procedure for Growth promotion test of microbiological media.
2.0 SCOPE :
- This procedure applies for growth promotion test of new lot of Microbiological media.
3.0 RESPONSIBILITY :
- Microbiologist
4.0 ACCOUNTABILITY:
- Head of Department
5.0 PROCEDURE :
Growth promotion test is carried out for every new batch/lot of media/medium to determine the suitability of culture media. Prepare approximately 100 cfu/ml dilution of respective culture for growth promotion testing of different microbiological culture media.
5.1 General precautions :
- Take clean, dried glassware of desired volume as per quantity of media.
- Use separate spatula for separate media to avoid cross contamination.
5.2 Preparation of media:
- After receiving new Lot / Batch of the media open each container and withdraw 5.00 gm quantity from container, Make a composite sample and use for further test.
- Take clean dried conical flask as per the requirement of media. Weigh and suspend the quantity of the dehydrated media as per volume required, as per manufacturer’s instruction.
- Check the pH of media; adjust the pH using 0.1 M HCl/ 0.1 M NaOH, if required.
- Autoclave it at 121°C, 15 lbs for 15 minutes.
5.3 Growth promotion test of microbiological media/medium:
- Select the organism as per annexure-2, Growth Promoting, Inhibitory and Indicative properties of Media, for growth promotion having count NMT 100 cfu/ml.
- Preparation of Inoculum:
- Streak the particular organism as per annexure-2, Growth Promoting, inhibitory and Indicative properties of Media, on sterile slant of SCDA for bacteria and SCA for fungi. Incubate the slant for 30°C to 35°C at 18 to 24 hours for bacteria and 20°C to 25°C for 72 to 120 hours for fungi.
- Transfer a loopful culture to 10 ml of sterile saline.
- Shake it properly so as to obtain uniform suspension of the organism.
- Take 0.1 ml of above suspension and add aseptically to another test tube containing 9.9 ml sterile saline.
- Shake the tube gently and uniformly between the palms, the final inoculum is supposed to contain 100 CFU/ml.
- For the growth promotion test of the Agar medium:
- Take 1.0 ml of final inoculum containing 100 CFU/ml into sterile empty petri plates in duplicate and pour 15-20 ml of sterile respective media to be examined (temperature not more than 450C) gently.
- Mix the suspension and media.
- Allow to solidify the media at room temperature.
- Keep the plates in inverted position in incubator at respective temperature and time as per annexure-2, Growth Promoting, Inhibitory and Indicative properties of Media.
- After completion of incubation period, count the colonies under colony counter.
- Positive control: Repeat the above steps with previously tested and approved batch/ lot of media.
- Negative control: Take 1 ml final inoculum without test organism.
- Compare the microorganisms growth of the microorganisms occurs comparable to that previously obtained with a previously tested and approved batch of medium.
- Calculate average CFU of the new lot of media and average CFU of the previously approved lot of media and % recovery by using following equation;
Mean cfu = P1 + P2 / 2
% Recovery = Mean cfu observed x 100 / Inoculated cfu/ml
- Acceptance criteria:
- The average CFU of the new batch of media should be comparable to the average CFU of the batch of previously approved media.
- The % recovery not less than 50%.
- For the growth promotion test of the Broth medium:
- Take 1.0 ml of the final inoculums suspension in to sterile tube containing medium.
- Gently, mix the suspension and keep the tube for incubation as per annexure-2, Growth Promoting, Inhibitory and Indicative properties of Media.
- After completion of Incubation period, compare result with positive and negative control.
- Positive control: Repeat the above step with previously approved media lot.
- Negative control: Take 1 ml final inoculum without test organism.
- Compare visually, the growth (Turbidity) in the tubes of the new media with previously approved medium.
- Acceptance criteria: The growth (turbidity) in the tube of the new batch of media should be comparable to the batch of previously approved media.
- For the growth promotion test of selective media (Indicative reactions):
- Take loopful of the final inoculums and streak on agar surface of respective media.
- Keep the plates in inverted position in incubator at respective temperature and time as per annexure-2, Growth Promoting, Inhibitory and Indicative properties of Media.
- After completion of incubation period compare the result with positive and negative control.
- Positive control: Repeat the above step with previously approved media lot.
- Negative control: Take 1 ml final inoculum without test organism.
- Compare visually, the colonies characters on the new media agar plates with the colonies characters on the previously approved media agar plates as per annexure-2, Growth Promoting, inhibitory and Indicative properties of Media.
- Acceptance criteria: The colonies characters on the new media agar plates should be comparable with the colonies characters on the previously approved media agar plates.
6.0 ABBREVIATIONS:
| Abbreviation | Expanded form |
| SCDA | Soyabean Casein Digest Agar |
| SCDM | Soyabean Casein Digest Medium |
| SCA | Sabouraud Chloramphenicol Agar |
| cfu | Colony Forming Unit |
| SOP | Standard Operating Procedure |
| °C | Degree Celsius |
| NCIM | National Collection of Industrial Microorganisms |
| NA | Not applicable |
| RVSE Broth | Rappaport Vassiliadis Salmonella Enrichment Broth |
7.0 ANNEXURES:
| Annex. No. | Title |
| 01 | Growth Promotion Test Record |
| 02 | Growth Promoting, Inhibitory and Indicative properties of media |
8.0 SOP REFERENCES
- Indian Pharmacopoeia
- United State Pharmacopoeia 38 NF 33 <61>, <62>
- British Pharmacopoeia 2015 Appendix XVI B.
END OF THE SOP
ANNEXURES :
Annex. No. 01 Growth Promotion Test Record
Annex. No. 02 Growth Promoting, Inhibitory and Indicative properties of media
