Growth Promotion Test-SOP

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:
AbbreviationExpanded form
SCDASoyabean Casein Digest Agar
SCDMSoyabean Casein Digest Medium
SCASabouraud Chloramphenicol Agar
cfuColony Forming Unit
SOPStandard Operating Procedure
°CDegree Celsius
NCIMNational Collection of Industrial Microorganisms
NANot applicable
RVSE BrothRappaport Vassiliadis Salmonella Enrichment Broth

7.0 ANNEXURES:
Annex. No.Title
01Growth Promotion Test Record
02Growth 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