Growth Promotion, Inhibition, Sterility and Indicative Test and its Acceptance Criteria

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Growth Promotion test: Growth promotion test is used for the qualification of the media. We use different media for microbiology testing. But before using that media in any testing media must qualify GPT. In growth promotion test, media are inoculated with different kinds of microorganisms as mentioned in pharmacopoeia. For example general purpose media like Soyabean casein digest agar (SCDA) and Nutrient agar (NA) are inoculated with different cultures. But for selective media particular microorganism is used for inoculation as mentioned in pharmacopoeia. Not more than 100 cfu culture is required for the growth promotion test. Spread plate method is used for solid agar media.

Duration of incubation is very important in GPT. Media should be incubated for the shortest period as the time mentioned in the pharmacopoeia. For example if 24 to 48 hours time is mentioned then for GPT, media should be incubated for 24 hours. As per pharmacopoeia, acceptance criteria for GPT is ±factor 2. Meaning of ±factor 2 is, standard inoculum size is multiplied by 2 and divided by 2. For example if we have standard  size of 50 cfu’s then acceptance criteria would be 25 cfu’s to 100 cfu’s. But 70% recovery is also good option for in house acceptance criteria for GPT. But in case of liquid broth media copious or luxuriant growth should be there in form of turbidity and should be comparable to the previous tested and approved lot of the media.

Inhibition test: In pharmacopoeia, in addition to growth promotion test, inhibition test is also mentioned. Inhibition test is used for selective media which can support the growth of particular microorganism and inhibit the growth of other type of microorganisms. In inhibition test, particular microorganism is inoculated in the media in a concentration of not less than 100 cfu’s and that media shouldn’t support the growth of that microorganism. For example in case of Cetramide agar media E.coli is used for inhibition and that media should not support the growth of E.coli and it should inhibit the growth of E.coli. This is called inhibition test. In inhibition test, media should be incubated for the longest period. For example if 24 to 72 hours are mentioned then media should be incubated for 72 hours and no growth should be found on the media at the end of incubation time. Spread plate method is used for solid agar media.

Sterility test: In media sterility test, prepared plate of media should be incubated with the other GPT and inhibition test plates. No microorganism added in that case. Sterility test plates should be incubated till the end of the test and no growth should be there on the media till the end of the test.

Indicative test: In indicative test, we check the characteristic growth and indication reaction on the media. For indicative test observation, we can consider GPT plates. Not more than 100 cfu’s culture suspension is used for that. Spread plate method is used for solid agar media. Indicative test plates should be incubated for a period of time within the range specified in the test. For example if 24 to 48 hours incubation time is mentioned then we can release the plates in between 24 to 48 hours. Colonies should be comparable in appearance and indication reactions to those previously obtained with a previously tested and approved batch of medium.

As per pharmacopoeia, every new received batch of the media should be tested for GPT and in that, on the basis of one container GPT we can approve the whole batch of the media. For example if we receive 10 container of media of same batch then we can perform GPT for only one container and can approve whole 10 containers of the media but in pharmaceuticals depending upon the criticality of the testing, every container of media should be qualified for GPT and every autoclaved lot of the media should be tested for Growth promotion test also.

 Table I: Growth Promotion Test requirement for Test for Specified Microorganisms

Medium Name  of Organism     Properties   Temperature Incubation Period
Enterobacteria enrichment broth mossel E.coli Growth Promotion 30-35oC 24 hours
P.aeruginosa Growth Promotion 30-35oC 24 hours
S.aureus Inhibitory 30-35oC 48 hours
Violet red bile glucose agar E.coli Growth Promoting & Indicative (purplish red colonies) 30-35oC Growth-18 hours

Indicative-18-24 hours

P.aeruginosa Growth Promoting & Indicative (colourless  colonies) 30-35oC Growth-18 hours

Indicative-18-24 hours

MacConky Broth E.coli Growth Promoting 42-44oC 24 hours
S.aureus Inhibitory 42-44oC 48 hours
MacConky Agar E.coli Growth Promoting & Indicative (Pink   colonies) 30-35oC Growth-18 hours

Indicative-18-24 hours

Rappaport vassiliadis salmonella enrichment broth S.enterica Growth Promoting 30-35oC 18 hours
S.aureus Inhibitory 30-35oC 24 hours
Xylose lysine deoxycholate agar S.enterica Growth Promoting & Indicative (red  colonies) with or without black centers 30-35oC Growth-18 hours

Indicative-18-48 hours

Cetrimide agar P.aeruginosa Growth Promoting 30-35oC 18 hours
E.coli Inhibitory 30-35oC 72 hours
Mannitol salt agar S.aureus Growth Promoting 30-35oC Growth-18 hours

Indicative-18-72 hours

E.coli Inhibitory 30-35oC 72 hours
Reinforced medium for     clostridia C.sporogenes Growth Promoting 30-35oC 48 hours (Anaerobic conditions)
Columbia agar C.sporogenes Growth Promoting 30-35oC 48 hours (Anaerobic conditions)
Sabouraud dextrose agar C.albicans Growth Promoting & Indicative (white  colonies) 30-35oC Growth-24 hours

Indicative-48 hours

Sabouraud dextrose broth C.albicans Growth Promoting 30-35oC 3 days

Table II: Growth Promotion Test requirement for Sterility Test

Medium Name of Organism Temperature  Incubation Period
Fluid thioglycollate medium C.sporogenes 30-35oC ≤3 days
S.aureus 30-35oC ≤3 days
P.aeruginosa 30-35oC ≤3 days
B.subtilis 30-35oC ≤3 days
Alternative Fluid thioglycollate medium C.sporogenes 30-35oC ≤3 days
Soyabean casein digest medium A.brasiliensis 20-25 oC ≤5 days
C.albicans 20-25 oC ≤5 days
B.subtilis 30-35oC& 20-25oC ≤3 days

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