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|
|Violet red bile glucose agar||E.coli||Growth Promoting & Indicative (purplish red colonies)||30-35oC||Growth-18 hours
|P.aeruginosa||Growth Promoting & Indicative (colourless colonies)||30-35oC||Growth-18 hours
|MacConky Broth||E.coli||Growth Promoting||42-44oC||24 hours|
|MacConky Agar||E.coli||Growth Promoting & Indicative (Pink colonies)||30-35oC||Growth-18 hours
|Rappaport vassiliadis salmonella enrichment broth||S.enterica||Growth Promoting||30-35oC||18 hours|
|Xylose lysine deoxycholate agar||S.enterica||Growth Promoting & Indicative (red colonies) with or without black centers||30-35oC||Growth-18 hours
|Cetrimide agar||P.aeruginosa||Growth Promoting||30-35oC||18 hours|
|Mannitol salt agar||S.aureus||Growth Promoting||30-35oC||Growth-18 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
|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|
|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|