| Growth Promotion Test and Inhibition Test of Media |
1.0 OBJECTIVE To lay down the Procedure for growth promotion test and inhibition property of the sterilized media. 2.0 SCOPE This procedure is applicable for evaluating the growth promotion test and inhibition property of the sterilized media for microbiological testing in the Quality Control Department of Microbiology Area. 3.0 RESPONSIBILITY • Microbiologist is responsible for perform growth promotion test and maintain record as per SOP. 4.0 ACCOUNTABILITY • Department Head 5.0 PROCEDURE 5.1 Safety considerations: 5.1.1 The dehydrated culture media as well their ingredients are highly hygroscopic and must be stored in a cool dry away from bright light. These media are meant for laboratory use only and shall be never used for human or animal 5.1.2 All the analysis should be carried out in an aseptic way in micro. 5.1.3 Use fresh sterile tips for each transfer. 5.1.4 There medium to be poured in petri plate should have a temperature about 40-45°C 5.1.5 Petri plate should be incubated in an inverted position to prevent collection on the plate surface. 5.1.7 If any spillage of cultures or media immediately wash with 70% filter IPA. 5.1.8 Examine the physical nature of dehydrated medium, if any unusual colour or physical appearance is noticed, discard the medium. 5.2 Preparation of Culture Suspension. 5.2.1 Preparation of microbial culture suspension as per current Sop of microbial inoculums. 5.3 Testing Procedures 5.3.1 Every new pack of media that is to be used tested for Growth Promotion, Inhibition and Indicative test, as applicable by using all challenge organisms as per media specification. 5.3.2 Received lot of media shall be qualified simultaneously along with the test. Analytical result shall be released only after getting the satisfactory result of GPT, if media disqualified in GPT test shall be invalidated and should be investigation as per SOP No. 5.4 Growth Promotion Test of solid media 5.4.1 Prepared the required solid media as per current SOP media preparation in micro lab. 5.4.2 Take required sterile plate and label them with culture name and date properly. 5.4.3 1.0ml Culture transfer having counts 10-100 cfu/ml into labelled plate. 5.4.4 Then pour media into the plates. 5.4.5 Swirl the plates and allow solidifying. 5.4.6 After solidifying plates incubated at specified temperature and period. 5.4.7 Observe the plate for number of colonies and counts observed on both plates and express the results in cfu/ml by following formula P1+P2 where P1 and P2 are plate 1 and plate2. 5.4.8 Calculate the microbial recovery in percentage by below formula. Mean(AVG) cfu observed• % Recovery = —————————- Inculcated cfu 5.4.9 Acceptance criteria recovery of challenge cfu organisms should be with in a factor of 2 from the challenge cfu. 5.4.10 Simultaneously run negative control to verify testing condition using the same procedure in place of the test organism use dilution 1.0ml of 0.9% saline water. 5.5 Growth Promotion Test for broth Media 5.5.1 Prepared required quantity of liquid media, as per SOP of media preparation in micro lab. 5.5.2 After sterilization transfer the media into bio-safety room and allow it to cool at room temperature. 5.5.3 Add 1.0ml of microbial culture to growth promotion properties containing 10-100cfu/ml into broth tube mark with permanent marker name of media, culture name and date of incubation. 5.5.4 Simultaneously run negative control to verify testing condition, using the same procedure in place of the test organism use dilution 1.0ml of 0.9ml saline water. 5.5.6 Observed the tubes for growth turbidity. 5.5.7 Acceptance criteria satisfactory growth should be observed within the predefined time period of incubation in the test. 5.6 Growth promotion test for selective solid media and broth media 5.6.1 Prepare the required media as per current SOP preparation of media preparation in microbiology department. 5.6.2 In case of solid media 5.6.2.1 After solidification of selective media pipette out microbial culture suspension (containing NMT 10-100cfu transfer microbial culture suspension onto the centre of the surface of an agar plate. 5.6.2.3 Incubate all the plate at temperature and time as per required by organism ( refer incubation table-I). 5.6.2.4 Take sterile pipette out sterile 0.1ml saline on media plates and spread on plate and plate as negative control plate incubate at appropriate time as per Table-I. To read this sop click here SOP for Media preparation Consumption and Sterilization in Microbiology Laboratory. 5.6.3 In case of broth Media 5.6.3.2 Simultaneously run negative control to verify testing condition, using the same procedure in place of the test organisms use dilution 1.0ml of 0.9% saline water. 5.6.3.3 Incubate all the tubes at specify temperature as per mentioned in Table-I. 5.6.3.4 Observed the tubes, Broth media are suitable if should show clearly visible growth in media tube. 5.6.3.5 Acceptance criteria satisfactory growth should be observed within the predefined time period of incubation in the test. 5.7 Growth inhibitory properties for selective solid media, sobouraud chlorampenicol agar and broth media 5.7.1 To check the inhibitory property of solid media and broth media, incubate the prepared media of new lot and previously approved lot media of culture suspension containing NLT 100cfu of specified microorganisms as mentioned in Table-II. 5.7.2 Incubate the media at specified temperature and time period as mentioned in Table-I. 5.7.3 After completion of incubation if no growth or turbidity observed on incubated media then the new lot of media shall be approved. 5.8 Growth indicative properties for selective solid media and broth media 5.8.1 To check the indicative properties of broth media, inoculated the prepared media of new media lot culture suspension NMT 100cfu of specified microorganism as mentioned in table-II. 5.8.2 Incubate the plate and tubes at specified temperature and period. 5.8.3 After incubation of growth characteristics of microorganisms and color change of media meets the pharmacopeia or other standard laboratory manual, colonies. 5.8.4 Record the details of observation in format no. XXX. 5.8.5 Indicative property test shall be carried out only for selective media which exhibit indicative property. 5.8.6 Note: plates used in GPT shall be further incubate for indicative properties, if same organism defines for promotion and indicate test. 5.9 Routine GPT procedure for selective solid media and broth media. 5.9.1 In routine media preparation GPT shall be carried out selective prepare media with microorganisms mentioned in Table-I to check the suitability of media used in routine analysis and record in format no. XXX. 5.9.2 Routine GPT of general media (like SCDA, SDA, SCA, R2A and SCDM) 5.9.3 In routine media preparation GPT shall be carried out for all regular prepared media with any microorganism mentioned in Table-I to check the suitability of media used in routine analysis and record in format no. XXX. 5.10 Pass in GPT 5.10.1 After getting the GPT release result enter the media bottle received in a media stock record. 5.10.2 In the case of media passed the growth promotion test, an approved label (green color label) shall be affixed on the media container, and then the same should be used for analysis. 5.10.3 Failure in GPT 5.10.4 In case the media fails for the growth promotion test, a rejected label (red label) shall be affixed on the media lots containers then follow bellow steps. 5.10.5 If the medium does not support of growth promotion testing, an investigation shall be done for the cause of failure follow sop of out of specification respective Sop No. XXX. |
