1.0 Purpose :
Test procedure represents the minimum standard for testing the Preservative Efficacy Test.
2.0 Scope :
This procedure covers all the activities related to the testing and interpretation of results of all liquid orals.
3.0 Responsibility :
4.0 Procedure :
4.1.0 Test Organisms :
4.1.1 For routine testing the following organisms should always be employed.
Aspergillus niger ATCC 16404
Candida albicans ATCC 10231
Pseudomonas aeruginosa ATCC 9027
Staphylococcus aureus ATCC 6538
Escherichia coli ATCC 8739
Zygosaccharomyces rouxii ATCC 14679
4.1.2 Culture Maintenance: Refer SOP No. QC/MB/xxx
Preparation of Bacterial master slopes.
Preparation of Yeast and Mould slopes.
If good growth has not occurred, do not incubate for a greater length of time, but re-inoculate a fresh agar slope from the resuscitated culture in broth.
4.1.3 Preparation of Culture suspension:
4.1.4 Prepare a fresh culture from the working culture by transferring a loop of culture to a 15ml agar slope of SCD Agar. Incubate at 30°C to 35°C for 18 to 24 hours.
4.1.5 From a glass bottle containing 50ml of sterile 0.1% peptone water pour an aliquot on to the agar slope surface. Make a suspension of the bacterial growth by gently rocking the diluent over the surface of the slope, or if necessary use a sterile loop to aid the removal of the growth from the agar surface.
4.1.6 Transfer all the suspension to the bottle containing the remainder of the diluent and mix for 30 sec. Dilute this with 0.1% peptone water to produce a suspension which has a viable cell count of approximately 2 x 10 8 per ml.
4.1.7 Candida albicans suspension Prepare a fresh culture from the working culture by transferring a loop of culture to 15ml agar slope of S D Agar. Incubate the slopes at 20° to 25°C for 48 hours.
4.1.8 Wash off all the surface growth from the slope with 5 ml sterile 0.1% peptone water by gently rocking the diluent over the surface of the slope. If necessary use a sterile loop to aid the removal of the growth from the agar surface. Transfer the suspension into a sterile 30ml glass universal container and mix for 30 seconds.
4.1.9 Dilute the suspension as necessary with 0.1% peptone water to produce a suspension having a viable count of approximately 2 x 10 8 per ml.
4.1.10 Aspergillus niger suspension Prepare a fresh culture from the working culture, by transferring a loop of culture to a 15ml agar slope of S D Agar, ensuring that a mixture of both surface and aerial hyphal growth is transferred.
Incubate the Slope at 20°C to 25°C for 7 days.
4.1.11 Wash off all the surface growth using a sterile loop as an aid with 2.0 ml ± 0.5 ml of 0.1% peptone water containing 0.05% v/v Polysorbate 80. Transfer the suspension to a sterile 30ml universal glass container and mix for 30 seconds. The solution is unlikely to require further dilution to obtain the desired cell count of approxi-mately 2 x 10 8 per ml
4.1.12 To find out the population of the above culture suspensions using Hemocytometer count the number cells, and dilute the culture suspension to desired cell count of approximately 2 x 10 8 per ml.
4.1.13 For confirmation the culture suspensions were further diluted to obtain 10 to 100cfu/ml using normal saline with 0.1% peptone .
4.1.14 Pipette out 1ml of above suspension in to two sterile petridishes and add about 15 to 20ml of SCDAgar for Bacterial culture suspension.
And add about 15 to 20ml of SDAgar for Fungal culture suspension.
4.1.15 Gently rotate to and fro motion 2 to3 times for uniform mixing of the culture and agar.
4.1.16 The pour plates were incubated at 30°-35°C for 3 days for the Bacteria and 20°-25°C for 5 days for the fungal count.
4.1.17 After incubation the number of colonies on each plate were counted and, taking the dilution factor into account, the number of cfu/ml of initial suspension calculated.
4.1.18 Use of culture suspension for Product inoculation
4.1.19 All culture suspensions are to be used on the day they are prepared.
During the day when they are not being used they should be stored at +4°C.
4.1.20 Inoculation volume to give 10 6 cfu/ml or gram in the product are as follows.
4.1.21 LIQUIDS SAMPLES: 0.1ml of the microbial suspension into 20ml of product. If the sample volume available is different from this then adjust the inoculum volume accordingly such that it is 0.5% of the sample volume. e . g. 0.05ml into 10ml.
4.2.0 Test Procedure:
The inoculation is to be prepared on a separate product sample for each organism. 1ml aliquots are then removed from each inoculated sample at each time point. Duplicate sets of dilution pour plates are necessary for each aliquot.
4.2.1 Preparation of Product samples:
Where the volume of the product unit is 10ml or more each test organism should be directly inoculated into individual product containers. (without removing the septum seals if present).
Providing this and repeated extraction of samples can be done aseptically. This will require at least 7 units for the test, with one extra unit being required for each additional challenge organism used in the test.
4.2.2 Alternative testing containers can be used:
Container requirements: The container should be a sterile glass bottle of approximately 50ml volume with a secure closure. It should be round with smooth sides.
4.2.3 Inoculation of product samples and controls:
Liquids: Inoculate each microbial suspension into separate container Of the product according to the guideline 4.1.21. In addition
Inoculate separate bottles containing an equal volume of 0.1% peptone Water with each microbial suspension these are inoculum count Controls according to the guideline 4.3.1
Mix all samples thoroughly, store all product containers for the entire duration of the test in dark room at room temperature 20° to 25°C.
4.2.4 Monitoring of Microbial survivors:
Sampling points: Microbial survivors in the challenged product should be enumerated at each of the following time points. The ‘0’ hour samples be extracted at the 10 –1 dilution prepared immediately after inoculation.
Inoculum count : 0 hours.
Inactivation controls : 0 hours.
Test for Bacteria : 0 hours , 24 hours, 7th day, 14th day ,21st day and 28th day.
Test for Fungi : 0 hours , 24 hours, 14th day ,21st day and 28th day.
4.2.5 Dilution of liquids:
Aseptically remove 1ml using a pipette and add this to 9.0 ml of 0.1% peptone water 0.3% Soyalecithin and 5% Polysorbate 80 in a sterile 30ml universal bottle mix thoroughly.
4.2.6 Enumeration by pour plate method:
Make serial dilutions to 10 –5. Transfer 1 ml aliquots of each dilutions to duplicate sterile 9 cm petridishes. Add approximately 15 ml of cooled agar to each of petridishes and mix by rotation. Leave the plates to set.
For the bacterial challenge organisms use SCD Agar and incubate at 30° to 35°C for 3 days.
For the Fungal challenge organisms use S D Agar and incubate at 20° to 25°C for 5 days.
4.3.1 Inoculum counts:
To be performed in all tests ‘0’ hour only.
This control procedure enumerates the total number of viable micro-organisms with which the product samples are challenged at time zero and provides the base-line for subsequent. “Log 10 Reduction”
Calculation: Dilute the control inoculum count samples at the same time as the product time zero samples. Remove 1ml from the control sample and serially dilute 9ml of 0.1% peptone water in 30ml containers.
Transfer 1ml aliquots of the 10-4 and 10-5 dilution to duplicate sterile 9cm petridishes. Add approximately 15ml of cooled agar (at approximately 45°C) to each petridish and mix by rotation.
Leave plates to set then incubate bacteria at 30° - 35°C for 3 days, Fungi at 20 – 25°C for 5 days.
4.3.2 Inactivation control:
To be performed at ‘0hr’ only.
For each challenge test organism prepare three 30ml bottles two(‘A’&’B’) containing 9ml of 0.1% peptone water with the inactivator/s used, and the third (‘C’) containing 10ml of 0.1% peptone water. If no inactivator is employed in the test, then bottle ‘C’ will not be required.
To ‘A’ add 1ml of 1gof test product and to ‘B’ add 1ml of 0.1% peptone water. Mix bottle thoroughly.
From the harvested microbial suspension prepared for challenging the product serial dilute in 9ml volumes of 0.1% peptone water to give between 103 and 104 CFU/ml of suspension. Add 0.1ml of this suspension to each of bottles A,B and C. Allow the bottles to stand at room temperature (20-25°C) for 30 minutes, then prepare a 10-1 dilution from each bottle by asceptically transferring 1ml into each of two sterile empty 9cm petridishes and pouring with 15ml of agar (at approximately 45°C )
For bottles ‘A’ & ‘B’ use SCD Agar for bacteria and SD Agar for A. niger, C. albicans & Z. rouseie.
These agars should contain inactivators at the levels employed in the test:
For bottle ‘C’ use the same agar media but containing no inactivators.
Incubate all plates as for the product sample test plates after incubation determine the means count per ml ofr each for bottles A,B, and C.
The means count of bottles ‘A’ & ‘B’ ditter by not more than a factor of 10 from of the count of bottle ‘C’ then the product is adequately neutralised and that the inactivating agents are not effecting the viability of the challenge organisms.
Examine the fungal plates after 2 days incubations and mark each developing colony on the back of the petridish. This facilitates counting of colonies in case of over growth after 5 days.
After the full incubation times for each organism, count the colonies on the plates. Record the results on raw data sheets.
Average the duplicate counts for each dilutions and calculate the number of C.F.U.’s per ml on gram of the original sample For each organism at each time point calculate the Log10 reduction as follows:
Log10(REDUCTION)=Log10 (INOCULUM COUNT)–Log10(SAMPLE COUNT)
INTERPRETATION OF RESULTS
Bacteria: The number of organisms recogered jper ml is reduced by a factor of not less than 102 within 7 days of challenge and there is no increase there after.
Moulds & Yeasts: There is no increase in the number of organisms recoved per ml with 14 days of challenge or there after.
Test extension (This applied to sample material under going development studies.)