sop for preservatives efficacy test

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sop for preservatives efficacy test

1.0 OBJECTIVE

1.1 To lay down a procedure for testing of Efficacy of Preservatives.

2.0 SCOPE

2.1 This SOP is applicable for testing of Efficacy of Preservatives added in products

3.0 RESPONSIBILITY

3.1 Microbiologist QC

4.0 ACCOUNTABILITY

4.1 QC Manager

5.0 PROCEDURE

5.1 Ensure the all the media employed should be tested for their growth promoting properties.
5.2
Inoculum Preparation:
5.2.1 Inoculate the surface of the Soybean casein Digest Agar and Sabouraud Dextrose Agar media plates / slants with 1 ml of the microorganisms from the recent culture suspension as given in Table-1.
5.2.2 Incubate the plates/ Slants at suitable conditions as given in the Table-1.

5.2.3 Harvest the cells except Aspergillus niger after the incubation by washing the growth on the surface of the respective solid agar media using Sterile Normal Saline (0.9%w/v), collect it in a sterile container and treat this as Stock solution.
5.2.4
5.2.5

For harvesting of Aspergillus Niger, Use Normal saline with 0.05% Polysorbate 80.
From the Stock solution, do tenfold serial dilutions using sterile normal Saline as diluent to get a count of 1 x 108 CFU / ml.
5.2.6 Determine the number of CFU per ml of these harvested suspensions by plate count Method using suitable Agar media and incubate at appropriate temperatures.
5.2.7 Use the initial count of suspensions to calibrate the size of inoculum to be used in the Test.
5.2.8 Use the Bacterial and Candida albicans suspensions within 24 hours of harvest.

5.2.9 Fungal suspensions shall be refrigerated and used up to 7 Days.

5.2.10 For microorganisms and media to be used, incubation conditions, microbial recovery time, refer to Table-1 given below.
Table-1
Organism Suitable Medium Incubation
Temperature Inoculum
Incubation Time Microbial Recovery
Incubation Time
Escherichia coli
(ATCC No. 8739) Soybean–Casein Digest Broth;
Soybean–Casein Digest Agar 32.5 ± 2.5 18 to 24 hours 3 to 5 days
Pseudomonas aeruginosa
(ATCC No. 9027) Soybean–Casein Digest Broth;
Soybean–Casein Digest Agar 32.5 ± 2.5 18 to 24 hours 3 to 5 days
Staphylococcus aureus
(ATCC No. 6538) Soybean–Casein Digest Broth;
Soybean–Casein Digest Agar 32.5 ± 2.5 18 to 24 hours 3 to 5 days
Candida albicans
(ATCC No. 10231) Sabouraud Dextrose Agar;
Sabouraud Dextrose Broth 22.5 ± 2.5 44 to 52 hours 3 to 5 days
Aspergillus niger
(ATCC No. 16404) Sabouraud Dextrose Agar;
Sabouraud Dextrose Broth 22.5 ± 2.5 6 to 10 days 3 to 7 days

5.3 Test Procedure:
5.3.1 Use five Product containers if the volume per container is sufficient.
5.3.2 If the volume is not sufficient, transfer the product into five sterile screw capped containers.
5.3.3 Reconstitute the product container if it is in dry powder form, as per the instruction on the label.
5.3.4 Standardize the volume of the inoculum to be between 0.5% and 1.0% of the volume of the product and the concentration to be between1 × 105 and 1 × 106 cfu per mL of the product.
5.3.5 Add the inoculum into the product containers using Sterile Syringe or Pipette.
5.3.6 Determine the initial count of the inoculated containers by Plate Count Method.
5.3.7 Incubate the inoculated containers at temperature 22.5C ± 2.5
5.3.8 Withdraw samples from containers at the intervals of 14 and 28 Days and determine the number of CFUs present in Sample containers by the Plate Count Method.
5.3.9 Observe and record any changes in appearance of the sample during the sampling as per Annexure-I.
5.3.10 Incorporate an Inactivator in the diluents such as sterile normal saline (0.9 % w/v) with polysorbate-80 (0.05 % w/v) and 0.05 % Soya lecithin or with peptone (0.1 % w/v) and 0.05 % Soya lecithin or sterile normal saline (0.9 % w/v) with 0.05 % polysorbate-80.
5.3.11 Using the Initial count of the test preparation, calculate the change in log10 values of the concentration of cfu per mL for each microorganism at the respective intervals.
5.3.12 Express this in terms of Log Reduction as per Annexure-I and report as Complies if results are complying to the criteria as given in Table-2.

Table –2
S.No. Micro organism Acceptance Criteria
1 Bacteria Not less than 1.0 log reduction from the initial count at 14 days, and no increase from the 14 days’ count at 28 days.
2 Yeast and Molds No increase from the initial calculated count at 14 and 28 days.
Note: No increase is defined as not more than 0.5 log10 unit higher than the previous value Measured.

Name of the Product
Batch No.
A.R.No
Date of analysis
Date of report

Day of Analysis	1^st Day	14^th day	28^th day
Media used
Lot No.:
Autoclave Lot No.:

Incubation Period		1^st Day	14^th day	28^th day
Incubation Started at / Date	Bacterial Cultures Incubator No: (30ºC – 35ºC)
Incubation Started at / Date	Fungal Cultures Incubator No: (20ºC – 25ºC)
Incubation Completed at / Date	Bacterial Cultures Incubator No: (30ºC – 35ºC)
Incubation Completed at / Date	Fungal Cultures Incubator No: (20ºC – 25ºC)

Name of the Microorganism	Day of Analysis	Dilution Considered in the Calculation	Test Count	% recovery *= Final Count x 100** Initial Count*
Escherichia coli ATCC 8739	1^st Day
	14^th Day
	28^th Day
Pseudomonas aeruginosa ATCC 9027	1^st Day
	14^th Day
	28^th Day
Staphylococcus aureus ATCC 6538	1^st Day
	14^th Day
	28^th Day
Candida albicans ATCC 10231	1^st Day
	14^th Day
	28^th Day
Aspergillus niger ATCC 10231	1^st Day
	14^th Day
	28^th Day
Day of Sample withdrawal		Appearance of the Product:
1^st Day
14^th Day
28^th Day

Acceptance Criteria
Bacteria	Not less than 1.0 log reduction from the initial count at 14 days, and no increase from the 14 days’ count at 28 days.
Yeast and Molds	No increase from the initial calculated count at 14 and 28 days.

Sr. No.	Reference Title
1	In House