1.0 OBJECTIVE:To lay down the procedure for the Microbial analysis of swab samples from equipment surfaces.
2.0 SCOPE:This SOP shall be applicable to Quality Control Dept.
4.0 ACCOUNTABILITY:Sr. Manager Quality Assurance
5.0 PROCEDURE:5.1 Collect swab sample from the equipment surface as per the procedure described in SOP for collection of swab samples and analyze without delay.
5.2 The method of analysis of swab sample for total microbial count, yeast mold count and pathogens is given below.
5.3 Total Microbial Count :5.3.1 Take four sterile petridishes and aseptically, transfer 1 ml of the sample into each petridish.
5.3.2 Add 20 ml of sterile, molten Soybean Casein Digest Agar, cooled to 40- 45ºC, to two of the above petridishes and allow to set.
5.3.3 Add 20 ml of sterile, molten Saborauds Agar, cooled to 40 - 45ºC, to the remaining two petridishes and allow to set.
5.3.4 Incubate the Soybean Casein Digest Agar plates at 30 - 35ºC for 5 days (for bacterial count) and the Saborauds Agar plates at 20 - 25ºC for 5 days (for fungi count).
5.3.5 At the end of the incubation period, count the number of colonies formed and report the results obtained.
5.4 Yeast and Mold Count :5.4.1 Follow the same method as that for Total Microbial Count, except that the medium used in this case is sterile, molten Potato Dextrose Agar.
5.4.2 At the end of the incubation period, count the number of colonies formed and report the results obtained.
5.5 Test for E.coli :5.5.1 Transfer 1 ml of the sample to 50 ml of sterile Nutrient Broth and incubate at 37ºC for 24 hrs. (Enrichment Culture I).
5.5.2 Transfer 1 ml of the Enrichment Culture I to a sterile tube containing 5 ml of sterile MacConkeys broth and an inverted Durham tube, and incubate at 35-37ºC for 48 hrs.
5.5.3 If acid and gas production is seen (the contents of the tube turn yellow and gas bubbles are seen in the durhams tube), carry out the secondary test.
5.5.4 If acid and gas production is not seen, the test complies for the absence of E.coli.
5.5.5 For the secondary test, add 1ml of the primary test contents to a) a sterile test tube containing 5 ml of sterile Mac Conkeys broth with an inverted durhams tube and b) 5 ml of Peptone Water
5.5.6 Incubate the tubes at 43.5º to 44.5º for 24 hours and examine tube (a) for acid and gas production and tube (b) for indole.
5.5.7 To test for indole, add 0.5 ml of Kovac’s reagent, shake well and allow to stand for 1 minute; if a red colour is produced in the reagent layer, indole is present.
5.5.8 The presence of acid and gas and of indole in the secondary test indicate the presence of E. coli
5.6 Test for Salmonella :5.6.1 Transfer 1 ml of the Enrichment Culture I to two sterile test tubes, each tube containing 10 ml Selenite F broth and Tetrathionate Brilliant Green Bile Broth.
5.6.2 Incubate the tubes at 37ºC for 24 hrs.
5.6.3 Isolate a loopful of the contents of the tube on any two of the following media (i). Bismuth Sulphite Agar, (ii). Xylose Lysine Deoxycholate Agar, (iii). Brilliant Green Agar, (iv). Deoxycholate Citrate Agar, and incubate at 35-37ºC for 24 hrs.
5.6.4 Look for typical Salmonella colonies, the characteristics.
5.6.5 If none of the colonies conform to the description, the test complies for the absence of Salmonella.
5.6.6 If colonies conforming to the description, carry out secondary test.
5.6.7 For secondary test, subculture a typical Salmonella colony on (a) a slant of Triple Sugar Iron Agar, by first inoculating the surface of the slant and then making a stab culture, (b) a tube containing 5 ml of urea broth.
5.6.8 Incubate the inoculated media at 36 - 38ºC for 24 hrs.
5.6.9 Formation of acid and gas in the stab culture and the absence of acid on the slant indicates the presence of salmonella.
5.6.10 No formation of red color in the urea broth also indicates the presence of Salmonella.
5.7 Test for Staphylococcus aureus :5.7.1 Transfer 1 ml of the sample to 100 ml of sterile Soybean Casein Digest Medium and incubate at 35-37ºC for 24 hrs. (Enrichment Culture II).
5.7.2 Isolate a loopful of Enrichment Culture II to sterile preincubated plates of Vogel Johnson Agar, Mannitol Salt Agar and Baird Parker Agar.
5.7.3 Look for typical S.aureus colonies, the characteristics.
5.7.4 If any colonies conforming to the description, carry out coagulase test
5.7.5 For coagulase test, transfer a typical S.aureus colony into a tube containing 0.5 ml of mammalian plasma.
5.7.6 Incubate in a water bath at 37ºC for 24 hrs.
5.7.7 If coagulation is seen, it indicates the presence of S.aureus
5.8 Test for Pseudomonas aeroginosa :5.8.1 Isolate a loopful of Enrichment Culture II to a preincubated plate of sterile Cetrimide Agar and incubate at 35-37ºC for 24 hrs
5.8.2 Look for typical P.aeroginosa colonies, the characteristics.
5.8.3 If any colonies conforming to the description, carry out pigment test.
5.8.4 For pigment test, streak a typical colony on sterile petridishes of Pseudomonas Agar for detection of fluorescein and Pseudomonas Agar for detection of pyocyanin.
5.8.5 Incubate the plates at 33 to 37ºC for not less than 3 days.
5.8.6 Examine the plates under ultraviolet light and look for typical colonies, the description.
5.8.7 For oxidase test, place 2 – 3 drops of freshly prepared solution of 1% N,N,N1,N1- tetra methyl-4 –phenylenediamine dihydrochloride on a filter paper and smear with the suspect colony.
5.8.8 Development of pink color, changing to purple, indicates the presence of Pseudomonas aeroginosa.
5.9 Record and report the results obtained.
5.10 The total microbial count should not be more than 100 cfu/100 cm2, fungi count should not be more than 10 cfu/100 cm2 and pathogens should be absent.
6.0 ABBREVIATIONS6.1 SOP : Standard Operating Procedure
6.2 QA : Quality Assurance
6.3 QC : Quality Control
6.4 Dept. : Department
Ankur Choudhary is India's first professional pharmaceutical blogger, author and founder of Pharmaceutical Guidelines, a widely-read pharmaceutical blog since 2008. Sign-up for the free email updates for your daily dose of pharmaceutical tips.
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