1.0 OBJECTIVE:To lay down the procedure for Palm swabbing of personnel and the subsequent analysis for microbial load.
2.0 SCOPE:This SOP shall be applicable to personnel of respective department who are in direct contact with raw materials and products at any stage of Sampling, Dispensing and Manufacturing.
4.0 ACCOUNTABILITY:Sr. Manager Quality Assurance
5.1 Preparation of cotton swabs :5.1.1 Take about 2 gms of absorbent cotton and wrap it around one edge of a glass rod.
5.1.2 Transfer this swab into a graduated, stoppered test tube, containing 5 ml of distilled water.
5.1.3 Sterilize by autoclaving at 15 lbs pressure (121ºC) for 15 minutes.
5.1.4 Take the sterilized swab to the sampling area.
5.2 Palm swabbing :5.2.1 Take out the sterile cotton swab, dipped in sterile distilled water and swab the palm of the person being examined, covering 5 x 5 sq. cms on each palm, in unidirectional movements and not to and fro movements.
5.2.2 Replace the swab in a test tube containing 10 ml of sterile peptone water.
5.2.3 Shake the tube gently and let it stand for 15 minutes.
5.2.4 Remove the swab from the test tube and analyze the peptone water for the total microbial count, E.coli and Salmonella, as follows.
Related: SOP for Collection of Swab Sample
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 Soyabean 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 Soyabean Casein Digest Agar plates at 30 - 35º C for 5 days (for bacterial count) and the Saborauds Agar plates at 20 - 25º C 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.
5.4 Test for E.coli :5.4.1 Transfer 1 ml of the sample to 50 ml of sterile Nutrient Broth and incubate at 37ºC for 24 hrs. (Enrichment Culture I).
5.4.2 Transfer 1 ml of the Enrichment Culture I to a sterile tube containing 5 ml of sterile Mac Conkeys broth and an inverted Durham tube, and incubate at 35-37ºC for 48 hrs.
5.3.3 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.
5.4.4 If acid and gas production is not seen, the test complies for the absence of E.coli.
5.4.5 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.
5.4.6 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.
5.4.7 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.
5.4.8 The presence of acid and gas and that of indole in the secondary test indicates the presence of E. coli.
Related: Different types of Culture Media
5.5 Test for Salmonella :5.5.1 Transfer 1 ml of the Enrichment Culture I to two sterile test tubes, each tube containing 10 ml of Selenite F broth and Tetrathionate Brilliant Green Bile Broth.
5.5.2 Incubate the tubes at 36-38º C for 24 hrs.
5.5.3 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 36-38ºC for 24 hrs.
5.5.4 Look for typical Salmonella colonies characteristics..
5.5.5 If none of the colonies conform to the description, the test complies for the absence of Salmonella.
5.5.6 If colonies conforming to the description in Table 1 are seen, carry out secondary test.
5.5.7 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
5.5.8 Incubate the inoculated media at 36-38º C for 24 hrs.
5.5.9 Formation of acid and gas in the stab culture (with or without concomitant blackening) and the absence of acidity on the surface of TSI slant indicates the presence of salmonella.
5.5.10 No formation of red color in the urea broth indicates the presence of Salmonella.
6.0 ABBREVIATION6.1 SOP : Standard Operating Procedure
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