SOP For microbiological analysis of swab sample of equipment's and surfaces is described in this post which you can follow in the section of microbiological lab.
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Objective:
This procedure has been established for microbiological analysis of swab sample of equipment's and surfaces.
Scope:
This procedure is applicable to microbiology lab.
Responsibility:
Microbiologist.
Accountability:
Quality Control
Manager.
Procedure:
Total Microbial Count (TMC)
Take four glass petri plates previously sterilized and transfer 1ml of the sample into each Petri plate aseptically under laminar airflow cabinet.
To above solution add 20-25ml of sterile nutrient agar to two petri plates and to remaining two plates add 20-25ml of sterilized sabouraud dextrose agar and allow solidifying.
Incubate the nutrient Agar plates at 32-35ºC for 2-3days (for the bacterial count) and sabouraud dextrose agar plates at 22- 25ºC for 5-7 days (for fungal count).
At the end of the incubation period, count cfu (Colon Forming Units) formed and record the results.
Test for E.coli
Primary Test:
Take 1ml of the sample and add to 50ml of the sterilized nutrient macconkeys broth and incubate at 35-37ºC for 24-48 hrs. During incubation period if the solution turns to yellow color and and gas bubble formation is seen then perform the secondary test and if solution remains the same then test complies for absence of E.coli.
Secondary Test:
Add 1ml of the primary test solution to 5ml of pre-sterilized MacConkeys broth and 5ml of Peptone Water. Incubate the media at 43.5ºC-44.5ºC for 24hrs and examine for acid and gas production and for indole.
Test for indole:
Take 1ml of the primary test solution and add in it 0.5ml of Kovac’s reagent, shake well and allow to stand for 1minute; if a red color is produced in the reagent layer, indole is present.
Test for Salmonella:
Primary Test:
Take 1ml of the sample and add it in 10ml Selenite F broth and Tetrathionate Brilliant Green Bile Broth and incubate the media at 37ºC for 24hrs. and observe for specific/typical salmonella colonies. if no colonies match the specific characteristics then the test complies for absence of salmonella and if colonies of salmonella observed then perform the secondary test.
Secondary Test:
For Confirmation of salmonella growth subculture a typical salmonella colony on triple sugar iron agar with 5ml of urea broth and incubate the media at 35-37ºC for 24 hrs. During incubation period if gas bubble formation is seen then the presence of salmonella is confirmed and No formation of red color in the urea broth also indicates the presence of Salmonella
Test for Staphylococcus aureus:
Take 1ml of the sample and add in 100ml of the TSB (Tryptic Soy Broth) and incubate at 35-37ºC for 24 hrs. and observe for specific/typical s.aureus colonies. if no colonies match the specific characteristics then the test complies for absence of s.aureus and if colonies of s.aureus observed then perform the secondary test.
Secondary Test:
Coagulase Test:
For Confirmation of s.aureus growth subculture a typical s.aureus colony on 0.5ml of mammalian plasma and incubate the media at 37ºC for 24 hrs. During incubation period if coagulation is seen then the presence of s.aureus is confirmed.
Test for Pseudomonas aeruginosa
Take 1ml of the sample and pour on pseudo-cetrimide agar and incubate at 35-37ºC for 24 hrs. and observe for specific/typical pseudomonas aeruginosa colonies. if no colonies match the specific characteristics then the test complies for absence of pseudomonas aeruginosa and if colonies of pseudomonas aeruginosa observed then perform the secondary test.
Secondary Test:
Pigment Test:
For Confirmation of pseudomonas aeruginosa growth subculture a typical pseudomonas aeruginosa colony on pseudo-cetrimide agar for detection of fluorescein and pyocyanin and incubate the media at 32-37ºC for not less than 3days. at the end of incubation period observe the plate for typical colonies of pseudomonas aeruginosa
Oxidase Test:
Add 2–3 drops of a freshly prepared solution of 1% tetra methyl-4 –phenylenediamine dihydrochloride on a filter paper and smear with the suspect colony. if pink color changes to purple then it indicates presence of pseudomonas aeruginosa.
Limits:
The TMC should NMT 100 cfu/100 cm2, and TYMC (total yeast mold count) should NMT 10 cfu/100 cm2 and all pathogens should be absent.
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