SOP for identification of pure bacterial culture in pharmaceuticals
1.0 PURPOSE:
To lay down a procedure for Identification of Culture in Microbiological Laboratory.
2.0 SCOPE:
3.0 RESPONSIBILITIES:
Microbiologist : To perform the procedure identification of culture as per SOP.
Head-QC : To review and implementation and ensure compliance of SOP.
Head-QA : To approve, implement and ensure compliance of SOP.
4.0PROCEDURE:
4.1 Identification tests are applicable for the following Cultures-
· Escherichia coli (ATCC – 8739)
· Salmonella abony (NCTC – 6017)
· Pseudomonas aeruginosa (ATCC – 9027)
· Staphylococcus aureus (ATCC – 6538)
· Bacillus subtilis (ATCC – 6633)
· Candida albicans (ATCC – 10231)
· Aspergillus brasiliensis (ATCC – 16404)
4.2 After receiving the pure culture from authorized agencies, identify the all cultures as below.
4.3 Tests for culture:
Take loop full of culture to 5 ml of sterile normal saline / sterile buffered sodium chloride peptone solution pH 7.0 for each culture in separate tube.
4.3.1 Gram’s staining:
4.3.1.1 Prepare a suspension of the microorganism in sterile saline solution.
4.3.1.2 Take a clean grease free microscopic slide & prepare a uniform smear of the suspension using wire loop.
4.3.1.3 Allow the smear to dry in air.
4.3.1.4 Heat fixes the slide.
4.3.1.5 Cool the slide & add few drops of crystal violet stain onto the smear & allow for 1 minute.
4.3.1.6 Drain off excess crystal violet stain & wash the slide with water.
4.3.1.7 Place Gram’s iodine onto the smear & allow for 1 minute.
4.3.1.8 Drain off excess iodine & wash the slide with water.
4.3.1.9 Decolorize the smear by adding drop wise Gram’s decolorize until no further violet color comes off.
4.3.1.10 Wash the slide with water.
4.3.1.11 Apply the counterstain like 0.5% Safranin solution for about 1 minute.
4.3.1.12 Drain off excess stain & wash the slide with water and let it dry.
4.3.1.13 Examine the slide first under objectives of the microscope for better magnification view the slide under oil immersion lens using cedar wood oil.
4.3.1.14 Determine the Gram nature of the organisms by viewing the Stained smear.
4.3.1.15 Gram’s positive organisms appear as violet to blue color while Gram’s negative organisms appear reddish or pinkish.
4.3.2 Test for Escherichia coli:
If you observe Gram’s negative short rods by Gram’s staining, perform tests as bellow:
4.3.2.1 Streak on selective agar plate: Take loop full of culture and streak on EMB (Eosin Methylene Blue) Agar plate. Incubate it at 30-35°C for 24 hrs. Observe plate for Metallic Sheen under reflected light and Blue-Black appearance under transmitted light.
4.3.2.2 Biochemical test: Take 1 ml of culture suspension to 5ml of Peptone Water for indole production. Incubate it at 30-35°C for 24 hrs. Now add 0.5ml of Kovac’s reagent in Peptone water and shake well and allow standing for 2 min.
4.3.3 Test for Salmonella abony:
If you observe Gram’s negative rods by Gram’s staining, perform tests as bellow:
4.3.3.1 Streak on selective agar plate: Take loop full of culture and streak on XLD (Xylose Lysine Deoxycholate) Agar plate. Incubate it at 30-35°C for 18-24 hrs.
4.3.3.2 Biochemical test: Subculture any colonies from XLD agar in Triple Sugar Iron (TSI) by first streaking the surface of the slope & making a stab culture with the same inoculating needle & incubate at 30-35°C for 18 to 24 hour.
4.3.4 Test for Pseudomonas aeruginosa:
If you observe Gram’s negative rods by Gram’s staining, perform tests as bellow:
4.3.4.1 Streak on selective agar plate: Take loop full of culture and streak on CA (Cetrimide Agar) plate. Incubate it at 30-35°C for 18-24 hrs.
4.3.4.2 Biochemical test: Transfer the representative colonies on Oxidase disc, or filter paper that impregnated with N, N dimethyl-p phenylene diamine dihydrochloride and wait for 10-15 second.
4.3.5 Test for Staphylococcus aureus:
If you observe Gram’s positive cocci with arranged in clusters, perform tests as bellow:
4.3.5.1 Streak on selective agar plate: Take loop full of culture and streak on MSA (Mannitol salt agar) plate. Incubate it at 30-35°C for 24 hrs. Observe plate for colonies having Yellow color with yellow zones.
4.3.5.2 Biochemical test: Transfer representative suspected colonies from the agar surface of the media to individual tube each containing 0.5ml of mammalian, preferably rabbit or horse plasma with or without additives. Incubate the tube in water bath at 37°C. Examine tubes after 3 hrs to suitable intervals up 48 hrs. Observe tube for coagulation occurs.
4.3.6 Test for Bacillus subtilis:
If you observe Gram’s positive slender rods, perform tests as bellow:
4.3.6.1 Spore staining:
· Prepare a thick heat fix smear. Allow the slide to cool, and flood it with 7.6 % malachite green stain for 10 minutes.
· Rinse with tap water for 10 seconds.
· Counter stain with 0.25 % safranin solution for 15 seconds.
· Rinse in tap water, let it dry and examine under oil immersing lens for green Spores with pink cytoplasm.
4.3.6.2 Biochemical test: Take 1 ml of culture suspension to 5 ml of Peptone Water for indole production. Incubate it at 30-35°C for 24 hrs. Now add 0.5ml of Kovac’s reagent in Peptone water and shake well and allow standing for 2 min. Observe for no red color ring in tube.
4.3.7 Test for Candida albicans:
If you observe Gram’s positive rods, perform tests as bellow:
4.3.7.1 Streak on selective agar plate: Take loop full of culture and streak on BiGGY (Bismuth Sulphite Glucose Glycine yeast agar) plate. Incubate it at 20-25°C for 4-5 days. Observe plate for chocolate brown colonies.
4.3.8 Test for Aspergillus brasiliensis:
Place one drop of picric acid in the center of the clean Glass slide. Take few fungal mycelia from culture to the slide. Put cover slip on it with no any air bubble in it and gently press the material until mycelium is separated. Clean the excess mounting medium before observing the slide. First observe under low power objective (10X) and then under high power objective (45X). Observe brown colored and round shaped conidia of Aspergillus.
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