1.0 OBJECTIVE
To determine the purity of microbial culture after subculturing.2.0 SCOPE
This SOP is applicable to verify the purity of microbial culture, used in the microbiological analysis after subculturing.3.0 RESPONSIBILITY
Microbiologist - Quality Control4.0 ACCOUNTABILITY
Manager - Quality Control5.0 PROCEDURE
5.1.1 Gram’s staining for Bacteria
5.1.2 Aseptically open microbial culture tube under LAF.5.1.3 Take a loop full of culture and make the suspension in sterile saline water.
5.1.4 Take a clean grease free slide and make a thin smear of the culture with saline.
5.1.5 Air dry, heat fix by passing the slide slowly through the flame. Avoid overheating.
5.1.6 Flood the smear with Crystal violet (Primary stain) for 1 minute. Wash with purified water.
5.1.7 Add Gram’s Iodine as a mordant for 1 minute. Wash with purified water.
5.1.8 Decolonize the smear with absolute alcohol for 10secs.
5.1.9 Add Safranin as the counterstain and leave it for 1 minute. Wash with purified water
5.1.10 Air dry, Blot dry the slide and observe under the microscope.
5.1.11 Organisms which would appear purple in color have taken primary stain and are Gram’s Positive Bacteria.
5.1.12 Organisms which would appear red/pink in color have taken counterstain and are Gram’s Negative Bacteria.
5.1.13 Compare the morphology of the culture observed in the microscope with the morphology mentioned in the point 5.3.
5.1.14 Verify the colony characteristics of microbial culture in respective media as per point no. 5.3
5.1.15 If colony characteristics do not match the point No 5.3. record the morphology only.
5.2 Lactophenol Cotton Blue Staining for Fungi
5.2.1 Aseptically open microbial culture tube under LAF.5.2.2 Take a clean grease free slide.
5.2.3 Take the mycelium (for filamentous fungi) of the fungi with the help of needle and forceps and place it on the slide. Carefully tease the mycelium and add few drops of Lactophenol cotton blue cover the whole mycelium, cover with a clean grease free coverslip and observe under the microscope.
5.2.4 Compare the morphology of the culture observed in the microscope with the morphology mentioned in the point 5.3.
5.2.5 Verify the colony characteristics of microbial culture in respective media as per point no. 5.3
5.2.6 If colony characteristics do not match the point No 5.3. Record the morphology only.
5.3 Morphology of Microbial Culture
5.3.1 E.coli: Coccobacilli Gram’s Negative –Indole production- positive (Colonies with Green Metallic Sheen on EMB agar)5.3.2 Salmonella sps: Gram’s Negative rods –Urease negative (Black colonies on XLDA, BSA)
5.3.3 Staphylococcus.aureus: Gram’s positive cocci in bunches-Coagulase positive(Black colonies with yellow zone on VJA, Black colonies with clearing zone on BPA)
5.3.4 Pseudomonas. aeruginosa: Gram’s negative short rods –motile, Oxidase positive (Fluorescent green color on CA)
5.3.5 Bacillus. Subtilis: Gram’s negative Bacilli spore-forming, non-capsulated, motile. (Non-mucoid, Irregular colonies on SCDA)
5.3.6 Aspergillus.niger: Filamentous fungi on microscopically show branching and septate hyphae.(Black spore-forming fungi)
5.3.7 Candida.albicans: Yeast Like/ Dimorphic Fungi oval/spherical budding yeast produces septate pseudohyphae & true hyphae (Whitish cream colored mucoid large colonies)
5.3.8 Shigella.flexeneri: Gram’s Negative rods (Off-white to colorless colonies on SS Agar)
5.3.9 Lactobacillus planthurum: Noncapsulated Gram’s positive rods which tend to occur singly or in chains.(Shows heavier cells in Pentothenate Inoculum Broth)
5.3.10 E.coli mutant: Gram’s negative rods. (Shows positive growth in presence of B12).
5.3.11 Enterobacteria: Gram’s Negative cocci (red to reddish colonies on VRBG).
Related: Identification of Microorganisms to Species Level
6.0 ABBREVIATIONS
6.1 SOP - Standard Operating Procedure6.2 SCDA - Soybean Casein Digest Agar
6.3 LAF - Laminar Air Flow
6.4 XLDA - Xylose Lysine Deoxycholate Agar
6.5 BSA - Bismuth Sulphite Agar
6.6 VJA - Vogel Johnson Agar
6.7 BPA - Baired Parker Agar
6.8 CA - Cetrimide Agar
6.9 VRBG - Violet Red Bile Broth
6.10 SS Agar - Salmonella Sheigella Agar
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