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SOP for Identification of Micoorganisms after Subculturing


Standard operating procedure to verify the microbial cultures after sub-culturing.

1.0  OBJECTIVE 

       To determine the purity of microbial culture after sub culturing. 

2.0  SCOPE 


       This SOP is applicable to verify the purity of microbial culture, used in microbiological analysis after sub culturing. 

3.0  RESPONSIBILITY 


       Microbiologist - Quality Control 

4.0  ACCOUNTABILITY


       Manager - Quality Control

5.0  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 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 counter stain and leave it for 1 minute. Wash with purified water 
5.1.10  Air dry, Blot dry the slide and observe under 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 counter stain and are Gram’s Negative Bacteria. 
5.1.13  Compare the morphology of the culture observed in 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 with 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 clean grease free cover slip and observe under microscope. 
5.2.4  Compare the morphology of the culture observed in 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 with 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 shows 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 Procedure 
6.2  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

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.
Email: .moc.enilediugamrahp@ofni Need Help: Ask Question


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