Question

B. Prepare a FLOWCHART for the isolation of the following: (conventional methods only: culture, through a by product and/or immune response) Include the specimen of choice for the indication (given disease).
1. Dysentery caused by Shigella dysenteriae

2. antibiotic associated diarrhea caused by Clostridium difficile (detection of toxin)

3. dermatophyte infection in the nails

4. Dengue fever by immune response

Answer 1

1.  Dysentery caused by Shigella dysenteriae is isolated using MacConkey Lactose agar and Xylose Lysine agar

2. Antibiotic associated diarrhea caused by Clostridium difficile (detection of toxin) is isolated using Clostridium difficile selective medium

3. Dermatophyte infection in the nails is isolated using potatoes dextrose agar

4.  Dengue fever by immune response is isolated using minimal essential medium (MEM)

1.  Dysentery caused by Shigella dysenteriae

• Collection of fecal specimens  and  poultry  (100)  fecal  samples  collected  from were processed according to published protocols.    
• Streak one loopful of fecal sample on  MacConkey  Lactose  Agar  (MLA)  and  Xylose  Lysine agar

• Incubated at 37 C for 24 hours  
• The MLA plates showing the presence of convex and colorless  colonies are considered for  further identification.
• Similarly, the XLD plates showing appearance of presence of translucent or red colored colonies are taken for further identification.

2. Antibiotic associated diarrhea caused by Clostridium difficile (detection of toxin)

• Mix equal parts of industrial absolute  alcohol and the fecal specimen.
• Homogenize using a vortex mixer.
• Leave at room temperature for 1 hr.
• Inoculate on Clostridium difficile  Selective Agar
• Incubate at  37ºC  in a conventional anaerobic gas jar
• Observe after 24-48 hours in anaerobic environment .  
• Use gram stain reagent and Malachite green for morphological study.
• Observe positive bacillus cell shape with single or chains(2-6)cell and sub terminal endo spore forming after gram staining

3. Dermatophyte infection in the nails

• Prepare potatoes dextrose agar (PDA)
• Collect clippings from the nail with a sterile scalpel after carefully washing the site with 70% alcohol.
• Dissolved in two drops of 10% KOH on a glass slide, if the sample is thick, use 20% KOH
• Heat the slide few seconds over a spirit lamp, do not allow to boil.
• Observe preparation under a low power of the microscope in reduced light.
• Look out for presence of mycelia fragments and the distribution of spores inside.

4.   Dengue fever by immune response

• Dilute blood samples in minimal essential medium (MEM) containing 2% fetal bovine serum (FBS)
• Add diluent to a cultured monolayer of C6/36 cells for 1 hour with gentle rocking in a 28 °C CO2 incubator.
• After 1 hour, remove the inoculum and cells
• Culture for 7 days in MEM with 2% FBS and antibiotics.
• Collect culture supernatant on day 7 and infect a new batch of C6/36 cells
• Repeat the procedure three times to achieve passage 3 (P3) isolates.

Learn more about culturing of microorganisms here:

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