Question

Design an experiment to examine the ancestry of the bacterial isolates.

Write the steps in detaiil

Answer 1

Procedure for examination of the ancestry of bacterial isolates is as follows:

steps 1:

Samples were collected aseptically, as a coherent mass and were frozen at −20°C within 10 h following collection. Based on the requirement, sub samples (fragments) were prepared aseptically from original samples by fracturing.

Step 2:

These fragments are placed in sterile air tight containers and stored at -20°C until examination and analysis.

Step 3:

Then the samples are treated with specified reagents like brilliant green and inoculated in suitable media culture like agar etc.

Step 4:

The number of organisms present in each sample was determined by using like acridine orange direct counting (AODC) and most-probable-number (MPN) viable counting. Aliquots of the initial sample suspensions were homogenized anaerobically with a sterile glass tissue grinder and serially diluted into serum bottles containing PRAS dilution fluid.

Step 5:

Direct viable counts were obtained by using filters like pore-size polycarbonate filters. MPN estimates of abundance were determined for separate five-tube inoculations containing either PRAS BHI or BHI medium. These media were inoculated with 0.1 ml of each serial dilution and incubated at 25°C until no changes in turbidity were observed.

Step 6:

Aliquots of each frozen sample were aseptically transferred into tubes containing ca. 7.0 ml of PRAS dilution fluid under an O2-free CO2 atmosphere and allowed to thaw at room temperature under strict anaerobic conditions and perform enrichment of samples with media.

Step 7:

Following incubation, all enrichments were streaked for isolation onto general and selective media. Aerobic enrichments were plated onto nonselective media and enteric-selective media. Single colonies, representing every unique colony type, were chosen from each plate based on colonial morphology or reactions to selective media. Anaerobic enrichments were streaked onto suitable media for the isolation of general anaerobic bacteria.

Example:

Anaerobic enrichments were streaked onto PRAS BHI and SE media for the isolation of general anaerobic bacteria and onto Clostridium egg yolk agar and Bacteroides bile esculin agar and for the selective isolation of Clostridium spp. and Bacteroides spp., respectively. All anaerobic isolations were performed in an anaerobic glove box under a 90% N2:10% H2 atmosphere. Presumptive obligately anaerobic isolates were confirmed by comparing growth on aerobic BHI agar and PRAS BHI agar plates. All isolates were purified by at least two transfers on either BHI or PRAS BHI agar. Purified isolates were stored as stock cultures in 10% glycerol–BHI medium or in PRAS BHI agar stabs at −70°C. Isolates were characterized by Gram stain, motility, cellular morphology, and oxidase and catalase reactions (aerobes only).

Note: The provided answer is as per my knowledge may be or may not be 100% correct, but definitely relevant to your question thank you so much.

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