Snake fecal analysis method notes

Posted byK of KyrenePosted inPersonal Snake Research Tips, Uncategorized
  1. Fecal samples were kept at -20 degrees Celsius
  2. Fecal prep: The feces were thawed and 1g of wet weight was added to 1000 microlitre double distilled water and vortexed for 30secs with stuart SA8. Serial dilutions were made of the fecal homogenate from 10^-1 to -10 with double distilled water. These dilutions were titrated and spread plated onto nutrient agar CM0003 and made using 100, 50 and 25 microlitres of fecal homogenate mix. Plates were aerobically incubated at 37 degrees Celsius and left for 24hrs. Heavy bacterial growth was seen at the lowest level so instructions were changed to 0.5g of feces in 1000 microlitres of double distilled water and using 50 and 25 microlitres of fecal homogenate for the plates.
  3. Bacterial Isolation: Different agars were used to isolate single bacterial species and they used a spread of 25 microlitres of 10^-6 fecal homogenate.
  4. Slanetz and Bartley Agar CM0377 = medium for fecal enterococci isolation. Plates were inverted and incubated aerobically at 44 celsius for 24hr but then left for 48hr to increase colony size. Higher counts are visible at incubation of 35 celsius but this allows growth of other organisms that are not enterococci. Deep red colonies were enterococcus and pinker colonies were also observced since some species of enterococcus interact with the agar to have a paler colour.
  5. Brilliant green agar CM0263 = medium for salmonella isolation excluding salmonella typhi. Inverted plates were incubated aerobically at 37 celsius and examined both after 24hrs and 48hrs. salmonella is non-lactose fermenting organisms and they grow red-pink-white opaque colonies on this type of agar with red clearing zones. Pseudomonas spp and proteus spp may also grow tiny red colonies on this agar so recommended to incubate the plate for 4 days to observe them. Lactose-fermenting organisms are usually inhibited but if you get yellow to green colonies with yellow-green clearance zones this may be signs of Escherichia coli or Klebsiella/Enterobacter group.
  6. MacConkey agar CM0115 = this is used to isolate coliforms and non lactose fermenters like Shigella spp. Gram-positive microcci like E. faecium are inhibited in this agar. Plates were inverted and incubated aerobically at 37 celsius for 18-24hr to isolate coliforms, plates were further incubated to a max of 48hrs if non-lactose fermenting colonies hadn’t apparated yet. Lactose fermenting species produce red/pink colonies and non lactose fermenting species are straw coloured or colourless.
  7. Gram staining: Single colonies were extracted from the agar and gram stained to further validated the organisms in the sample. This was done on microscope slides where a drop of double distilled water and a single colony of bacteria was added. More exact steps in the write up.
  8. Drug Susceptibility Testing: I think she found 3 different colonies of the same type of bacteria, enterococcus, and then tested to see if they had different minimal inhibition concentrations to different drugs. Maybe if there were treatment induced differences she wanted to see if they had an effect on the susceptibility of the fecal bacterial produced.

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