Soil Sample Collection andEnvironmental data• The soil was collected using a wrapped spoon.• Environmental data was recorded; time ofsampling, location, airtemperature, depth, moisture content, andfeatures of the site.
Enrichment• With a clean spatula 0.5 grams of the sample were weightedand then added to a 50 mL tube.• Inside the flask were; 8mL of sterile H2O, 1mL of sterile 10x7H9/glycerol broth, 1mL of AD supplement, 0.1mL of 100 mMCaC12, and 1mL of late long stationary phase of M.smegmatisculture.• The flask was incubated at 37˚ C shaking at 220 rpm, for 24hours.
Harvesting and Preparing theEnriched Sample• The flask was balanced and spin at 3,000 rpm for 10 minutesto pellet particulate matter.• The liquid from the centrifuge sample was poured into a fresh50 mL conical tube.• The enrichment sample was filter sterilized.
Plaque Streak Protocol• A sterile wooden stick was removed from the package.• After opening the tube with the centrifuged filteredenrichment it was gently streaked across the top third of theagar plate.• This was repeated three times from the streaked area to theunstreak.• 4.5 mL of Top Agar were added to 0.5 mL aliquot of M.smegmatis.• At the end it was dispensed onto the most dilute point to themore concentrated areas by titling the plate.
Plaque Streak Control• After the Top Agar was hard, the plates were incubated at37˚ C for 24 hours.