This process is done aseptically. First, the soil slurry is made by suspending 0.1 g of the collected dry soil in 10 ml distilled water. The slurry is mixed by vortexing for 2 min and four 1 in 10 fold serial dilutions is made from the slurry. These dilutions are done in duplicates (A and B). Next, 3 ml volumes of the provided top agar are poured onto the bottom agar plates. The plates are allowed to set, after which 0.1 ml portions of each dilution, are then plated by spreading on the set chitin agar plates. All spread plates are labelled and incubated at 25 ̊C for a period of 14 da

Soil microorganisms provide an excellent resource for the isolation and identification of therapeutically important products. Among them, actinomycetales are an important group. Soils samples are serially diluted and plated on actinomycete isolation agar media. Potential colonies can be screened, purified, and stored in glycerol stock. Isolates can be morphologically and biochemically characterized. These isolates are subjected to extraction for production of the antibacterial compound. 31 actinomycete isolates are tested for antagonistic activity against 12 pathogenic microorganisms. Isolates AS14, AS27, and AS28 are highly active, while AS1 showed less activity against the pathogenic microorganisms. Isolate AS7 exhibited the highest antagonistic activity against Bacillus cereus (24 mm) and AS16 showed the highest activity against Enterococcus faecalis (21 mm). MIC was also determined for actinomycete isolates against all the tested microorganisms. MIC of actinomycete isolates was found to be 2.5 mg/ml against Shigella dysenteriae, Vancomycin-resistant enterococci, and Klebsiella pneumoniae, and was 1.25 mg/ml for Staphylococcus saprophyticus, Streptococcus pyogenes, Staphylococcus epidermidis, Methicillin-resistant Staphylococcus, Bacillus cereus, Staphylococcus xylosus, Methicillin-resistant Staphylococcus aureus, Enterococcus faecalis, and Staphylococcus aureus. Thirty-one actinomycete strains are isolated as pure culture by using standard microbiological method. One gram of dried soil is suspended in 99 ml sterile water and serially diluted in sterile water up to 10−7. An aliquot of 0.1 ml of each dilution was taken and spread evenly over the surface of actinomycete isolation agar (AIA) medium supplemented with cyclohexamide (50 μg/ml) and nystatin (50 μg/ml). Plates are incubated at 30°C for 7 days. After 7 days incubation, whitish pin-point colonies, characteristic of actinomycetes, with a clear zone of inhibition around them are observed. The whitish pinpoint colonies with inhibitory or clear zone of inhibition are selected and purified. The purified actinomycetes isolates are preserved in International Streptomyces Project (ISP) 1 (tryptone-yeast extract broth) at 4°C and 25% v/v glycerol stocks stored at 40°C for long time preservation. 

Prepare 500 ml sterile distilled water for dilutions, also prepare a series of tubes with 9 ml water in each. Sterilize tubes after water is added. The number of tubes prepared should depend on the initial number of samples. Cool dilution water to room temperature before use.

1.      Divide the sample in half: half for organic matter content analysis and half for Actinomycetes isolation if organic content analysis is to be done.

2.      Prepare plates of Actinomycete Isolation Agar or starch-casein agar. These are made by preparing the agar as per the package directions and adding 50 µg/ml Nystatin to the agar solution after the liquefied agar solution has cooled enough to touch the outside of the bottle.

3.      Dilute raw samples of water up to dilution 1:10,000; for treated water, direct plating is suitable. Soil samples should be diluted from 1:1000 to 1:1,000,000. No pH adjustment is required.

4.      When the plates are cool and solidified, add 0.5 ml of diluted inoculum to each plate and spread aseptically with a glass rod. Use three dilution levels for each sample. Be sure to label each plate with the sample number and dilution plated.

5.      Incubate plates at room temperature for 10-14 days.

6.      Count actinomycetes colonies on each plate. Actinomycete colonies can be identified by the fuzzy appearance as compared to other bacterial colonies, which appear smooth and glossy.

Report actinomycetes by C.F.U. per gram of dry weight soil or mL of water.

Serial dilution of soil sample - plate in starch-casein agar.

using Starch Casein Agar