Isolation of fungi by Warcup method The isolation and identification of soil fungi is an example in which a mycological ecosystem (a particular habitat with is interacting, associated community both biotic and abiotic) can be studied. It is usually too difficult to separate fungi from soil by directly picking them to place under a microscope for observation. Therefore, various techniques have been devised for stimulating growth and consequently easing fungal isolation and subsequent examination. Techniques used for isolation of fungi from soil include serial dilution agar plate, Warcup soil plate, syringe inoculation, immersion tube method, screened immersion plates, plate profile, hyphal isolation, soil washing, partial presterilization, soil sieving, floatation, baiting efc. A single method cannot be used to count all the different types of fungi present in a given sample. So to have a complete spectrum of fungi present, a sample is processed by a variety of techniques. The dilution plates and soil plate methods are two most widely used methods for the fungal isolation from soil. Dilution plate method has already been described in the previous experiment. Direct soil plate or Warcup soil plate method was developed by Warcup in 1950 and is superior to dilution plate method in that it is easier to use, less time consuming, requires less glassware and provides more qualitative information. Requirements # Soil sample ‘ : wee Cea ord © Sabouraud agar medium (100 mL) ?* ese Ae Streptopenicillin Aga - (Saxo © Rose bengal De - | o00rt Syringe with needle « Inoculating loop or needle Spirit lamp/Bunsen burner. Procedure - Collect soil at random in sterilized polythene bags, make a composite sample by mixing the above samples. Rv . Add 0.005 to 0.15 g (approximately) air dried soil each in 5 sterile Petri plates with - the help-of-a sterilized cooled loop or transfer needle w . Add 15-20 mL of melted, cooled (45°C) medium, supplemented with streptopent and rose bengal, to each soil inoculated Petri plate. in 5 Dispense soil particles throughout the medium by gentle rotation of the Petri dishes. . Allow the plates to solidify. Incubate, the plates at 25°C in an inverted position for 15 days. J staldivation Gopairs cat ly D,160 Experiments in Microbiology, Plant Pathology, Tissue Culture and Microbial Observations 1, Observe the plates for the appearance of the fungal colonies after 3 days of i and continue till 15 days. 2. Purify various fungal isolates for identification up to specific level and Results Record your results, ie., various fungal species isolated and identified. The comr encountered in tropical soil will include: Mucor, Rhizopus, Aspergillus, Penicillium, Cladosporium, Alternaria, Curvularia and Drechslera (© Helminthosporium). Precautions 1. Transfer needle or loop must be cooled either in soil or sterile medium whe for inoculating soil in the plates. 5 2. Streptopenicillin (used for inhibiting the growth of bacteria) should not b a medium which is too hot, it is always added just prior to pouring of th (at 45°C) into the plates. t 3. Soil particles are to be mixed to the medium immediately after po plates for the uniform distribution of spores. 4. If mucoraceous fungi appear in the plate they are to be killed with the help of transfer needle, otherwise they will not allow the growth of other fun; or isolation difficult. : : o 5. Plates are to be incubated in an inverted position. SiMe (ean eee eo eA 20