Kwon-Chung in 1976 has mentioned that C. neoformans exists as two mating types, MATa and MATα which is determined by single locus with diomorphic alleles.(Halliday CL et al, 1999). The MAT locus of C. neoformans is larger and consists of 100 kb having more than 20 conserved genes. These mating types play an important role in sexual development changes. These also play a vital role in epidemiology by knowing the prevalence and virulence of the isolate (Lengeler KB et al, 2000). Based on the epidemiological studies it has been found that clinical as well as environmental isolates of MATα strains are more predominate strains (98-99%) than MATa (Kwon-Chung KJ and Bennett JE 1978; Halliday CL et al, 1999; Yan Z et al, 2002; Litvitnseva AP et
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As a negative control, the test strain should be inoculated alone. Inoculate in the centre of the plate (V8 juice agar) or Eucalyptus tree and incubate at 25oC in the dark for two weeks.
The plate or bark is observed periodically for the sexual development i.e perfect state. Mating can be observed by seeing the dense filament growth surrounding the colony and is confimed by observing dikaryotic filaments, clamp connections, basidia and chain of basidiospore under light microscope as compatible mating type (Wicks BL et al, 1996; Halliday CL et al, 1999) or stereoscope Niko- SZ-PT (Ohkusu M et al, 2002).
Slide Mating Methd (Fraser JA et al, 2003):- In this method a sterile glass slide is placed in a sterile petri-dish at an angle of 20o. Then molten V8 juice agar is overlaid to form a thin film on it. Five ml of liquid V8 medium is added to the bottom of plate. The test strain is resuspended in distilled water and 10µl is inoculated with pipette from the top of slide. Incubate the slide vat room temperatue for three to seven days in the dark. The observation of sexual development can be done as …show more content…
Then observed after staining with DAPI (4, 6-diamino-2phenylindole-2HCl, a fluorescent dye) which gives strong fluorescence when bounds DNA (Wicks BL et al, 1996). The other method for observantion of septa and DNA i. e. Calcofluor white-Sytox Green staining method wherer the slide washed three times with PBSand then fixed by using 1% Triton 100X in PBS. The agar section from the slide should be mounted on another slide with antifade (1mg of p-phenylenediamine per mlin 50% glycerol). The fluorescent stained preparation agar blocks on slide should observe under differential interference microscope and Ziess Axioskop 2 plus fluorescent microscope with attached digital camera (Fraser JA et al, 2003). Calcofuor white stains the fused clamps of filaments while DAPI stains the nucleus of cells and detects uni-nuclear or bi-nuclear cells (Fraser JA et al, 2003; Wicks L et al,
As a negative control, the test strain should be inoculated alone. Inoculate in the centre of the plate (V8 juice agar) or Eucalyptus tree and incubate at 25oC in the dark for two weeks.
The plate or bark is observed periodically for the sexual development i.e perfect state. Mating can be observed by seeing the dense filament growth surrounding the colony and is confimed by observing dikaryotic filaments, clamp connections, basidia and chain of basidiospore under light microscope as compatible mating type (Wicks BL et al, 1996; Halliday CL et al, 1999) or stereoscope Niko- SZ-PT (Ohkusu M et al, 2002).
Slide Mating Methd (Fraser JA et al, 2003):- In this method a sterile glass slide is placed in a sterile petri-dish at an angle of 20o. Then molten V8 juice agar is overlaid to form a thin film on it. Five ml of liquid V8 medium is added to the bottom of plate. The test strain is resuspended in distilled water and 10µl is inoculated with pipette from the top of slide. Incubate the slide vat room temperatue for three to seven days in the dark. The observation of sexual development can be done as …show more content…
Then observed after staining with DAPI (4, 6-diamino-2phenylindole-2HCl, a fluorescent dye) which gives strong fluorescence when bounds DNA (Wicks BL et al, 1996). The other method for observantion of septa and DNA i. e. Calcofluor white-Sytox Green staining method wherer the slide washed three times with PBSand then fixed by using 1% Triton 100X in PBS. The agar section from the slide should be mounted on another slide with antifade (1mg of p-phenylenediamine per mlin 50% glycerol). The fluorescent stained preparation agar blocks on slide should observe under differential interference microscope and Ziess Axioskop 2 plus fluorescent microscope with attached digital camera (Fraser JA et al, 2003). Calcofuor white stains the fused clamps of filaments while DAPI stains the nucleus of cells and detects uni-nuclear or bi-nuclear cells (Fraser JA et al, 2003; Wicks L et al,