DEPARTMENT OF BIOCHEMISTRY, CELL AND MOLECULAR BIOLOGY
ISOLATION, PURIFICATION AND CULTURING OF NON-PATHOGENIC FUNGI (OOMYCETES) FROM A NATURAL SOURCE.
Picking out a fungi source.
The first part of the experiment which was done in practical one involved scouting the area to identify leaf’s that showed visible signs of fungus infection. A good example of such a leaf was identified, plucked and brought into the laboratory for testing. Before plucking however, photographs of the parent plant in its natural environment was taken.
Growth Medium and Antibiotic preparation.
A second team who stayed in the laboratory were concerned with preparing the growth media and the antibiotic solution used to prevent bacterial growth in the culture.
To prepare the media, 65g of Sabouraud dextrose agar that contained 15g of agar in 1 liter giving a concentration of 1.5% was used. However, because this growth media was too soft for the purposes of this experiment 5g of normal agar agar was added to harden the media.
An amount of antibiotic stock was prepared, sterilized and added to the molten agar. This was used to prevent bacterial growth since we aimed at growing only fungi.
Surface sterilization of the leaf
A piece of the infected portion of the plant material (leaf) was cut. The portion was dropped in bleach and washed for 20 seconds to remove microorganisms except fungi. Next, it was removed and dropped in de-ionized water and washed for 20 seconds to rinse out the bleach. The leaf was then taken and dropped din 10% ethanol and washed for 20 seconds to kill bacteria. Water was squirt onto the now sterilized plate to moisturize it and provide the environment for fungal growth. The portion was the placed in a moist chamber where ethanol evaporates. The chamber was then covered and left for a few days to check for fungal growth using a hand lens. Any fungal growth present was taken and used as an inoculum to culture. All the fungi.
The next practical session inoculation of the media prepared with fungal spores that could be located on the leaf. Taking a toothpick (alcohol treated) and a hand lens to magnify the magnify the leaf revealing spores, a piece of the spores was scrapped off and streaked on the growth medium A, B and C. All procedures were carried out using aseptic techniques. Before scrapping the leaf for fungal spores, a photograph of the leaf was taken. The agar plate inoculated with fungi was then stored in a dark cabinet at room temperature till the next day.
In a third practical session, the task was the preparation of subculture media with antibiotic materials. In a volume of 500 ml of water, 32.5g of sabouraud dextrose agar containing 7.5g of agar and 2.5 g of pure agar gar was dissolved to give 2%, 500 ml agar. For antibiotic solution containing Tetracycline, we wanted to prepared a concentration f 0.15 mg/ml to add to the 500 ml agar