3.1 Fungal Strains
The main objective of this research was to determine the effects of PA on the growth of C.neoformans var. grubii (serotype A) with H99 strain. The strains were obtained from American Type Culture Collection (ATCC) which is a nonprofit and research organization in the biotechnology field. They were stored in glycerol stocks at -80°C. Before starting our experiment, the strains were cultured in Sabouraud Dextrose Agar (SDA) plates with a total of 3 passages in 37°C incubator. This is to restore the cells functionalities to the maximum. Few colonies of fungal growth from the third passage were then cultivated overnight in Sabouraud Dextrose Broth (SDB) inside a conical centrifuge tube (Falcon tube) in 37°C shaking…show more content… There are several ways of cell counting using hemocytometer, for this experiment top and left ruling is used. This means that cells fall on the ‘line’ on the right and bottom of the square will not be counted (Figure A). Besides, the total number of cells will be counted as two or more if the cell appears to be in budding stage. The time taken for the whole cell counting procedure should be no more than 30 minutes as the cell are multiplying over time.
Figure A: Schematic diagram showing cells included and excluded in counting for top and left ruling (1956). Note: From Value @ Amrita. Copyright 2014 by NME ICT Initiative of MHRD.
3.4 Preparing sample
For the first experiment, 27 samples were prepared in 96 well plates. Each sample consists of 100 µl of cell suspension, which is a mixture of C.neoformans H99 cells and RPMI-1640 media with a total of 1x105 cells/ml and 100 µl of PA with different concentration accordingly. PA with concentration of 0 µg/ml , 2 µg/ml , 4 µg/ml , 8 µg/ml , 16 µg/ml , 32 µg/ml , 64 µg/ml and 128 µg/ml will be prepared first ,followed by adding in the cells in a triplicate manner. The wells without PA concentration will be the positive control of this experiment whereas there were 3 wells filled with only RMPI-1640 media to function as negative control for