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 ...view middle of the document...
These include ensuring the cover slip and hemocytometer are carefully clean with ethanol and lens paper to remove any foreign materials or grease on it, and also carefully placed the cover slip onto hemocytometer to prevent any air bubbles being trapped that may be mistaken as part of the cells. 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 absorbance studies. The same procedure was repeated for the second experiment with different C.neoformans seeding densities, which are 1x103 cells/ml, 1x105 cells/ml, and 1x107 cells/ml. Besides, the chosen PA concentrations were 0 µg/ml, 4 µg/ml, 32 µg/ml and 128 µg/ml. The samples were run in triplicates as well. Hence a total of 39 samples were prepared in 96 well plates, including the 3 wells for negative control.
The cell growth with PA, either increases the growth, no effects on the growth or inhibits the growth was calculated by using microplate spectrophotometer or also known as microplate reader with the concept of cell absorbance in the settings of 450nm for 60 seconds. This means that the higher the absorbance value reflects the higher is the cell density. To prevent the error in cell absorbance readings which is most probably due to formation of biofilm, all samples will be resuspended and homogenized carefully prior to absorbance examination. The readings were taken in a manner of every 4 hours intervals for the first experiment and every 12 hours for the second experiment.
3.6 Statistical analysis
Effects of PA concentration on the growth of C.neoformans H99 were analyzed by using the absorbance values obtained. Absorbance values were analyzed and growth curves for each concentration compared to the control were...