Effects of Pantothenic Acid on the Growth of C.neoformans Essay

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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
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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 incubator at 240 rpm. The main purpose of storing in a shaking incubator is to ensure the mixture is well mixed so as to provide sufficient nutrients to all fungal cells.

3.2 Cell preparation

The cells were then centrifuged at 25 °C at 3000 rpm for 5 minutes. The SDB or the supernatant was then discarded. Then, the fungal cells were washed and resuspended in 5ml of phosphate buffered saline (PBS), followed by centrifugation at the same settings. This process was repeated twice to completely wash off any impurities or excess SDB that may affect the outcomes of the experiment. After that the cells were resuspended in 5ml of RPMI-1640 media.

3.3 Using hemocytometer for cell counting

The sample was then diluted for 1:100 in RPMI-1640 media,
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