Methods
This study compared the clinical isolates, containing varying levels of chitin, to the survival status and immune response of the patient that the cells were extracted from. To test whether the amount of chitin correlated with an increased ability of Cryptococcus neoformans to worsen human disease, the chitin levels of the clinical isolates were measured and analyzed with the clinical data for the patients. The findings of the study will contribute to the understanding of the relationship between chitin and pathogenesis in humans, so that the importance of pursuing the development of antifungal drugs that specifically target chitin synthesis can be determined.
Preparation of Clinical Strains: We have 40 clinical strains of Cryptococcus …show more content…
The sample was microfuged at 15000xg for 1 minute. The supernatant was removed, and 999µl PBS and 1µl of calcofluor white stain was added to the sample. The cells were vortexed, and remained at 25°C for 5 minutes away from direct light. After the staining, the solution was microfuged at 15000 xg for 1 minute. The supernatant was removed, and 1 mL PBS was added. The sample was microfuged again at 15000 xg for 1 minute, and then the supernatant was removed, except for about …show more content…
There was an extra control slide so that the microscope could be adjusted without harming the samples. The cover slip was placed on top and nail polish was added to the sides of the cover slip to prevent the sample from drying out. With the 40x lens of the Zeiss Axiovision Widefield Fluorescence Microscope, images of the cells were captured. The two channels that were used were DAPI and DIC. DAPI had an exposure time of 960,000ms per picture and DIC had an exposure time of 7200ms per picture. About 300-500 cells were imaged. Afterwards, in ImageJ (software from the NIH), mean gray values, mode gray values, maximum gray values, and minimum gray values were measured. The images were left in their original bit depth and file format (.czi) to avoid data clipping or rounding. Then, the images were duplicated and had a threshold to subtract the background. Since the cells with a greater quantity of chitin have a higher gray value, statistical analysis was completed to find any correlations between fluorescence and human host immune
This study compared the clinical isolates, containing varying levels of chitin, to the survival status and immune response of the patient that the cells were extracted from. To test whether the amount of chitin correlated with an increased ability of Cryptococcus neoformans to worsen human disease, the chitin levels of the clinical isolates were measured and analyzed with the clinical data for the patients. The findings of the study will contribute to the understanding of the relationship between chitin and pathogenesis in humans, so that the importance of pursuing the development of antifungal drugs that specifically target chitin synthesis can be determined.
Preparation of Clinical Strains: We have 40 clinical strains of Cryptococcus …show more content…
The sample was microfuged at 15000xg for 1 minute. The supernatant was removed, and 999µl PBS and 1µl of calcofluor white stain was added to the sample. The cells were vortexed, and remained at 25°C for 5 minutes away from direct light. After the staining, the solution was microfuged at 15000 xg for 1 minute. The supernatant was removed, and 1 mL PBS was added. The sample was microfuged again at 15000 xg for 1 minute, and then the supernatant was removed, except for about …show more content…
There was an extra control slide so that the microscope could be adjusted without harming the samples. The cover slip was placed on top and nail polish was added to the sides of the cover slip to prevent the sample from drying out. With the 40x lens of the Zeiss Axiovision Widefield Fluorescence Microscope, images of the cells were captured. The two channels that were used were DAPI and DIC. DAPI had an exposure time of 960,000ms per picture and DIC had an exposure time of 7200ms per picture. About 300-500 cells were imaged. Afterwards, in ImageJ (software from the NIH), mean gray values, mode gray values, maximum gray values, and minimum gray values were measured. The images were left in their original bit depth and file format (.czi) to avoid data clipping or rounding. Then, the images were duplicated and had a threshold to subtract the background. Since the cells with a greater quantity of chitin have a higher gray value, statistical analysis was completed to find any correlations between fluorescence and human host immune