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Week 4

My Last Week Solely in the Ayscough Laboratory

This week was my last week in the Ayscough laboratory, as I will be doing experiments in the Johnston laboratory starting from Monday. However, I will still be growing up my Candida with Harriet and would like to squeeze in another cell wall microscopy session if possible; so it is not a permanent farewell just yet.

Laboratory Work

Experiment 1: Caspofungin Growth Curve Take Two
This weeks experiment was almost identical to that of last week, except I used a different concentration of caspofungin, namely 0.2 µg/mL instead of 5µg/mL. Remarkably, despite the staggered timings and attempting the experiment solo, everything ran smoothly. Unfortunately, my results were not the same as last weeks. Although I got very similar looking trends, these were the reverse of last weeks findings, in that the wild-type strain grew "better" than the apm4Δ mutant strain this time around. However, once again none of the differences were statistically significant and so I concluded that the loss of apm4 in Candida yeast forms does not alter caspofungin sensitivity. 

The loss of apm4 does not alter the sensitivity of Candida albicans to caspofungin. Wild-type and apm4Δ/Δ Candida strains were cultured in YPD supplemented with or without 0.2 µg/mL and 0.5 µg/mL caspofungin. Samples were incubated at 28oC shaking; and 1 mL samples collected at t = 0, 2, 6 and 24 hours.

Experiment 2: Calcofluor White Microscopy 
This week when I repeated my calcofluor white staining, I did not see such an obvious difference in chitin distribution between wild-type and apm4Δ cells as last time.
Wild-type (top) and apm4Δ (bottom) hyphae stained with calcofluor white.

 We also decided to try staining hyphae induced on an agar microscope slide instead of in liquid culture. This seemed preferable, as using the agar slide technique resulted in much better looking less-clumped together hyphae. In these images, the wild-type hyphae showed pretty uniformly distributed fluorescence, whereas apm4Δ hyphae displayed fluorescence everywhere but the tip, despite taking images with solely the tip in focus. 
Chitin Distribution in Wild-type and apm4Δ/Δ Hyphae in Candida albicans. Wild-type (top) and apm4Δ/Δ (bottom) hyphae were induced and stained with calcofluor white on agar slides.
Clearly, the calcofluor white staining protocol needs to be optimized in order to ensure any results obtained are reproducible.



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