Skip to main content

Week 2

BMS Away Day
Firth Court Entrance

At the beginning of the week I attended the Biomedical Science (BMS) research away day, which was held in Firth Hall. The day consisted of numerous talks and poster presentations given by various members of the four research areas which exist within the BMS department at the University of Sheffield. Namely, cell biology and cancer, developmental biology and disease, neuroscience, and stem cell and regenerative medicine. The day was really interesting as it allowed me to explore the work that is going on within the department; in addition to giving me an indication as to how to approach poster and flash talk presentations when I (hopefully) become a PhD student.  

Laboratory Work

Experiment 1: Microscopy
This week I had my first go at microscopy; using standard transmitted light before moving on to stain some samples and use fluorescence. However, I wasn't able to gain any results from these latter experiments as our cells were contaminated. As the fluorescent dye itself appeared to be working fine, we are hoping to simply repeat the staining procedure and hopefully get some usable data next week.
The Microscope

Candida Hyphae Visualized under Transmitted Light using 150X Objective

 Experiment 2: Caspofungin Concentration Sensitivity Plates
This experiment was very similar to that of the cell wall sensitivity plate assay; however, this week we simply focused on only one chemical agent (caspofungin) and tested a broader range of concentrations.  

 Experiment 3: Alcian Blue Assay Take Two
I ran through the alcian blue staining experiment again on Friday, this time including 3 biological repeats for each Candida strain in both yeast and hyphal growth conditions. This meant having 12 tubes in total, which equated to somewhat of a lengthy cell counting session! Although I did get results for both yeast and hyphal cells, I decided not to conduct statistics on the latter, as they did not pellet very well and re-suspended almost immediately following centrifugation. This made it very hard to actually collect supernatant following alcian blue incubation, which made the relevance of the results questionable. This was in stark contrast to using yeast cells, which didn't seem to cause any trouble at all. As alcian blue binds to phosphomannan, I used my results to infer differences in phosphomannan content between wild-type and mutant strains.
The loss of apm4 increases phosphomannan content in C. albicans. Yeast cells from wild-type (WT) and apm4Δ/Δ mutant strains of C. albicans were incubated with 30µg/mL alcian blue. Values plotted are the mean and standard deviation obtained from 3 refreshed cultures. *p = 0.0117 (unpaired t test, n=3)


Comments

Post a Comment

Popular posts from this blog

Introduction to the Project: The Role of Endocytosis in Regulating Cell Wall Composition in Candida Albicans

Background Candida Albicans (C. albicans) C. albicans is the most common human fungal pathogen, which causes various types of Candidiasis depending on the anatomical site of disease manifestation. It is perhaps most readily associated with thrush, which occurs in the throat and vagina. Both in vitro  and in vivo, Candida are capable of growing in a variety of morphogenic forms, namely yeast, pseudohyphae and true hyphae. These forms can be selectively induced by particular experimental conditions; for example, growing C. albicans at 28 o C and 37 o C induces yeast and true hyphal forms, respectively. Pseudohyphal, Yeast, and True Hyphal Forms of Candida albicans . Source: Sudbery, 2011   Endocytosis, AP-2 and Polarised Growth Yeast budding and hyphal growth are highly polarised events, that are co-ordinated in-part by the regulation of cell surface composition via endocytosis (membrane internalisation) and exocytosis (secretion). Both processes are ...
Post a Comment
Read more

Week 3

Laboratory Work Experiment 1: Caspofungin Growth Curve The caspofungin plate spotting assay we set up last week did not show any differences between wild-type and apm4 Δ  mutant strains in their ability to grow in the presence of various concentrations of caspofungin. Wild-type and apm4Δ Strains Appear to Grow Similarly  in the Presence of Caspofungin. Wild-type (1st and 3rd rows) and apm4Δ mutant (2nd and 4th rows) strains were grown at 37˚C on a YPD plate supplemented with 0.16 µg/mL caspofungin. Because of this, we decided to do a literature search to find other caspofungin sensitivity assays in order to double check our results. With help from Harriet, I adapted an experiment which measured the OD 600  of cells growing in YPD media supplemented with various concentrations of caspofungin. As it would not be accurate to measure the OD of hyphal cells as an indication of total population size, we could only do this experiment with yeast. In additio...
Post a Comment
Read more