Lab 1

Objective

The overall objectives our first lab session was to orient us on how to properly and efficiently use microscopes and prepare slide mounts in such a way that excludes contamination.

Sterile techniques

1.      Surface clean table with bleach or 30% alcohol

2.      Turn on Bunsen burner to a sterile environment through convection of hot air.

3.       Work close to the burner as any area outside the “convection zone” is not sterile

4.      Flame needle and allow it to cool before transferring specimen to slide.

Use of microscopes

It was demonstrated to us on how safely microscopes. In addition there was a tutorial on how best to set up the microsopes to ensure clarity on sample observation. Of emphasis was the need to ensure that Köhler was set up on a compound microscope before use.

The demonstrations also covered how best to manipulate the amount mainly light and color balances so as to take good specimen pictures using QCapture software

Preparation of squash and tape mounts

a)      Squash mount

1.      Squash mounts were prepared by getting a small specimen sample especially on the edge of the hyphal growth in a petri dish.

2.      The small piece of specimen was placed on a slide.

3.      A drop of sterile water was added

4.      The specimen was covered with a cover slip and the using the back of the needle handle to flatten the specimen under the cover slip and observed under a microscope.

 

b)      Tape mounts

1.      A 1-0.5 of an inch one sided tape was cut

2.      The tape was then used to slightly touch fungal specimen in a petri dish

3.      The tape was then placed on a slide and slightly pressed so that it was on a fixed position on a slide

4.      A drop of water was used put on top of the tape and it was observed under a microscope

Hemocytometer

The protocol followed was as the one described in the provided on the class lab

Results

Discussion

Emphasis was given on how best to handle and use microscopes, taking of sample pictures using Q Capture software. In addition other safety issues in the lab were also highlighted. From trying different slide mount types it was clear that squash mounts did not always result in observable intact structures on the other hand tape mounts preserved the intact structures in most cases. Spore enumerations using Hemocytometer requires a consistency throughout the counting process with respect to spores on border lines, which squares to count, spores that are tightly attached to each other and on levels (color intensity) for accepting spores as  living or dead spores. Cell suspensions should be dilute enough so that the cells do not overlap each other. In addition extra care should be taken on calculations as significant figures are supposed to be kept within the range of the Hemocytometer accuracy, minor error may reflect huge differences when spore numbers are corrected by dilution factor.

Personally I think this lab is a fundamental step for any proper fungal diagnosis as it dealt with issues that usually can make diagnosis of fungi frustrating namely a failure to effectively use the microscope and poor slide mounts which makes it difficult to observe important fungal diagnostic features or structures.