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.
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