microbiology lab report

Ex 5 The Gram Stain
Ex 8 Phase Contrast Microscopy
Ex 9 Environmental Conditions Impacting Microbial Growth
Ex 10 Oxidation Fermentation: Oxygen Requirement

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Exercise 5
Gram Staining
Technique
Purpose: To practice staining technique and to be able to
differentiate between gram positive and gram negative bacteria.
Procedure:
Part One
– Heat Fixing Bacterial Smear
1.
Using sterilized wire loop, place one droplet of
water on slide.
2.
Following aseptic technique, pull from the stock
bacterial culture and place a small amount in the water on the slide. Smear
bacteria to a quarter size area on the slide.
3.
After all of the water dries from the slide,
quickly run the slide through the flame twice with the bacterial sample facing
away from the flame.
Part Two – Gram Staining Process
1.
Place heat fixed slide on staining rack in a
tray to catch excess chemicals.
2.
Place enough crystal violet on slide to cover
bacterial sample. Let sit for one minute.
3.
After one minute, take the slide with a
clothespin and flush with water being through and getting all excess crystal
violet off the slide.
4.
Place slide back on the rack and put enough iodine
on sample to cover. Let sit for one minute.
5.
Flush with tap water on both sides of the slide.
6.
Place slide back on rack and use the Alcohol
(gram decolorizer) on the slide letting it sit for 10-12 seconds before rinsing
it off with tap water.
7.
Place the slide back on the rack and place
enough safranin on the sample to cover. Let sit for one minute.
8.
Flush the slide with
tap water and blot the slide with the bibulous paper.
Results:

Microbe

Color

Gram reaction

Morphology

S. marcescens

Pink

Gram Negative

Bacilli

Discussion:
Based on the results, I can determine this bacterial sample
is gram negative because it held the safranin counterstain. Crystal violet
holds on to the thicker peptidoglycan on gram positive cells while being washed
away with alcohol from the gram negative cells that have thinner peptidoglycan.
If these steps were to be reversed and the safranin was used first then the
crystal violet, everything would appear blue because there would be no alcohol
rinse to get rid of the excess crystal violet. In addition, the iodine plays a
major role in cementing the crystal violet to the gram positive cells and
allows it to resist the alcohol wash.

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