BIOL240L Study Guide - Final Guide: Gram Staining, Crystal Violet, Methylene Blue

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5 Aug 2016
Biol 240L
Fundamentals of Microbiology
Experiment 1 – Simple Staining:
A coloured stain is used to provide contrast between the specimen and the background
Simple Stain:
Single stain is used. ALL cells and structures are the same colour
Allows you to see the shape of the cells
Common dyes: methylene blue, basic fuchsin, and crystal violet
Two types:
oPositive: stain is basic, and has positively charged chromophore. Bacteria have slightly
negatively charged outer surfaces
1. Sterilize inoculating loop in Bunsen burner (45o angle). Wait until it cools.
2. Take broth culture and take off lid with pinky finger. DO NOT LET IT TOUCH ANYTHING
3. Sterilize the air in and around the opening of the test tube by quickly passing it through the fire
2-3 times to prevent cool air from entering the pure culture
4. Put the loop into the tube and remove some of the culture. Re-flame the test tube and replace
the cap
5. Put the loop of bacteria onto a clean microscope slide. Repeat 2-4 times. Spread the culture out
to a dime-sized spot. Heat-kill bacteria before returning loop to stand
6. Wait until the smear dries, then heat-fix the slide by quickly passing it through the fire 2-3
times. Do not overheat
7. Place slide on staining rack and put 1-3 drops of the stain on the bacteria. Leave on for 1-2
minutes. Don’t let it dry out
8. Pour gentle stream of water to clean the dye off. Keep adding water until it flows colourless off
of the slide.
9. Gently blot excess water from the slide
Experiment 2 – Operation of a Microscope:
Light microscope consists of lens system, controllable light source and geared mechanism for
adjusting distance between lens and object
2 different lenses:
oObjective: nearest specimen; magnifies specimen to make a real image
oOcular: eyepiece lens; magnifies the specimen to create the virtual image we see
Experiment 3 – Basic Bacterial Shapes
3 General Shapes
Coccus Bacillus Spirillium
Shape of cell Round Rod Spiral
Motility Most nonmotile Most motile Most motile
Arrangement Singly (micrococcus)
Pairs (diplococcus
Chains (streptococcus)
Groups of 4 (Gaffkya)
Groups (cubes) of 8 (sarcina)
Clusters or packets
Pairs (diplobacillus)
Chains (streptobacillus)
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Spore None Produced by several different
Other shapes:
oStalks – Caulobacter
oClub shaped – Corynebacterium
oComma-shaped – vibrio
Experiment 4 – The Gram Stain:
Gram stain differentiates types of bacteria based on their ability to retain a specific stain (i.e. it is
a differential staining technique)
Only fresh culture should be used in Gram stain
Gram-positive: Will not be decolourized; appear purple due to crystal violet
Gram-negative: Will be decolourized by alcohol; appear red or pink
1. Stain fixed smear of organism with primary stain of crystal violet (1-3 drops for 1 minute); rinse
with tap water
2. Add Gram’s iodine stain or mordant (substance that fixes the primary stain in bacterial cells) (1-
3 drops for 1 minute); rinse with tap water
3. Wash with a decolourizing agent, 95% ethyl alcohol, at 45o angle, drop by drop until alcohol
runs clear (4-5 drops should be sufficient)
4. Counterstain with 1-3 drops safrinin for 1 minute; rinse with tap water, and blot to dry
Experiment 5 – The Acid-Fast Stain (Kinyoun Method):
Acid-fast stain is a differential stain
Used for cells that have waxy cell walls that contain large amounts of lipoidal material (mycolic
acids). Walls are impermeable to most stains
1. Smear is flooded with 4-5 carbolfuchsin for 5-10 mins (dark red dye containing 5% phenol);
Rinse with tap water and drain by touching corner to paper towel
2. Decolourize with acid alcohol applied drop by drop until it runs clear; rinse with tap water
3. Counterstain with methylene blue for 2 minutes; rinse with tap water and blot to dry
Acid-fast organisms will not be decolourized and turn red
Non-acid-fast organisms will be decolourized and turn blue
Kinyoun modification increases concentration of phenol and carbolfuchsin + detergent so no
heat needed
Experiment 6 – The Spore Stain (Schaeffer-Fulton Method):
When environmental conditions become too harsh to permit vegetative growth and
reproduction, some species are able to condense vital cellular components into endospores
Spores are highly resistant to extreme dessication, high temperature, ionizing radiation and
many chemicals
Spores need vigorous treatment for staining; Resist ordinary dyes
Primary stain is malachite green and is driven into cell by heating. Counterstained afterwards
with safranin to show vegetative cells as red. Spores will be green
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