Download MCB 3020L Lab Experiment 6 Differential and Cytological Stains A

MCB 3020L
Lab Experiment 6
Differential and Cytological Stains
A one lab session experiment
In Experiment 2, simple and Gram stains were done. The Gram stain is a differential stain: it
stains one type of cell one color and other types of cells a different color. In this experiment several
differential stains are done: some reveal different cell types but others stain one part of the cell one
color and other parts a different color.
THE SPORE STAIN
Bacterial endospores are resistant to most staining procedures and appear as clear areas in the
Gram stain. Poly-beta hydroxybutyrate (bacterial fat) and other intracellular inclusions can also not
stain in the Gram stain and appear as clear areas. The spore stain allows you to clearly demonstrate
that the clear area in a Gram stain is or is not a spore. Since spores are refractile to stains, the spore
stain Malachite Green must be "driven" into the spore by heat. That is, during the spore stain, you will
heat the slide being stained to steaming....this causes breaks in the spore such that the stain can
penetrate the spore coat, cortex and spore wall. After cooling the slide is rinsed in water and
counterstained with safranin. The cells stain red and the spores stain green.
Materials per Pair
1. Two Nutrient Agar slant cultures of Bacillus species grown for 48 hours.
from Experiment 3.
2. Shaeffer-Fulton spore stain: 0.5% Malachite Green in water.
3. Safranin.
4. Slides and slide holders.
And your pure culture
Procedure
1. Prepare two slides with each of the known Bacillus culture and your pure culture from Experiment 3
(heat fixed smears).
Bacillus
24 hour
Bacillus
48 hour
Your
Culture
Crystal Violet Slide
2.
3.
Bacillus
24 hour
Bacillus
48 hour
Your
Culture
Spore Stain Slide
Simple stain one with Crystal Violet. DO NOT do a full Gram Stain.
Spore Stain the other:
a. Drop Spore stain onto the smear and cover with a piece of paper towel. The paper towel must
be cut so that it does not extend beyond the sides of the slide.
b. Carefully heat the slide to steaming for 3-5 minutes. Do not let the stain boil!
AND, do not let the stain dry on the slide. To prevent this add stain when it gets close to
drying out.
c. Allow the slide to cool. Remove the paper towel and place in the waste bucket (not the sink!).
d. Rinse slide with water.
e. Counterstain with safranin for at least 10 seconds.
f. Rinse with water, dry and observe with the oil immersion lens.
ACID FAST STAIN
Acid fast bacteria are Gram positive bacteria that possess waxes in their cell walls. They are
generally difficult to stain, but the Acid Fast Stain uses heat to drive the stain through the waxy wall.
This stain also uses a challenge step (like the Gram stain): a rinse in acid alcohol (3% HCl in 95% ethyl
alcohol) which washes out the acid fast stain primary stain (carbol fuchsin) from cells that do not
possess waxes in their walls. The acid fast stain is an important diagnostic tool in the diagnosis of
tuberculosis and leprosy.
Materials per Pair
1. Cultures of Mycobacterium species and Staphylococcus aureus and your pure culture from
Experiment 3.
2. Zielhl's Carbol Fuchsin.
3. Acid Alcohol.
4. Methylene Blue.
Procedure
1. Prepare on two slides a smear of S. aureus and a smear of one of the Mycobacterium species
provided and a third smear of your pure culture. Allow the smears to air dry and then heat fix.
S.
aureus
Mycoba
cterium
Your
Culture
Crystal Violet Slide
S.
aureus
Mycoba
cterium
Your
Culture
Acid Fast Stain Slide
2. Simple stain one slide with Crystal Violet. DO NOT do a full Gram Stain.
3. Stain the other slide by covering the smear with Carbol Fuchsin and a paper towel as was done in
the spore stain. Heat to steaming for 5 minutes making sure to not let the slide dry out....add more
stain if the slide appears to becoming dry.
4. Cool and then rinse the slide with tap water.
5. Rinse with acid alcohol until color no longer runs out of the smears. Avoid over rinsing.
6. Rinse with water.
7. Counter-stain with Methylene Blue for 1 minute.
8. Rinse with water, dry, and observe with the oil immersion lens.
Questions:
1. What differences can you identify between the simple stains and the differential stains?
2. What color should spores stain?
3. What color should acid fast bacteria stain?
Note:
Next Lab is the Yogurt Lab (check the Lab Schedule)...you will need to:
1. Design the experiment - please read in advance and do the homework (read section of the
textbook), check this experiment out...you have to make up the procedure
2. BRING IN Yogurt (worth TWO bonus points). Make sure you know the difference between
Set-type and Stirred-type yogurts so that you will be able to design a valid experiment using this
fermented food.