Scribd
Upload
74 views2 pages

Acid-fast and Endospore Staining Guide

The document outlines the steps and expected results of two staining techniques: 1) Acid-fast staining will result in acid-fast bacteria appearing red and non-acid fast bacteria appearing colorless or blue after being treated with carbolfuchsin dye, acid-alcohol, and methylene blue. 2) Endospore staining will result in vegetative cells appearing red and endospores appearing green after treatment with malachite green, steam heating, and safranin counterstain. The document also notes that different dyes and reagents are used to identify bacteria based on color differentiation and that the cell wall impacts how bacteria stain.

Uploaded by

Jirou Balcita
Copyright
© © All Rights Reserved
We take content rights seriously. If you suspect this is your content, claim it here.
Available Formats
Download as DOCX, PDF, TXT or read online on Scribd
74 views2 pages

Acid-fast and Endospore Staining Guide

The document outlines the steps and expected results of two staining techniques: 1) Acid-fast staining will result in acid-fast bacteria appearing red and non-acid fast bacteria appearing colorless or blue after being treated with carbolfuchsin dye, acid-alcohol, and methylene blue. 2) Endospore staining will result in vegetative cells appearing red and endospores appearing green after treatment with malachite green, steam heating, and safranin counterstain. The document also notes that different dyes and reagents are used to identify bacteria based on color differentiation and that the cell wall impacts how bacteria stain.

Uploaded by

Jirou Balcita
Copyright
© © All Rights Reserved
We take content rights seriously. If you suspect this is your content, claim it here.
Available Formats
Download as DOCX, PDF, TXT or read online on Scribd

1. Steps of Acid-fast Staining and their expected result.

Step 2: Smear Preparation


a. Add one loopful sterile water to a microscope slide.
b. Make a heavy smear of Mycobacterial smegmatis. Mix thoroughly with your loop. Then transfer
a small amount of Staphylococcus epidermidis to the same drop of water. You will now have a
mixture of M. smegmatis and S. epidermidis.
c. Air dry and heat fix.

Step 3:
a. Cover the smear with carbolfuchsin dye. Carbolfuchsin a potential carcinogen. Wear gloves and
work the stain in the hood.
b. Place a piece of paper towel on top of the dye. Be sure the paper towel is saturated with the
dye.

Step 4: Dry heat for 2 minutes.

Step 5:
a. Cool and rinse with water
b. Decolorize with acid –alcohol for 15-20 seconds.

Step 6: Wash the top and bottom of slide with water and clean the slide bottom well.

Step 7:
a. Counterstain with Methylene Blue for 30 seconds to 1 minute.
b. Wash and blot the slide with bibulous paper.

ACID-FAST STAIN Cell Color


Procedure Reagent Acid-fast Bacteria Nonacid-fast Bacteria
Primary dye Carbolfuchsin RED RED
Decolorize Acid-alcohol RED COLORLESS
Counterstain Methylene blue RED BLUE

2. Steps of Endospore Staining and their expected result.

Endospore Stain Procedure:


a. Prepare smears of organisms to be tested for endospores.
2. Heat fix the smears.

3. Cover the smears with a piece of absorbent paper cut to fit the slide and place the slide on a wire
gauze on a ring stand.
4. Saturate the paper with malachite green and holding the Bunsen burner in the hand heat the slide
until steam can be seen rising from the surface. Remove the heat and reheat the slide as needed to
keep the slide steaming for about three minutes. As the paper begins to dry add a drop or two of
malachite green to keep it moist, but don't add so much at one time that the temperature is appreciably
reduced. DO NOT OVERHEAT. The process is steaming and not baking.

5. Remove the paper with tweezers and rinse the slide thoroughly with tap water.

6. Drain the slide and counterstain 45 seconds with 0.5% safranin.

7. Wash, blot, and examine.

8. The vegetative cells will appear red and the spores will appear green.

3. The importance of the different dyes and reagents used in Staining.

Bacteria can be identified by the color differentiation of stains (dyes) with the help of different staining
techniques and the use of different reagents. Stains are classified broadly as basic, acidic, or neutral
stains.

4. Significance of the cell wall on Staining

You might also like