Microbiology Lab-Practicals-No3 Staining

Microbiology and Parasitology – Laboratory
Laboratory Practical no. 3

Staining
Section: _________ Date: ______________

Group no:________
Group Members:
Introduction:
Staining is the process of giving color to a clinical specimen in order to determine the presence or
absence of a microorganism. In this experiment you will perform ACID FAST STAIN, to be more precise the
Ziehl-Neelsen Method. This staining procedure is designed to determine acid fast organism (ex. M.
tuberculosis). Acid Fast organism will produce Pink/Red and Non-Acid Fast will produce Blue/Purple. In
order to produce the desired results, it is important that you will the instructions given.
Materials:
 Acid Fast Stain Kit  3cc Syringe

 Glass Slide  Alcohol
 Plastic Pipet  Plastic Bottle with a wide opening (1 per
section)
 Alcohol Lamp  Bleach (1 per section)
 Forceps  Match/Lighter
 Stand  Sterile cotton swab
 Microscope
* Materials highlighted in Red are already provided and are not required to be brought by group.
Important to Remember:
 Wear proper PPE when performing the experiment this is to prevent contamination to your
product and to protect yourself from the microorganism.
 Exercise the STANDARD PRECAUTION
 You are using fire in this experiment, exercise proper caution.
 Read the instructions before performing the experiment.
Procedure:
I. Ziehl-Neelsen Acid Fast Stain

1. Using tap water, dilute the bleach inside the plastic bottle. The mouth of the plastic bottle
should be wide enough for the glass slide to pass through. This is where you will throw
the slides after the experiment.
2. Take out your agar plate from the box. Note the color of the bacterial colonies.
Microbiology and Parasitology – Laboratory
Laboratory Practical no. 3
Staining
3. Break the cotton-end of the cotton swab so that the wooden stick is exposed.
4. Using the wooden-end of the cotton swab select a bacterial colony from the agar plate.
When opening the agar plate DO NOT FULLY OPEN.
5. Scrape your selected bacterial colony from the agar plate to the glass slide. And make a
smear.
6. After smearing, use the forceps and the alcohol lamp to heat fix the bacteria to the glass
slide with the bacteria facing upward. Heat for about 1-2 minutes.
7. Once heat fixed, use the plastic pipet to FLOOD the glass slide with Carbol Fuchsin. Again
heat the glass slide. DO NOT BOIL OR DRY OUT. Stop if you see fumes.
8. Don’t throw the Carbol Fuchsin yet, wait 10-20 minutes.
9. After that, remove the needle of the 3cc syringe and fill it with distilled water. Pour the
water on the slide removing the Carbol Fuchsin. The color of the smear should be red.
Perform this step slowly so that the bacteria will not be washed off only the excess stain.
10. After that use a different plastic pipet and decolorize the smear using acid alcohol until
the color disappears.
11. After decolorizing, use the 3cc syringe you used earlier and fill it with distilled water to
remove the excess acid alcohol.
12. After that use a different pipet and counterstain using Aklaline Methylene Blue for 5-10
minutes.
13. After that wash the slide again using the 3cc syringe with distilled water.
14. After washing air dry the slide.
15. Examine under the microscope and determine if acid fast or non-acid fast.
16. After examining throw the slides on the plastic bottle. And sanitize your area using
alcohol.
Results and Discussion:
1. What is the colony morphology/shape of the bacteria?

______________________________________________________________________________
______________________________________________________________________________
2. What is the color of your bacteria? Is it acid fast or non-acid fast?
______________________________________________________________________________
______________________________________________________________________________
3. What are the 3 reagents of Acid Fast Stain – Ziehl Neelsen Method?
______________________________________________________________________________
______________________________________________________________________________
4. What is the mordant used in the experiment?
______________________________________________________________________________
______________________________________________________________________________
5. What is the result of an acid fast stain? How about non-acid fast?
______________________________________________________________________________
______________________________________________________________________________

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Microbiology and Parasitology – Laboratory

Laboratory Practical no. 3

Section: _ Date: ______

 Acid Fast Stain Kit  3cc Syringe

I. Ziehl-Neelsen Acid Fast Stain

Results and Discussion:

1. What is the colony morphology/shape of the bacteria?

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