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Acid Fast Staining

Lab on Acid fast stain

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Tammi Gabriel
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12 views

Acid Fast Staining

Lab on Acid fast stain

Uploaded by

Tammi Gabriel
Copyright
© © All Rights Reserved
We take content rights seriously. If you suspect this is your content, claim it here.
Available Formats
Download as PDF, TXT or read online on Scribd
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STUDENT NAME: LTTAMI GABRIEL

STUDENT ID: 94508


TITLE: ACID FAST STAINING
INTRODUCTION

Acid-fast staining is a differential staining technique used to identify bacteria with a high
lipid content in their cell walls, specifically mycobacteria such as Mycobacterium
tuberculosis. These bacteria are resistant to conventional staining methods due to their waxy
cell walls.
The Ziehl-Neelsen and Kinyoun methods are two common acid-fast staining techniques. The
Ziehl-Neelsen method involves heating the primary stain, carbolfuchsin, to penetrate the
waxy cell wall. The Kinyoun method, a cold staining technique, utilizes a more concentrated
primary stain to achieve similar results.
In this laboratory exercise, we will perform either the Ziehl-Neelsen or Kinyoun method to
identify acid-fast bacteria in a given sample. By understanding the principles of acid-fast
staining and the characteristics of acid-fast organisms, we can gain valuable insights into the
diagnosis of infectious diseases.

OBJECTIVE
To identify Bacillus using Acid Fast Staining (Ziehl-Nellsen method) and Endospore Staining.
HYPOTHESIS
Bacillus is a spore-forming, non-acid fast bacteria and would be negative on the Acid-Fast and positive on
the Endospore Stains respectively.
METHOD

1. Firstly, the slides were prepared by passing them both over the flames from the Bunsen
burner in order to sterilize them.
2. Then, the loop was also passed into the flames from the Bunsen Buner also for
sterilization purposes.
3. Using the wax pencil, a circle was drawn in the middle of both slides giving a guideline
of where everything was placed.
ACID-FAST STAINING

1. One of the prepared slides was taken and then the sterile loop was dipped into the
bacteria and gently smeared into the circle drawn by the wax pencil.
2. This stain was then placed over the Bunsen burner for the smear to evaporate.
3. The slide was then gently rinsed under running tap water.
4. Next, a napkin was used to gently dry off the slide by tapping it on the slide (bottom of
the slide as well as the top of the slide).
5. After this, the smeared was then covered in carbol fuchsin and then placed under heat
for 5 minutes.
6. Then, the slide was gently rinsed off.
7. Acid alcohol was then added onto the smeared part of the slide and left for 15 seconds.
8. The slide was then rinsed off.
9. Next, crystal violet was added onto the smeared part of the slide and left for 1 minute.
10. The slide was then rinsed off and left to blot dry.
11. The slide was then placed under the microscope and the results was recorded.
ENDOSPORE STAIN

1. The other prepared slide was also sterilized under the Bunsen burner and then the sterile
loop was dipped into the bacteria and smeared over the circle drawn using the wax pencil.
2. The slide with the bacteria smeared was then placed over the heat for it to evaporate.
3. The slide was then rinsed off.
4. Next, when the slide was dried off with the paper towel, some malachite green was added
into the circle.
5. The malachite green was left under heat for 5 minutes and then gently rinsed off.
6. Safranin was then added and left for 1 minute.
7. After, the slide was rinsed and blot dried.
8. The slide was then placed under the microscope and the results was then recorded.
MATERIALS

1. Sterile loop
2. Bunsen burner
3. Light microscope.
4. Ziehls carbol fuchsin
5. Glass slides
6. Crystal Violet
7. Safranin
8. Malachite Green
9. Bacteria
10.Paper Towels
11.Running water
12.Wax pencil
13.Acid Alcohol
RESULTS
DISCUSSION

Acid-fast staining is a crucial technique in microbiology for identifying bacteria with a


unique cell wall structure, particularly Mycobacterium tuberculosis. This technique relies on
the principle that certain bacteria, such as mycobacteria, are resistant to decolorization by
acid-alcohol due to the presence of mycolic acids in their cell walls.
In this experiment, we successfully performed the acid-fast stain to identify acid-fast
bacteria. The red-stained bacilli observed under the microscope confirmed the presence of
acid-fast organisms. This staining technique is invaluable in clinical microbiology for the
diagnosis of tuberculosis and other mycobacterial infections.
Heat fixation is essential to adhere the bacteria to the slide and ensure proper staining. Carbol
fuchsin, the primary stain, penetrates the waxy cell wall of acid-fast bacteria. Acid-alcohol is
used to decolorize non-acid-fast bacteria, while methylene blue, the counterstain, stains non-
acid-fast bacteria blue.
To ensure accurate results, it is important to avoid overheating the slide during the staining
process, as this can damage the bacterial cells. Additionally, adequate decolorization is
crucial to prevent false-positive results. Proper smear preparation, with a thin and even
smear, is also essential for optimal visualization.
Future experiments could explore the use of different staining techniques, such as the
Kinyoun method, or the use of fluorescent stains to improve the sensitivity and specificity of
detection. By understanding the principles of acid-fast staining and mastering the technique,
microbiologists can effectively identify and characterize acid-fast bacteria, contributing to
the accurate diagnosis and treatment of infectious diseases.

CONCLUSION

In conclusion, this experiment successfully demonstrated the acid-fast staining technique, a


crucial tool for identifying acid-fast bacteria. The procedure, involving the use of primary
stain, decolourizer, and counterstain, effectively differentiated acid-fast bacteria from non-
acid-fast bacteria.
The ability of acid-fast bacteria to retain the primary stain, even after decolorization, is a
characteristic feature that sets them apart from other microorganisms. This unique property is
due to the presence of mycolic acids in their cell walls, which make them resistant to
conventional staining techniques.
By mastering the acid-fast staining technique, microbiologists can accurately identify and
diagnose infections caused by acid-fast bacteria, such as tuberculosis. This knowledge is
essential for effective treatment and prevention of these diseases
REFERENCES
https://www.researchgate.net/figure/Figure-2-Micrograph-showing-the-Acid-Fast-Bacilli-AFB-
organisms-under-a-microscope_fig2_274249041

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