Background

The purpose of this experiment is to show the differentiation of bacteria in acid-rapid and also non-acid quick staining groups. Many bacterial organisms can be stained by either straightforward or Gram staining steps. The features displayed between mycobacteria and various other microorganisms are incredibly various because of the presence of a thick, waxy wall that provides the stain penetration incredibly tough. Once the stain is penetrated, it is not able to be conveniently removed even with the vigorous acid-alcohol agent, unfavor the 95% ethyl alcohol that is offered in gram staining. This an extremely necessary building bereason it is what distinguishes these organisms as acid-quick, versus non- acid-rapid, which are conveniently decolorized by acid alcohol (Cappuccino 81).

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The acid-fast staining offers three various reagents which consists of the major stain, the decolorizing agent, and the counterstain. The main stain being provided is carbolfuchsin which is a dark red stain 5% phenol that is soluble in the lipoidal materials. It penetrates most of the cell wall, is preserved, and also enters the bacteria. Acid-alcohol is offered as the decolorizing agent, bereason acid-quick cells will be resistant to decolorization because of the reality that the main stain is even more soluble in the cellular waxes than in the decolorizing agent. The last reagent supplied in this procedure is methylene blue, which is offered to stain previously decolorized cells, and serves as the counterstain.

Materials and Methods

In completing this experiment, we supplied a Bunsen burner, hot plate, 250 ml beaker, inoculating loop, glass slides, bibulous paper, lens paper, staining tray, and a microscopic lense. We likewise supplied 72-96 hour soy broth culture of M. smegmatis and 18-24 hour culture of S. aureus and also for our reagents, carbolfuschin, acid-alcohol, and methylene blue. In our smear preparation, we took 3 glass slides and making use of aseptic strategy, we ready a bacterial smear of each organism plus a 3rd smear which was a mixture of the two onto the third slide. We then permitted the smears to dry, and also as soon as they we dry we warm solved them by conveniently running then over the flame of the Bunsen burner.

After the smears were completely dry and also warm resolved, we flooded the smears via carbolfuchsin and inserted the slide over a beaker on a warmth hot plate, and also permitted it to steam for 5 minutes. Once the slides were totally cooled, we waburned them through tap water. Next, we decolorized them by adding the acid alcohol, drop by drop onto the slides till the alcohol was nearly clear via a red tinge, then waburned it with tap water again. Then we counterstained via methylene blue for 2 minutes and also waburned with tap water. Once that part was done, we blotted the slides through bibulous paper and also examined it using the microscope under oil immersion.

Results

The acid-quick stain revealed that S. aureus stained blue, which intended that it was non acid-quick. S. aureus was circular-shaped and also had actually a clustered (almost favor grapes) setup. Antithetically, M. smegmatis stained red, which supposed that it was acid rapid. M. smegmatis was irregular-shaped and also had actually a pseudofilamentous setup. The mixed society of S. aureus and M. smegmatis stained pink and also blue.

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S. aureus

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M. smegmatis

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Mixture of M. smegmatis and S. aureus

Review Questions:

1. Why should warmth or a surface-active agent be used via application of the primary stain during acid-fast staining?

Heat is supplied in acid-quick staining to drive the carbolfuchsin lipoidal cell wall and right into the cytoplasm. Alternatively, a surface-active agent can be offered to mitigate the surconfront tension between cell wall of the bacteria and the stain.

2. What is the specific diagnostic value of this staining procedure?

A specific diagnostic value of acid-rapid staining is its use to confirm the existence of Mycobacterium tuberculosis is patients suspected of suffering from pulmonary tuberculosis.

3. Why is the application of warmth or a surface-active agent not forced throughout the application of the counterstain in acid-fast staining?

The respond to stain is used to colorize the non-acid-rapid cells existing in the specimen. These cells execute not have the kind of cell wall that calls for heat application for the stain to penetrate.

4. A boy presents symptoms suggestive of tuberculosis, namely a respiratory infection through a productive cough. Microscopic examination of the child’s sputum reveals no acid-fast rods. However before, examicountry of gastric washings reveals the existence of both acid-rapid and also non-acid fast bacilli. Do you think the child has active tuberculosis? Exsimple.

It does not appear most likely that the kid has active tuberculosis considering that the sputum test is a reliable implies of diagnosis pulmonary tuberculosis. It is recommfinished that 3 specimens of sputum be gathered from suspect people and also analyzed (duhscme.com)

Works Cited

Cappuccino, James G. Microbiology: a laboratory hand-operated – 10th ed. Glenwatch, IL: Pearkid, 2014. Print.

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Duhscme.com. “Tuberculosis.” duhscme.com. Ojha Institute of Chest Diseases, 2011. Net. 5 April 2014.