The purpose of this experiment is to show the differentiation the bacteria in acid-fast and also non-acid quick staining groups. Many bacterial organisms have the right to be stained by either simple or Gram staining procedures. The characteristics displayed between mycobacteria and also other microbe are very different due to the fact that of the visibility of a thick, waxy wall surface that provides the stain penetration really difficult. Once the stain is penetrated, it is not able to be conveniently removed also with the vigorous acid-alcohol agent, unlike the 95% ethyl alcohol that is supplied in gram staining. This a really important property because it is what distinguishes this organisms together acid-fast, matches non- acid-fast, i m sorry are easily decolorized by acid alcohol (Cappuccino 81).

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The acid-fast staining supplies three various reagents which consists of the main stain, the decolorizing agent, and the counterstain. The main stain being provided is carbolfuchsin i beg your pardon is a dark red stain 5% phenol that is dissolve in the lipoidal materials. That penetrates many of the cabinet wall, is retained, and enters the bacteria. Acid-alcohol is offered as the decolorizing agent, since acid-fast cells will certainly be resistant to decolorization as result of the fact that the primary stain is more soluble in the cellular waxes 보다 in the decolorizing agent. The final reagent used in this process is methylene blue, i m sorry is provided to stain previously decolorized cells, and also serves as the counterstain.

Materials and Methods

In perfect this experiment, we offered a Bunsen burner, warm plate, 250 ml beaker, inoculating loop, glass slides, bibulous paper, lens paper, staining tray, and a microscope. We also used 72-96 hour soy broth culture of M. Smegmatis and also 18-24 hour culture of S. Aureus and also for our reagents, carbolfuschin, acid-alcohol, and methylene blue. In ours smear preparation, we took three glass slides and also using aseptic technique, we ready a bacterial smear of each organism to add a third smear which to be a mixture of the two onto the 3rd slide. Us then permitted the smears come dry, and also once they us dry we warmth fixed lock by quickly running climate over the fire of the Bunsen burner.

After the smears were fully dry and also heat fixed, us flooded the smears v carbolfuchsin and placed the slide end a maker on a heat hot plate, and enabled it to steam for 5 minutes. When the slides were completely cooled, us washed them with tap water. Next, us decolorized them by adding the mountain alcohol, drop by drop onto the slides till the alcohol was practically clear v a red tinge, then washed it v tap water again. Then we counterstained v methylene blue for 2 minutes and washed v tap water. As soon as that part was done, us blotted the slides through bibulous record and examined it utilizing the microscopic lense under oil immersion.


The acid-fast stain revealed that S. Aureus stained blue, which supposed that it to be non acid-fast. S. Aureus was circular-shaped and had a clustered (almost favor grapes) arrangement. Antithetically, M. Smegmatis stained red, which expected that it was acid fast. M. Smegmatis to be irregular-shaped and also had a pseudofilamentous arrangement. The mixed culture of S. Aureus and also M. Smegmatis stained pink and also blue.

S. Aureus

M. Smegmatis

Mixture of M. Smegmatis and S. Aureus

Review Questions:

1. Why must heat or a surface-active agent be offered with application of the primary stain throughout acid-fast staining?

Heat is offered in acid-fast staining to journey the carbolfuchsin lipoidal cell wall surface and into the cytoplasm. Alternatively, a surface-active agent have the right to be provided to reduce the surface tension between cell wall of the bacteria and the stain.

2. What is the particular diagnostic worth of this staining procedure?

A particular diagnostic worth of acid-fast staining is its use to check the visibility of Mycobacterium tuberculosis is patients suspected of experiencing from pulmonary tuberculosis.

3. Why is the application of warm or a surface-active agent no required throughout the application of the counterstain in acid-fast staining?

The respond to stain is provided to colorize the non-acid-fast cells existing in the specimen. These cells do not have the kind of cell wall surface that requires heat application because that the stain to penetrate.

4. A son presents symptom suggestive of tuberculosis, specific a respiratory tract infection with a fertile cough. Microscopic examination of the child’s sputum reveals no acid-fast rods. However, check of gastric washings expose the visibility of both acid-fast and non-acid fast bacilli. Carry out you think the kid has active tuberculosis? Explain.

It does not appear likely that the boy has energetic tuberculosis because the sputum check is a reliable means of diagnosis pulmonary tuberculosis. The is encourage that 3 specimens the sputum be gathered from doubt individuals and analyzed (

Works Cited

Cappuccino, James G. Microbiology: a laboratory hands-on – 10th ed. Glenview, IL: Pearson, 2014. Print.

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