Rober Koch was the first person who found the tubercle bacillus in 1882. And he gave the path to the microbiologist about complex staining procedures. He also said that there is an appearance of bacilli in the procedure. There were other researchers as well, like Nelson, Rindfleisch, Ziehl, and Ehrlich. And they were trying to improve Koch’s method.
At the same time, Franz Ziehl used carbolic acid for the mordant. And he was the first person to use that. On the other hand, Friedrich Neelsen changed the procedure a little bit. He kept the mordant same, but instead of using the primary stain, he used basic fuchsin. And after that, people started to call the method the Ziehl-Neelsen method. This thing happened in the mid-1890s.
There was a difference between these two methods. In the second method, the heat was one of the key factors. It helped the primary stain to drive the waxy cells, which were difficult for stain cells. Therefore, the method was called hot staining or acid fast staining procedure because Ziehl used heat in that method. From that, the Ziehl-Neelsen method started to become popular, and still, it is an effective and reliable way to give a demonstration about acid fast bacteria.
But, What Was The Purpose?
So, what is the purpose of the acid fast stain? There was this other method where there was no heat in the method. Then, in 1915, Kinyoun used a different method and name cold staining. In that method, he didn’t use heat. He removed the step and used a carbol fuchsin primary stain. The only difference was that he used the compound in a higher concentration. But what was the purpose of the method and test?
The main purpose of the method is to find acid fatness in bacteria and what are the characteristics of them. The method also proves the cysts of Isospora and Cryptosporidium. And this is a method to know Mycobacterium tuberculosis and its characteristics. If a person tests their sputum sample, then the method shows if that person has tuberculosis or not.
How Do Bacteria Take Up The Stain?
It makes you wonder, right? It is quite exciting to think about. Suppose you have experience with bacterial or fungal staining. You will know how beautiful these microorganisms look under the microscope, with all the colors surrounding them.
So, how do bacteria take up the stain? And why do we stain them? Even though the latter does not need a separate answer, the basic logic of staining bacteria, or any other microorganism, is for better visualization.
The microorganisms that can take up stains usually have a cell structure that can hold that stain within. This is common in bacteria with a cell wall type with components that allow its retention. For example, in the Ziehl Neelsen method of staining, microbiologists check the acid fastness of the bacterium.
Like Gram staining, where the bacteria can either be Gram positive or Gram negative, acid fastness is a property of the bacteria to not release the stain even after acid wash.
This is because the bacteria are not just mildly Gram-positive or Gram variable; their cell wall contains a thick peptidoglycan layer that can hold the Gram stain. It also has an additional layer of glycolipids. This layer of glycolipids is quite large, retaining the stain and preventing its loss despite the decolorizing step with acid.
You would think, how does it happen? When the bacteria can hardly take up the Gram stain, how can it take the primary stain in this method? This bacterium has a thick waxy layer that does not open up. This is why the Ziehl-Neelsen method uses heat to allow the primary stain to penetrate the acid-fast bacteria.
This property of the bacteria allows it to protect itself in harsh conditions. However, when the bacteria are to be stained through the method, they undergo exposure to steam that allows their waxy layers to loosen up.
This loose layer allows the primary stain to penetrate the cell walls, after which it does not decolorize easily.
Acid Fast Staining Procedure: Theory
All three tests were successful, and they showed the acid fast mycobacterium. These are as follows.
Ziehl-Neelsen Method (Hot)
To this test, there are certain things needed. To make the carbolfuchsin stain, there are a couple of things to mix up. These are- 10 ml Ethanol 95%, 0.3g basic fuchsin, 5 ml phenol (it should be heat-melted crystals), and 95 ml distilled water. After mixing the compounds, there is a need to stand it for several days. And lastly, filter the mixture before using.
The second is decolorizing solvent. To make it, hydrochloric acid and ethanol are needed. Then Counterstain, which is a mixture of distilled water and methylene blue chloride. The method was so old, but they both were successful in making it happen.
Auramine-Rhodamine Fluorochrome (Truant)
This method is different than the rest two methods—the main this to make a fluorescent staining reagents. And to make this, the things needed are- distilled water, phenol, glycerol, rhodamine B, and auramine O. The decolorizing solvent is a must thing to use. In this method, there is another thing used, which is the counterstain. To make the counterstain, the things used were distilled water and potassium permanganate.
Kinyoun Method (Cold)
In this method, Kinyoun used different things, and he made those compounds by himself. The main ingredients were solution A (20 ml ethyl alcohol and 4 g fuchsin) and solution B (100 ml distilled water and 8 g phenol), and then he mixed these two solutions. The other compounds were methylene blue counterstain (distilled water, methylene blue chloride) and acid-alcohol decolorizing agent (hydrochloric acid and ethanol).
Important Notes
There are a couple of things that you should know about the mycobacterium acid fast stain procedure. If you want to do the test, then you must take some precautions. First, phenol is a dangerous compound. It is combustible, corrosive, and poisonous. So when you will use phenol, be careful about handling it.
Always use a material safety data sheet. Do not forget to wear gloves while you are doing the test. Also, there will be a heating process, so the chances of inhaling phenol are high. So you will need a mask too.
Frequently Asked Questions (FAQs):-
Here are popular questions that most people ask. You may find these questions interesting.
Ans: The main principle behind the acid fast staining is based upon a simple theory. Heat softens wax, so when the cell wall gets heated, the wall gets melted. And then, the stain can access the cell. Normally fuchsin is not soluble in water or alcohol, but it is highly soluble in phenol. And phenol is soluble in waxes or lipids. So the dye phenol can access the cell.
Ans: The 3 main acid fast stain steps are as follows.
➼ You need to air dry the microorganisms and then heat them up.
➼ There will be a slide with Carbolfuchsin, flood that.
➼ Then you need to acid alcohol. The process will go for 30 seconds.
➼ Then the mixture needed to flood for 3 seconds with the counterstain.
➼ After that, dry the slide and put it on a bibulous paper.
Ans: Here are the three steps of staining.
➼ First, the cells get stained with violet dye.
➼ Then there is a need to add a decolorizer such as acetone or ethyl alcohol. Then there will be a peptidoglycan layer through dehydration. After that, it will tighten and shrink.
➼ Lastly, counterstain will be added to the mixture, and it will show a red color.
To Conclude
The method is highly popular and a remarkable work. Through the acid fast bacillus test shows acid fast bacteria, so the diagnosis of tuberculosis can be possible. Also, remember to wear gloves and masks. And follow all the safety parameters. Always be careful while handling such dangerous compounds. Please let us know if you find this article interesting in the comment section below.
Thank You.
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