Acid-Fast (AF) is an important special staining technique used in the histology laboratory. It is a differential staining techniques which was first developed by Ziehl and later on modified by Neelsen 1883. This method is used for those microorganisms which are not staining by simple or Gram staining method, particularly the member of genus Mycobacterium and Nocardia, are resistant and can only be visualized by acid-fast staining.
Most of the bacteria can be stained, either by simple basic staining or by gram staining, as their cells take up stains easily.However, some bacteria possess a thick waxy cell wall made of lipid materials.Such bacteria are extremely difficult to be stained, as ordinary aqueous stains cannot enter into their cells easily, but once stained; it is equally difficult to remove the stain out of their cells, even with vigorous use of acid-alcohol as decolorising agent. These bacteria are stained by acid-fast staining.Thus, acid-fast staining is a differential staining method, which differentiates bacteria, based on the nature of their cell wall. The staining differentiates bacteria into two groups as follows:
(1) Acid-fast Bacteria
A bacteria, which is extremely difficult to be stained, but once stained, it is equally difficult to remove the stain out of its cells, even with vigorous use of acid-alcohol as decolourising agent, is an acid-fast bacteria (acid-loving bacteria).
Examples: Mycobacterium spp. [M. tuberculosis (TB bacteria), M. leprae (leprosy bacteria), M. smegmatis (natural bacteria of smegma) and M. marinum (TB bacteria of marine fishes). They possess a thick waxy cell wall made of lipid materials.
(2) Non-acid-fast Bacteria
A bacteria, which is easy to be stained and is also easy to be decolourised by acid-alcohol as decolourising agent, is a non-acid-fast bacteria (non-acid-loving bacteria). Examples: All bacteria except Mycobacterium spp. In these bacteria, the cell wall is not thick and waxy.Acid-fast staining is useful in differentiating acid-fast and non-acid-fast bacteria and also in the identification of bacteria belonging to the genus Mycobacterium.
The acid-fast bacteria are different from the non-acid-fast bacteria in that, they possess a thick waxy cell wall made of lipid materials. Ordinary aqueous stains, such as methylene blue or crystal violet cannot enter into their cells through this waxy cell wall.
However, the red coloured phenolic primary stain carbol fuchsin (carbolic acid or phenol + basic fuchsine); which is soluble in the lipid materials of the cell wall, can enter into their cells through the cell wall and can be retained inside the cells imparting red colour to them.
Penetration is further augmented by application of heat, which drives carbol fuchsine through the lipoidal wall into the cytoplasm. (A slight modification of this Ziel- Neelsen method describes the addition of a wetting agent, Turgitol, to the stain, which reduces the surface tension between the cell wall and the stain, thereby requiring no heating). The cells are allowed to cool, so as to harden the waxy cell wall.
When the cells are exposed to the decolourising agent, acid- alcohol, they resist decolourisation, as the primary stain is more soluble in the cellular waxes than in the decolourising agent. Subsequently, when counter-stained with methylene blue, it cannot enter into the cells through the waxy cell wall.
Thus, finally the acid-fast bacteria retain the red colour of the primary stain and appear red. On the other hand, the non-acid-fast bacteria take up the primary stain easily and undergo decolourisation easily. When counter-stained with methylene blue, these colourless cells also take up the counter-stain easily and appear blue, unlike the acid-fast bacteria, which appear red.
Reagent And Materials Required
- Primary stain (carbol fuchsine)
- Decolourising agent (acid-alcohol)
- Counter stain (methylene blue)
- Hot plate, broth/slant/plate culture of bacteria
- Immersion oil
- A slide is cleaned properly under tap water, such that water does not remain as drops on its surface.
- The adhering water is wiped out with bibulous paper and the slide is air-dried.
- A smear of bacteria is prepared at the center of the slide in two methods as follows: (a) If the bacteria grown on agar plate or agar slant are to be observed, a drop of water is put at the center of the slide and a loop of bacteria from the plate or slant is transferred to it by a loop sterilized over flame. Then, by slow rotation of the loop in the drop, a bacteria suspension is made and it is spread till a smear is obtained.(b) If bacteria grown in liquid broth are to be observed, a drop of the bacteria suspension is directly placed at the center of the slide by a flame-sterilized loop and a smear is made by spreading.
- The smear is air-dried.
- The smear is fixed by heating. Heating results in the coagulation of the cellular proteins, due to which the cells stick to the slide surface and do not get washed away during staining. Heat- fixation is done by quickly passing the slide high above a flame 2-3 times, with the smear surface facing upward, so that the smear does not get heated up.
- The smear is flooded with carbol fuchsin.
- The slide is placed on a warm hot plate, allowing the preparation to steam for 5 minutes. The temperature of the hot plate is so adjusted that, the preparation does not boil and get evaporated quickly. The evaporation loss is replenished, so that the smear does not dry up. (For heatless method, the smear is flooded for 3-5 minutes with carbol fuchsin containing Turgitol).
- The slide is removed from the hot plate and cooled.
- Excess stain is washed away from the smear under gently-flowing tap water, in such a way that, water does not fall directly on the smear.
- The decolourising agent, acid-alcohol, is added on the smear drop by drop, until carbol fuchsin fails to wash from the smear.
- The acid-alcohol is washed away from the smear under gently-flowing tap water, in such a way that, water does not fall directly on the smear.
- The smear is flooded with the counter-stain, methylene blue, for 2 minutes.
- Excess counter-stain is washed away from the smear under gently-flowing tap water, in such a way that, water does not fall directly on the smear.
- The slide is blotted dry with bibulous paper.
- The slide is clipped to the stage of the microscope and the smear observed under low power and high dry objectives.
- A drop of immersion oil is put on the smear.
- The smear is observed under oil-immersion objective.
Result Interpretation of Acid Fast Stain
Acid fast: Bright red to intensive purple, Red, straight or slightly curved rods, occurring singly or in small groups, may appear beaded.
Non-acid fast: Blue color; In addition, background material should stain blue.
Other Observations (Under Oil-immersion 100X Objective)
Shape of bacteria
- Spherical (coccus)
- Rod-shaped (bacilli)
- Comma-like (vibrio)
- Spiral (spirochetes
Arrangement of bacteria
- Pairs (diplobacillus / diplococcus)
- In fours (tetrads)
- In chains (streptococcus/streptobacillus)
- Grape-like clusters (staphylococcus)
- Cuboidal (sarcinae or octet)
Examples Of Acid Stain
Acid-fast: Mycobacterium tuberculosis, Mycobacterium smegmatis.
Non-Mycobacterial bacteria: Nocardia
Coccidian Parasites: Cryptosporidium
Limitations Of Acid Fast Stain
- The filter paper must remain moist and in contact with the specimen during heating to allow for proper penetration of the primary stain.
- Organisms cultivated on blood agar may experience nutrient deprivation, resulting in a lower lipid content in the outer membrane resulting in poor staining.