Principle:

Acid fast staining is a differential staining technique which differentiate acid fast and non-acid fast bacteria. Mycobacterium species contains large amount of mycolic acid in its cell wall. Due to high lipid content, most dyes cannot enter easily through the cell wall, so Mycobacterium species cannot be stained by Gram staining. A special staining method is developed to stain such organism called as Acid-fast staining method. It is also known as Ziehl-Neelsen method.

In this method, bacteria is stained with carbol fuchsin combined with phenol. The stain binds to the mycolic acid in the mycobacterial cell wall. After staining, an acid alcohol is applied as decolorizing agent which removes the stain from the background cells, tissue fibres, and any organisms in the smear except mycobacteria which retain (hold fast to) the dye and are therefore referred to as acid fast bacilli, or simply AFB.  After decolorization, the smear is counter stained with malachite green or methylene blue which stains the background material green or blue, providing a contrast colour against which the red AFB can be seen.

Note: Some Actinomycetes, Corynebacteria, and bacterial endospores are also acid fast.

The acid-fast stain uses three different reagents.

1.Primary Stain (Carbol Fuchsin): Unlike cells that are easily stained by ordinary aqueous stains, most species of mycobacteria are not stainable with common dyes such as methylene blue and crystal violet. Carbol fuchsin, a dark red stain in 5% phenol that is soluble in the lipoidal materials that constitute most of the mycobacterial cell wall, does penetrate these bacteria and is retained. Penetration is further enhanced by the application of heat, which drives the carbol fuchsin through the lipoidal wall and into the cytoplasm. This application of heat is used in the Ziehl-Neelsen method. The Kinyoun method, a modification of the Ziehl-Neelsen method, circumvents the use of heat by addition of a wetting agent (Tergitol) to this stain, which reduces surface tension between the cell wall of the mycobacteria and the stain. After primary staining, all cells will appear red.

2. Decolorizing Agent (Acid-Alcohol; 3% HCl + 95% Ethanol): Prior to decolorization, the smear is cooled, which allows the mycolic acid to solidify such that AFB will be resistant to decolorization. Primary stain is more soluble in the cellular waxes than in the decolorizing agent. So, in this event, the primary stain is retained and the mycobacteria will stay red while non-acid fast bacteria become decolorized (colourless).

3. Counterstain (Methylene Blue or malachite green): This is used as the final reagent to stain previously decolorized cells. As only non–acid-fast cells undergo decolorization, they may now absorb the counterstain and take on its blue color or green, while acid-fast cells retain the red of the primary stain.

Requirements:

1. Fresh culture sample: 72-96 hour trypticase soya broth culture of Mycobacterium smegmatis/24-hour agar culture of Staphylococcus epidermidis
2. Primary stain: carbol fuchsin
3. Decolorizing agent: acid alcohol (3% HCl +95% ethanol)
4. Counter stain: methylene blue/ malachite green
5. Bunsen burner
6. Inoculating loop
7. Microscope
8. Distilled water
9. Soft cotton or tissue paper
10. Microscopic Slides
11. China-marking pencil or permanent marking pen

Procedure

Procedure for Smear preparation

1. Take a clean glass slide.
2. Using aseptic technique, prepare a smear of given sample organisms
3. Heat-fix the dried smear
4. Alcohol-fixation: This is recommended when the smear has not been prepared from sodium hypochlorite (bleach) treated sputum and will not be stained immediately. M. tuberculosisis killed by bleach and during the staining process. Heat-fixation of untreated sputum will not kill M. tuberculosis whereas alcohol-fixation is bactericidal.

Procedure for Acid-fast staining

1. Flood the smear with carbol fuchsin stain.
2. Heat the stain until vapour just begins to rise (i.e. about 60C). Do not allow stain to evaporate; replenish stain as needed. Also, prevent stain from boiling. Do not overheat. Allow the heated stain to remain on the slide for 5 minutes.
3. * For a COLD METHOD, flood the smear with carbol fuchsin containing Tergitol for 5 to 10 minutes.
4. Wash off the stain with clean distilled water. Heated slides must be cooled prior to washing
5. Decolorize the smear with 3% v/v acid alcohol for 5 minutes or until the smear is sufficiently decolorized, i.e. pale pink by adding the reagent drop by drop until the alcohol runs almost clear with a slight red tinge.
6. Wash well with clean water.
7. Flood the smear with malachite green or methylene blue stain for 1–2 minutes.
8. Wash off the stain with clean water.
9. Wipe the back of the slide clean, and place it in a draining rack for the smear to air-dry (do not blot dry).
10. Examine the smear microscopically, using the 100X oil immersion objective.
11. Scan the smear systematically.
12. Note: Do not touch the smear with the end of the oil dispenser because this could transfer AFB from one preparation to another.
13. After examining a positive smear, the oil must be wiped from the objective.

References

1. Cappuccino, J.G and Welsh C. Microbiology: a laboratory manual (2018). Pearson education limited, England. 11 edition.
2. Manandhar S, Sharma S (2006): practical approach to microbiology, Graphic plus printers, Kathmandu
3. Shah P.K, Dahal P.R, Amatya J (2009): Practical microbiology, Delta offset press, Thapathali, Kathmandu

Acid-Fast staining (Ziehl-Neelsen technique): principle, requirements and procedure

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Introduction

Staining techniques are of different types, such as simple staining, negative staining and differential staining. Gram staining is an example of differential staining techniques.

Differential staining differentiates bacteria into different groups or which can differentiate different structure cell. In differential staining, specimen is subjected to series of stains in which different organism or different parts of cell stained differently. So that they can be distinguished from each other.

For differential staining, at least four chemical reagents are required, primary stain, mordant, decolorizing agent and counter stain.

Primary stain is the first stain. Its function is to impart its color to all cells. Mordant is second stain which intensify the color of the primary stain. In order to establish a color contrast, the third reagent used is the decolorizing agent. Based on the chemical composition of cellular components, the decolorizing agent may or may not remove the primary stain from the entire cell or only from certain cell structures. The final reagent, the counterstain, has a contrasting color to that of the primary stain.

Principle:

Gram staining is the most important differential stain used in bacteriology. It was introduced by Dr. Hans Christian Gram (1884). It divides bacterial cells into two major groups, gram positive and gram negative, which makes it an essential tool for classification and differentiation of microorganisms.

The Gram stain reaction is based on the difference in the chemical composition of bacterial cell walls. Gram-positive cells have a thick peptidoglycan layer, whereas the peptidoglycan layer in gram-negative cells is much thinner and surrounded by outer lipid containing layers.

Peptidoglycan is mainly a polysaccharide composed of two chemical subunits found only in the bacterial cell wall. These subunits are N-acetylglucosamine (NAG) and N-acetylmuramic acid (NAM). With some organisms, as the adjacent layers of peptidoglycan are formed, they are cross-linked by short chains of peptides by means of a trans-peptidase enzyme, resulting in the shape and rigidity of the cell wall.

In the case of gram-negative bacteria and several of the gram-positive such as the Bacillus, the cross-linking of the peptidoglycan layer is direct because the bacteria do not have short peptide tails. Early experiments have shown that a gram-positive cell denuded of its cell wall by the action of lysozyme or penicillin will stain gram-negative.

The Gram stain uses four different reagents.

1. Primary Stain (Crystal Violet): This violet stain is used first and stains all cells purple.
2. Mordant (Gram’s iodine): This reagent serves not only as a killing agent but also as a mordant, a substance that increases the cells’ affinity for a stain. The reagent does this by binding to the primary stain, thus forming an insoluble complex. The resultant crystal-violet–iodine (CV-I) complex serves to intensify the color of the stain. At this point, all cells will appear purple-black.
3. Decolorizing Agent (Ethyl Alcohol; 95%): This reagent serves a dual function as a protein-dehydrating agent and as a lipid solvent. Its action is determined by two factors, the concentration of lipids and the thickness of the peptidoglycan layer in bacterial cell walls. In gram-negative cells, the alcohol increases the porosity of the cell wall by dissolving the lipids in the outer layers. Thus, the CV-I complex can be more easily removed from the thinner and less highly cross-linked peptidoglycan layer. Therefore, the washing-out effect of the alcohol facilitates the release of the unbound CV-I complex, leaving the cells colorless or unstained. The much thicker peptidoglycan layer in gram-positive cells is responsible for the more stringent retention of the CV-I complex, as the pores are made smaller due to the dehydrating effect of the alcohol. Thus, the tightly bound primary stain complex is difficult to remove, and the cells remain purple. Note: Be careful not to over-decolorize the smear with alcohol.
4. Counter stain (Safranin): This is the final reagent, used to stain pink those cells that have been previously decolorized. Since only gram-negative cells undergo decolorization, they may now absorb the counterstain.

Requirements:

1. Fresh culture sample: 24-hour agar culture of Staphylococcus epidermidis/ 24-hour agar culture of Bacillus subtilis/ 24-hour agar culture of Escherichia coli
2. Primary stain: crystal violet
3. Mordant: gram’s iodine
4. Decolorizing agent: alcohol (95% ethanol)
5. Counter stain: safranin
6. Bunsen burner
7. Inoculating loop
8. Microscope
9. Distilled water
10. Soft cotton or tissue paper
11. Microscopic Slides
12. China-marking pencil or permanent marking pen

Gram staining procedure.

1. Take a clean glass slide.
2. Using aseptic technique, prepare a smear of given sample organisms. Do this by placing a drop of water on the slide, and then transferring sample organism to the drop of water with a sterile, cooled loop. (Note: If bacteria are taken from a broth culture, the drop of water is not required. Place a loop of bacterial suspension directly on the glass slide.)
3. Allow smears to air-dry and then heat fix in the smear
4. Gently flood smears with crystal violet and let stand for 1 minute.
5. Gently wash with distilled water.
6. Gently flood smears with the Gram’s iodine mordant and let stand for 1 minute.
7. Gently wash with distilled water.
8. Decolorize with 95% ethyl alcohol. Note: Do not over-decolorize. Add reagent drop by drop until the alcohol runs almost clear, showing only a blue tinge.
9. Gently wash with distilled water.
10. Counterstain with safranin for 45 seconds.
11. Gently wash with distilled water.
12. Let the slide for air dry
13. Examine under 10X, 40X and under oil immersion.
14. As you observe each slide under oil immersion, record your observation in record file.
15. Draw a representative microscopic field.
16. Describe the cells according to their morphology and arrangement.
17. Describe the color of the stained cells.
18. Classify the organism as to the Gram reaction: gram positive or gram negative.

References

1. Cappuccino, J.G and Welsh C. Microbiology: a laboratory manual (2018). Pearson education limited, England. 11 edition.
2. Manandhar S, Sharma S (2006): practical approach to microbiology, Graphic plus printers, Kathmandu
3. Shah P.K, Dahal P.R, Amatya J (2009): Practical microbiology, Delta offset press, Thapathali, Kathmandu

Gram staining technique

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Introduction

It is very difficult to observe microorganisms by our naked eyes because they are very minute and transparent as well as colorless when they are suspended in aqueous medium. The refreactive index of microorganism is not very different from the medium in which they grow, due to which they cannot be observed in unstained preparation. Staining helps to observe the organism clearly.

Chemically a stain (dye) can be defined as an organic compound containing an aromatic compound ie, benzene ring, chromphore and auxochrome group. According to the nature of stain, they are classified into three groups.

1. Acidic dye: those dyes which ionizes to give anionic chromogen portion and has a strong affinity towards positively charged constituents of the cell wall are called acidic dyes. Eg. eosin, picric acid, india ink etc
2. Basic dye: those dyes which ionizes to give cationic chromogen portion and therefore has strong affinity for negatively charged constituents of the cell wall are called basic dyes. Eg. Crystal violet, methylene blue
3. Neutral dye: they are formed by the suitable mixing of two types of dyes, so they contain both cationic and anionic chromogens. They produce precipitate with the cellular components. They are used to stain nucleic acids and cytoplasm. Eg. Sudan IV, eosinate of methylene blue.

Principle                                                                                     

Simple staining technique uses a single stain to visualize the bacteria, which produces a distinctive contrast between the organism and its background. Basic stains (such as methylene blue, crystal violet, and carbol fuchsin) with a positively charged chromogen are preferred in simple staining because bacterial nucleic acids and certain cell wall components carry a negative charge that strongly attracts and binds to the cationic chromogen. They stains the microbial cell, not the background.

The purpose of simple staining is for visualization of morphological shape, size, and arrangement of microbial cells.

Requirements:

1. Fresh culture sample: 24-hour agar culture of Staphylococcus epidermidis/ 24-hour agar culture of Bacillus subtilis/ 24-hour agar culture of Escherichia coli
2. Stains (Methylene blue or Safranin or crystal violet)
3. Bunsen burner
4. Inoculating loop
5. Microscope
6. Distilled water
7. Soft cotton or tissue paper
8. Toothpicks
9. Microscopic Slides
10. China-marking pencil or permanent marking pen

Procedure

1. Take a clean, grease free glass slide. In order to make the slide grease free, wash it, rinse with alcohol and then clean with filter paper to make it dry.
2. Prepare separate bacterial smears of the given sample organism.
3. Smears must be heat fixed prior to staining.
4. Place a slide on the staining tray and flood the smear with one of the indicated stains, using the appropriate exposure time for each: carbol fuchsin, 15 to 30 seconds; crystal violet, 20 to 60 seconds; methylene blue 1 to 2 minutes.
5. Gently wash the smear with distilled water to remove excess stain. During this step, hold the slide parallel to the stream of water; in this way you can reduce the loss of organisms from the preparation.
6. Using bibulous paper, blot dry, but do not wipe the slide. Or let it for air dry sometime
7. Examine the stained slides under 10X, 40X and then under oil immersion respectively.
8. Observe the morphology of the organisms with reference to their shapes (bacilli, cocci, spirilla) and arrangements (chains, clusters, pairs).
9. Record your observation in record file.

References

1. Cappuccino, J.G and Welsh C. Microbiology: a laboratory manual (2018). Pearson education limited, England. 11 edition.
2. Manandhar S, Sharma S (2006): practical approach to microbiology, Graphic plus printers, Kathmandu
3. Shah P.K, Dahal P.R, Amatya J (2009): Practical microbiology, Delta offset press, Thapathali, Kathmandu.

Simple staining technique

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