Albert Staining- Principle, Reagents, Procedure, Results, Interpretation

The objective of this article is to explain the principle, reagents, procedure, results, and interpretation of Albert staining, a differential staining technique used to stain and observe metachromatic granules from a Corynebacterium diphtheriae culture.

Corynebacterium diphtheriae is a Gram-positive, non-spore-forming, club-shaped bacterium that causes diphtheria, a serious respiratory infection that can lead to suffocation and death. One of the distinctive features of this bacterium is the presence of metachromatic granules in its cytoplasm. These granules are composed of polyphosphate and serve as energy reserves for the bacterium. They are called metachromatic because they change color when stained with certain dyes.

Albert staining is a simple and rapid method to detect these granules and differentiate Corynebacterium diphtheriae from other similar bacteria that lack them. It involves two staining solutions: Albert solution 1, which contains toluidine blue and malachite green as the primary stains, and Albert solution 2, which contains iodine and potassium iodide as the mordant. The mordant enhances the binding of the primary stains to the granules and makes them more visible under the microscope.

By using Albert staining, one can observe that the metachromatic granules appear as bluish-black dots at the poles or along the sides of the green-stained bacilli. This contrast helps to identify Corynebacterium diphtheriae and confirm its diagnosis.

In this article, we will describe how to prepare the Albert staining solutions, how to perform the staining procedure, how to interpret the results, and what are the applications and limitations of this technique. We hope that this article will be useful for students, researchers, and health professionals who are interested in learning more about Albert staining and Corynebacterium diphtheriae.

Albert`s staining technique aims at detecting the presence of metachromatic granulated bodies of Corynebacterium diphtheriae. These granules are also known as volutin granules or Babes-Ernst granules, and they are composed of polymetaphosphate, a type of inorganic phosphate. The granules are located in the cytoplasmic membrane of the bacteria and serve as energy reserves.

Albert staining technique uses two staining solutions: Albert Solution 1 and Albert Solution 2. Albert Solution 1 contains toluidine blue and malachite green as the primary stains and glacial acetic acid and alcohol as the solvents. Albert Solution 2 contains iodine and potassium iodide as the mordant, which enhances the binding of the stains to the granules.

The principle of Albert staining is based on the phenomenon of metachromasia, which means a change in the color of a dye when it binds to certain substances. Toluidine blue is a basic dye that normally stains blue, but when it binds to the metachromatic granules, it changes its color to bluish-black. Malachite green is an acidic dye that normally stains green, but when it binds to the bacterial cell wall, it changes its color to light green. Iodine acts as a mordant that fixes the toluidine blue to the granules and intensifies their color.

The result of Albert staining is that the metachromatic granules appear as round-shaped blue-black dots at the bottom of L-shaped or V-shaped green bacilli. This indicates the presence of Corynebacterium diphtheriae, which is the causative agent of diphtheria, a serious respiratory infection. Albert staining technique helps to differentiate Corynebacterium diphtheriae from other non-pathogenic diphtheroid that lack metachromatic granules.

Albert Solution 1 is the primary staining solution that contains two dyes: toluidine blue and malachite green. These dyes are mixed with glacial acetic acid and alcohol to form a solution that can stain the metachromatic granules of Corynebacterium diphtheriae.

To prepare 100 ml of Albert Solution 1, follow these steps:

• In a beaker, measure 100 ml of distilled water and add 0.1 ml of glacial acetic acid. Stir well to mix.

• In a separate container, measure 2 ml of 95% ethanol and add it to the water-acid mixture. Stir well to mix.

• In a mortar and pestle, grind 0.15 g of toluidine blue to a fine powder and transfer it to the water-acid-ethanol mixture. Stir well to dissolve the dye.

• In the same mortar and pestle, grind 0.2 g of malachite green to a fine powder and transfer it to the same mixture. Stir well to dissolve the dye.

• Filter the solution through a filter paper or a muslin cloth to remove any undissolved particles.

• Store the solution in a dark glass bottle away from light and heat.

Albert Solution 1 is ready to use for staining the smear of Corynebacterium diphtheriae culture.

Albert solution 2 is the mordant that enhances the staining of the metachromatic granules by forming a complex with the dye in Albert solution 1. The mordant consists of iodine and potassium iodide in water. To prepare 300 ml of Albert solution 2, follow these steps:

• Dissolve 2 g of iodine crystals in 50 ml of distilled water in a beaker. You may need to heat the solution gently to dissolve the iodine completely.

• Add 250 ml of distilled water to the iodine solution and mix well.

• Dissolve 3 g of potassium iodide in a small amount of water in another beaker. Potassium iodide helps to stabilize the iodine and prevent its evaporation.

• Add the potassium iodide solution to the iodine solution and stir well. The solution should turn dark brown in color.

• Transfer the Albert solution 2 to a labeled glass bottle and store it in a dark place.

Albert solution 2 is ready to use as a mordant for Albert staining. You can also buy ready-made Albert Solution 2 from some suppliers.

To perform Albert staining, you will need the following reagents:

• Albert staining Solution 1

• Albert staining solution 2

• Distilled water

You will also need the following materials:

• A loopful culture of Corynebacterium diphtheriae

• A clean, sterile glass slide

• A Bunsen burner or a spirit lamp

• A staining rack

• A dropper or a pipette

• A blotting paper or a tissue paper

• Cedarwood oil

• A microscope

The steps for Albert staining are as follows:

1. Aseptically, take a loopful culture of Corynebacterium diphtheria and make a thin smear at the center of a clean, sterile glass slide.

1. Heat fix the smear by passing it over the flame of a Bunsen burner or a spirit lamp three times. Do not overheat the smear, as it may damage the cells.

1. Place the smeared glass slide on a staining rack and cover the smear with Albert staining Solution 1. Leave it for 3 to 5 minutes. This is the staining step, where the metachromatic granules and the cell wall are stained with different colors.

1. Wash the smeared slide with gently flowing tap water or distilled water to remove excess stain. Do not wash for too long, as it may decolorize the smear.

1. Cover the smear with Albert staining solution 2 and leave it for 1 minute. This is the mordanting step, where the stain is fixed on the metachromatic granules by forming an insoluble complex.

1. Wash the slide again with gently flowing tap water or distilled water to remove excess mordant.

1. Blot to dry the smeared glass slide with blotting paper or tissue paper. Do not rub the smear, as it may damage the cells.

1. Add a drop of cedarwood oil to the smear and place a coverslip over it. This is the mounting step, where the smear is prepared for observation under a microscope.

1. Observe the smear under a microscope by oil immersion at 1000x magnification. You should see round-shaped blue-black dots at the bottom of L-shaped or V-shaped green bacilli. These are the metachromatic granules of Corynebacterium diphtheriae.

The result of Albert staining can be observed under a microscope by oil immersion at 1000x magnification. The metachromatic granules stain bluish black while the rest of the microbial cell stains green. This is because the toluidine blue in Albert solution 1 binds to the phosphate groups in the granules and forms a complex that turns dark blue or black when exposed to iodine in Albert solution 2. The malachite green in Albert solution 1 stain the cytoplasm and cell wall of the bacteria green.

The image below shows an example of Albert`s staining results for Corynebacterium diphtheriae:

The appearance of round-shaped blue-black dots at the bottom of L-shaped or V-shaped green bacilli indicates the presence of metachromatic granules in Corynebacterium diphtheriae. These granules are also known as volutin granules or Babes-Ernst granules and are composed of polyphosphate, which is a storage material for energy and phosphorus in some bacteria. The metachromatic granules are a characteristic feature of Corynebacterium diphtheriae and help to differentiate it from other non-pathogenic diphtheroid that lack these granules.

Albert`s staining technique is used to detect the presence of metachromatic granules in the cytoplasm of Corynebacterium diphtheriae. Metachromatic granules are also known as volutin granules or Babes-Ernst granules. They are composed of polyphosphate, a polymer of inorganic phosphate that serves as a reserve of energy and phosphate for the cell. Metachromatic granules exhibit a phenomenon called metachromasia, which means that they stain a different color from the dye used. In Albert staining, metachromatic granules stain bluish black when exposed to toluidine blue and iodine, while the rest of the bacterial cell stains green.

The appearance of round-shaped blue-black dots at the bottom of L-shaped or V-shaped green bacilli indicates the presence of metachromatic granules in Corynebacterium diphtheriae. This is a characteristic feature of this bacterium, which causes diphtheria, a serious infection of the respiratory tract and other organs. Metachromatic granules can also be found in some other bacteria, yeasts, fungi, and protozoa, but they are not as prominent or abundant as in Corynebacterium diphtheriae. Therefore, Albert staining can help to identify and differentiate Corynebacterium diphtheriae from other microorganisms that lack or have fewer metachromatic granules.

Albert staining is a useful technique for identifying and differentiating metachromatic granules in certain microorganisms. Metachromatic granules are intracytoplasmic storage forms of complexed inorganic polyphosphate, which appear as round-shaped blue-black dots when stained with Albert solutions. These granules are characteristic features of some bacteria, yeasts, fungi, and protozoa, and they may have various functions such as energy and phosphate reserve, stress response, and virulence factor.

One of the main applications of Albert staining is to detect the presence of metachromatic granules in Corynebacterium diphtheriae, the causative agent of diphtheria. Diphtheria is a serious infection of the respiratory tract or skin, which can lead to severe complications such as myocarditis, neuritis, and death. Albert staining can help to distinguish C. diphtheriae from other non-pathogenic diphtheroid that lack metachromatic granules. This can aid in the diagnosis and treatment of diphtheria, especially in resource-limited settings where more sophisticated methods may not be available.

Another application of Albert staining is to identify metachromatic granules in other disease-causing microorganisms, such as Mycobacterium tuberculosis, the causative agent of tuberculosis. Tuberculosis is a chronic infection of the lungs or other organs, which can cause cough, fever, weight loss, and death. Albert staining can reveal the presence of metachromatic granules in M. tuberculosis, which are thought to be involved in its resistance to antibiotics and survival within macrophages. This can provide insights into the pathogenesis and drug susceptibility of M. tuberculosis and help to develop new strategies for its control and eradication.

Albert staining can also be used to study the distribution and function of metachromatic granules in other microorganisms that are not necessarily pathogenic, such as Saccharomyces cerevisiae, a common yeast used for baking and brewing. S. cerevisiae contains metachromatic granules that are influenced by the age and condition of the culture. Albert staining can help to visualize these granules and investigate their role in the metabolism and stress response of S. cerevisiae. This can enhance our understanding of the physiology and ecology of this important microorganism.

In summary, Albert staining is a simple and effective technique for detecting metachromatic granules in various microorganisms. It has several applications in microbiology, such as identifying and differentiating pathogenic bacteria, studying their virulence and resistance mechanisms, and exploring their metabolic and environmental adaptations.

: Diphtheria - Symptoms and causes - Mayo Clinic

: Diphtheria: Diagnosis | CDC

: Tuberculosis (TB) - Symptoms and causes - Mayo Clinic

: Metachromasia in Mycobacterium tuberculosis: A novel method for ... (http://nih.gov/)

: Tuberculosis: Diagnosis | CDC

: Formation of volutin granules in Corynebacterium glutamicum (http://nih.gov/)

• Albert staining is a specific technique that can only be used to stain the metachromatic granular bodies and not any other inclusions in the cytoplasmic membrane. Metachromatic granules are composed of polyphosphate molecules that have a high affinity for basic dyes like toluidine blue and malachite green. Other cytoplasmic inclusions, such as glycogen, lipid droplets, sulfur granules, or carboxysomes, may not react with these dyes and remain unstained or faintly stained.

• Albert staining is not a very sensitive or reliable method for detecting Corynebacterium diphtheriae in clinical specimens. The presence of metachromatic granules is not a definitive criterion for identifying this bacterium, as some strains may lack them or produce them in small numbers. Moreover, some other bacteria, such as Nocardia, Rhodococcus, or Mycobacterium, may also produce metachromatic granules that can be confused with those of Corynebacterium diphtheriae. Therefore, Albert`s staining should be supplemented with other tests, such as biochemical tests, culture methods, or molecular techniques, to confirm the diagnosis of diphtheria.

• Albert staining is a time-consuming and labor-intensive technique that requires careful preparation of the staining solutions and the smear. The staining solutions must be freshly prepared and filtered before use to avoid precipitation and contamination. The smear must be thin and evenly spread to avoid overlapping of cells and ensure proper staining. The staining time and temperature must be controlled to avoid over-staining or under-staining the granules. The smear must also be washed gently and dried thoroughly before observation under the microscope to prevent distortion or loss of the granules.

Albert staining is a useful technique for identifying and characterizing bacteria that contain metachromatic granules, especially Corynebacterium diphtheriae, the causative agent of diphtheria. However, like any other staining method, it also has some advantages and disadvantages that should be considered.

Some of the advantages of Albert staining are:

• It is a simple and rapid method that requires only two staining solutions and a few steps.

• It is a differential stain that can distinguish Corynebacterium diphtheriae from other non-pathogenic diphtheroid that lack metachromatic granules.

• It can detect the presence of volutin granules, which are intracellular inclusion bodies that store complexed inorganic polyphosphate (poly-P) and enzymes. These granules are important for the virulence and survival of Corynebacterium diphtheriae.

• It can visualize the characteristic morphology of Corynebacterium diphtheriae, which are L-shaped or V-shaped bacilli with round-shaped blue-black dots at the bottom.

Some of the disadvantages of Albert staining are:

• It can only be used to stain the metachromatic granular bodies and not any other inclusions in the cytoplasmic membrane.

• It can give false-positive results if the smear contains non-pathogenic corynebacterium species (diphtheroids) that also have metachromatic granules. This could lead to a presumptive diagnosis that needs to be confirmed by other methods.

• It can be affected by the quality and concentration of the staining solutions, the duration and intensity of staining and mordanting, and the thickness and fixation of the smear. These factors could influence the color and clarity of the stain.

Therefore, Albert staining is a valuable technique for demonstrating metachromatic granules in Corynebacterium diphtheriae, but it also has some limitations that should be taken into account. It is advisable to use Albert staining along with other methods such as culture, biochemical tests, and molecular techniques for the definitive identification and diagnosis of diphtheria.

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