Albert stain Introduction

Different stains have been developed over time to distinguish bacteria species, separating them morphologically and the specific characteristics they possess. The most popular stain is Gram staining, acid-fast staining, and endospore staining. Each stain aims at identifying and defining bacteria according to their forms and morphologies.

Albert stain is not different. It is used to detect bacteria with special structures called metachromatic Granules. Other staining techniques employed to determine that there are granules inside the cytoplasmic membrane of bacteria include Pugh’s stain as well as Nessers’s. Albert stain clearly identifies metachromatic granules found in the Corynebacterium Diphtheriae.

Corynebacteria are Gram-positive, non-spore-forming, non-motile bacteria that have metachromatic (Volutin) Granules that constitute intracellular inclusion bodies that are found in the cytoplasmic layer of some bacterial cells to allow storage of inorganic polyphosphate (poly-P) as well as enzymes. When these granules undergo to staining using Methylene blue dye, they show a reddish-purple hue and not blue.

The most well-known Corynebacterium can be described as Corynebacterium diphtheriae. It is responsible for diphtheria which is a nasopharyngeal disease (affecting the throat, nose, and nasal) that may also affect the skin following the bacterial colonization process and infection.

The way it works is that the bacteria are first cultivated in specific media, either Loeffler agar or Mueller-Miller agar and Tinsdale tellurite agar. Then, the colony is isolated to create liquid cultures that are later used to stain. Albert stain acts only as an affirmative stain for the bacteria. As a differential stain it only stain the granules of volutin, so bacteria that do not have these granules will not be identified or stained by this method.

Objective of Albert stain 

• To detect and color metachromatic granules of Corynebacterium diphtheriae.

Principle of Albert stain 

Albert staining is a method of finding metachromatic granulated bodies in Corynebacterium diphtheriae. Albert stain is comprised of two staining solutions; such as Albert Solution 1 and Albert Solution 2. Albert Solution 1 is made up of malachite blue, blue toluidine green, glacial acetic acids, and alcohol. Albert Solution 2 is made up of Iodine as well as Potassium Iodide found in water.

To make use of Albert’s staining solution both solutions needs to be made with the appropriate percentages of ingredients to show the granules in the proper hue after staining.

Albert staining solution 1 functions as the staining agent, while Albert solution 2 functions as mordant i.e an ion element that bonds and holds the chemical dye, in order so that it is able to stick to the microorganism.

Toluidine blue”O” and malachite-green both of which are fundamental dyes with high affinity for tissues with acidic components such as the cytoplasm. Albert stain’s pH can be adjusted to 2.8 through the use of acetic acid. Which is then converted to a base for volutin granules since the pH of volutingranules is very acidic.

When applying Albert’s stain on the smear, the toluidine blue’ O’ stains volutin particles. are the most acidic cell part and malachite-green stains the cell’s cytoplasm blue-green. In the event of adding Albert’s Iodine because of the iodine’s effect, the metachromatic effect is not evident and the granules appear blue-colored.

Composition of Albert Stain

Albert’s 1 solution

Ingredients	Amount
Toludine blue	0.15 gm
Malachite green	0.20 gm
Glacial acetic acid	1 ml
Alcohol (95% ethanol)	2 ml

Albert’s solution 2

Ingredients	Amount
Iodine	2 gm
Potassium iodide (KI)	3 gm

Preparation of Albert Stain

Preparation of 100ml Albert stain 1

1. Add 0.1ml of glacial acetic acids in 100ml of water. 
2. Incorporate 2ml of ethanol that is 95% into the solution.
3. Then dissolve 0.15g of blue toluidine into the solution.
4. Then take a moment to dissolve 0.2g of green malachite into the solution

Preparation of 300ml of Albert stain 2

1. Dissolve 2g of Iodine in 50ml of distillate water
2. Add 250ml water to the solution.
3. Dissolve 3g Potassium Iodide into the solution.

Procedure for Albert Stain

The Albert staining process is divided into two phases, such as;

A. Staining:

1. Aseptically, collect one of the loops of Corneybacterium diphtheriae
2. Create a smear in the centre of clear glass slide.
3. Apply heat to fix the smear slowly.
4. Take a staining rack and place the smear glass on it. 
5. Add the staining solution of Albert 1 to the smear, and let it sit for 3-5 mins.
6. The slide should be cleaned by gently squeezing tap water.

B. Mordanting

1. Add the staining solution 2 and allow it to sit for 1 minute.
2. The slide can be cleaned with gentle running tap water.
3. Blot dry to dry the smeared glass slide.
4. Apply cedarwood oil to the Smear.
5. Then, observe under a microscope using the immersion of oil at 1,000x.

Result and Interpretation of Albert Stain

Metachromatic granules stain blueish black, whereas the remaining cells of the microbiome stain green.

The cytoplasmic membrane of the Corynebacterium diphtheriae contains the volutin granules known as metachromatic, which are distinctive features of this bacterium. The staining process, which is based on Albert solutions, stain the granules, resulting in them appearing as black dots of a round shape at the bottom of an L-shaped or V-shaped green Bacilli.

Applications of the Albert Stain

• It is commonly used to detect metachromatic granules that are found in micro-organisms responsible for causing disease such as Corynebacterium diphtheriae
• As a differential stain, it assists in distinguishing Corneybacterium diphtheriae from nonpathogenic diphtheroids without metachromatic granules.

Limitations of Albert Staining

• It is used to stain metachromatic granular body and not to detect inclusions in the cytoplasmic membrane.

FAQ on Albert Stain

References

• https://www.microrao.com/alberts_staining.htm
• https://www.pro-lab.co.uk/wp-content/uploads/2021/01/Alberts-Stain-IfU-2020-10.pdf
• https://www.yashodahospitals.com/diagnostics/alberts-stain-test/
• https://www.researchgate.net/publication/334986058_Albert’s_Staining
• https://www.microxpress.in/uploads/product/albert%E2%80%99s-stain-kit_technicaldetails_75520220412.054131.pdf