Albert Stain Principle, Procedure, Result

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

Toludine blue0.15 gm
Malachite green0.20 gm
Glacial acetic acid1 ml
Alcohol (95% ethanol)2 ml

Albert’s solution 2

Iodine2 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.

Result and Interpretation of Albert Stain

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

What is Albert stain?

Albert stain is a type of staining technique used in microbiology to identify and differentiate between gram-negative and gram-positive bacteria.

What are the steps involved in Albert stain?

The steps involved in Albert stain include heat fixation, staining with the primary stain, decolorization, staining with the counterstain, and observation under a microscope.

How does Albert stain work?

Albert stain works by using two dyes, a primary stain and a counterstain. The primary stain is used to stain the bacterial cells, while the counterstain is used to provide contrast and make the bacteria more visible under a microscope.

What is the purpose of Albert stain?

The purpose of Albert stain is to identify and differentiate between gram-negative and gram-positive bacteria based on their staining characteristics.

What are the dyes used in Albert stain?

The dyes used in Albert stain are typically crystal violet for the primary stain and safranin for the counterstain.

How does Albert stain compare to other staining techniques?

Albert stain is similar to other differential staining techniques, such as the Gram stain, but it has some key differences. It is less complex and less time-consuming compared to the Gram stain, but it is not as specific and may not provide as much information about the bacteria being studied.

What are the advantages of Albert stain?

The advantages of Albert stain include its simplicity, low cost, and ability to differentiate between gram-negative and gram-positive bacteria.

Can Albert stain be used in food and water quality control?

Yes, Albert stain can be used in food and water quality control to identify the presence of gram-negative and gram-positive bacteria, which can indicate contamination or spoilage.

What are the applications of Albert stain in bacterial research?

The applications of Albert stain in bacterial research include the study of bacterial morphology, classification, and identification, as well as the investigation of bacterial growth and metabolic processes.

What precautions should be taken when performing Albert stain?

Precautions that should be taken when performing Albert stain include wearing gloves and protective clothing, using a fume hood to prevent exposure to toxic chemicals, and properly disposing of used materials to avoid contamination.



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