Metachromatic Granule Staining

Metachromatic Granule Staining

Aim: To stain the metachromatic granules by Albert’s staining method

Approach:

        Metachromatic granules were first discovered in Spirillum volutans, so the name volutin granules. They are also called Babes-Ernst granules, after their inventors and metachromatic granules, because they show metachromatic effect. (Gr. Meta = change; chroma = color) appear red when stained with blue dyes such as Methylene blue or Toludine blue. Chemically, volutin granules are polyphosphates and strongly basophilic demonstrated by using special staining techniques such as, Albert's method. By electron microscopy they appear as electron dense bodies.

       Starvation of the cells for any nutrient leads to volutin formation. Sulphate starvation is particularly effective and leads to a rapid and massive accumulation of polyphosphate.
Volutin granules have been observed in a wide variety of bacteria and blue green algae. The bacteria include species of Lactobacillus, Corynebacterium, Spirillum, Pseudomonas, and Desulfovibrio.

        The formation of polyphosphate occurs by the sequential addition of phosphate residues to pyrophosphate, ATP serves as phosphate donor.

       Metachromatic granules have a strong affinity towards basic dye, Toludine blue, Methylene blue. When basic dye is used to stain the organism that contain the metachromatic granules are stained much more intensely (dark) than the other part of the cell. The cell typically looks like a string of beads because of more deeply stained granules. In some cells granules occur at the end of the cell resulting in bipolar staining appearance.

Principle:
Albert staining is a type of differential stain used for staining the volutin granules also known as Metachromatic granules or food granules found in Corynebacterium diphtheriae.
Albert stain is basically made up of two stains that is Toluidine blue ‘O’ or Methylene Blue and Malachite green both of which are basic dyes with high affinity for acidic tissue components like cytoplasm. The pH of Albert stain is adjusted to 2.8 by using acetic acid which becomes basic for volutin granules as pH of volutin Granule is highly acidic.
Therefore, on applying Albert’s stain to the smear, Toluidine blue ‘O’ or Methylene Blue, stains Volutin Granules i. e the most acidic part of cell and Malachite green stains the cytoplasm blue-green. On adding Albert’s iodine due to effect of iodine, the metachromatic property is not observed and granules appear blue in colour.

Requirements:

1.      Bacterial Culture or curd sample

2.      Albert’s stain

Solution A

    Methylene blue / Toludine blue        -       0.15 gm

    Malachite green                            -       0.2 gm

    95 % ethanol                                -       2 ml

Solution B

    Glacial acetic acid                         -       1 ml

    Distilled water                            -       100 ml

Mix solution A & B

 

3.      Albert’s iodine

        Iodine                                        -       2 gm

        Potassium iodide                           -       3 gm

        Distilled water                             -       300 ml

Procedure:                                 

1. Prepare the smear of the given bacterial suspension or curd sample, air dry and heat fix it.

2. Treat the smear with xylene fir 30 second to 1 minute, if curd sample is used.

3. Discard the xylene & cover the smear with Albert’s stain for 5 - 7 minutes.

4. Drain of the excess stain do not water wash the slide with water.

5. Cover the smear with Albert’s iodine for 1 to 2 minutes.

6. Wash smear with tap water, air dry it & observe under the oil immersion objective.

Observation table:

Result:

        By Albert’s method, if Corynebacterium diphtheria is present in the sample it appears green colored rod-shaped bacteria arranged at angle to each other, resembling English letter ‘L’, ‘V’ or Chinese letter pattern along with bluish black metachromatic granules at the poles.

Conclusion:

        Metachromatic granules are reserved food materials. They are highly acidic than the cytoplasm. Due to this character, the metachromatic granules get stained with Toludine blue or M. B. present in the Albert’s stain which is basic in nature. The cytoplasm is less acidic so it gets stained by malachite green. The Albert’s iodine acts as mordant. The acetic acid acts as fixative.