Albert stain is a type of differential stain used for staining the volutin granules also known as Metachromatic granules or food granules found in Corynebacterium diphtheriae. It is named as metachromatic because of its property of changing colour i.e when stained with blue stain they appear red in colour. When grown in Loffler’s slopes, C. diphtheriae produces large number of granules
Principle of Albert Staining:
Albert stain is basically made up of two stains that is Toluidine blue’ O’ 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’ 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.
Composition of Albert stain:Albert stain is composed of two reagents:
Albert’s A solution consist of
- Toludine blue 0.15 gm
- Malachite green 0.20 gm
- Glacial acetic acid 1 ml
- Alcohol (95% ethanol) 2ml
Dissolve the dyes in alcohol and add to the distilled water and acetic acid.
Allow the stain to stand for one day and then filter.
Add Distilled water to make the final volume 100ml
Albert’s B solution consist of
- Iodine 2gm
- Potassium iodide (KI) 3 gm
Dissolve KI in water and then add iodine. Dissolve iodine in potassium iodide solution
Requirements: Smear on glass slide, staining rack, Albert’s A solution , Albert’s B solution, blotting paper, immersion oil, microscope
- Prepare a smear on clean grease free slide.
- Air dry and heat fix the smear.
- Treat the smear with Albert’s stain and allow it to react for about 7 mins.
- Drain of the excess stain do not water wash the slide with water.
- Flood the smear with Albert’s iodine for 2 minutes.
- Wash the slide with water, air dry and observe under oil immersion lens.
If Corynebacterium diphtheria is present in the sample it appears green coloured 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.
This helps to distinguish Corynebacterium diphtheriae from most of the short nonpathogenic diphtheroides which lack granules.