ALBERT STAINING - CORYNEBACTERIUM DIPTHERIAE

ALBERT STAINING – PRINCIPLE, REQUIREMENTS, PROCEDURE & INTERPRETATION

INTRODUCTION TO ALBERT STAINING

Corynebacterium is the genus of Gram +ve, non-acid-fast, non-sporing, non-motile bacilli. The most important member of the genus is Corynebacterium diphtheriae, the causative agent of Diphtheria disease in children. The C. diptheriae or diphtheria bacillus was first described by Klebs but Loffler was the first to cultivate it in the laboratory and also called as the Klebs-Loffler’s bacillus (KLB)

They form metachromatic granules, these granules are formed by many other types of bacteria. They are made up of Polyphosphate and considered as the food reserve in bacteria. These food reserves are involved in all metabolic processes.

In sputum slide, Albert’s staining is used to differentiate the gram +ve bacteria forming metapositive chromatic granules or Volutin granules. These bacteria are present in CLUB or Chinese letter arrangement.

PRINCIPLE OF ALBERT STAINING

The bacterial cell of Corynebacterium diphtheriae & Yersinia pestis has volutin granules in their cytoplasm which are highly acidic while the cytoplasm is neutral. Albert stain I have two dyes ‘Toluidine blue O’ and ‘Malachite green’ both of which are basic dyes with high affinity for neutral tissue components like cytoplasm and the pH of Albert stain I is adjusted to 2.8 by using ‘Glacial acetic acid’, which is acidic for cytoplasm (as it is neutral) but basic for volutin granules (as the pH of volutin granules are highly acidic).

Therefore, When Albert stain I applied to the cell the volutin granules stain by Toluidine blue O while cytoplasm is green by Malachite green. Due to the metachromatic property of volutin granules when stained with Toluidine blue O dye they appear Red in color.

When Albert stains II i.e. the Iodine solution is applied due to the effect of Iodine the metachromatic property is not observed and Granules appear blue-black in color.



REQUIREMENTS FOR ALBERT STAINING

  • Glass Slides
  • Specimen/Bacterial culture
  • Tissue paper
  • Inoculating loop
  • Spirit lamp/Bunsen burner
  • Staining tray
  • Microscope
  • Wash Bottle
  • Albert’s stain A
  • Albert’s Stain B

The composition of Albert stain:

Albert’s A Solution consist of

  • Toluidine 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 then add the acetic acid to the thus obtained solution. Allow the stain to stand for one day and then filter. Add Distilled water to make the final volume 100ml.

Albert’s B Solution consists of

  • Iodine                                    2gm
  • Potassium iodide (KI)          3 gm

Dissolve the KI in water and then add iodine. Dissolve iodine in thus formed potassium iodide solution.

PROCEDURE OF ALBERT STAINING

  • Take an inoculum of bacterial culture or sputum sample with help of inoculating loop.
  • Prepare a thin smear of the specimen by mixing the cell with a drop of water / Normal saline on the glass slide.
  • Heat fix the smear onto the slide by gently passing it over the flame.
  • Flood the smear with Albert’s Stain A solution and wait for 5 min.
  • Remove the dye and flood it with Albert’s Stain B for 1 min.
  • Wash the smear with tap water / Distilled water.
  • Air dry the slide and observe under the microscope at 100X objective.

INTERPRETATION OF THE ALBERT STAINING

Metachromatic / Volutin granules appeared as a bluish-black in the green colored bacterial body arranged at an angle to each other, resembling English letter ‘L’, ‘V’ or Chinese letter pattern.



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