This staining technique is used to stain the volutin or metachromatic granules of corynebacterium diphtheriae,other corynebacterium species and occasionally Bacillus species especially when the organism is cultured on a protein rich medium such as dorset eggs,loeffler serum or Egg yolk agar which makes the volutin granule most numerous.
Principles of Albert staining technique
This stain demonstrates the presence of metachromatic granules which are found in Corynebacterium diphtheriae and other corynebacterium species.
- The granules stain purple-black against the light green counterstained cytoplasm.
- This helps to distinguish Corynebacterium diphtheriae from most of the short nonpathogenic diphtheroids species lacking volutin granules
Albert’s stain A (Composition)
|Ingredients||Grams and ml|
|Toluidine blue||0.150 gm|
|Malachite green||0.200 gm|
|Glacial acetic acid||1.000 ml|
|Ethyl alcohol, 95%||2.000 ml|
|Distilled water||100.000 ml|
Albert’s stain B (Composition)
|Iodine crystals||2.000 gm|
|(S002) Iodine crystals||3.000 gm|
|Distilled water||300.000 ml|
Material Required for Albert staining technique
- Albert’s stain A
- Albert’s stain B
- Inoculum or specimen
- Clean slide
- 70% alcohol
Procedure for Albert staining technique
- Make a thin smear of the specimen on a clean slide
- Allow to air dry
- Fix the dried smear with 70% alcohol or with gentle heat
- Cover the dried smear Albert’s Stain A for 3-5 minutes.
- Do not wash instead drain the slide
- Flood the smear with Albert’s Stain B for 1 minute
- Rinse the slide with clean water
- Blot dry the slide with tissue paper and examine starting with 40X objectives in order to check the staining and the distribution of the cells then switch to 100X oil immersion objective
Observation and interpretation
|Bacteria cells||Pale green|
|Volutin or metachromatic granules||Black|
The metachromatic granules of Diphtheria bacilli will stain black while their cytoplasm stains light green or green
- Monica cheesbrough Part 2,p44.