Medical Analysis

Introduction – Albert’s Staining Procedure and Metachromatic Granules Principle

Albert’s staining is used to demonstrate metachromatic granules found in the bacteria Corynebacterium diphtheriae [4, 6]. The granules appear purple-black when exposed to Albert’s stain and against the light green cytoplasm [5]. Albert stain only acts to stain metachromatic granules and no other granules in the bacteria [9]. The test utilizes the property of metachromasia exhibited by the granules i.e. the granules appear a colour different from the colour used for staining [1]. The volutin granules are highly acidic in nature and in comparison, the cytoplasm is almost neutral [11]. When Albert’s A solution is applied, toluidine blue stains the volutin granules i.e the most acidic part of the cell and they appear red in colour due to their metachromatic property, while Malachite green stains the cytoplasm [7]. Therefore, the bacilli appears green in colour [8]. When Alberts solution B is applied, it suppresses the metachromatic property of the granules and the granules appear blue – black in colour [10].

Oropharyngeal Swab and Throat Swab Collection Protocol

Oropharyngeal swab / throat swab collection requires specific steps to ensure an accurate sample [15]. Have the patient open the mouth and relax the tongue by saying “aaaah.” Press the tongue down using a tongue depressor. Gently rub the swab against both tonsils and the posterior pharynx. Place the swab in the culture medium, transport medium, or sterile test tube [12].

Procedure and Preparation of Albert’s Stains Reagents

ReagentsConstituentsAlbert’s A SolutionInto 100ml of water, add 0.1ml of glacial acetic acid. Add 2ml of 95% ethanol into the solution. Then, dissolve 0.15g of toluidine blue into the solution. Lastly, dissolve 0.2g of malachite green into the solution [1]. Albert’s B SolutionIodine crystals 2.0 g Potassium iodide 3.0 g Distilled water 300 mL Formula adjusted, standardized to suit performance parameters [4]. Albert’s Stain B: Dark amber coloured solution. Store at 15-30°C in tightly closed container and away from bright light [5]. Place the slide on the staining rack with smear facing upwards. Cover the smear with Albert’s A solution for 5 minutes. Pour off the stain and wash the slide with few drops of Albert’s B solution (DO NOT wash with tap water). Cover the smear with Albert’s B solution for 1 minute [6]. Pour off the stain and wash the slide with tap water. Dry the slide using blotting paper or allow it to air dry. Focus under oil immersion objective [7].

Microscopic Interpretation and Cellular Characteristics

Green coloured, rod shaped bacteria arranged at angles to each other, resembling English letter ‘L’, ‘V’ or Chinese letter pattern along with bluish black metachromatic granules at either poles are seen [13].

Applications of Albert’s Staining in Medical Microbiology

Used in presumptive diagnosis of Diphtheria [14]. It helps to distinguish C. diphtheriae from most of the non-pathogenic diphtheroids (other Corynebacterium spp) as they lack these granules [12]. Only used for staining metachromatic bodies [9]. Cannot be used to stain other inclusions in the bacteria [10].

Other Similar Staining Techniques in Diagnostic Pathology

StainingProcedurePugh’s StainStaining techniques utilized in parallel for specialized microbiological observations [3]. Neisser’s StainStain the smear with Neisser’s methylene blue for 3 minutes. Wash off with dilute iodine and leave some of this solution on the slide for 1 minute. Wash in water [2]. The Neisser stain is a microbiological stain for visualizing the polar bodies in the cytoplasm of gram-positive bacteria [4]. This staining is used to identify diphtheria bacteria [6]. Cells are stained with methylene blue and crystal violet in acidic solution and then counterstained with chrysoidin solution [7].

Clinical Applications and Public Health Significance

Rapid demonstration of C. diphtheriae in throat swabs [15]. Useful in early presumptive diagnosis of diphtheria [13]. The stain helps differentiate C. diphtheriae from other nonpathogenic bacteria, making it a valuable frontline diagnostic tool in public health and laboratory settings [14].

For Non-Medicos

Understanding Diphtheria Testing and Albert’s Stain Basics

Albert’s stain is a special laboratory technique used by doctors to find bacteria called Corynebacterium diphtheriae, which causes diphtheria [4]. When throat samples are stained, the main body of the bacteria turns green, while specific internal spots called granules turn a dark blue-black color, allowing technicians to identify them easily under a microscope [5].

How Throat Swab Samples Are Collected and Analyzed

To perform this test, a medical professional gently rubs a cotton swab against your tonsils and the back of your throat after asking you to say “ah” [15]. This sample is then placed on a glass slide, treated with special chemical solutions (Albert’s A and B), and examined to quickly check for signs of infection [6].

References:

1. Albert, H. (1896). A simple method of staining the diphtheria bacillus. Journal of the American Medical Association, 27(13), 643–644.

2. Neisser, M. (1888). Über die Färbung der Diphtheriebacillen. Deutsche Medizinische Wochenschrift, 14(42), 858–860.

3. Pugh, R. (1936). A modification of Albert’s stain for diphtheria bacilli. Journal of Medical Laboratory Technology, 4, 120–121.

4. Brooks, G. F., Carroll, K. C., Butel, J. S., & Morse, S. A. (2015). Jawetz, Melnick, & Adelberg’s Medical Microbiology (27th ed.). McGraw-Hill Education.

5. Cheesbrough, M. (2005). District Laboratory Practice in Tropical Countries, Part 2 (2nd ed.). Cambridge University Press.

6. Koneman, E. W., & Allen, S. D. (2006). Koneman’s Color Atlas and Textbook of Diagnostic Microbiology (6th ed.). Lippincott Williams & Wilkins.

7. Murray, P. R., Rosenthal, K. S., & Pfaller, M. A. (2020). Medical Microbiology (9th ed.). Elsevier.

8. Topley, W. W. C., & Wilson, G. S. (1998). Topley and Wilson’s Microbiology and Microbial Infections (9th ed.). Arnold.

9. MacFaddin, J. F. (2000). Biochemical Tests for Identification of Medical Bacteria (3rd ed.). Lippincott Williams & Wilkins.

10. Barrow, G. I., & Feltham, R. K. A. (1993). Cowan and Steel’s Manual for the Identification of Medical Bacteria (3rd ed.). Cambridge University Press.

11. Pappenheimer, A. M. (1952). Diphtheria toxin; a review. Symposia on the Society for General Microbiology, 2, 41–56.

12. Holmes, R. K. (2000). Biology of Corynebacterium diphtheriae. The Prokaryotes, 1–25.

13. Hadfield, T. L., McEvoy, P., Polotsky, Y., Tzinserling, V., & Yakovlev, A. (2000). The pathology of diphtheria. The Journal of Infectious Diseases, 181(Suppl 1), S116–S120. https://doi.org/10.1086/315532

14. Mattos-Guaraldi, A. L., Formiga, L. C., & Camello, T. C. (2000). Corynebacterium diphtheriae: an underestimated pathogen in systemic infections. Brazilian Journal of Medical and Biological Research, 33(6), 637–642. https://doi.org/10.1590/s0100-879×2000000600003

15. Efstratiou, A., & George, R. C. (1999). Laboratory guidelines for the diagnosis of infections caused by Corynebacterium diphtheriae and related species. World Health Organization, 1–32.

FAQ’s:

• What does Albert’s stain demonstrate?
  Metachromatic granules found in the bacteria Corynebacterium diphtheriae.

• How do the granules appear?
  They appear purple-black against a light green cytoplasm under microscopy.

• What is metachromasia property?
  Granules appear a color different from the color used for staining.

• What stains volutin granules red?
  Toluidine blue in Albert’s A solution stains the acidic volutin granules.

• How is a throat swab collected?
  Gently rub the swab against both tonsils and the posterior pharynx.

• What is Albert’s B solution?
  An iodine and potassium iodide solution stored in a dark amber container.

• How are cells arranged?
  Green rod-shaped bacteria resemble English letters ‘L’, ‘V’, or Chinese letters.

• What is its main application?
  Used in the presumptive diagnosis of diphtheria and differentiating non-pathogenic diphtheroids.

• What are its disadvantages?
  It is only used for staining metachromatic bodies, not other bacterial inclusions.

• What is Neisser’s stain used for?
  A microbiological stain for visualizing polar bodies in gram-positive bacteria.

Related Tests 

• Gram Stain Test