Staining: Introduction, Types, Gram stain, AFB stain, Principle, Procedure, Result Interpretation

Staining is a method used to enhance contrast in specimens,  normally  at the microscopic level. Stains and dyes are frequently used in histology and in the medical fields of microbiology  to stain microorganisms like bacteria, fungus, parasites etc

Introduction of Staining

Staining is a method used to enhance contrast in specimens,  normally at the microscopic level. Stains and dyes are frequently used in histology and in the medical fields of microbiology to stain microorganisms like bacteria, fungus, parasites, etc., histopathology, hematology, and cytopathology that focus on the study and diagnoses disease at a microscopic level. Stains may be used to define biological tissues, cell populations e.g. blood cells, or cell organelles within individual cells. Similarly in biochemistry, it involves adding a class-specific like DNA, proteins, lipids, carbohydrates dye to a substrate to qualify or quantify the presence of a specific compound. Staining and fluorescent tagging is also useful that can serve similar purposes. Biological staining is also used to mark cells in flow cytometry, and to flag proteins or nucleic acids in gel electrophoresis but here we concern with the field of medical or clinical microbiology.

❖Stain: to leave a mark on something that is difficult to remove


❖Dye: A dye is a colored substance that has an affinity
to the substrate to which it is being applied.

❖Staining: Staining is an auxiliary technique used in
microscopy to enhance contrast in the microscopic
image

.

Types of  staining 

  1. Simple Staining
  2. Gram’s Staining
  3. Acid Fast Staining
  4. Albert’s Staining
  5. Capsule Staining
  6. Spore Staining
  7. Negative Staining

Simple Staining

Simple staining employs staining of bacterial smear with a single staining reagent. The commonly used simple stains are the basic stains such as methylene blue, crystal violet, and carbol fuchsin. They provide good color contrast and impart the same color to the stained organisms. Example of simple staining -WBCs stained by New methylene blue as shown below

Pus cells in supravital stain ( New methylene blue)

Pus cells, RBCs, and C L crystals in the pleural fluid under the microscope ( without any stain)

Gram’s Staining

It is a differential stain and thus used to differentiate Gram-positive and Gram-negative bacteria. It was originally devised by a Danish bacteriologist, Hans Christian Joachim Gram (1884) as a method of staining bacteria.

Principle of Gram stain

The reaction is dependent on the permeability of the bacterial cell wall and cytoplasmic membrane, to the dye–iodine complex. In Gram-positive bacteria, the crystal violet dye iodine complex combines to form a larger molecule which precipitates within the cell. Also, the alcohol /acetone mixture which acts as a decolorizing agent causes dehydration of the multi-layered peptidoglycan of the cell wall. This causes a decrease in the space between the molecules causing the cell wall to trap the crystal violet iodine complex within the cell. Hence the Gram-positive bacteria do not get decolorized and retain primary dye appearing violet. Also, Gram-positive bacteria have more acidic protoplasm and hence bind to the basic dye more firmly. In the case of Gram-negative bacteria, the alcohol, being a lipid solvent, dissolves the outer lipopolysaccharide membrane of the cell wall and also damages the cytoplasmic membrane to which the peptidoglycan is attached. As a result, the dye-iodine complex is not retained within the cell and permeates out of it during the process of decolonization. Hence when a counterstain is added, they take up the color of the stain and appear pink.

Requirements for Gram stain

  1. Compound light microscope
  2.  Reagents and glasswares
    Bunsen flame
    Wire loop
    Clean grease-free slides
    Marker pen
    Crystal violet (Basic dye)
    Gram’s iodine(mordant)
    95% ethanol (decolorizing agent)
    1% safranine or dilute carbol fuchsin or neutral
    red
  3. Specimen

Preparation of bacterial smear: from liquid culture

  1. Take a clean, and grease-free slide for making a smear.
  2. Take one or two loopful of the bacterial cell suspension and place them on the slide with a bacteriological loop.
  3. Then with a circular movement of the loop, spread the cell suspension into a thin area.
  4. Allow the smear to air dry.
  5. Heat fix the smear while holding the slide at one end, and by quickly passing the smear over the flame of the Bunsen burner two to three times.

Preparation of bacterial smear: from the solid medium

  • Take a clean, and grease-free slide for making a smear.
  • Take a loopful of 0.85% saline i. e. physiological saline and place it on the Center of the slide.
  • With a straight wire touch the surface of a well-isolated colony from the solid media and emulsify in the saline drop forming a thin film.
  • Allow the smear to air dry.
  • Heat fix the smear while holding the slide at one end, and by quickly passing the smear over the flame of the Bunsen burner two to three times.

Procedure

  1. Cover the smear with crystal violet and allow it to stand for
    one minute.
  2. Rinse the smear gently under tap water.
  3. Cover the smear with Gram’s iodine and allow it to stand for
    one minute.
  4.  Rinse smear again gently under tap water.
  5. Decolorize the smear with 95% alcohol.
  6. Rinse the smear again gently under tap water.
  7. Cover the smear again gently with safranine for one minute.
  8. Rinse the smear again gently under tap water and air dry it.
  9. Observe the smear first under the low power (10 X) objective, and then under the oil immersion (100X) objective.

Escherichia coli Gram-stained smear under a microscope
Rod-shaped
pink in color
that’s why Gram-negative Bacilli

Bacillus species growth on CLED agar and gram stain as shown below-

Gram-positive cocci in pairs, short chains, and long chains under a microscope

Gram-positive cocci under microscope | Gram-positive bacteria | GPC in pair, chain, and cluster possible organism (Staphylococcus aureus) as shown below-

Yeast cell identification-
Colonial morphology
on blood agar from urine specimen

Specimen: High Vaginal Swab (HVS)
Heavy growth of Streptobacillus was seen on blood agar from which Gram stain performed and finally result shown in the video.

Diphtheroids | growth on blood agar | gram stain

Urethral discharge /Gram’s negative diplococci/ Neisseria gonorrhoeae /Gonococcus in Gram stain as shown below-

Growth of Candida albicans on  SDA, Gram stain and its germ tube test (GTT) positive as shown below-

A patient was 53 years old with Chronic Otitis Media (COM) having a failure of antibacterial drugs- Pus swab from ear discharge sent to microbiology section for Gram staining- result found – Fungal spores with plenty of pus cells and lacking bacteria as shown in video-

Haemophilus Influenzae in Gram stain is Gram-negative coccobacillus or short bacilli to a long thread-like and pleomorphic forms as shown below-

Pus under the microscope showing Gram-positive cocci in singles, pairs, and clusters।। Staphylococcus as shown below-

Ziehl- Neelsen /AFB Staining

Principle

Presence of higher alcohol, glycerol, fatty acid, and especially mycolic acid
in the cell wall have been found responsible for keeping the acid-fast
property of bacteria.

Requirements

  • Compound light microscope
  • Reagents and glasswares
    Bunsen flame
    Wire loop
    Clean grease-free slides
    Marker pen
    Sprit lamp
    Carbol fuchsin
    20% Sulphuric acid
    Methylene blue
  • Specimen

Procedure

  1.  Make smear on a clean glass slide.
  2. Dry and fix the smear.
  3. Cover the smear with a strong carbol fuchsin solution.
  4. The heat from underneath the slide until just steam comes from the stain. Do not boil.
  5. Wait for five minutes.
  6. Rinse with water.
  7. Decolorize by 20% Sulphuric acid or 3% acid alcohol until the smear becomes pale pink in color. (wait for nearly five minutes)
  8. Rinse with water.
  9. Counterstain with methylene blue for one minute.
  10. Rinse with water.
  11. Drain and dry.
  12. Observe the smear first under low power (10 X) objective, and
    then under oil immersion (100 X) objective.

Acid-fast staining | AFB stain | Z -N staining a fully practical microbiology | AFB positive

Observation of Acid Fast Bacilli(AFB) /Z- N stained slide under the microscope showing pink, beaded, thin slender rod with sometimes curved having size about 1 -8 x 0.2 -0 .6 µm i.e Mycobacterium tuberculosis as shown below-

Acid alcohol Fast stained slide of Mycobacterium tuberculosis on counter stain methylene blue showing pink, beaded, thin slender rod with sometimes curved having size about 1 -8 x 0.2 -0 .6 µm as shown below-

Auramine phenol stain/ Mycobacterium/ positive/ Fluorescence microscope as shown below-

Modified Ziehl-Neelsen stain-

Modified Ziehl-Neelsen for Leprosy causative agent Mycobacterium leprae as shown below-

Mycobacterium leprae on microscope/cold Ziehl-Nelsen stain/AFB Positive:
Red solid bacilli, some are beaded forms, occurring singly also in masses i.e. globi and thus grade is  6+ because more than 1,000 bacilli, clumps and globi in every field as shown below-

Various stages of Cyclospora cayetanensis in Modified Ziehl-Neelsen stained smear as shown below-

Nocardia asteroides under acid-fast stained slide

Kinyoun’s stained slide 0f Cryptospoidium parvum under the Microscope

Albert’s Staining

It is used to stain Corynebacterium diphtheriae causative agent of diphtheria.

Capsule Staining

It is used to stain capsules of organisms like the fungus Cryptococcus neoeormans and bacteria Streptococcus pneumoniae , Klebsiella pneumoniae, etc.

Spore staining

It is used to stain spores.

Negative staining

Negative staining is so-called because the background gets stained and the organism remains colorless. e.g.  India ink, nigrosin, or eosin are the stains used.

Here below. you can see capsules of Cryptococcus neoformans in nigrosin preparation.

Staining techniques

Cryptococcus capsules in Nigrosin preparation of CSF as shown below-

Encapsulated Klebsiella pneumoniae from drain sample under India ink preparation as shown in video-

CSF I Cryptococcus India ink Positive I KOH I Nigrosin I  LPCB I Gram stain I Giemsa  I chlorazole black E as shown below-

Further Reading

  1. Bailey & Scott’s Diagnostic Microbiology. Editors: Bettey A. Forbes, Daniel F. Sahm & Alice S. Weissfeld, 12th ed 2007, Publisher Elsevier.
  2. Bailey & Scott’s Diagnostic Microbiology. Editors: Bettey A. Forbes, Daniel F. Sahm & Alice S. Weissfeld, 12th ed 2007, Publisher Elsevier.
  3. Clinical Microbiology Procedure Handbook Vol. I & II, Chief in editor H.D. Isenberg, Albert Einstein College of Medicine, New York, Publisher ASM (American Society for Microbiology), Washington DC.
  4. Colour Atlas and Textbook of Diagnostic Microbiology. Editors: Koneman E.W., Allen D.D., Dowell V.R. Jr, and Sommers H.M.
  5. Cowan & Steel’s Manual for identification of Medical Bacteria. Editors: G.I. Barron & R.K. Felthani, 3rd ed 1993, Publisher Cambridge University Press.
  6. Jawetz, Melnick and Adelberg’s Medical Microbiology. Editors: Geo. F. Brook, Janet S. Butel & Stephen A. Morse, 21st ed 1998, Publisher Appleton & Lance, Co Stamford Connecticut.
  7. Mackie and Mc Cartney Practical Medical Microbiology. Editors: J.G. Colle, A.G. Fraser, B.P. Marmion, A. Simmous, 4th ed, Publisher Churchill Living Stone, New York, Melborne, Sans Franscisco 1996.
  8.  Manual of Clinical Microbiology. Editors: P.R. Murray, E. J. Baron, M. A. Pfaller, F. C. Tenover and R. H. Yolken, 7th ed 2005, Publisher ASM, USA
  9.  Textbook of Diagnostic Microbiology. Editors: Connie R. Mahon, Donald G. Lehman & George Manuselis, 3rd edition2007, Publisher Elsevier.
  10. Topley & Wilsons Principle of Bacteriology, Virology, and immunology Vol I, II, III, IV & V. Editors: M.T. Parker & L.H. Collier, 8th ed 1990, Publisher Edward Arnold publication, London.
  11. Medical Microbiology-The Practice of Medical Microbiology Vol-2-12th Edn. –Robert Cruickshank
  12. District Laboratory Practice in  Tropical Countries  –  Part-2-   Monica Cheesebrough-   2nd Edn Update

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