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Techniques in Microbiology: Staining Techniques

Source: World Health Organization.

Staining of the clinical material or the bacteria from colonies on laboratory media provide a direct visualization of the morphology of the organisms as well as their reactions to the chemicals present in stains. This is an invaluable and easy-to-use tool for establishing the identity of various microorganisms. Some of the commonly-used staining techniques are:

Methylene blue staining
Gram staining
Albert staining
Ziehl Neelsen staining (Acid fast staining)
India ink staining
Iodine staining for ova and cysts in faeces

Methylene blue staining

Ingredients and preparation

Methylene blue 0.3 gm
Distilled water 100 ml

Dissolve the dye in water. Filter through a filter paper.

Staining procedure

• Make a smear on a glass slide, dry in air and fix by passing it over the flame of a burner 3-4 times.
• Stain for one minute by pouring methylene blue solution over the smear.
• Wash with water, blot dry and examine under the oil immersion of light microscope.

Uses

The stain is used to make out clearly the morphology of the organisms e.g. Yersinia pestis in exudate, Haemophilus influenzae in CSF and gonococci in urethral pus.

Gram staining

This is the most extensively used differential stain that divides bacteria into two major groups. Those which retain crystal violet dye after treatment with iodine and alcohol appear purple or bluish purple and are designated as Gram positive. Those bacteria which lose the crystal violet show the colour of the counter stain employed. The commonly-used counter stain is saffranin which gives a pink/red colour to bacteria and these organisms are labelled as Gram negative.

Ingredients and preparation

Crystal violet

1. Solution A

Crystal violet 2.0 gm
Ethanol, 95% 20 ml

2. Solution B

Ammonium oxalate 0.8 gm
Distilled water 80 ml

Mix solutions A and B. Store for 24 hours before use.

3. Gram iodine

Iodine crystals 1.0 gm
Potassium iodide 2.0 gm
Distilled water 300 ml

Grind the dry iodine and potassium iodide in a mortar. Add water, a few ml at a time, and grind thoroughly after each addition until the iodine and iodide dissolve. Rinse the solution into an amber glass bottle with the remainder of the distilled water.

Saffranin solution

1. Stock solution

Saffranin O 2.5 gm
Ethanol, 95% 100 ml

2. Working solution

Stock solution 10 ml
Distilled water 90 ml

Staining procedure

• Make a thin smear on a clean glass slide, dry it in air and fix by passing through flame of a burner.
• Cover the smear with crystal violet, keep for one minute.
• Wash the slide with water, then cover with Gram iodine and let it stand for one minute.
• Wash the slide with water.
• Decolour with acetone/alcohol, rocking the slide gently for 10-15 seconds till the violet colour comes off the slide.
• Wash with water immediately.
• Counterstain with saffranin. Let the counterstain stand for 30 seconds.
• Wash with water, blot dry and examine under the oil immersion lens of a microscope.

Uses

Widely used in diagnostic bacteriology mainly to differentiate organisms on the basis of morphology and Gram reaction.

Albert staining

Ingredients and preparations

1. Albert stain I

Toluidine blue 0.15 gm
Malachite green 0.20 gm
Glacial acetic acid 1.0 ml
Alcohol(95%) 2.0 ml
Distilled water 100 ml

Grind and dissolve the dyes in alcohol, add water and then add acetic acid. Let the mixture stand for 24 hours and then filter.

2. Albert stain II

Iodine 2.0 gm
Potassium iodide 3.0 gm
Distilled water 300 ml

Dissolve iodine and potassium iodide in water by grinding in a mortar with a pestle. Filter through a filter paper.

Staining procedure

• Cover the heat-fixed smear with Albert stain I. Let it stand for two
• minutes.
• Wash with water.
• Cover the smear with Albert stain II. Let it stand for two minutes.
• Wash with water, blot dry and examine.

Uses

To demonstrate metachromatic granules in C. diphtheriae. These granules appear bluish black whereas the body of bacilli appear green or bluish green.

India ink staining

Staining procedure

• Place a loopful of India ink on the side of a clean slide.
• A small portion of the solid culture is suspended in saline on the slide
• near the ink and then emulsified in the drop of ink, or else, mix a loopful
• of liquid culture of specimens like CSF with the ink.
• Place a clean cover slip over the preparation avoiding air bubbles.
• Press down, or blot gently with a filter paper strip to get a thin, even film.

Use

To demonstrate the capsule which is seen as an unstained halo around the organisms distributed in a black background. This is employed for fungal diagnostics especially for Cryptococcus neoformans.

Ziehl Neelsen staining

Ingredients and preparations

Carbol fuchsin 1%
Sulphuric acid 25%
Methylene blue 0.1%

• Select a new, unscratched slide and label the slide with a Laboratory Serial number.
• Make a smear from yellow purulent portion of the sputum using a bamboo stick. A good smear is spread evenly, 2 cms x 3 cms in size and is neither too thick nor too thin. The optimum thickness of the smear can be assessed by placing the smear on a printed matter, the print should be readable through the smear.
• Let the smear air-dry for 15-30 minutes.
• Fix the smear by passing the slide over the flame 3-5 times for 3-4 seconds each time.
• Place the fixed slide on the staining rack with the smeared side facing upwards.
• Pour filtered 1% carbol fuchsin over the slide so as to cover the entire slide.
• Heat the slide underneath until vapours start rising. Do not let carbol fuchsin to boil or the slide to dry. Continue the process up to five minutes.
• Allow the slide to cool for 5-7 minutes.
• Gently rinse the slide with tap water to remove the excess carbol fuchsin stain. At this point, the smear on the slide looks red in colour.
• Decolor the stained slide by pouring 25% sulphuric acid on the slide and leaving the acid for 2-4 minutes.
• Lightly wash away the free stain. Tip the slide to drain off the water.
• If the slide is still red, reapply sulphuric acid for 1-3 minutes and rinse gently with tap water.
• Counter stain the slide by pouring 0.1% methylene blue solution onto the slide and let it stand for one minute.
• Gently rinse the slide with tap water and tip the slide to drain off the water.
• Place the slide in the slide tray and allow it to dry.
• Examine the slide under a microscope using 40 x lens to select the suitable area of the slide and examine under 100 x lens using a drop of immersion oil.

Uses

Iodine staining for ova and cysts

• On a clean glass slide place one drop of normal saline and one drop of 2% iodine solution at two different sites.
• Mix a portion of stool first with normal saline and then with iodine solution with the help of a wire loop or applicator.
• Place coverslips on both the emulsions.
• Examine the preparations under 10x and 40x of the microscope for various ova and cysts.

Quality control of stains

Test all stains at appropriate intervals for their ability to distinguish positive and negative organisms and document the results. The performance standards for Ziehl-Neelsen and Gram staining are as given below:

Stain	Control organism/ material	ATCC No*	Expected result
Ziehl-Neelsen	Mycobacterium spp. E. coli	25177 25922	Pink red bacilli Blue bacilli
Gram	E. coli S. aureus	25922 25923	Gram -ve bacilli Gram +ve cocci
Iodine solution	Formalin treated stool specimen with cysts		Visible cyst nuclei

* If no standard strains are available, known laboratory strains should be used as controls.

The quality control procedure for stains needs to be performed on a weekly basis and also as and when a new lot of reagents for staining is procured/prepared.

Further reading

1. Manual of basic techniques for a health laboratory, WHO, 1980.

2. Bailey & Scott1s Diagnostic Microbiology by Baron, Peterson and Finegold, 9th Ed, Mosby, 1994.

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