Stains in Bacteriology - The Definitive Guide

Name of Stain

Diagnostic Use


Acridine Orange

Trichomonas vaginalis

Positive: Trophozoites of Trichomonas vaginalis stain brick red with green banana-shaped or rounder nucleus.

Negative: Yeasts stain red with a bright green nucleus.

Auramine Phenol Stain-1

AAFB/Mycobacterial species

Positive: Acid fast bacilli vary from 0.5 to 10 μm in length and stain bright yellow-green against a dark background.

Negative: No fluorescence observed.

Auramine Phenol Stain-2

Cryptosporidium spp.

Positive: Cryptosporidium oocysts (4 to 6 μm diameter) are ring or doughnut-shaped and fluoresce greeny-yellow (depending on the filter wavelengths) against a dark background. Putative oocysts may be measured by increasing the bright field light intensity and measuring the oocysts with a calibrated eye-piece graticule.

Negative: No fluorescence observed.

Calcofluor Stain


Positive: Spores of microsporidia are typically ovoid or piriform and fluoresce brilliant bluewhite. Dimension of spores vary by species and range from 1 to 20 μm.

Negative: No fluorescence observed.

Field’s Stain

Plasmodium spp.

Positive: Plasmodium species.

Negative: A proven negative smear may be used as the negative control.

Giemsa’s Stain

Dientamoeba fragilis and Blastocystis

hominis in faeces and Pneumocystis jirovecii in broncho-alveolar lavage.

Positive: Dientamoeba fragilis and Blastocystis hominis.

Negative: A proven negative smear may be used as the negative control.

Gram’s Stain

Gram positive vs Gram negative

Beware of over decolourisation

Lugol’s Iodine


Positive: Protozoan nuclei take up the iodine and stain pale brown while cytoplasm remains colourless.

Negative: N/A

Modified Trichome stain

Microsporidia in faeces

Positive: Spores of species of microsporidia that infect mammals including humans tend to be small, ranging in size from 1.0 to 3.0 μm X 1.5 to 4.0 μm22. They are ovoid and refractile. The spore walls stain bright pink-red. Occasionally the spores stain with a red “belt” across the centre of the spore.

Negative: No spore material observed.

Modified Cold Ziehl-Neelsen’s Stain

Cryptosporidium spp.

Positive: Cryptosporidium species are 4 to 6 μm and spherical. Oocyst staining is variable, and some oocysts may appear unstained. Internal structures may take up the stain to varying degrees. Sometimes the crescent shape of the sporozoites may be seen under high power magnification. Isospora species stain red, measure 32 x 16 μm and are elongated oval bodies tapered at both ends, containing a granular zygote or two sporoblasts. Cyclospora species oocysts stain pinkish red, are spherical 8 to 10 μm and contain a central morula. Staining is variable and some oocysts may appear unstained. The oocysts seen in faeces are usually unsporulated. Yeasts, other biota and faecal debris may also take up the stain.

Negative: Parasite not detected.

India Ink Preparation

Cryptococcus neoformans

Positive: Cryptococcus neoformans, or other capsulate organisms.

Negative: A proven negative smear may be used as the negative control

Rapid Field’s Stain

Dientamoeba fragilis, Blastocystis hominis and Pneumocystis Jirovecii

Positive: Dientamoeba fragilis, Blastocystis hominis and Pneumocystis jirovecii.

Negative: A proven negative smear may be used as the negative control.

Vincent’s Stain

Borrelia vincentii

Positive: Borrelia vincentii appear as pale pink staining spirals together with pink cigar shaped fusiforms.

Note: Presence of both organisms is needed for establishing the diagnosis of Vincent’s


Negative: N/A

Ziehl-Neelsen’s Stain


Modified for Nocardia spp.

Positive: Acid fast bacilli vary from 0.5 to 10 μm in length6 and stain red. Some may appear beaded.

Negative: All other organisms and background material stain green if malachite green counterstain is used or blue if methylene blue counterstain is used.

Loeffler’s methylene blue

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.

Metachromatic granules appear dark blue in a light blue cytoplasm.

Albert’s Stain

For identification of genus Corynebacterium by differential staining method.

The cytoplasm appears light green and the granules blue/black.

Neisser’s Stain

Corynebacterium spp.

The cytoplasm appears light brown and the granules brown/black.

Spore stain

Spore formers

Bacterial bodies stain red & spores stain green.

Gimenez stain

Gimenez technique may be valuable for detecting certain slow-growing or fastidious bacteria (e.g. Legionella)

Basic fuchsin stain in aqueous solution with phenol and ethanol colours many bacteria (both gram positive and Gram negative) red, magenta, or pink. A malachite green counterstain gives a blue-green background cast to the surrounding tissue.


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