Stains for Microbiology Specimens

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STAINS FOR SPECIMEN PROCESSING

Acridine Orange Stain - Hardy Catalog Number: 313B8
Acridine orange is a fluorescent stain used for the detection of bacteria and fungi in clinical specimens. The dye
intercalates into nucleic acid in both the native and denatured states. At neutral pH, bacteria, fungi and cellular material
(e.g., leukocytes, squamous epithelial cells) stain red-orange. At acid pH (pH 4.0), bacteria and fungi remain red-orange
but background material stains green-yellow. It is a rapid stain that may be particularly useful in thick or purulent
specimens for which the Gram stain is not interpretable.
Auramine-Rhodamine Stain - Hardy Catalog Number: 789D8
Auramine and rhodamine are fluorochromes that bind to mycolic acids and are resistant to decolorization with
acid-alcohol (acid-fast stain). Acid-fast organisms fluoresce orange-yellow with UV light. Potassium permanganate is
used as a counterstain. It is a strong oxidizing agent that inactivates the unbound fluorochrome dyes to produce a black
background for the stained specimens. It allows rapid screening of specimens and is used by laboratories who routinely
perform acid-fast examinations. Fluorochrome-stained smears can be restained by the Kinyoun or Ziehl-Neelsen methods.
Calcofluor White Stain - Hardy Catalog Number: SC15100 or SC15250
Calcofluor white is a non-specific fluorochrome that bind to cellulose and chitin in the cell walls of fungi, including yeast
cells, hyphae, pseudohyphae, and spherules.(1) Because of the rapidity and specificity with which specimens can be
observed, the stain has become commonplace in microbiology laboratories. The dye can be mixed with 10% potassium
hydroxide so that mammalian cells can be dissolved, thus facilitating visualization of fungal elements. Fungi,
Pneumocystis spp., and Acanthamoeba spp. appear green or blue against a dark background when the stained slide is
examined under UV illumination. Care must be used to distinguish specific staining from stained debris.
Cryptosporidium Stain - Hardy Catalog Number: 482K or 482KMG
Cryptosporidium Stain is used to stain smears made from clinical specimens for the purpose of detecting the oocysts of
Cryptosporidium spp. The test employs the use of the dimethyl sulfoxide (DMSO) acid-fast staining procedure. Of the
various acid-fast staining procedures, it is the easiest and quickest to perform and provides excellent results. DMSO, a
wetting agent with tremendous penetrating qualities, is mixed with carbol fuchsin stain. DMSO transports the carbol
fuchsin through the oocyst wall and rapidly stains the red cell, eliminating the need to heat the slide for stain penetration.
A mild acetic acid is added to the malachite green counterstain solution combining the decolorization and counterstain
steps into one. Oocysts stain red while most other fecal material appears green. Other structures that may stain red can be
easily distinguished from oocysts.
Direct Fluorescent-Antibody Stain
Specific fluorescein-labeled antibodies are used to directly detect a variety of organisms (e.g., Streptococcus pyogenes,
Bordetella pertussis, Francisella tularensis, Legionella spp., Chlamydia trachomatis, Cryptosporidium parvum,
Giardia lamblia, influenza virus, herpes simplex virus) in clinical specimens. Specimens are fixed to a glass slide and
incubated with fluorescein-labeled antibody. The labeled antibodies bind to the organisms and fluoresce green under UV
light. The sensitivity and specificity of the stain are determined by the quality of the antibodies used in the reagents.
Dobell & O'Connor Iodine - Hardy Catalog Number: Z66
A weak iodine solution such as Dobell and O'Connor (diluted Lugol's) is recommended for use in staining protozoan
cysts. This preparation is a 1:5 dilution of Lugol's Iodine. Iodine Solution is often used in wet mounts of concentrated

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fecal material. It is useful for staining glycogen and making nuclei visible in protozoan cysts. Protozoan cysts correctly
stained with iodine contain yellow-gold cytoplasm, brown glycogen material, and paler refractile nuclei.
Flagella Stain - Hardy Catalog Number: Z87
Hardy Diagnostics Flagella Stain is a simple, rapid, qualitative method for detecting bacterial flagella and their shape,
length, curvature, arrangement and number on the cell.(2,4) This method was developed by Ryu in 1937, and also later
described by Kodaka, et al. in 1982.(3,5) This test has been found especially useful in providing taxonomic and identifying
information about motile bacteria, and more recently, anaerobic bacteria.(2,3,4) The Flagella Stain provides a method for
viewing bacterial flagella by employing crystal violet in an alcoholic solution as the primary stain.The Flagella Stain also
contains tannic acid and aluminum potassium phosphate as mordants, and phenol as an antifungal agent.
Giemsa Stain - Hardy Catalog Number: 591A16
Giemsa Stain, like Wright Stain, is a modification of the Romanowsky Stain, which combines methylene blue and eosin.
Both stains are typically used by hematology laboratories for demonstrating the differences of nuclei and cytoplasmic
features of the blood cell components. Both stains are also used for detection of blood parasites (e.g., Plasmodium,
Babesia, and Leishmania spp.), fungi (e.g., Histoplasma spp., yeast cells, Pneumocystis spp.), rickettsiae, chlamydiae,
and viral inclusions. A protozoan trophozoite has a red nucleus and a gray-blue cytoplasm; intracellular yeasts and
inclusions typically stain blue (basophilic); and rickettsiae, chlamydial elementary bodies, and Pneumocystis spp. stain
purple.
Gram Stain Kit - Hardy Catalog Number: GK400
Gram Stain is the most commonly used stain in clinical microbiology laboratories. It is used to separate bacteria into
gram-positive (blue) and gram-negative (red) groups as well as to detect fungi and many parasites. The stain also provides
information about the state of infection and quality of a specimen by allowing differentiation of epithelial and
inflammatory cells. Variations in the performance of this stain are commonplace, but the staining principle is constant.
After fixation of the specimen to a glass slide (by heating or treatment with 95% methanol), the specimen is exposed to
the basic dye, crystal violet. Iodine is added, and the iodine forms a complex with the primary dye. During the
decolorization step, this complex is retained in gram-positive organisms but lost in gram-negative organisms. The
gram-negative organisms are detected with a counterstain (e.g. safranin). The degree to which an organism retains the
stain is a function of the species, culture conditions, and staining skills of the microbiologist. Older cultures tend to
decolorize readily.
Gram Stain Kit Advanced - Hardy Catalog Number: GK400A
Hardy Diagnostics Gram Stain Kit Advanced is comprised of Advanced Crystal Violet, Stabilized Iodine, Fast
Decolorizer and Advanced Counterstain. Advanced Crystal Violet has been shown to provide superior, consistent
and brighter staining of gram-positive organisms, especially those which decolorize easily. Advanced Counterstain, a
stronger counterstain than Safranin, will stain the gram-negative organisms a very deep red. Contrast between
gram-positive organisms and gram-negative organisms in a mixed field is greatly enhanced. The advanced system helps to
guard against accidental over-decolorization, thus reducing the possibility of mistaking a gram-positive organism for a
gram-negative one.
India Ink Stain (Nigrosin) - Hardy Catalog Number: Z64
The use of India Ink is not technically a staining method. The polysaccharide capsule of encapsulated organisms (e.g.,
Cryptococcus neoformans) excludes the ink particles. Organisms are detected by the appearance of a distinctive halo
surrounding the encapsulated fungi. Care in interpretation is required because artifacts (e.g., leukocytes, erythrocytes,
powder, bubbles) may be confused with yeast cells. The morphologic characteristics of the yeast cells must be recognized
before the preparation can be interpreted.
Iron Hematoxylin Stain
Iron Hematoxylin Stain is used for the detection and identification of fecal protozoa. Helminth eggs and larvae generally
retain too much stain and are more easily identified with wet-mount preparations. Iron Hematoxylin Stain can be applied
to either fresh stool specimens or ones preserved with polyvinyl alcohol or a similar preservative. Formalin-fixed
specimens cannot be used. Background material and organisms stain gray-blue to black, with cellular inclusions and
nuclei appearing darker than the cytoplasm.

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Kinyoun Stain for AFB - Hardy Catalog Number: AF900

The presence of long-chain fatty acids (e.g., mycolic acid) in some organisms make these organisms both difficult to stain
with water-soluble dyes and resistant to decolorization with acid solutions (i.e., the organisms are considered acid-fast).
The Kinyoun method of staining uses high concentrations of basic carbol fuchsin and phenol to facilitate penetration of
the dye into the cells. Increased concentration of these two components avoids the need of heating the slide, and is
therefore referred to as a cold acid-fast stain. Basic carbol fuchsin is used as the primary stain, 3% sulfuric acid in 95%
ethanol (acid-alcohol) is the decolorizing agent, and methylene blue is the counterstain. Acid-fast organisms appear
pink-red on a pale blue background. The contrast between organisms and background is sometimes poor, and
fluorochrome stain is generally preferred for specimen examination. Acid-fast stains are used for detecting bacteria,
including Mycobacterium, Nocardia, Rhodococcus, Tsukamurella, and Gordona spp., and the oocysts of
Cryptosporidium spp., Isospora belli, Sarcocystis spp., and Cyclospora spp. Because some of these organisms lose the
primary stain when they are exposed to 3% sulfuric acid, the decolorizing agent can be reduced to 0.5 to 1%. Organisms
that retain this modified stain are referred to as being partially acid-fast.
Lactophenol Cotton Blue - Hardy Catalog Number: Z68
Lactophenol Cotton Blue Stain is recommended for mounting and staining yeast and molds. Lactophenol Cotton Blue
Stain is formulated with lactophenol, which serves as a mounting fluid, and cotton blue. Organisms suspended in the stain
are killed due to the presence of phenol. The high concentration of the phenol deactivates lytic cellular enzymes thus the
cells do not lyse. Cotton blue is an acid dye that stains the chitin present in the cell walls of fungi.
Lugol's Iodine Stain - Hardy Catalog Number: VIG008
Iodine, a non-specific contrast dye, is added to wet preparations of parasitology specimens to enhance the contrast of the
internal structures (e.g., nuclei, glycogen vacuoles). Parasitic cysts take up dye and appear light brown, and are thereby
differentiated from WBCs. One disadvantage of this method is that protozoa are killed by the iodine and thus motility
cannot be observed.
Methenamine Silver Stain
Methenamine silver staining is generally performed in surgical pathology laboratories rather than microbiology
laboratories. It is primarily used for the detection of fungal elements in tissues, although other organisms (e.g.,
Legionella spp.) can be detected. Silver staining requires skill, because non-specific staining can render the slide
uninterpretable. Fungi are delineated in black against a pale green background. Inner parts of hyphae appear charcoal gray.
Methylene Blue Stain - Hardy Catalog Number: Z88
Methylene blue is another contrasting dye commonly used in the laboratory, primarily for detection of bacteria and fungi.
It can be mixed with potassium hydroxide and used to examine skin scrapings for fungal elements. Methylene blue is also
used for detecting fecal WBCs. The presence of WBCs in stool is suggestive of invasive bowel disease.
Periodic Acid-Schiff (PAS) Stain
Periodic Acid-Schiff Stain is used to detect yeast cells and fungal hyphae in tissues. Periodic acid (5%) hydrolyzes the cell
wall aldehydes, which then combine with the modified Schiff reagent and stain the cell wall carbohydrates pink-magenta
against a light green background. The stain is time consuming and also requires the specimens to be digested. Because this
staining procedure is complex, most laboratories have replaced it with the calcofluor white stain.
Sudan Black B Stain - Hardy Catalog Number: Z89
Sudan Black B Stain is a qualitative method for detecting the presence of fat granules in Bacillus species and other
bacteria. This procedure, when performed carefully and consistently, aids in the detection of poly-beta-hydroxybutyrate
granules in bacteria. Sudan Black B Stain was originally prepared in Germany in the early 1930's. Lison and Dagnelie
proposed its use as a myelin stain in 1935. Gerard, also in 1935, first developed its use as a fat stain. In 1940's, both
Hartman and Burdon further established its value as a bacterial fat stain. It has also been proposed for staining Golgi
apparatus and leukocyte granules.(6)
Sudan Black IV Stain - Hardy Catalog Number: Z83
Sudan Black IV Stain is used as a qualitative method to detect the presence of fecal fat. In the case of steatorrhea, fat

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malabsorbtion occurs, and high quantities of fat are detected in the stool. This procedure, when performed carefully and
consistently, is a simple method of detecting this condition in the patient. Sudan Black IV Stain, also known as scarlet red,
was introduced by Michaelis in 1901 as a fat stain.(6) It is a dimethyl derivative of Sudan III, which makes it a deeper and
more intense stain, yet it has similar physical properties and is fat soluble.(6) This stain has been widely used as a screening
method because it is easy to use, and correlates well with quantitative methods.(7)
Trichrome Stain - Hardy Catalog Number: T016
Trichrome Stain, like Iron Hematoxylin Stain, is used for the detection and identification of protozoa. Trichrome Stain
produces well-stained smears from both fresh and PVA-preserved material. When staining is done properly, the specimen
background is green and the protozoa have blue-green to purple cytoplasms with red or purple-red nuclei and inclusions.
Uristain - Hardy Catalog Number: Z74
The stain contains various dyes that aid in differentiating the abnormal and normal cellular elements found in urine.
UriStain is a one solution modification of the Sternheimer and Malvin procedure. This combination stain includes:
Ammonium Oxalate, Safranin, Crystal Violet, and Ethanol.
Wright Stain - Hardy Catalog Number: 926A32
Wright Stain is used in the differential staining of basophilic and acidophilic material. It is a polychromatic stain that
contains a mixture of methylene blue, azure B (from the oxidation of methylene blue), and eosin Y dissolved in methanol.
The eosin ions are negatively charged and stain the basic components of the cells orange to pink, while the other dyes stain
the acidic cell structures various shades of blue to purple. Like Giemsa Stain, it is primarily used for the differentiation of
intracellular and extracellular circulating blood parasites (e.g., Plasmodium, Babesia, and Leishmania spp.), fungi (e.g.,
Histoplasma spp., yeast cells, Pneumocystis spp.), rickettsiae, chlamydiae, and viral inclusions.
Wrights Stain, One Step - Hardy Catalog Number: SS016 or SS032
One Step Wrights Stain is a hematology stain that is used for differential staining of blood smears, bone marrow and
blood parasites. The traditional Wrights stain is diluted 1:1 with giordano buffer before use. One Step Wrights Stain
contains the buffer already dissolved in the stain. The slides are stained in the undiluted stain and differentiated by
decolorizing in purified water.
Ziehl-Neelsen Stain for AFB - Hardy Catalog Number: 484K
Ziehl-Neelsen, an acid-fast stain, requires that the specimen be heated during staining so that the basic carbol fuchsin can
penetrate into the organisms. Once the dye is forced into the cell, decolorization and counterstaining are the same as with
the Kinyoun method. The sensitivity and specificity of this stain are essentially the same as those of the Kinyoun method.
The Kinyoun Stain procedure, however, is less time-consuming and easier to perform.

REFERENCES
1. Forbes, B.A., et al. 2007. Bailey and Scott's Diagnostic Microbiology, 12th ed. C.V. Mosby Company, St. Louis, MO.
2. Murray, P.R., et al. 2003. Manual of Clinical Microbiology, 8th ed. American Society for Microbiology, Washington,
D.C.
3. Kodaka, H., et al. 1982. Practical procedure for demonstrating bacterial flagella. J. Clin. Microbiol.; 16:948-952.
4. Koneman, E.W., et al. 2005. Color Atlas and Textbook of Diagnostic Microbiology, 6th ed. J.B. Lippincott Company,
Philadelphia, PA.
5. Ryu, E. 1937. A simple methos of staining bacterial flagella. Kitasato Arch. Exp. Med.; 14:218-219.
6. Lillie, R.D. 1977. H. J. Conn's Biological Stains, 9th ed. Williams & Wilkins Company, Baltimore, MD. Reprint by
Sigma Chemical Company, 1991.
7. Henry, J.B., ed. 1979. Clinical Diagnosis and Management, 16th ed. Vol. I. W.B. Saunders Company, Philadelphia,
PA.

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8. Isenberg, H.D. Clinical Microbiology Procedures Handbook, Vol. I, II & III. American Society for Microbiology,
Washington, D.C.

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