Staining of Fungi
Direct microscopic examination without stain lacks sensitivity, especially when hyphae are sparse in the specimen. A variety of differential stains are commonly used like Gram, Giemsa, India Ink Stain, Lactophenol cotton blue stain, etc to stain fungi.
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Methods of Staining Fungi
India Ink stain
Introduction
This stain was previously known as the “Nigrosin stain”. India ink staining is a negative staining technique used to determine an organism’s cellular morphology. The background is stained whereas the organism remains unstained and the morphology is not distorted in any way. Capsules displace the dye and appear as halos surrounding the organism.
This fungi stain provides a high degree of contrast not available in most other staining procedures. This technique is particularly recommended for the demonstration of the capsule of the yeast, Cryptococcus neoformans and it can also be used to demonstrate the presence of bacterial capsules.
Method
- place a drop of India ink on to a clean glass slide
- Add 1 drop of the specimen or liquid culture or rub a speck of material on the slide surface just beside the ink before mixing it into the ink. Sputum or pus can be cleared with KOH and heat and then mixed with India ink
Note: If preparation is too dark, it may be diluted with a small drop of water
- Place a cover slip over the smear avoiding air bubbles, press it down gently through a sheet of blotting paper so that the film becomes very thin and pale in colour
- Examine with a high-power lens (phase-contrast microscope) for the presence of encapsulated cells. Bright field microscopy may also be used
Interpretation
Positive result
Organisms possessing a capsule appear highly refractile, surrounded by a clear zone or halo against a dark background.
Leucocytes may also appear haloed due to leakage of the cytoplasm but the halo has a fuzzy, irregular appearance at the periphery and the cell within the halo has a paler cell wall.
Note: Some Cryptococcus neoformans strains have been reported to be India ink negative.
Negative result
No clear zone around the organism is observed.
Technical information
Sensitivity
The cryptococcal latex antigen test has been proven to be significantly more sensitive than the India ink preparation and is therefore recommended for the initial diagnosis of cryptococcal disease.
Errors with India ink stain
- Common errors with this stain are:
- The use of diluted ink. The correct concentration of India ink is critical for showing the capsular zone
- The smear on the slide is too thick. Some practice is required by laboratory staff in making satisfactory smears
Lactophenol cotton blue stain
Introduction
The lactophenol cotton blue (LPCB) stain is the most widely used staining solution in the examination of yeasts and molds and serves as both a mounting fluid in wet mounts and a stain. It is simple to prepare. The preparation has three components: phenol, which will kill any live organisms; lactic acid which preserves fungal structures, and cotton blue which stains the chitin in the fungal cell walls. Upon the addition of lactophenol cotton blue, fungi stain blue allowing for easier visualization and examination.
Other alternative stains that can be used are the Lactofuchsin or aniline blue stains and these have the same principles as the LPCB stain. The Lactofuchsin stain, if performed correctly, can preserve the structure and arrangement of the hyphae, if present for several weeks.
Safety considerations
- Lactophenol cotton blue is acidic while Lactofuchsin is corrosive. They can be toxic if inhaled, in contact with skin and if swallowed.
- Adhesive tape preparations from fungal cultures must be prepared under a biological safety cabinet to ensure safety of laboratory personnel. Attention must be paid to patient travel history and any suspected Hazard Group 3 organisms must be processed in a Class 1 exhaust protective cabinet in a Containment Level 3 room.
- Phenol kills any fungus present, allowing the microscope preparations to be examined out with the biological safety cabinet.
Skin scraping, Fluid exudate or Tissue
- Mix the specimen whether a skin scraping, fluid exudate or tissue with two drops of 10% KOH on a clean slide.
- add one, or at most two drops of the lactophenol cotton blue mountant/stain to the slide
- Press a cover slip gently to make a thin mount avoiding air bubbles. Gentle warming can also aid in clearing the mount
- Examine the prepared slide under low power (x10) with reduced lighting. Switch to high power (x40) to check for the presence of suspected fungal structures.
Culture
If examining a fungal culture, direct microscopic mounts can be made. Alternatively, the adhesive tape method may be used.
Direct microscopic mount
- place one drop of lactophenol cotton blue mountant to a microscope slide
- using a mounted needle, gently remove a small portion of the colony and place in the LPCB drop
- cover with a coverslip, pressing gently to make a thin mount avoiding air bubbles
- blot off any excess LPCB stain
- examine the prepared slide under low power (x100) with reduced lighting. Switch to high power (x400) to examine the fungal structures in more detail
Adhesive tape
This technique may be a quick and easy alternative to direct preparation; it often allows fungal structures to remain intact in the slide preparation. There are many variations of this technique; the standard procedure is given below:
- Place one or two drops of lactophenol cotton blue mountant to a clean glass microscope slide
- Take a 40mm length of clear adhesive tape and place the sticky side on to the surface of the culture, gently applying pressure allowing fungal elements to become attached to the tape. Forceps may be used.
- Carefully lift the tape and place on to the LPCB mountant on the slide gently pressing down
Note: The preparation may also be examined directly without the use of a coverslip; however, another drop of LPCB stain may be added on top of the tape and a cover glass placed on top
- examine the prepared slide under low power (x100) with reduced lighting. Switch to high power (x400) to examine the presence of suspected fungal structures in more detail
Note: Commercial preparations are available and if used, manufacturer’s instructions should be adhered to.
Interpretation
Positive result
Yeast cells, mycelia, and fruiting structures stain a delicate blue color while the background appears a faint, pale blue.
Negative result
The absence of fungal structures indicates a negative result.
Note: For culture, the fungal colony may be identified using macroscopic and microscopic characteristics.
Technical information/limitations
Lactophenol cotton blue stain is only useful in the staining of yeasts and moulds and when used as a mounting medium. However, this staining procedure does not always preserve the original position and structure of the conidia, spores, and other characterizing elements.
Modified Giemsa’s stain (Pneumocystis jirovecii)
Introduction
Giemsa’s fungi stain has been used routinely to demonstrate the presence of Pneumocystis jirovecii in bronchoalveolar lavage (BAL) smears from patients with pneumonia or who are immunocompromised. The trophozoites and intracystic bodies in intact cysts can be stained with Giemsa, but the cyst wall does not take up this stain.
But in recent years, a modification of this stain was developed, where sulphation of smears before staining with Giemsa apparently modifies the surface of P. jirovecii cysts in a way that enables the Giemsa stain to react and allows both cysts and trophozoites of P. jirovecii to be visualized. It also shows all the stages in BAL or sputum, which is particularly useful, considering the prevalence of P. jirovecii pneumonia in conjunction with the spread of AIDS.
Safety considerations
Follow local COSHH and risk assessments when performing all staining procedures.
Method
- prepare a 1 in 10 dilution of Giemsa’s stain in buffered water pH 7.2. This should be freshly prepared
- prepare a smear of the centrifuged BAL fluid sediment and allow to air dry
- fix BAL smear with either ethanol or by using heat
- dip slide in sulphation reagent* (using forceps) for 10min
- wash in running tap water for 5min
- flood the slide with diluted Giemsa’s stain and leave for 30min
- run tap water on to the slide to float off the stain and to prevent precipitation on the smear and allow to air dry
- mount a coverslip on the slide using any suitable mountant or examine using a low power oil immersion objective without adding a coverslip
* 15mL of concentrated sulphuric acid is added slowly to 45mL of glacial acetic acid in a Coplin jar. The Coplin jar should be standing in a container of cool tap water (not below 10°C). The solution is gently mixed and the jar sealed with petroleum jelly.
Interpretation
Positive result
Parasite nuclei and chromatin stain red. The cysts are oval to circular, about 5μm in diameter. The outline of the cyst is generally reddish purple and the central portion of the cyst purple, though the exact colour varies throughout the smear with the red tints predominating in some areas and the blue in others.
Negative result
Leucocyte nuclei stain purple, cytoplasm stains bluish-grey, bacteria and yeasts stain dark-blue.
Technical information
Staining for Pneumocystis jirovecii is more commonly done by specific immunofluorescence antibody methods or by Grocott- Gomori methanamine silver staining. Alternative diagnostic methods such as polymerase chain reaction (PCR) are used increasingly.