Direct Microscopic Examination

Direct microscopic examination

Direct microscopic examination of collected clinical sample has been preferred for many years; however, its use should not be for all the case. The mission of clinical microbiology laboratory is to provide rapid and accurate diagnosis, which is done by direct microscopic examination primarily gram staining technique of the clinical specimen submitted for culture.

Almost all the specimens for laboratory diagnosis are processed for direct microscopic examination. This provides the presumptive diagnosis for the physician and also helps in the selection of appropriate culture media.The table shows the presumptive identification of fungus by direct examination technique:

Different microscopic techniques performed are:

1. KOH mount methods
2. Gram staining technique
3. India ink stain
4. Lactophenol cotton blue stain
5. Calcofluor white stain
6. Periodic Acid-Schiff stain (PAS stain)

KOH Mount

Traditionally, potassium hydroxide has been the recommended method for the direct microscopic examination of a specimen; however, a superior method applied now a day is calcofluor white stain. In the KOH mount, usually 10% (w/v) KOH is used as the mounting medium particularly for hard specimens like hair and nail. The collected specimen are directly mounted in 40% and 10% KOH for skin and nail sample respectively. KOH helps in dissolving the epithelial cells and thus help in fungal visibility.The alkaline solution potassium hydroxide digests keratin but has no effect on fungi. An alkali- glycerin solution can also be used for such specimen.

ProcedureE

1. Place a drop of mounting fluid on a clean and dry slide.
2. Mix the specimen with KOH solution.
3. Place a cover slip on the preparation.
4. Place the slide in a petri-dish or the other container with lid together with a damp piece of filter paper to prevent the preparation from drying out.
5. After 5-10 min, observe under low power microscope and then high powder to identify the hyphae structure.

Staining technique

Before visualizing under the microscope, the clinical specimen is stained by a different technique for identifying the causative agent. The techniques implied are:

1. Gram staining technique
2. India ink preparation
3. Calcofluor white stain
4. Giemsa stain
5. Periodic Acid-Schiff(PAS) stain
6. Lactophenol blue stain
7. Lactophenol cotton blue stain

Gram staining

Gram staining technique is specially used for detection of yeast cells. Yeast cell results in the gram-positive reaction. For example, Candida albicans which are observed in vaginal discharge results in the gram-positive test.

Gram stain is usually a poor stain use to examine a specimen for a fungus. Gram stain commonly used when examining smears of Candida, Malassezia, and Sporothrix but should not be relied upon to demonstrate the yeast of the other dimorphic fungi.

India ink preparation

It is a negative staining technique where the organism appears as clear disc against a dark (black) background. India ink preparation is done for detection of a capsule containing the fungus which is the extracellular polysaccharide. India ink can be used to specimens such as spinal fluids or exudates to provide a dark background that will highlight hyaline yeast cells and capsular material (halo effect). Hence, it should be used to determine specimens suspected of containing Cryptococcus neoformans.The encapsulated yeast cells are mostly detected against the black background. The presence of encapsulated yeast cells in CSF sample; most of the time is an indicator of Cryptococcal meningitis. It is also used in the identification of Cryptococcus neoformans which is also present in CSF sample. For demonstrating the presence of fungal cells in clinical sample dye is useful because it helps to binds polysaccharides.The procedure for India ink preparation will be performed only in particular instances with the approval of the director or supervisor.

Calcofluor white stain

It is a fluorochrome which can be used to rapidly detect yeast cells, pseudohyphae and hyphae in the specimen when examined by fluorescence microscopy. A fluorescence microscope is necessary for identifying fungal cells prepared with calcofluor White. Yeast cells, pseudohyphae, and hyphae display a chalk-white or brilliant apple-green fluorescence with the application of calcofluor white stain. The fluorochrome binds to the cellulose and chitin present in the fungal cell walls but calcofluor used for a fluorescence microscope cannot identify the endospores, and also the difficult to detect vaginal secretions.

The fungi appear light apple green or blue-white, depending upon the wavelength of exciter light.

Giemsa stain

It is preferred to Pneumocystis cavinii which is the causative agents of pneumocystis associated in pneumonia especially in the immunocompromised host. The organism was initially named as pneumocystis cavinii and categorized under protozoan. Recently, the use of 16s rRNA gene categories this organism under fungi.

Periodic Acid-Schiff’s (PAS) stain

It is a staining method used to detect polysaccharides such as glycogen and mucosubstances such as glycoproteins, glycolipids, and mucin in tissues. The PAS stain is commonly used stains for fungal histopathology in a direct examination of the clinical specimen. This PAS stain is sometimes used when a KOH preparation does not identify fungi that are doubt to be present in the clinical sample. For the working of PAS stain requires 20 to 25 minutes. Light green is used as the counterstain because the fungus appears deep purplish red against the contrasting background color. The PAS reaction stains the fungal polysaccharide.

The reaction of periodic acid Schiff's forms a pair of aldehyde at the two few ends of each broken monosaccharide ring. This aldehyde then reacts with Schiff’s reagent to give a purple color compounds.

Lactophenol blue stain

The Lactophenol Blue Stain is used as a mounting medium for the examination of fungi. Lactophenol Blue Stain is a mounting medium and staining technique is used in the preparation of slides for microscopic determination of fungi. The specimen mounts for microscopic examination of mold include teased, mashed, and slide culture preparations; however, the slide culture is limited to organisms with low virulence. Internal quality control of the Lactophenol Blue stain must be performed timely on known reference organisms to ensure the performance of the mounting solution. Lactophenol Blue Stain should be stored at room temperature and protected from light. Under these conditions, it has a shelf life of 52 weeks from the date of manufacture.

Procedure

1. At first place a drop of Lactophenol Blue on a clean microscope slide.
2. Using an inoculating needle, gently remove a small portion of growth midway between the colony center and edge.
3. Then, place the growth from that needle in the drop Lactophenol Blue on the slide.
4. With two sterile dissecting needles, gently tease the fungus apart in order to thinly spread out in the Lactophenol.
5. Place a cover slip edge of the Lactophenol and slowly lower ii but avoid trapping air bubbles under the coverslip.
6. If desired, seal the edges of the coverslip with nail polish or per-mount to preserve the mount as a reference slide.
7. Finally, examine the slide under the microscope.

Lactophenol cotton blue stain

Lactophenol cotton blue stain is a simple and widely used staining method for fungi.The main component of Lactophenol cotton blue (LCB) staining are:

1. Phenol- fungicidal in nature
2. Lactic acid- preserves fungal structure
3. Cotton blue- stains the chitin in the fungal cell wall and the cytoplasm in light blue appearance.

LCB stain consists of:

cotton blue 0.05g

Phenol crystal 20g

Glycerol 40ml

Lactic acid 20ml

Distilled water 20ml

Procedure

1. Place a drop of 70% alcohol on the slide.
2. Add the specimen to the drop of alcohol.
3. Before alcohol gets off, add few drop of lactophenol cotton blue stain on a slide.
4. Then place the coverslip on the drop without formation of an air bubble.
5. Finally, examine under the microscope.

REFERENCE

Cheesbrough, M. Medical Laboratory Manual for Tropical Countries. Vol. Vol 2. ELBS London, 2007.

Tille, P. Diagnostic Microbiology. 13th. Elsevier, 2014.

D, Grenwood, Slack RCB and Peutherer J. Medical Microbiology. Dunclude Livingstone: ELBS, 2001.