Staining Procedure for Rapid Identification of Fungi

Scrapings from involved ocular site complemented with the appropriate staining method can offer the ophthalmologist circumstantial as well as definitive information concerning the identity of the invading organism.

STAINING METHODS 

Light Microscopy

• KOH

• Gram’s stain

• Giemsa stain

• Grocott-Gomori Methenamine Silver (GMS) stain.

Fluorescent Microscopy

• Calcofluor white staining

• KOH-Calcofluor white procedure.

KOH mount

Principle: Potassium hydroxide is used as a mounting fluid for visualization of fungal filaments as it helps in the clearance or lyses of all surrounding tissues. In addition, Acanthamoeba cysts and Nocardia filaments can also be visualized.

Preparation of 10% KOH

• Approximately 1 gm (8 pellets) of KOH is weighed

• It is dissolved in 10 ml of distilled water

• One drop of 10% glycerol is added

• Fresh stock should be prepared every week

Can be kept at room temperature in a dropper bottle like a penicillin bottle (Fig. 4.1).

Fig. 4.1: KOH pellets and preparation of 10% KOH

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Ocular specimen collection

Standard procedures followed for ocular specimen collection, culture isolation and identification of the organisms are described below.

SPECIMEN COLLECTION KIT TO BE KEPT IN THE OPHTHALMOLOGIST OFFICE

The following are the items that must be available for collecting the corneal specimens.

• Culture plates (blood agar, potato dextrose agar)

• Sterile Kimura’s spatula or scraper

• Topical anesthetic agents

• Bunsen burner

• Clean glass slides

• Clean cover slips.

Corneal Scraping

Corneal scraping is performed under aseptic conditions by an ophthalmologist using a sterile Kimura spatula. The procedure is performed under magnification of a slit lamp or binocular loupe following instillation of topical anesthetic agents such as 0.5% proparacaine or 4% lignocaine.

Material obtained from scraping the leading edge and the base of each ulcer specimen are inoculated directly onto sheep blood agar, potato dextrose agar (PDA) and Brain Heart Infusion broth (BHI) without gentamycin sulphate. The material from the corneal scraping is also smeared and labeled onto slides in a thin, even manner to prepare a 10% KOH wet mount and Gram staining. In cases of suspected actinomycetes keratitis Kinyoun’s metho of acid fast staining is performed (Fig. 3.1). Corneal scrapings collected by an opthalmologist by using an ophthalmic microscope in shown in Figures 3.4A to F.

In some of the patients, the ulcer may be predominately in the deeper stromal lesion, with inflammatory outpourings in the anterior chamber. In such a case, a paracentesis is made using a 26 gauge needle mounted on a 2cc plastic disposable tuberculin syringe (Figs 3.2A and B).

Fig. 3.1: Materials used for collecting specimens from corneal ulcers

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Antibiotics and Other Chemotherapeutic Agents

Technically, the antibiotic era began with the discovery of penicillin by Sir Alexan- der Fleming in 1929. However, its development could occur only during World War II. By that time, an energetic soil scientist, Dr. Selman Waksman, had established a school of soil microbiology in New Jersey’s Rutgers University. Focusing on soil- borne aerobic actinomycetes, his group started a systematic program that lead to the discovery of streptomycin, an antibiotic credited with saving lives of millions of tuberculosis patients all over the world. Since then, his group at Rutgers as well as his students in various educational and industrial research laboratories went on to discover thousands of antibiotics, which include almost all the powerful drugs, such as tetracycline, erythromycin, chloramphenicol, amphotericin B, and vancomycin. Traditionally, the term “antibiotics” has been used for the antimicrobial agents derived from microorganisms. Since a number of antibiotics currently in use are actually synthetic, the term antibiotics has become synonymous with antimicrobial agents used for the treatment of infectious diseases.

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Host-Microbe Interactions

The human body reacts in many different ways to microorganisms. These interac- tions can be summarized in the following categories:

RESIDENT MICROBIOTA

All surfaces of the human body, including the skin as well as the mucous membranes that surround the inner parts of the mouth, nostrils, genitals, and gastrointestinal tract, are inhabited by a distinct set of microbial communities, which are specifically adapted to the local physical and chemical environment. Such normal microorgan- isms, called resident microbiota, perform extremely important roles.

1. Resident microbiota engage all available binding sites on the host cell sur- faces, thus invaders have a diminished possibility of attaching to the host cell surfaces.

2. Many microbes secrete vitamins that are absorbed by the host and serve important nutritional needs.

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TRANSMISSION OF INFECTIOUS DISEASE (MODE OF DISSEMINATION)

Airborne (Inhalation of Bioaerosols) 

A bioaerosol contains bacteria in its center, surrounded by air and a small amount of liquid, generally saliva. Bioaerosols may be produced due to sneezing, coughing, or talking. Depending on the force of sneezing or coughing, the bioaerosol-borne microorganism can travel up to several meters in air. Almost all respiratory tract infections are airborne; some can also pass from person to person through the inhala- tion of bioaerosols. Some of the examples of airborne infections include tuberculo- sis, strep throat, diphtheria, pertussis, legionellosis, influenza, and chicken pox, and a wide range of mycotic diseases such as aspergillosis, zygomycosis, cryptococcosis, histoplasmosis, and coccidioidomycosis.

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Assessing White Cells and Platelets

White cells and platelets may be increased or decreased in number. They may also show morphological abnormalities, either inherited or acquired. Assessing whether the numbers of individual types of white cell are increased or decreased requires a differential count. However, the differential count is of little importance in itself and should only be used to calculate the absolute numbers of each cell type. The absolute counts are then compared with those expected in healthy people of the same age, sex and ethnic group. The terms used in describing numerical abnormalities in white cells and platelets are defined in Table 3.1.

Table 3.1  Terminology used  for abnormalities of white cell  and  platelet numbers

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Assessing Red Cells

Red cells should be assessed as to their:

• number

• size

• shape

• degree of haemoglobinization

• distribution in the blood film.

Their appearance should be described using a standard terminology.

Assessing red cell number and distribution (anaemia, polycythaemia, rouleaux formation, red cell agglutination)

The thickness of a film of blood spread on a glass slide is deter- mined by how thick the blood is, i.e. by its viscosity. This in turn is determined by the Hb. In a normal blood film it is possible to find a part of the film which is ideal for microscopic examination where the red cells are touching but not overlapping. If the Hb is abnormally high (a condition referred to as polycythaemia) the blood has a high viscosity and the film of blood on the glass slide is thick. The red cells therefore appear packed together through- out the whole length of the film. The term ‘packed film’ is often used. Conversely, when a patient is anaemic the viscosity of the blood is low, the blood film is very thin and there are large spaces between the red cells. The effect of Hb on the blood film can be seen by comparing Figs 2.1, 2.2 and 2.3.

Usually red cells are distributed fairly regularly on the slide. Two abnormalities of distribution may occur. When there is an

Fig. 2.1  Anaemia (caused by iron  deficiency).

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