Laboratory diagnosis of fungi Mrs. Dalia Kamal Eldien Msc in Microbiology Lecture NO (3)

Objectives To know General steps will be done for the isolation and identification of the pathogenic fungi, include the type of specimen, direct microscopy, culture, staining techniques& serological test

Laboratory diagnosis of fungi General steps will be done for the isolation and identification of the pathogenic fungi:- 1) Specimen collection:- Specimen depends on the site affected, Include hair, skin scrapings, nail clippings, sputum, blood, CSF, urine, corneal scraping, discharge or pus from lesions and biopsy. 2) Macroscopic examination:- Especially in sub-cutenous mycosis, the macro examination give an idea on the etiological agent

Skin scrapping collection by scalpel

CSF collection

3) Microscopy:- Microscopy is used to examine the clinical specimens for the presence of fungal elements, direct examination can be stained or unstained. 20% KOH mount (unstained): Several specimens are subjected to KOH mount for direct examination. The material is mixed with 20% KOH on a slide and a cover slip is placed, the slide is then gently heated by passing through the flame 2-3 times. KOH serves to digest the protein debris and clears keratinized tissue to increases the visibility. Addition of Dimethyl sulphoxide (DMSO) permits rapid clearing in the absence of heat.

KOH preparation

B. Calcofluor white: C. India Ink: This is a fluorescent dye, which binds selectively to chitin of the fungal cell wall. The specimen then can be observed under fluorescent microscope. C. India Ink: This is negative staining technique, stain the back ground of the smear Use for demonstration of Capsules Example the Cryptococcus neoformans capsule

D. Periodic Acid-Schiff (PAS) stain: On staining by this stain, fungal elements appear bright magenta color, while the background stains green. It is useful in staining tissue specimens. E. Giemsa’s stain: It is particularly useful in the detection of Histoplamsa capsulatum in the bone marrow smears.

Calcofluor white stained smear

Cryptococcus neoformans capsule demonstrated by India ink

F. Haematoxylin and Eosin (H&E) stain: Useful for staining tissue sections Candida and Aspergillus may be missed in H&E stained G. Gram stain: Candida is best demonstrated in clinical specimen by Gram stain, it is gram positive cell. Direct microscopy is diagnostic in some fungal infection mainly Dermatophytes

Gram stain for candida

4) Culture: One of the most common media used to culture fungi in laboratory is Sabouraud’s Dextrose Agar (SDA). It consists of peptone, dextrose and agar. High concentration of sugar and a low pH (4.5-5.5) prevents growth of most bacteria and makes it selective for fungi. Addition of antibiotics such as Chloramphenicol, or mixture of penicillin &Streptomycin to SDA serves to inhibit bacterial multiplication. The saprophytic fungi can be inhibited by the addition of cycloheximide (actidione) to the SDA

An example of SDA with cycloheximide and Chloramphenicol is Mycosel agar. Other basic media fungi growth include Potato Dextrose Agar, Malt Extract Agar etc… For the fastidious fungi we use Brain Heart Infusion

Sabouraud’s Dextrose Agar

Brain heart infusion media

Incubation methods:- Most fungi are able to grow at room temperature 22oC, while few pathogenic fungi (e.g, Cryptococcus, dimorphic fungi) can grow at 37oC, So we need duplicate plate Saprophytic fungi grow much quickly than pathogenic fungi Since some fungi grow slowly cultures should not be discarded for 4-6 weeks& incubated aerobically.

Colonial morphology:- Fungi are identified on the basis of colony morphology, and pigmentation(color of pigment, and if diffused or localized).

Pigmented& non pigmented fungi on DSA

5)Needle mount:- The first rule to remember in mould studies is to examine young, actively growing material. Older parts of colonies on natural material will often be partially decomposed or so covered with spores as to be unrecognizable. The best way to begin is to examine the growth from the margin of the colony . The lactophenol cotton blue (LPCB) wet mount preparation is the most widely used method of staining and observing fungi and 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. under the microscope examine hyphae, conidia, spore or any other structure

Preparation of lactophenol cotton blue (LPCB) slide mounts Place a drop of 70%alcohol on a clean microscope slide. Material from cultures of filamentous fungi should be removed using a stiff inoculating wire. Remove a small amount of the culture. For fungal cultures, Immerse the fungal material in the drop of 70%alcohol Before the alcohol dries out add one or at most two drops of the stain lactophenol cotton blue to the slide

Cover by cover slip& examine under the microscope Make the initial examination using a low power objective lens. The thinner parts of the preparation, generally around the edges of the mounted material, will yield the best images. Switch to a higher power 40X objective for more detailed examination of spores and other structures.

 Lactophenol Cotton blue mount of the isolated pathogen, showing septate hyphae

6)Serology: - Detection of anti-fungal antibody is helpful in diagnosis of sub-cutaneous and systemic mycoses& for the prognosis and response to anti-fungal drugs. Different serologic techniques that are used include agglutination, immunodiffusion, counter-immunoelectrophoresis, complement fixation test, immuno fluorescence, RIA and ELISA.

immunoelectrophoresis

7)Molecular techniques:- Newer techniques such as DNA hybridization, PCR are useful in diagnosis of mycoses in a shorter period as well as detect those fungi that are difficult or dangerous to cultivate in vitro.