ISOLATION AND IDENTIFICATION OF CLINICALLY IMPORTANT FUNGI –Candida albicans, Aspergillus sp.,

 ISOLATION AND IDENTIFICATION OF CLINICALLY IMPORTANT FUNGI –Candida albicans, Aspergillus sp., 

AIM: 

 To isolate and identify the Aspergillus sp, Candida albicans from clinical samples. 

PRINCIPLE: 

Aspergillus sp., 

 Aspergillus species are ubiquitous fungi, commonly occurring in soil, water, and decaying vegetation. Route of acquiring infection being inhalation of fungal spores. From lungs, dissemination takes place leading to systemic aspergillosis. Occurrence of Aspergillus infection in association with chronic lung disease like pulmonary TB, lung abscess, bronchopneumonia, with residual lung cavity, asthma and lung malignancy has been documented. Out of 185 species of genus Aspergillus, only 20 can cause human infection, Some are Aspergillus flavus, Aspergillus fumigates, Aspergillus niger and Aspergillus nidulans. 

Candida albicans., 

Fumigatus is the most common species found in human infection all over the world. Germ tube test is a screening test for the presumptive identification of Candida albicans. Candida dubliniensis can also form germ tubes and Candida tropicalis can produce pseudohyphae that closely resemble germ tubes Germ tube formation was first reported by Reynolds and Braude and hence the germ tube test is also known as Reynolds-Braude phenomenon.

 Buds and pseudo-hyphae can be distinguished from germ tubes by the constricted attachment at the point of origin. Germ tubes don’t show constriction at the point of origin. germtube appears as a short lateral extension from the yeast cell and does not have a constriction (septum) where it meets the yeast cell or any constriction at the septum along the tube. 

The formation of germ tubes is associated with increased synthesis of protein and ribonucleic acid. Various media like fetal bovine serum may be used as a substitute for human pooled serum. Germ tubes are short outgrowths, non-septate germinating hyphae. They are ½ thewidth and 3 – 4 times the length of the cell from which they arise. When cells of Candida are incubated in serum at 37°C for 2-4 hours, Candida albicans and Candida dubliniensis produce short, slender, tube-like structures called germ tubes 

MATERIALS REQUIRED: 

Aspergillus sp: 

Clinical sample Sabouraud dextrose agar Lacto phenol cotton blue staining 

Candida albicans: 

Culture: Suspected Candida colonies from sabouraud dextrose agar (SDA). 

Reagent: Serum (human, sheep, fetal bovine) or other commercially produced media for germ tube testing 

Others: Test tubes, loop or wooden applicator, Pasteur pipettes, slides, cover slips.

PROCEDURE: 

1. Clinical specimens were streaked on SDA plate and it was incubated at 37 °C for 5-7 days. 

2. Fungal growth was identified by colony morphology on plate. 

3. Microscopic observations: Lacto phenol cotton blue staining Growth of Fungus were taken from culture medium on clean grease free sterile microscopic slide and mixed with 1-2 drops of LPCB stain. Focused the slide first under 10x to find the area and then under 40x for identification of fungus. 

4.Slide culture technique 10 mm square block from Potato dextrose agar (PDA) was cut using sterile coverslip or scalpel. The growth of Fungus were taken from culture medium i.e. Sabouraud’s dextrose agar (SDA) with the help of L shaped wire and inoculated into four corner of the block and then a sterile coverslip was kept over the block. The inoculated side culture was incubated in a wet Petri dish at 25 ±5°C in Biological Oxygen Demand incubator for 48 to 72 hours. Then the coverslip was removed and placed over a drop of Lactophenol Cotton Blue (LPCB) stain in clean sterile glass slide and then focused under 10x to find the area and then under 40x for identification of fungus. 

Procedure for Candida albicans: 

• Lightly touch a yeast colony with a wooden applicator stick.
•  Suspend the yeast cells in an appropriately labeled tube of fetal bovine serum (make a light suspension). Incubate the tube for 2-3 hours in a 35 – 37°C incubator. 
• Place a drop of the suspension on a slide using a Pasteur pipette. 
• Place a coverslip over the suspension. 
• Examine the wet mount microscopically (at 40X) for the presence or absence of germ tubes.

RESULT AND INTERPRETATION: 

 Obtained fungal culture from clinical sample was identified as Aspergillus niger based on Colony morphology and microscopic characterization. Negative germ tube test: No hyphal extension arising from a yeast cell or a short hyphal extension with constriction at the point of origin. A constriction where the lateral extension meets the yeast cell is produced by pseudohyphae or budding cells. Positive germ tube test: A short hyphal (filamentous) extension arising laterally from a yeast cell with no constriction at the point of origin is identified as C. albicans or C. dubliniensis. Some C. tropicalis isolates produce pseudohyphae that require careful observation to discriminate from those of C. albicans. 

A minimum of five germ tubes should be observed before calling an organism positive, because rare tubes may be produced by other species. Growth at 45°C separates C. albicans from Candida dubliniensis.