isolation and identification of Candida sp.and Aspergillus sp.

Isolation and identification of clinically important fungi Candida albicans & Aspergillus sp.

AIM: To isolate and to identify Candida albicans and Aspergillus sp, from the given specimen

INTRODUCTION: The most common oral fungal infection in human beings is caused by the Candida species. The term Candida originates from the Latin word candid, meaning white. The spores of Candida are a commensal, harmless form of a dimorphic fungus. Its prevalence in healthy human oral cavities. However, when appropriate conditions such as local or systemic deficiencies in the host defenses supervene they become invasive and pathogenic pseudohyphae. Mycotic infections have become a major cause of morbidity and mortality in clinically debilitated or immunocompromised patients. The co-existence of Candida species are humans either as commensals or pathogens. The genus Candida includes several species C. albicans is by far the most common species causing infections in humans. The emergence of non-albicans Candida species as significant pathogens has however been well recognized. Although they are closely related they differ from each other with respect to epidemiology, virulence characteristics, and antifungal susceptibility.

Aspergillus species are filamentous fungi that are commonly found in soil, decaying vegetation, and seeds and grains, where they thrive as saprophytes. Aspergillus species can be occasionally harmful to humans. Most Aspergillus species are found in a wide variety of environments and substrates on the Earth throughout the year. Only a few well-known species are considered as important opportunistic pathogens in humans

MATERIAL REQUIREMENTS:

Sabouraud’s dextrose agar medium, Pre sterilized cotton swabs, Potassium hydroxide, Gram stain kit, Corn meal agar and required glass wares.

PROCEDURE:

Methods of sample collection & Isolation: Candida albicans

1. Smear technique: Scraping and smearing directly on the slide

 2. Plain swab: Using cotton swab sample is collected from the lesional tissue

 3. Impression culture technique: Impression casting in agar fortified with broth. 

4. Concentrated oral rinse: 10 ml of sterile phosphate buffered saline rinsed for 1minute. The solution is then concentrated (10-fold) by centrifugation and 50 ml, inoculated on an agar medium. 

Aspergillus sp.

1.      PDA (peptone dextrose agar) was used as the culture medium while collecting the samples.

2.      30 mg/L streptomycin had been added to the culture to prevent bacteria reproduction.

3.      Rose-bengal stain was added to the culture in order to prevent faster reproduction of moulds

4.      Peptone Dextrose Agar which was used for isolation was put into 7 days of incubation in laboratories at room temperature (22-26 ºC).

5.      After the incubation, pure cultures of microfunguses were obtained. Lactophenol solution stained by picric acid and lactophenol solution stained by cotton blue were used for investigation of microscopic structures of moulds.

Identification of Candida sp.:

 1. Direct microscopy Morphological features of Candida sp. need to be examined for identification.

2. Potassium hydroxide (KOH) preparation of the specimen reveals non-pigmented septate hyphae with characteristic dichotomous branching.

3. A smear taken from the lesional site is fixed on to microscope slides and then stained either by the gram stain or by the periodic acid Schiff (PAS) technique.

4. Laboratory culture of Swab: The sampling approach involves gently rubbing a sterile cotton swab over the lesional tissue and then subsequently inoculating a primary isolation medium such as Sabouraud’s dextrose agar (SDA)

5. Culture media: The most frequently used primary isolation medium for Candida is SDA which, although permitting growth of Candida, SDA is incubated aerobically at 37°C for 24–48 hrs.

6. Morphological criteria: The germ-tube test is the standard laboratory method for identifying C. albicans. The test involves the induction of hyphal outgrowths (germ-tubes) when subcultured in serum at 37 °C for 2-4 hours.

7. Chlamydospores are refractile, spherical structures generated at the termini of hyphae following culture of isolates on a nutritionally poor medium such as cornmeal agar. Agars are incubated for 24-48 hours at 37°C and then examined microscopically for chlamydospore presence

8. Biochemical identification: Candida species is largely based on carbohydrate utilization. Traditional testing would have involved culture of test isolates on a basal agar lacking a carbon source. Carbohydrate solutions would then be placed within wells of the seeded agar or upon filter paper discs located on the agar surface. Growth in the vicinity of the carbon source would indicate utilization.

RESULTS: Morphological characteristics of Candida species.

S.no	Morphological characteristics	Features
1.	Size (μm	3–6.2
2.	Shape	Spherical or oval
3.	Number of buds	Single; chains
4.	Attachment of buds	Narrow
5.	Thickness	Thin
6.	Pseudohyphae &/or hyphae	Characteristic
7.	Number of nuclei	Single

Candida develops as cream, smooth, pasty convex colonies on SDA and differentiation between species is rarely possible.