Lab 7: Isolation & Identification of Fungal Infection PDF

Basic Microbiology Practical No.: 87 Isolation and identification of fungal infection Intended learning Objective (ILOs): By the end of this lab the student will be: 1. Introduced to most commonly used fungal media. 2. Introduced to fungal i...

Basic Microbiology Practical No.: 87 Isolation and identification of fungal infection Intended learning Objective (ILOs): By the end of this lab the student will be: 1. Introduced to most commonly used fungal media. 2. Introduced to fungal inoculation techniques and incubation conditions. 3. Introduced to different growth characteristic of fungi. 4. Introduced to wet mount KOH and Lactophenol Cotton Blue (LPCB) techniques. Precaution: 1. Wear appropriate personal protective equipment for the laboratory procedure. 2. Maintain aseptic condition 3. Select standard setting of autoclave (No over or under sterilization) Page 1 Introduction/Background: Microorganisms The term "mycology" is derived from Greek word "mykes" meaning mushroom. Therefore mycology is the study of fungi. The ability of fungi to invade plant and animal tissue was observed in early 19th century but the first documented animal infection by any fungus was made by Bassi, who in 1835 studied the muscardine disease of silkworm and proved the that the infection was caused by a fungus Beauveria bassiana. In 1910 Raymond Sabouraud published his book Les Teignes, which was a comprehensive study of dermatophytic fungi. He is also regarded as father of medical mycology. Fungi are classified based on their morphology to four different categories: 1. Moulds (Molds): Filamentous (hyphae) fungi e.g: Aspergillus spp. 2. Yeasts: Single celled cells that buds e.g: Saccharomyces cerviciae 3. Yeast-like: Similar to yeasts but produce pseudohyphae e.g: Candida albicans 4. Dimorphic: Fungi existing in two different morphological forms at two different environmental conditions. They exist as yeasts in tissue and in vitro at 37°C and as moulds in their natural habitat and in vitro at room temperature. E.g: Histoplasma capsulatum. Fungal specimen collection depends on the site affected. Different specimens include hair, skin scrapings, nail clippings, sputum, blood, CSF, urine, corneal scraping, discharge or pus from lesions and biopsy. After the transportation of the specimen to the lab, samples can be either cultured or investigated via direct microscopy. One of the most common media used to culture fungi in laboratory is Sabouraud’s Dextrose Agar (SDA) along with Page 2 modified 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. Most fungi are able to grow at room temperature while few pathogenic fungi (e.g, Cryptococcus, dimorphic fungi) can grow at 37°C. Fungal inoculation represented in to main techniques: 1. For moulds, a piece of sample or agar with mycelial growth is removed with a sterile scalpel, inverted, and placed in a sterile petri dish. 2. For yeast, streaking technique is used for isolation of yeast. One of the basic techniques to observe fungal infection in the specimen is the direct microscopy using potassium hydroxide (KOH) wet mount preparation. This mainly used for specimens like skin hair and nail and some body fluids. Different stains can be also used to observe fungi in a specimen. Some of these stains are Gram stain and Lactophenol cotton blue (LPCB). In this practical, you will be introduced to two different fungi (mould and yeast) culture them and know how to stain them and observe under microscope. Principle of Lactophenol cotton blue Staining Lactophenol cotton blue is used in medical mycology for the examination of fungal cell structure, by preparing a wet mount. Its principle mainly rely on two important components: methyl blue and lactophenol. Methyl blue is a histological stain which stains collagen blue in tissue sections, and lactophenol is a mixture of four components; phenol, lactic acid, and glycerol in water. Where phenol helps to kill any microorganisms, lactic acid preserves fungal structures, and glycerol inhibits the cellulolytic activity of the fungus. Page 3 When preparing the microscopic slide, methyl blue stains the chitin in the fungal cell walls, as a bright cerulean color, and lactophenol acts as a mountant. PRACTICAL Part One: Culture of fungal specimen: MATERIALS: (Per pairs) - 1x Sabouraud’s Dextrose Agar (SDA) - 1x Loop PROCEDURE: - Each student should prepare a culture from yeast using streaking technique on SDA. - Record your observations of plate. Page 4 RESULTS : RECORD COLONY MORPHOLOGY OF MOULDS AND YEAST MORPHOLOGY YEAST MOULD Part Two: Staining of the fungi and LPCB staining MATERIALS: (per pairs) - 1x Glass slide PROCEDURE: - Prepare Gram stain slide. - Your instructor will demonstrate the mould inoculation techniques and the LPCB staining technique. - Observe both slides under the microscope. - Record your observations Page 5 RESULTS (WEEK 5) RECORD FUNGAL MORPHOLOGY Observation YEAST MOULD Page 6 The important characteristics in the identification of fungi are: Rate of growth Pigmentation Colony characteristics 1. Rate of growth Based on the rate of growth, fungi are grouped into three. a. Rapid growers: Produce colonies in less than 5 days. e.g: Saprophytic fungi b. Intermediate growers: Produce colonies in 6-10 days. e.g: Opportunistic fungi and dermatophytes c. Slow growers: Require 11 days or more, sometimes even up to 8 weeks to produce colonies. e.g: Systemic and subcutaneous fungi. 2. Pigmentation Pigmentation which is seen on the surface of the colony is referred to as surface pigmentation and on the reverse side of the colony reverse pigmentation. Page 7  Hyaline fungi: Colourless fungi.(non pigmented) Dematitious fungi: Produce dark brown to black or olive green colonies. 3. Colony characteristics a. Texture: Is defined as the way the colony looks. Texture is influenced by the length of aerial mycelium and the number of conidia of the spores produced. Page 8  Different descriptive terms are used for texture. i. Glaborous: Glaborous colonies appear leathery or waxy. They have very few aerial mycelium. ii. Velvety: Resemble velvet fabrics. They have short aerial hyphae. iii. Yeast like colony: They resemble colonies of Staphylococcus (Bacterial colony). Page 9 iv. Cottony colonies: Cottony colonies are produced when the fungus produce large quantities of aerial mycelium v. Granular colonies : Powdery colonies mostly produced by sporulating fungi. Page 10 b. Topography: Topography of a colony (surface morphology) is a way in which the colony surface is arranged. Different terms are used for different arrangements such as: i.Flat ii.Rugose: have radial grooves from centre to edge iii.Folded: Have random folds iv. Crateriform colonies: Have central depression surrounded by raised edges. v. Verrucose colonies: wart like or rough knobs vi. Cerebriform colonies: look like brain Rugose: have radial grooves from center to edge Cerebriform colonies: look like brain Page 11