DEGEE O. GONZALES,R.M.T. College of Med. Tech.

A battery of culture media should be used for optimal recovery of fungi in clinical specimens
Addition of enhancement supplements in culture media for fungal recovery

1.

Media with or without CYCLOHEXIMIDE (0.5mg/ml)

1.1 Cycloheximide

- a.k.a ACTIDIONE
- NOT an antibiotic for any treatment of the disease

Uses of Cycloheximide in Culture Media

used primarily in fungal culture dimorphic fungi are primarily recovered partially or completely inhibits the growth of rapidly growing molds and some fungi that are known to be pathogenic

Fungi Inhibited by Cycloheximide

Cryptococcus neoformans Candida krusei other Candida species Trichosporon cutaneum Pseudollescheria boydia Aspergillus species

2. Media with or without Antimicrobial Agents

2.1 Used for specimens that are likely to contain contaminating bacteria (UNSTERILE SPECIMENS) 2.2 NOT necessarily used on specimens from sterile sites

Gentamicin (5g/ml) + Chloramphenicol (16g/ml)

provides better results in inhibiting the growth of contaminating molds and bacteria inhibits growth of Pseudomonas

Ciprofloxacin (5g/ml)

3. Media with or without Blood Enrichment

3.1 Certain fungi seem to have a requirement for 5-10% sheeps blood

 Agar plates and Screwcapped tubes are SATISFACTORY culture media

1.

Agar Plates (Culture Dishes)

1.1 preferred culture media:
- provide better aeration of cultures - provide large surface area for isolation - easier to prepare microscopic mounts

1.2 DISADVANTAGES - easily contaminated - less safe to handle in the lab - apt to dry out unless deep pour plates (40ml of culture medium or more) are used

2.

Screw-capped Tubes
2.1 After inoculation, tubes are placed in a horizontal position for at least 1-2 hours to allow specimen to absorb to the agar surface 2.2 prevents growth of fungi at the bottom of the tube

ADVANTAGES

lower dehydration rate and less hazardous to handle because lids are kept tight easily stored since it require less space for incubation more easily to handle

DISADVANTAGES

poor isolation of colonies

reduced surface area for culturing promotes ANAEROBIOSIS which causes culture to become nonviable due to caps are tightened

 MEDIA FOR THE IDENTIFICATION AND ISOLATION OF FUNGI

 Cultures should be incubated at RT (25C) or PREFERRABLE at 30C for 21-30 days before reporting negative (-)
30C is the recommended incubation temperature because nearly all pathogenic fungi grow better and rapidly

 Humidity in the range of 40-50% can be achieved by placing an open pan of water in the incubator

Prevents the agar from drying out over time

 Fungal Cultures should be examined 3x weekly during incubation

 Often provide the first microbiologic proof of the etiology of disease of patients with fungal infection Most rapid method currently available for the detection of fungi in clinical specimens

1.

Gram stain
1.1 most commonly performed on most clinical specimens and will detect fungi, if present 1.2 Some fungi stain well however others show only stippling (e.g. C. neoformans) and stain weakly in some instances

2.

Periodic Acid Schiff (PAS)

2.1 stains fungal elements well, and hyphae of molds and yeasts can be readily distinguished 2.2 stains fungi red

2.3 Blastomyces dermatitidis appears pleomorphic

3.

Potassium hydroxide (KOH)

3.1 recommended traditionally for the direct microscopic examination of specimens using Bright Field Microscopy

3.2 acts as clearing agent since it dissolves human cellular elements and debris for easier visualization of fungal elements
3.3 used for opaque materials and scrapings from skin

4.

Calcoflour White Stain (CFW)

4.1 An industrial textile brightener, which nonspecifically binds to chitin and other elements in the fungal cell wall 4.2 believed to be superior over that of KOH since slides may be observed using Fluorescent Microscopy

4.3 CW fluorescence occurs maximally at an excitation wavelength of 440 nm 4.4 Fungal elements will fluoresce blue-white stain

5.

India Ink
5.1 visualization of C. neoformans in CSF because of its large polysaccharide capsule 5.2 A drop of CSF sediment is mixed with one-third volume of India ink until a smooth suspension is obtained

5.3 A coverslip is applied to the drop and the preparation is examined under HPO (400x) for characteristic encapsulated cells which can be confirmed under OIO 5.2 Positive in less than 50% of cases of meningitis and not reliable in non-HIV infected patients

6. Lactophenol Cotton Blue (LPCB)

6.1 stain used for microscopic mounts 6.2 contains lactic acid (used to preserve fungal structures); phenol (acts as killing agent); and cotton blue (stains the fungi blue)

7. Molecular Detection Methods

7.1 becoming popular in all areas of Clinical microbiology 7.2 None of the methods has been accepted as a routine diagnostic tool in clinical mycology

1.

Wet Mount
1.1 suitable method for observing the presence of fungal spores but the characteristic arrangement of spores are not determined since spores are disrupted when pressure is applied to the coverslip 1.2 method not adequate in making a definitive ID

Procedure:
1. A small portion of an isolated colony containing a small amount of the supporting agar is cut out using a wire bent at a 90-degree angle
1.1 Portion should be removed from a point intermediate between the center and the periphery

2. The portion is placed onto a slide to which a drop of LPCB has been added 3. A coverslip is placed into position and a gentle pressure is applied using a pencil eraser is applied to disperse the growth and the agar. 4. Examine microscopically

2.

Scotch (Cellophane) Tape Preparation

2.1 Traditionally used by most laboratories since it can be prepared easily and quickly and often is sufficient to make the ID for most of the fungi since spores are usually intact

2.2 Allows one to observe the fungus microscopically approximately the way it sporulates in culture 2.3 Allows observation for the characteristic shape and morphology of the spores

Procedure:

1. Touch the adhesive side of a small length of transparent tape to the surface of the colony

2. Adhere the length of tape to the surface of a microscope slide to which a drop of LPCB has been added
3. Observe microscopically

3.

Slide Culture
3.1Used when greater detail of the morphologic features is required for identification
3.2 Ideal for making a definitive ID of a fungus since its undisturbed morphology is demonstrated

Procedure:
1. Cut a small block of a suitable agar medium that has been previously poured into a culture to a depth of approximately 2mm using a sterile scalpel blade or with a sterile test tube that has no lip (produces a round block)

2. In a separate culture dish, place a sterile filter paper or paper towel moistened with a sterile paper at the bottom.

3. Add two applicator sticks and position the microscope slide on top. Add the agar block to the surface of the microscope slide.

4. Inoculate the four quadrants of the agar plug with the organism using a right-angle wire. 5. Apply a sterile coverslip onto the surface of the agar plug and incubate at 30C for 5-10 days

6. After the incubation period, observe microscopically.

3.4 DO NOT MAKE SLIDE CULTURES of Histoplasma, Coccidioides, Blastomyces, Paracocciodioides, and Sporothrix because it is TOO HAZARDOUS

 The question of WHEN AND HOW far to go with the identification of Fungi recovered from clinical specimens

 Ideally , ALL laboratories SHOULD identify and REPORT ALL FUNGI recovered from clinical specimens so that their clinical significance can be determined

Factors Affecting in the ID of Fungi in Clinical Specimens

The limits of practicality and economic considerations (cost-containment) Ever-increasing number of opportunistic fungi causing infection in compromised patient

Basis in Decision-Making of How Far to go with the ID

Patient Population Laboratory Practice

Economic Impact

 Extent of Identification of Yeasts Recovered from Respiratory Specimens of Compromised Patients

- Respiratory Specimens are the most commonly submitted specimens for fungal culture

1. Routine ID of yeasts recovered in culture from respiratory secretions is not warranted, but all yeasts should be screened for the presence of Cryptococcus neoformans.

2. All respiratory secretions submitted for fungal culture, regardless of the presence or absence of oropharyngeal contamination, should be cultured because common pathogens may be recovered such as Histoplasma capsulatum, Blastomyces dermatitidis, Coccidoides immitis, and Sporothrix schenckii

3. Routine ID of yeasts in respiratory secretions is of little or no value to the clinician and probably represents normal flora except for Cryptococcus neoformans.

 Extent of Identification of Molds

1. All commonly encountered molds should be identified and reported if recovered from patients at risk for invasive fungal disease

- In immunocompromised patients, organisms fail to sporulate after a reasonable time should be reported as being present but the ID need not to be attempted if the dimorphic fungi have been ruled out or if the clinician believes that the organism is not clinically significant

 Identification of Molds is made using a COMBINATION of the following:

The Growth Rate Colonial Morphologic Features Microscopic Morphologic Features

1.

Growth Rate
1.1 Determination can be the most helpful when examining a mold culture
1.2 Of limited value since growth rate of certain fungi is variable, depending on the amount of inoculum present in a clinical specimen

1.3 Dimorphic fungi are slow, 1-4 weeks are usually required before colonies become visible 1.4 In some instances, some dimorphic fungi (B. dermatitidis, H. capsulatum; and C. immitis) may be detected within 3-5 days when large numbers of organisms are present in the specimen

1.5 colonies of Zygomycetes may appear within 24 hours 1.6 other hyaline and dematiaceous fungi often exhibit growth within 1-5 days

2. Colonial Morphologic Features

2.1 colonial morphology is variable among molds because of natural variation among isolates and colonies grown on different culture media

2.2 considered an unreliable criterion and should be used only to supplement the microscopic morphologic features since incubation conditions and culture media should be considered

2.3 includes color, size, texture, and topography of the colony

3.

Microscopic Morphologic Features

3.1 provides the most definitive means for identification 3.2 microscopic morphologic features of molds are stable and exhibit minimal variation

3.3 Definitive identification is based on the characteristic shape, method of reproduction, and arrangement of spores

 A Common sense approach concerning the handling of fungi to protect the lab from contamination and workers from becoming infected

 All mold cultures and clinical specimens must be handled in a CLASS II BIOLOGICAL SAFETY CABINET with NO EXCEPTION

 Cultures of organisms suspected of being pathogens should be sealed with tape (Oxygenpermeable Tape) to prevent lab contamination and should be autoclaved as soon as definitive ID is made

 Hyphae
- Helpful in placing an organism into a certain group

1. Antler Hyphae
curved, freely branching, and antlerlike in appearance

2. Racquet Hyphae
Enlarged, clubshaped structures

3. Spiral Hyphae
- Coiled or exhibits corkscrew-like turns seen within the hyphal strand

 Hyphae
- Antler, Racquet, and Spiral Hyphae are found most commonly in Dermatophytes

 Spores
- Produced by species of fungi and can either be sexual or asexual

1. Sexual Spores
- associated with formation of specialized structures that facilitate fertilization and nuclear fusion in the production of specialized spores

1.1 Ascospores
produced in a large saclike structure called ASCOCARP Ascoscarp contains smaller sacs called ASCI Each asci contains 4-8 ascospores

 Uncommonly seen in the fungi recovered in clinical microbiology laboratory

May not have been observed yet on artificial culture media

1.2 Zygospores
Rough-walled spores produced as a result of union of 2 matching types of Zygomycetes

2. Asexual Spores
- Primary means for the identification of molds is by characterization of asexual reproductive structures

Conidia
Represent the asexual reproductive cycle and are produced by most fungi
Type of conidia, their morphology, and arrangement are important criteria for establishing definitive ID of fungi

 The simplest type of sporulation is the development of a spore directly from the vegetative hyphae: Arthroconidia Chlamydoconidia

2.1 Arthroconidia
Formed directly from the hyphae by fragmentation through points of septation

When mature, appear as square, rectangular, barrelshaped, thick walls

 Results from the simple fragmentation of the hyphae into spores which are easily dislodged and disseminated into the environment

2.2 Chlamydoconidia
A.k.a. Chlamydospores Round, thick-walled spores formed directly from the differentiation of hyphae in which there is a concentration of protoplasm and nutrient material

 Appear to be resistant resting spores produced by rounding up and enlargement of the terminal cells of the hyphae

May be INTERCALARY (within the hyphae) or TERMINAL (end of hyphae)

 Conidia are asexual spores produced singly or in groups by specialized hyphal strands called CONIDIOPHORES:

Aspergillus Genus Acremonium Penicillus

 In some instances, the conidia are freed from their point of attachment by pinching off or by abstriction

2.3 Aspergillus Fruiting Structure

Conidiophores terminate in a swollen vesicle From the surface of the vesicle are formed secondary small, flask shaped PHIALIDES, which in turn, give rise to long chains of Conidia

2.4 Penicillus Structure

Conidiophores form a branching structure termed as Penicillus

Each branch terminates in secondary branches (METULAE), and Phialides from which chains of conidia are borne

 Representative fungi: Penicillium and Paecilomyces

2.5 Single, simple, slender, tubular conidiophore (Phialide) that produces a cluster of conidia held together as a gelatinous mass
Characteristic of a certain fungi including the Genus Acremonium

2.6 Sporangiospores
Spores produced within the SPORANGIUM, a saclike structure produced at the tip of a long stalk (SPORANGIOPHORE)
Sporulation takes place by progressive cleavage during maturation within the Sporangium

 Spores are released by the rupture of the sporangial wall Observed in Zygomycetes

 In other instances, fungi may produce conidia of two sizes: Microconidia Macroconidia
- both are seen in some fungal structures and are not specific; may be used to differentiate a limited number of genera

2.7 Microconidia
Small, unicellular, round, elliptical, or pyriform in shape Borne directly on the side of a hyphal strand or at the end of a conidiophore

2.8 Macroconidia
Large, usually multiseptate, and club- or spindle shaped Usually borne on short to long conidiophore and may be smooth or rough walled