Excercise4 Sample Answer Sheet MICROBIOLOGY LAB PRACTICAL Name Date 1.Specimen Number 2. Description of specimen Cultural Characteristics Microscopic and Staining Characteristics 5 Test Results(report results of relevant and applicable tests you chose to perform) 6. Presumptive identification(give genus and species) Tests requested for confirmation file://CWINDOWS/Desktop/clown/Microb Lab Web 2001/Exercise4.htm (2 of 3)[8/31/20027:48:07 PM
Sample Answer Sheet MICROBIOLOGY LAB PRACTICAL Name________________ Date ________________ 1. Specimen Number _______ 2. Description of specimen ______________________________________________________________ _______________________________________________________________ 3. Cultural Characteristics _______________________________________________________________ _______________________________________________________________ 4. Microscopic and Staining Characteristics _______________________________________________________________ _______________________________________________________________ _______________________________________________________________ 5. Test Results (report results of relevant and applicable tests you chose to perform) ______________________________________________________________ ______________________________________________________________ 6. Presumptive identification (give genus and species) _______________________________________________________________ 7. Tests requested for confirmation ________________________________________________________________ ______________________________________________________________ _______________________________________________________________ Excercise4 file:///C|/WINDOWS/Desktop/clown/Microb Lab Web 2001/Exercise4.htm (2 of 3) [8/31/2002 7:48:07 PM]
Cover EXERCISE #5 DERMATOPHYTES Exercisel Examination of Dermatophytes,Selective and Differential Media,and Clinical Specimens Exercise2 Focus: Exercise3 Identification of dermatophytes by colony and microscopic morphology,and Exercise4 dermatophyte culture medium Exercise5 Exercise6 Superficial Dermatophytoses The dermatophytes comprise a special group of fungi(about 30 species)capable of Exercise7 degrading and utilizing keratin(nonliving tissue)found inskin hair and nails.These Clinical fungi are classified into three genera based on their microscopic appearance and include case studies the Microsporum,Trichophyton,and Epidermophyton.Infections with these microorganisms involve the colonization of the keratinized layers of the skin,hair,and Suggestions nails without invasion of living tissue,and these are referred to as tinea capitis(scalp), tinea corporis(ringworm-like infections of trunk,legs,and arms),tinea cruris(groin infection),tinea unguium(nail infection),and tinea pedis(foot infection).The severity of these superficial diseases depends on the strain or species of dermatophyte and the sensitivity of the host to the particular fungus The majority of tinea pedis infections are caused by Trichophyton mentagrophytes. Trichophyton rubrum,and Epidermophyton floccosum which are anthropophilic species (spread from man to man).Other dermatophytes that are endemic to specific geographic areas (Trichophyton tonsurans and Microsporum cnis,.)may be involved in etiology of a small percentage of cases(<10%).The infection frequently starts in the toe webs and soles,and results in development of lesions that may be mild or chronic and scaling Acute spreading lesions with vesicles and inflammation presenting as erythematous painful irritations(rash)may also occur as well as inapparent infection without lesions and inflammation.Transmission of tinea pedis is apparently enhanced under conditions of repeated exposures to desquamated skin contaminated with dermatophytes especially on floors of locker rooms and showers.In addition,moisture and warmth and anatomical problems that occur between the toes when shoes are worn predispose the individual to infections. Tinea corporis is a cutaneous disease that can involve the legs,arms,chest,and back The infection characteristically spreads in a circular fashion from the initial site and produces rings of inflammation.Spontaneous healing occurs at the center of the scaling lesions and the fungus is usually demonstrable in the advancing margin.T.rubrum and T.mentagrophytes are consistently and frequently isolated as etiologic agents but zoophilic species(contacted from animals)such as M.canis and T.verrucosum are occasionally implicated in such infections.Microsporum gypseum is an example of a geophilic species(contacted from soil)that can cause tinea corporis p/clown/Microb Lab Web 2001/Exercise5/E 5.htm(1of4)[8/31/20024:30:55PM
Cover Exercise1 Exercise2 Exercise3 Exercise4 Exercise5 Exercise6 Exercise7 Clinical case studies Suggestions Hit Counter EXERCISE #5 DERMATOPHYTES Examination of Dermatophytes, Selective and Differential Media, and Clinical Specimens Focus: Identification of dermatophytes by colony and microscopic morphology, and dermatophyte culture medium Superficial Dermatophytoses The dermatophytes comprise a special group of fungi (about 30 species) capable of degrading and utilizing keratin (nonliving tissue) found in skin, hair and nails. These fungi are classified into three genera based on their microscopic appearance and include the Microsporum, Trichophyton, and Epidermophyton. Infections with these microorganisms involve the colonization of the keratinized layers of the skin, hair, and nails without invasion of living tissue, and these are referred to as tinea capitis (scalp), tinea corporis (ringworm-like infections of trunk, legs, and arms), tinea cruris (groin infection), tinea unguium (nail infection), and tinea pedis (foot infection). The severity of these superficial diseases depends on the strain or species of dermatophyte and the sensitivity of the host to the particular fungus. The majority of tinea pedis infections are caused by Trichophyton mentagrophytes, Trichophyton rubrum, and Epidermophyton floccosum which are anthropophilic species (spread from man to man). Other dermatophytes that are endemic to specific geographic areas (Trichophyton tonsurans and Microsporum canis, e.g.) may be involved in etiology of a small percentage of cases (<10%). The infection frequently starts in the toe webs and soles, and results in development of lesions that may be mild or chronic and scaling. Acute spreading lesions with vesicles and inflammation presenting as erythematous painful irritations (rash) may also occur as well as inapparent infection without lesions and inflammation. Transmission of tinea pedis is apparently enhanced under conditions of repeated exposures to desquamated skin contaminated with dermatophytes especially on floors of locker rooms and showers. In addition, moisture and warmth and anatomical problems that occur between the toes when shoes are worn predispose the individual to infections. Tinea corporis is a cutaneous disease that can involve the legs, arms, chest, and back. The infection characteristically spreads in a circular fashion from the initial site and produces rings of inflammation. Spontaneous healing occurs at the center of the scaling lesions and the fungus is usually demonstrable in the advancing margin. T. rubrum and T. mentagrophytes are consistently and frequently isolated as etiologic agents but zoophilic species (contacted from animals) such as M. canis and T. verrucosum are occasionally implicated in such infections. Microsporum gypseum is an example of a geophilic species (contacted from soil) that can cause tinea corporis. Exercise 5 file:///C|/WINDOWS/Desktop/clown/Microb Lab Web 2001/Exercise5/Exercise5.htm (1 of 4) [8/31/2002 4:30:55 PM]
Exercise 5 The dermatophytes may differ in their origin of infection(body area invaded)but they are similar in their mode of skin invasion.A microscopic examination of skin scrapings may reveal mycelium and arthrospores,thus indicating a fungus infection without differentiating between the organisms.For specific identification the lesion must be cultured and the fungus identifiedon the basis of the type of spore and pigmentation Most of these infections respond to topical treatment but oral griseofulvin and ketoconazole are used for persistent cases Structure of Dermatophytes The dermatophytes are rather ubiquitous microorganisms with some species being widespread throughout the world and others being limited to certain geographic and climatic areas.These fungi are usually found in soil or in association with animals and man.Different species may produce different clinical types of disease or diseases of different severity.Zoophilic and geophilic strains of dermatophytes generally produce more inflammation than anthropophilic strains. The cultural characteristics of dermatophytes are quite similar with many species Mi otee ot macroconidia which are rough,thick-walled,spindle-shaped,and abundant in the genus.The infrequent to rare appearance of smooth,thin-walled.cigar- shaped or distorted macroconidia exemplifies the Trichophyton genus,whereas Epidermophyton floccosum produces thin walled,club-shaped macroconidia usually in small bunches.The absence of microconidia and production of abundant chlamydospores further characterized E.floccosum,whereas various amounts of round to rodlike microconidia aids in identification of Trichophyton species Further characterization includes the fact that Microspo species donot infect nails (tinea unguium being primarily caused by T.rubrum,T.mentagrophytes,and E floccosum)and Epidermophyton does not infect hair(tinea capitis being primarily caused by M.audouinii,T.tonsurans,and M.canis in the U.S.). Examine and record the various macroscopic characteristics of each dermatophyte culture and prepare lactophenol cotton blue mounts for microscopic examination using the following tease prep procedure: 1.Aseptically remove some of the fungal growth with a teasing needle(dissecting pins) and place this sample on a clean microscope slide. 2.Add 1 to 2 drops of lactophenol cotton blue stain(LPCB)to the sample and tease or break it apart.Ideally,you should gently breakup one mycelial mat with the dissecting probes into several smaller pieces. 3.Place a cover slip over the preparation and examine it with your microscope using the high dry (40x)objective. 4.Please note that at times you may have to make more than one LPCB mount before you obtain a good representative slide of any particular fungus file://C/WINDOWS/Desktop/clown/Microb Lab Web 2001/Exercise5/Ex se5.htm(2of4)[83120024:30:55PM
The dermatophytes may differ in their origin of infection (body area invaded) but they are similar in their mode of skin invasion. A microscopic examination of skin scrapings may reveal mycelium and arthrospores, thus indicating a fungus infection without differentiating between the organisms. For specific identification the lesion must be cultured and the fungus identified on the basis of the type of spore and pigmentation. Most of these infections respond to topical treatment but oral griseofulvin and ketoconazole are used for persistent cases. Structure of Dermatophytes The dermatophytes are rather ubiquitous microorganisms with some species being widespread throughout the world and others being limited to certain geographic and climatic areas. These fungi are usually found in soil or in association with animals and man. Different species may produce different clinical types of disease or diseases of different severity. Zoophilic and geophilic strains of dermatophytes generally produce more inflammation than anthropophilic strains. The cultural characteristics of dermatophytes are quite similar with many species presenting as white and fluffy type molds on Sabouraud dextrose medium. Microscopically, the 3 genera can be distinguished based on the appearance of macroconidia which are rough, thick-walled, spindle-shaped, and abundant in the Microsporum genus. The infrequent to rare appearance of smooth, thin-walled, cigarshaped or distorted macroconidia exemplifies the Trichophyton genus, whereas Epidermophyton floccosum produces thin walled, club-shaped macroconidia usually in small bunches. The absence of microconidia and production of abundant chlamydospores further characterized E. floccosum, whereas various amounts of round to rodlike microconidia aids in identification of Trichophyton species. Further characterization includes the fact that Microsporum species do not infect nails (tinea unguium being primarily caused by T. rubrum, T. mentagrophytes, and E. floccosum) and Epidermophyton does not infect hair (tinea capitis being primarily caused by M. audouinii, T. tonsurans, and M. canis in the U.S.). Examine and record the various macroscopic characteristics of each dermatophyte culture and prepare lactophenol cotton blue mounts for microscopic examination using the following tease prep procedure: 1. Aseptically remove some of the fungal growth with a teasing needle (dissecting pins) and place this sample on a clean microscope slide. 2. Add 1 to 2 drops of lactophenol cotton blue stain (LPCB) to the sample and tease or break it apart. Ideally, you should gently breakup one mycelial mat with the dissecting probes into several smaller pieces. 3. Place a cover slip over the preparation and examine it with your microscope using the high dry (40x) objective. 4. Please note that at times you may have to make more than one LPCB mount before you obtain a good representative slide of any particular fungus. Exercise 5 file:///C|/WINDOWS/Desktop/clown/Microb Lab Web 2001/Exercise5/Exercise5.htm (2 of 4) [8/31/2002 4:30:55 PM]
Exercise 5 5.Since the student will be expected to identify each microorganism by its genus and species name,a record of the macroscopic and microscopic characteristics should be kept for every fungal culture.Permanent LPCB mounts can be made by encircling the cover slips with clear nail polish and allowing thes to air dry.Howe er,the studen must be sure that their LPCB preparations are good representative slides.Therefore,each used as a reference for descriptions and photographs of the various fungal cultures. Microsporum gypseum Microsporum canis Trichophyton rubrum Trichophyton mentagrophytes Trichophyton tonsurans Epidermophyton floccosum Identification of Dermatophytes on Special Media Dermatophyte Test Medium 1 Dermatophyte Test Medium(DTM)is a color indicator medium for the selective growth of dermatophytes.DTM contains the antibiotics,cycloheximide,gentamicin sulfate,and chlortetracycline-HCl,which inhibit the growth of most saprophytic fungi and bacteria.Only dermatophytes should grow on DTM and produce alkaline by products which cause a change in medium color (from yellow to red). 2. The presence of a dermatophyte in clinical specimens is noted by a color change in DTM after 24 to 72 hours of growth.Full color change may require up to 7 days,bu development of red color after 14 days of growth should not be considered significant. 3 Microorganisms such as Candida albicans,Pseudomonas aeruginosa,and Pseudallescheria boydii have been found to grow on DTM and produce a red color. Cultural and microscopic characterization of any growth on DTM is thus required for presumptive identification of the etiologic agent. 4.DTM agar slants may be available for student inoculation with one of the various dermatophytes Clinical Fungal Specimens and Cultures The following procedure should be used clinically for the microscopic examination of infected skin scrapings and nails(KOH procedure) clown/Microb Lab Web 2001/Exercise5/E e5.htm(3of4)[8/31/20024:30:55PM
5. Since the student will be expected to identify each microorganism by its genus and species name, a record of the macroscopic and microscopic characteristics should be kept for every fungal culture. Permanent LPCB mounts can be made by encircling the cover slips with clear nail polish and allowing these to air dry. However, the student must be sure that their LPCB preparations are good representative slides. Therefore, each preparation should be examined microscopically prior to making permanent LPCB slides. The Medical Mycology Manual can be used as a reference for descriptions and photographs of the various fungal cultures. Microsporum gypseum Microsporum canis Trichophyton rubrum Trichophyton mentagrophytes Trichophyton tonsurans Epidermophyton floccosum Identification of Dermatophytes on Special Media Dermatophyte Test Medium 1. Dermatophyte Test Medium (DTM) is a color indicator medium for the selective growth of dermatophytes. DTM contains the antibiotics, cycloheximide, gentamicin sulfate, and chlortetracycline-HCl, which inhibit the growth of most saprophytic fungi and bacteria. Only dermatophytes should grow on DTM and produce alkaline by products which cause a change in medium color (from yellow to red). 2. The presence of a dermatophyte in clinical specimens is noted by a color change in DTM after 24 to 72 hours of growth. Full color change may require up to 7 days, but development of red color after 14 days of growth should not be considered significant. 3. Microorganisms such as Candida albicans, Pseudomonas aeruginosa, and Pseudallescheria boydii have been found to grow on DTM and produce a red color. Cultural and microscopic characterization of any growth on DTM is thus required for presumptive identification of the etiologic agent. 4. DTM agar slants may be available for student inoculation with one of the various dermatophytes. Clinical Fungal Specimens and Cultures The following procedure should be used clinically for the microscopic examination of infected skin scrapings and nails (KOH procedure). Exercise 5 file:///C|/WINDOWS/Desktop/clown/Microb Lab Web 2001/Exercise5/Exercise5.htm (3 of 4) [8/31/2002 4:30:55 PM]
Exercise 5 1 Prior to obtaining a specimen,thoroughly sponge the affected site with 70% alcohol to remove surface contamination and medication. 2.After the alcohol has evaporated,scrape the active edge of a lesion or the top of a vesicle with a sterile(flame-sterilized)scalpel.Skin scales or nail scrapings may be placed in a sterile paper envelope(or Petri dish)until microscopic mounts can be made 3.Mount part of the specimen on a clean microscope slide in a drop of 10-20% potassium hydroxide(KOH)Apply a coverslip and allow to stand at room temperature for 5 to 10 minutes.The student may gently heat the preparation over a low flame but overheating may distort fungi (unnecessary for skin scrapings). 4.Examine the specimen microscopically using the low-dry and high-dry objectives. The KOH digests cellular components more rapidly than fungi and thus clears the specimen leaving fungal elements more clearly visible.Fungi should appear as fragments of mycelia with or without branches and septa(conidiospores may be present in some instances). Perform the KOH procedure on clinical skin and nail scrapings,which may be available in the lab module,as described above. file:///CJ/WINDOWS/Desktop/clown/Microb Lab Web 2001/Exercise5/E 5.htm(4of4④831/20024：30：55P
1. Prior to obtaining a specimen, thoroughly sponge the affected site with 70% alcohol to remove surface contamination and medication. 2. After the alcohol has evaporated, scrape the active edge of a lesion or the top of a vesicle with a sterile (flame-sterilized) scalpel. Skin scales or nail scrapings may be placed in a sterile paper envelope (or Petri dish) until microscopic mounts can be made. 3. Mount part of the specimen on a clean microscope slide in a drop of 10-20% potassium hydroxide (KOH). Apply a coverslip and allow to stand at room temperature for 5 to 10 minutes. The student may gently heat the preparation over a low flame but overheating may distort fungi (unnecessary for skin scrapings). 4. Examine the specimen microscopically using the low-dry and high-dry objectives. The KOH digests cellular components more rapidly than fungi and thus clears the specimen leaving fungal elements more clearly visible. Fungi should appear as fragments of mycelia with or without branches and septa (conidiospores may be present in some instances). Perform the KOH procedure on clinical skin and nail scrapings, which may be available in the lab module, as described above. Exercise 5 file:///C|/WINDOWS/Desktop/clown/Microb Lab Web 2001/Exercise5/Exercise5.htm (4 of 4) [8/31/2002 4:30:55 PM]