Laboratory Manual for Mycology PDF

Preview

Summary

The material in this manual is designed to be a supplement to the textbook, Bailey and Scott’s Diagnostic Microbiology, 14th Edition Mycology section...

Description

Tille: Bailey & Scott’s Diagnostic Microbiology, 14th Edition Laboratory Manual—Part D The material in this manual is designed to be a supplement to the textbook, Bailey and Scott’s Diagnostic Microbiology, 14th Edition.

PART V: Mycology This section of exercises in mycology is designed to accomplish three major objectives. The first objective, which is addressed in the first exercise, is to provide the opportunity to learn and practice various laboratory techniques required to identify clinically significant yeast and molds. The exercises that follow are intended to guide you in accomplishing the second objective, which is recognizing the macroscopic and microscopic morphologic characteristics of some of the common fungal saprophytes, opportunists, and pathogens. When you are able to recognize the diagnostic features of the fungi, you will have accomplished the third objective, which is to combine newly acquired skills and knowledge to process clinical specimens and to recover and identify fungi that may be present in sputum, skin, tissue, and other types of specimens. Safety in the Mycology Laboratory Inhaling aerosols containing conidia and the accidental inoculation from contaminated sharps (e.g., needles, broken glass) are the two most common ways to acquire a fungal infection in the laboratory. The use of a biological laminar flow safety cabinet is essential when working with mold cultures. Be especially cautious with any rapid-growing, white, fluffy fungus; it may be the systemic pathogen Coccidioides immitis. Slow-growing molds should also be handled with caution; they also may be systemic pathogens. All standard safety guidelines for the microbiology laboratory should also be implemented when working through these exercises.

Laboratory 27: Basic Techniques in Mycology Objectives: 1. Prepare and examine a cellophane tape prep and a tease mount of a mold culture to determine the morphologic characteristics of conidiophores and conidia. 2. Prepare and examine a slide culture of a mold specimen to determine the morphologic characteristics of conidiophores and conidia. 3. Prepare and examine a wet prep from a yeast or yeast-like culture to determine the morphologic characteristics of the blastoconidia or yeast cells. 4. Inoculate agar slants or plates with mold specimens. Reference: Bailey and Scott’s Diagnostic Microbiology, 14th Edition. Chapter 58: Overview of Fungal Identification Methods and Strategies. Methods for Direct Microscopic Examination of Fungal Specimens Any specimen submitted for fungal culture may be examined microscopically for fungal elements. This examination is made in addition to, not instead of, a culture. As well as providing

Laboratory Manual—Part D

2

the physician with early information regarding the possible need for treatment, it may be helpful in determining the significance of the organism that will later be identified on culture. The microscopic examination of a fungus should be completed when the culture first begins to grow and develop conidia or spores and again 48 to 72 hours later. In many instances, the microscopic morphology of conidiation or sporulation is important to the identification of the fungus and may be obscured in old cultures. Potato flake or potato dextrose agar (PDA) typically promotes conidiation better than Sabouraud dextrose agar (SDA). There are multiple methods to examine the microscopic morphology of a fungus. The cellophane tape preparation utilizes a small piece of cellophane tape to touch the fuzzy or mold looking growth of a fungal culture. (Bailey and Scott’s Diagnostic Microbiology Procedure 58-2) A tease prep is a procedure that utilizes two wires to remove some of the fungal colony from a culture plate. The colony is then placed on a microscope slide, stained, cover-slipped, and viewed using a bright-field microscope. (Bailey and Scott’s Diagnostic Microbiology Procedure 58-4)

Tease Mount Preparation on the Microscope Slide. The third method is a basic wet mount from a culture. This is simply using a colony that may appear yeast like, and applying it to saline, adding stain, cover-slipping, and observing under the microscope. (Bailey and Scott’s Diagnostic Microbiology Procedure 58-3) Finally, a Microslide culture may be used to examine the fungus. This technique uses a specialized commercially prepared plate that has a glass coverslip placed over an agar block or maybe manually set-up in the microbiology laboratory. The coverslip is removed from the agar block that contains the fungal growth and is placed on a slide for staining and viewing the microscopic morphology. (Bailey and Scott’s Diagnostic Microbiology Procedure 58-5)

Microslide Manual Materials

Copyright © 2017, Elsevier Inc. All Rights Reserved.

3

Laboratory Manual—Part D

Materials: 4-day-old Candida albicans culture on a rice extract-polysorbate (Tween) 80 or cornmeal-Tween 80 yeast agar plate 4-day-old culture of Aspergillus spp. or Penicillium spp. on potato dextrose agar 4-day-old culture of Aspergillus spp. or Penicillium spp. on a microslide culture plate Microscope slides Clear cellophane tape Cover slips Inoculating needles and loop Lactophenol cotton blue or Calcofluor white 0.85% sterile saline. Procedure Laboratory Report and Study Guide

Name: _____________________

In this laboratory exercise, the procedure, report, and study guide are combined to enhance the process for the identification and performance of laboratory identification procedures. Microscopic Identification of Yeast and Molds 1. Wet mount (Bailey and Scotts Procedure 58-3) Obj. 3 Complete a wet mount from the Candida albicans culture provided by the instructor. Utilize lactophenol cotton blue (bright field microscopy) or calcofluor white (fluorescent microscopy) if available. Draw and label yeast cells, budding yeast, and/or hyphal or pseudohyphae present in the wet mount. Observe preparation using both the 10 (low objective lens) and high dry objective. 10 Magnification High Dry Magnification

10 points 2. Cellophane Tape Mount (Bailey and Scott’s Procedure 58-2) and Tease Mount (Bailey and Scott’s Procedure 58-4). Obj. 1 Using the 4-day-old culture of Aspergillus spp. or Penicillium spp. on potato dextrose agar provided by the instructor complete a cellophane tape and a tease mount preparation. Using your textbook, draw and label the microscopic morphologic features of each of the following fungi. Aspergillus sp. Penicillium sp. Septate hyphae Conidiophore Conidia

Copyright © 2017, Elsevier Inc. All Rights Reserved.

4

Laboratory Manual—Part D Phialide Vesicle Penicillus 12 points Draw and label the fungal elements present in the cellophane tape and tease mount. Observe the preparations using both the 10 and high dry objective. (Obj. 1) Cellophane Tape Preparation 10  Magnification High Dry Magnification

Tease Mount Preparation 10  Magnification

High Dry Magnification

20 points 3. Microslide Culture (Bailey and Scott’s Procedure 58-5) Obj. 2 Using the 4-day-old culture of Aspergillus spp. or Penicillium spp. on a microslide culture plate provided by the instructor, complete the microscopic examination of the isolate. Draw and label the microscopic morphologic structure of the mold growing on the slide culture of Aspergillus sp. or Penicillium sp. in the space provided. (5 points) Is there a significant difference in the morphologies observed using the cellophane tape, tease mount, or microslide culture preparation? (5 points) 4. Agar Inoculation Obj. 4 Using the 4-day-old culture of Aspergillus spp. or Penicillium spp. on potato dextrose agar, you will practice inoculating an agar slant and agar plate with a fungal isolate. 1. Label the SDA slant and the SDA agar plate provided by the instructor. 2. Select a portion of the colony that appears granular or powdery. This will provide the best chance of obtaining conidia for further growth and propagation. 3. Remove a small piece of the colony, approximately the size of a pinhead, with a teasing needle. Avoid picking up any excess agar. 4. Place the piece of mold in the center of the agar surface on the slant. 5. Loosely replace the tube cap to provide air circulation around the growing colony. 6. Repeat steps 2 and 3 using an inoculating loop, stab the agar plate midway between the center of the plate and the outer rim of the petri dish. Repeat this procedure three times as indicated in the diagram. Stabbing the agar instead of streaking a plate as in bacteriology provides a means to clearly separate fungal growth and visualization of the colony morphology and any reverse morphologic characteristics that are relevant to the identification of the fungus.

2 1

Inc. All Rights Reserved.

3 5

Laboratory Manual—

Stabs in the agar that would ensure separate colonial growth of a mold. Each number represents an individual agar inoculation point. 7. Incubate the tube and agar plate at room temperature or 30C and observe daily for growth. Allow the subcultures to grow for 4-5 days. Compare the original culture with the subculture of Aspergillus sp. or Penicillium sp. Original Subculture SDA Slant Subculture SDA Plate Medium Age Color Texture Topography 15 points Grading Rubric General Laboratory Safety Uses appropriate laboratory safety and sterile technique Uses proper personal protective equipment Disposes of all biohazard/waste materials properly Laboratory Report and Study Guide Wet mount (10 points) Cellophane tape and tease mount (32 points) Microslide culture (10 points) Subculture inoculations (15 points) Total Possible Student Score

15 points

67 points

82 points

Laboratory 28: Identification of Common Opportunistic and Saprophytic Mycoses Objectives: 1. Perform, describe the method and principle for each technique and interpret (identify fungal isolates in) microscopic smears (actual, photographs, or digital images) of fungal specimens using the following techniques: wet-prep, cellophane tape, or tease mount using lactophenol cotton blue or calcofluor white.

Copyright © 2017, Elsevier Inc. All Rights Reserved.

Laboratory Manual—Part D

6

2. Differentiate and identify yeasts and molds from bacteria on routine media and using mycology media using appropriate morphologic descriptions related to the following:  Hyphae: Septate, aseptate, hyaline, dematiaceou  Mucorales: Sporangiophore, Sporangium, Sporangiospore, Stolons  Hyaline: Conidiophore, conidium, macroconidia, microconidia, vesicles, phialides  Texture: Glabrous, granular, velvety, cottony  Topography: Rugose, umbonate, verrucose, cerebriform. 3. Prepare and examine a cellophane tape prep and a tease mount of a mold culture to determine the morphologic characteristics of conidiophores and conidia. 4. Prepare and examine a slide culture of a mold specimen to determine the morphologic characteristics of conidiophores and conidia. 5. Describe and follow a systematic process for the identification of molds. Reference: Bailey and Scott’s Diagnostic Microbiology, 14th Edition. Chapter 58: Overview of Fungal Identification Methods and Strategies, Chapter 59: Hyaline Molds, Mucorales, Dermatophytes, and Opportunistic and Systemic Mycoses, Chapter 60: Dematiaceious (Melanized) Molds, and Chapter 61: Opportunistic Atypical Fungus: Pneumocystis jiroveci. Mucorales The mucorales (zygomycetes) characteristically produce large, ribbonlike hyphae that are irregular in diameter and contain occasional septa. Because the septa may not be apparent in some preparations, this group sometimes has been characterized as aseptate. The specific identification of these organisms is confirmed by observing the characteristic saclike fruiting structures (sporangia), which produce internally spherical, yellow, or brown spores (sporangiospores). Each sporangium is formed at the tip of a supporting structure (sporangiophore). During maturation, the sporangium becomes fractured and sporangiospores are released into the environment. Sporangiophores are usually connected to one another by occasionally septate hyphae called stolons, which attach at contact points where rootlike structures (rhizoids) may appear and anchor the organism to the agar surface. Identification of the mucorales is partly based on the presence or absence of rhizoids and the position of the rhizoids in relation to the sporangiophores. Entomophthorales The newly created phylum Entophythoromycota contains more than 250 species distributed worldwide. However, only four species have been identified as significant in clinical samples: Conidiobolus coronatus, C. lamprauges, C. incongruous, and Basidiobolus ranarum. Opportunistic and Saprophytic Mycoses The tissue-invasive opportunistic mycoses are a group of fungal infections that occur almost exclusively in immunocompromised patients. Opportunistic fungal infections are typically identified in a host compromised by some underlying disease process, such as lymphoma, leukemia, diabetes mellitus, or another defect of the immune system. Many patients, particularly those who undergo some type of transplantation, are often placed on treatment with corticosteroids, cytotoxic drugs, or other immunosuppressive agents to control rejection of the transplanted organ. Many fungi previously thought to be nonpathogenic are now recognized as etiologic agents of opportunistic fungal infections. Because most of the organisms known to cause infection in this

Copyright © 2017, Elsevier Inc. All Rights Reserved.

Laboratory Manual—Part D

7

group of patients are commonly encountered in the clinical laboratory as saprobes (saprophytic fungi), it may be impossible for the laboratorian to determine the clinical significance of these isolates recovered from clinical specimens. Therefore, laboratories must identify and report completely the presence of all fungi recovered, because each is a potential pathogen. Many of the organisms associated with opportunistic infections are acquired during construction, demolition, or remodeling of buildings or are hospital acquired. Other information about the specific clinical aspects of the opportunistic fungal infections is discussed with the individual organism. Identification of Opportunistic Molds Is the mold aseptate? If yes, consider the Mucroales. Are rhizoids present? If yes, where are they in relation to the sporangiophore? If rhizoids are prominent and directly beneath the sporangiophore, then check Rhizopus. If rhizoids are fine and hairlike between sporangiophores, then check Lichtheimia. If rhizoids are absent, then check Mucor. Is the mold septate? If yes, consider the groups that cause mycetoma, chromoblastomycosis, or phaeohyphomycosis. Is the mold dematiaceous? If yes, compare its conidiophores and conidia with illustrations of dematiaceous opportunists such as Bipolaris, Cladosporium, and Alternaria. Is the mold hyaline? If yes, compare its conidiophores and conidia with illustrations of hyaline opportunists such as Aspergillus, Penicillium, and Fusarium. What additional tests are needed to confirm the identity of the mold? Materials Microscope slides Clear cellophane tape Cover slips Inoculating needles and loop Lactophenol cotton blue or Calcofluor white 0.85% sterile saline. Four-day-old potato dextrose agar cultures (Petri dish or slants) of saprophyte molds, which should include but not be limited to the following:  Mucurales: Rhizopus spp., Mucor spp.  Dematiaceous: Alternaria spp., Bipolaris spp.  Hyaline: Aspergillus fumigatus, A. flavus, Penicillium spp., Scopulariopsis spp., Fusarium spp., Acremonium spp. Procedure

Copyright © 2017, Elsevier Inc. All Rights Reserved.

Laboratory Manual—Part D

8

Laboratory Report and Study Guide

Name: _____________________

In this laboratory exercise, the procedure, report, and study guide are combined to enhance the process for the identification and performance of laboratory identification procedures. 1. Utilizing your textbook complete the following table. Obj. 1 and 2. Microscopic Morphologic CHARACTERISTICS of Common Opportunistic and Saprophytic Mycoses Study Chart 28-1

Clinical Disease and Mode of Transmission

Culture: Growth Rate and Colony Morphologic Characteristic s

Microscopic Morphologic Characteristics

Mucorales & Hyaline Apophysomyces spp. Lichtheimia sp. Rhizomucor spp. Rhizopus sp. Mucor sp. Cunninghamella sp. Syncephalastrum sp. Entomophthorales Basiodiobolus sp. Conidiobolus sp. Hyaline Acremonium sp. Aspergillus flavus Aspergillus fumigatus Aspergillus niger Fusarium sp. Geotrichum sp. Paecilomyces sp. Purpureocillium sp. Penicillium sp. Copyright © 2017, Elsevier Inc. All Rights Reserved.

Identification Pathway and Notes

9

Laboratory Manual—Part D Scopulariopsis Dematiaceous Alternaria sp. Bipolaris sp. Curvularia sp. Misc. Pneumocystis jiroveci 92 points 2. Cellophane Tape Mount (Bailey and Scott’s Procedure 58-2) and Tease Mount (Bailey and Scott’s Procedure 58-4). Obj. 1-5 Obtain a culture of each of the following: aseptate mold, hyaline mold, dematiaceous mold, and an unknown culture. Record the fungus name (or unknown identification number), age, types of media, front and reverse pigmentation, and colony texture in the appropriate tables. Note whether the mold is hyaline or dematiaceous. Describe the front and reverse topographies of the mold. Prepare and examine a cellophane tape or tease mount of each culture. Draw and label the hyphae, spores, structures associated with spore formation, and any other features that are observable. Note: If the structures that you are observing do not resemble your reference materials, then you may be working with a contaminated culture and should consult your instructor. ASEPTATE MOLD Table 28-1 (25 points) Fungus name: ___________________________________ _______________________________ Age: ____________________ Pigmentation: Size and shape: ___________________________________ _____________________________ Texture: ___________________________________ __________________________________

Medium: _________________________________ Front: __________________________ Reverse: _______________________

Describe the topographies, front and reverse:

Copyright © 2017, Elsevier Inc. All Rights Reserved.

10

Laboratory Manual—Part D ________________________________________________________________________ ________________________________________________________________________ ________________________________________________________________________ Draw and label the hyphae, conidiophores, conidia, and/or other structures: This fungus may cause what diseases? ________________________________________________________________________ ________________________________________________________________________ ________________________________________________________________________ From what clinical specimens would this fungus be isolated? ________________________________________________________________________ ________________________________________________________________________ ________________________________________________________________________ HYALINE MOLD Table 28-2 (25 points) Fungus name: ___________________________________ _______________________________ Age: ____________________ Pigmentation: Size and shape: ___________________________________ _____________________________ Texture: ___________________________________ __________________________________

Medium: ___________________________________ Front: __________________________ Reverse: _______________________

Describe the topographies, front and reverse: ________________________________________________________________________ ________________________________________________________________________ ________________________________________________________________________ Draw ...