Lab Manual Volk-nelson 2011

7/18/2019 Lab Manual Volk-nelson 2011

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Laboratory Manualfor

Medical Mycology

Based on a Lab Manual by Dr. Allen C. Nelson,Professor at University of Wisconsin-La Crosse !""-!!"

Modified by #o$ %ol& '(ring )*B+ /0



Preface

The purpose of this manual is to present laboratory techniques for the identification of

fungi causing mycotic and actinomycotic diseases in humans. The first three exercises are an

introduction to the fungi. This includes a survey of a variety of fungi, which will introduce you

to various fungal structures, both vegetative and reproductive. You will later find that many of

these can act as opportunistic human and animal pathogens. All subsequent exercises will study

fungi and actinomycetes causing diseases in humans.

Equipment for each student or on each table

1. icroscope

!. "ens paper

#. icroscope slides

$. icroscope slide cover slips %glass&

'. (noculating needle

). (noculating loop

*. Teasing needle

+. orceps

-. ' ml bea/er with 0uatsyl disinfectantall used slides should be placed in here to

minimi2e possible release of pathogens

1. 34"5 with aniline blue and other stains.

11. 6lide box for your slide collection

!



General Laboratory Procedures

The following list of procedures is for the benefit of each student. 7bservance of theseminimal regulations will enhance your learning opportunity and reduce personal ha2ard.

8emember you are wor/ing with viable organisms, mostly pathogens.

1. Each student is responsible for the equipment provided. (t will be chec/ed bac/ to theinstructor at the end of the semester.

!. All contaminated slides should be disposed of in a bea/er containing disinfectant.#. All contaminated glassware, tubes, and petri dishes will be disposed of in designated

containers. 9o not discard any contaminated material in the trash can or waste bas/et.$. (f a culture is dropped and bro/en, do not attempt to clean the area: rather consult with the

laboratory instructor.'. Avoid breathing in spores. 9o not open plates to sniff to chec/ for odors.). The laboratory wor/ing area for each student must be cleaned with 0uatsyl disinfectant

before and after every laboratory period.*. or laboratory assignments, students will be divided into groups of two or four, dependingupon the procedure to be completed. To maximi2e the learning process, it is necessary thatthe wor/load be evenly distributed among the group members.

+. All culture transfers should be done in biological safety cabinets %hoods&, unless specificallyallowed by the instructor

-. (f you have any temporary or permanent problems with your immune system, please informthe instructor privately.

Slide Collection

1. 6hould have one slide of each organism that we examine this semester, in the proper slot.

!. The slide collection will be graded on the following criteria;

a. "abels %) pts.& < "abel with labeling tape, the name neatly printed.

b. 6ample 0uality %) pts.& < A good example of the fungus should be on the slide.

c. 6lide 0uality %) pts.& < The slide should be properly and neatly prepared. 34"5

should be completely dry. =sually a few days at )>* °? will dry the slide to

permanency.d. ?ollection ?ontent %) pts.& < There should be a slide of each organism that we study.

@@@6lide boxes can be purchased for 1 %our cost& at the end of the semester, or you may ta/e

the slides in some other box for your future reference.

#



Laboratory I - Survey of Fungal Structure

7bBectives;1. To learn techniques for the ma/ing of wet mounts.

!. To become familiar with fungal vegetative structures.#. To become familiar with fungal reproductive structures.

7rganisms; %1 agar plate for each $ students& MorchellaSchizophyllum commune

Mucor

Saccharomyces cerevisiae

Schizosaccharomyces octosporus Aspergillus cristatus

Neurospora terricola Aspergillus Penicillium chrysogenum

3repared slides of Rhizopus 2ygospores7ther prepared slides

You will be responsible for identifying all organisms in this lab to genus.

Equipment, edia and 8eagents;6lides C coverslips, a/e permanent mounts using 34"5 with aniline blue. 7bserve

these next wee/. or some we will only ma/e temporary mounts with Aniline blue inlactophenol

$



3rocedures;1. 3repare 34"5 Dwith lactophenol aniline blue preparations of each organism as directed by

the instructor and as below. These will be mostly tape mounts.

Morchella drop coverslip on culture to observe hyphae in

situSchizophyllum commune mount a tiny piece of the culture in 34"5

Gilbertella for sporangia;or 2ygospores; twee2e out from center

interaction 2one. ount in 34"5

Mucor tape mount of culture, mount in 34"5

Saccharomyces cerevisiae scrape, mount in 34"5

Schizosaccharomyces octosporus scrape, mount in 34"5

Aspergillus cristatus tape mount of culture, mount in 34"5

Neurospora terricola tape mount of culture, mount in 34"5

Aspergillus tape mount of culture, mount in 34"5

Penicillium chrysogenum tape mount of culture, mount in 34"5

!. Examine each preparation for the structures indicated by the instructor. 8emember thatFycology is an exercise in contemplative observationG %H. 8ippon&, so spend a lot of timestudying each culture and each slide you ma/e. a/e sure your drawings are large enough anddetailed enough to help your studying later. The more times you spend on this now the betteryou will learn the fungi and the better you will do in the course.

#. 3ure culture isolation. Ta/e three 3etri dishes containing 39A or 6AI agar home with you.(noculate the 3etri dishes with something interesting from somewhere you go in the next coupledays. After inoculation seal the petri dishes with parafilm and leave at room temperature infiltered light %not direct sunlight&, incubated agar side up. Iring the 3etri dishes bac/ to the nextlab. You will %eventually& be responsible for isolating three filamentous fungi in pure culture andidentifying them to species.

$. 7bserve boo/s and other materials on demonstration. You should understand what the contentof each boo/ isi.e. what /inds of information you would expect to find there. 6ometimes/nowing where to find the information is almost as important as /nowing a piece of information.

'



Morchella Schizophyllum commune

Gilbertella Mucor

)



Saccharomyces cerevisiae Schizosaccharomyces octosporus

Aspergillus cristatus Neurospora terricola

*



Aspergillus fumigatus Penicillium chrysogenum

Rhizopus sporangia prep slide Rhizopus 2ygospores prep slide

+



Penicillium conidia on orange p.s. Penicillium cleistothecium p.s.

Aspergillus conidia p.s. Morchella asci p.s.

-



Coprinus basidia p.s.

Rhizopus spot plate Aspergillus cristatus cleistothecia

1



Neurospora terricola perithecia. Morchella asci whole mount.

11



1!



1#



Laboratory II - Common Fungal Contaminants

7bBectives;

1. To learn how to prepare slide cultures.

!. To become familiar with and be able to identify some common fungal laboratorycontaminants.

7rganisms; %1 slant culture for each $ students&

Jee/ 1 > Absidia

Circinella

Mucor Rhizopus stolonifer

Syncephalastrum

Aureobasidium pullulansGeotrichum candidum

Rhodotorula gracilis

Cunninghamella

Jee/ ! < Alternaria

Aspergillus nigerCladosporium

Fusarium roseum

rechslera

Paecilomyces Penicillium chrysogenum

Scopulariopsis

!richoderma Acremonium

Curvularia

"erticillium

You will be responsible for identifying the organisms in these two labs to the taonomic

level listed here

Equipment, edia, and 8eagents;

6lides C coverslips6terile glass petri dishes for slide cultures6terile water 34"5 with aniline blue

Iegin pure culture isolation as directed by your instructor. You will eventually be responsible for identifying two filamentous un/nowns.

1$



3rocedure;

1. 3repare a slide culture for each organism as described here and as demonstrated byyour instructor.

!YC"L"GY SLI#$ C%L&%'$ &$C()I*%$ %6ee also Iene/e and 8ogers&

Principle

(n the study of fungi, it is often necessary to observe the undisturbed relationship betweenreproductive structures and the mycelium. This may be accomplished by growing the fungi onglass slides in a moist chamber. You should constantly be aware of the possible infectiousha2ards of this procedure. ?onsult the procedure described in Iene/e and 8ogers %1--)&

1. 3lace a slide on a bent>glass rod in the bottom of a petri dish, add a cover slip and a piece of brown paper or filter paper, cover and sterili2e.

!. 3repare 6abouraud dextrose agar plates with about 1 ml of agar per plate. Allow to solidifyand dry. ?ut agar bloc/s about 1 cm square or puncture with a test tube and lift.

#. 3lace the bloc/ of agar, using sterile technique, on the slide in a petri dish. "ift out withflamed loop.

$. (noculate the sides of the bloc/ with a small fragment of fungus being studied by cutting intoit.

'. . 6terili2e coverslip by passing it briefly through a flame. ?over inoculated bloc/ with thesterile cover slip.

). Add sterile water to bottom of petri dish.

*. (ncubate at !' °? until sporulation occurs %usually 1>! wee/s, occasionally faster&. The slide

preparation may be chec/ed periodically under the low power of a microscope.

+. Jhen sporulation is complete, carefully lift off cover slip and place onto a drop of polyvinylaniline blue %34"5& on a clean slide, ma/ing your first slide.

-. ?ut away agar blac/ from any hyphae that are coming from it. "ift agar bloc/ from slide and

discard.

1. 3lace a drop of 34"5 in the center of growth on the slide and cover with a fresh cover slip.

11. Ilot away excess mounting fluid from the two preparations, allow to dry, and place in oven

%)>*° ? & to dry for a minimum of ! days. =sually drying for a wee/ is best.

1'



Absidia Circinella

Mucor Rhizopus stolonifer

1)



Syncephalastrum Aureobasidium pullulans

Geotrichum candidum Rhodotorula gracilis

1*



Alternaria Aspergillus niger

Cladosporium Fusarium roseum

1+



#ipolaris or rechslera Paecilomyces

Penicillium chrysogenum Scopulariopsis

1-



!richoderma Acremonium

Curvularia "erticillium

!



Laboratory III - Superficial !ycoses

The superficial mycoses include a few diseases caused by a heterogeneous group of organismsthat are usually saprophytes, but occasionally produce lesions on the s/in or hair.

1. 3iedraA disease of the hair. Ilac/ 3iedra is caused by Piedraia hortai and Jhite 3iedra is caused by!richosporon beigelii. The fungus infects the hair producing stony hard nodules along the hairshaft. 7bserve demo slides.

!. Tinea versicolor %3ityriasis&?ausal organism > Malassezia furfur A s/in disease. The disease is characteri2ed by fawn to brownish>colored lesions, usually arms,nec/, face, scalp, thighs, and groin area. Ko attempt is made to culture this lipophilic fungus inthe laboratory. ?linical diagnosis is confirmed by a microscopic examination of a potassiumhydroxide preparation of the scales from a lesion. 7bserve demo slide.

#. Tinea nigra palmaris?ausal organism > $%ophiala &ernec'iiA superficial infection of the s/in on the palm of the hand which is characteri2ed by brown to blac/ macules resembling silver nitrate stains on the s/in. The lesions are smooth, flat, withoutraised borders, are not scaly or vesiculate. Irown hyphae will be seen in the stratum corneumonly.

9iagnosis; 6crape pigmented areas on palm and examine in a L7M preparation for brownish,septate, branching hyphal segments.

(n culture; ?ulture s/in scrapings from lesion on 6abouraudNs glucose agar and incubate atroom temperature. 7n 6abouraudNs glucose agar the organism will develop into shiny, moist, blac/ yeastli/e colonies with continued growth areas of grayish mycelium. Iudding cellsdevelop from the slides of the hyphae, remain clustered and appear similar to clusters of blastospores along the pseudohyphae of Candida sp.

$. Erythrasma?ausal organism > Corynebacterium minutissimumA superficial infection with lesions primarily occurring in various intertriginous areas such as perineal, crural, axillary, and submammary regions. Although the causal organism is a bacterium, the infection is diagnosed by mycological methods > microscopic examination of s/in

scrapings from the lesions. The organism appears short, delicate, branching filaments %less than1 um in diameter& which are easily bro/en into shorter bacillary segments. ?ulturing of the pathogen is not a routine laboratory procedure.

'. Trichomycosis axillaris?ausal organism > Corynebacterium tenuis

Another superficial dermatophytic infection caused by a bacterium. (t is an infection of theaxillary and pubic hairs producing yellow, red, or blac/ nodules which are usually scattered

!1



along the hair shaft. ?linical diagnosis is by microscopic examination of potassium hydroxide preparations of infected hair. The nodules on the hair consist of short, delicate, bacillaryelements. Ko attempt is made to culture the organism. 7bserve demo slide.

7bBectives;

1. To become familiar with the superficial mycotic diseases listed above by observingmaterials from boo/s, displays and demo materials..!. To become familiar with the cultural and microscopic characteristics of !richosporonbeigelii and $%ophiala &ernec'ii.

7rganisms; %1 slant culture for each ! students&

$%ophiala &ernec'ii!richosporon beigelii

Cladosporium sp. % a proteolytic saprophytic species&

Piedraia hortai prepared demo slide only

Equipment, edia and 8eagents;

6lides C coverslips34"5 with aniline blue1!O 5elatin tubes %! for each ! students&

+, Gelatin

Irain Meart (nfusion Iroth !' g5elatin 1! g9istilled water 1 gAdBust pM *.$ > *.)Tube > ) ml in screw cap tubes6terili2e, 1' min., 1' p.s.i.

9emonstration materials for Ilac/ 3iedra, Erythrasma, and Trichomyces axillaris

3rocedure;1. (noculate one 1!O gelatin tube with a large fragment of $%ophiala &ernec'ii and onetube with Cladosporium sp. %6aprophytic species of Cladosporium are proteolytic, while

pathogenic species e.g. $( &ernec'ii and C( carronii do not brea/down protein.&!. Examine and study demonstrations.

!!



)( $%ophiala &ernec'ii a. ?olony characteristics >

b. 5elatin test

1. !richosporon beigelii a. ?olony characteristics >

!#



!. Ilac/ 3iedra > on hair. demo slide

$. 3ityriasis versicolor > slide demonstration

!$



'. Erythrasma > slide demonstration

). Trichomycosis axillaris > slide demonstration

!'



Laboratory I. - #ermatophytes

The dermatophytes are pathogenic fungi that produce cutaneous infections of the s/in, hair andnails, and are the most common and widely distributed of the fungal diseases of humans. Themaladies are referred to as a ringworm or tinea and are incited by species of $pidermophyton*

Microsporum* and !richophyton. These organisms rarely invade subcutaneous tissues or internalorgans. The isolation and identification of the fungus involved is essential, especially in casesthat have been difficult to treat. Jith the advent of a2ole compounds, "amisil, and their differentefficacies in treating infections, it has become essential to identify the fungus to species. anyinsurance companies will not pay for prescriptions if the fungus is not identified.

?linical isolation >3late out scrapings from lesions or fragments of infected hair on 6abouraud glucose agarcontaining chloramphenicol and cycloheximide %can use ycosel or ycobiotic agar&.

"aboratory identification <

(dentification is based upon colony and microscopic characteristics. The three genera aredifferentiated by the shape of the macroconidia. 6pecies identification is based upon variousmorphological characteristics. (n addition, sometimes physiological characteristics are also used.(t is also helpful to /now from where on the body the scrapings or hair came from.

7rganisms; %7ne slant culture for each $ students& Kot all species may be available for ma/ingslides. 9emonstration slides are available for most of these.

Jee/ 1 >> $pidermophyton floccosum

Microsporum audouinii Microsporum canis

Microsporum fulvum Microsporum gypseum

Microsporum nanum Microsporum vanbreuseghemii

Microsporum gallinae

Jee/ ! >>!richophyton a+elloi

!richophyton verrucosum

!richophyton megninii

!richophyton mentagrophytes!richophyton rubrum

!richophyton schoenleinii!richophyton terrestre

!richophyton tonsurans

!richophyton e,uinum!richophyton simii

!)



You are responsible for identifying the fungi on the preceding page to genus. "earn the variationwithin each genus by loo/ing at as many species as possible.


6lides C coverslips6terile glass petri dishes for slide cultures6terile water 34"5 with aniline blue8ice grain plates5uinea pig hair %sterile&Morse hair or human hair %sterile&1O sterile yeast extract

3rocedure;

1. 3repare a slide culture of each dermatophyte.!.. (noculate rice grain plates.a. 3lace a few small fragments of Microsporum audouinii on one rice plate and M( canis or M(distortum on the second rice plate. Ta/e care not to transfer any agar with the inoculum.

(noculate hair. Ta/e a couple pieces of your hair and inoculate M( gypseum and one otherspecies in two slide culture setups.

7bserve demo slides of Ectothrix and Endothrix invasion of hair.

7bserve demo materials of infected nails and whatever else is available.

!*



9E8AT7Y?76E69iagnosis

A direct microscopic examination of hair, s/in and nails suspected of beinginfected with fungi offers the most important and best means of diagnosis. The material

is collected between two glass slides that have been previously wrapped in paper andsterili2ed. To examine the s/in or nail scrapings or small FstubsG of hair, place thematerial in a drop of 1>$O L7M on the end of a glass slide, apply a cover glass andheat gently over a low flame. (f the material fails to clear sufficiently, add more L7M tothe edge of the cover glass and gently heat again.

Although a clue to the genus !richophyton or Microsporum may be obtained fromthe characteristic appearance of the infected hair, the species can be determined only after the fungus has been isolated in culture. 6ince the three genera !richophyton* Microsporum* and $pidermophyton are identical in appearance in infected s/in and nails,this material must be collected and cultured to obtain both a generic and specificidentification of the invading fungus. The material is cut into small fragments and three

or four pieces placed short distances apart on each of two or three 6abouraudNs glucoseagar slants. ?ultures are maintained at room temperature, and must be /ept for two orthree wee/s before they can be considered negative.

any of the dermatophytes do not produce distinguishing spore forms orconsistent pigment on 6abouraudNs medium. 8ecent nutritional studies have shown thatdeficiencies play an important part in the growth of these fungi and the morphologywhich they display.A. !( rubrum < the reddish to purple pigment in the agar distinguishes this species from

!( mentagrophytes( Two media may be used for pigment production; 3otato dextroseagar %9ifco& and corn meal dextrose %!O& agar.

icroscopic morphology is more consistent on Meart infusion agar plus tryptose %Iloodagar base, 9ifco&.I. ?hristensenNs =rea Agar. !( mentagrophytes is positive immediately while !( rubrum

ta/es some days to become positive.?. urther differentiation of !( mentagrophytes from !( rubrum is based on the ability of

!( mentagrophytes to perforate hair in culture while !( rubrum does not. 6terili2edhuman hair is placed in a petri dish with sterile distilled water containing yeast>extract %.1 ml of 1O yeast extract in !' ml distilled water&. (noculate with culture

to be tested, incubate at !'°? for two wee/s and examine hair in lactophenol cotton

blue.9. !( verrucosum -!( faviforme. < isolate best on blood agar base %9ifco& plus thiamine

%1 mlPliter& with incubation at #*°?. 6abouraudNs glucose thiamine agar may also be

used.icroscopic morphology, spore production, best on 6abouraudNs glucose thiamine agar atroom temperature.E. !( tonsurans < characteristic gross morphology on 6abouraudNs glucose agar.icroscopic morphology best on Jort agar at *>1 days and again at !>!$ days.. !( violaceum < slow growing, glabrous pigmented culture on 6abouraudNs glucose

agar characteristic of this species.

!+



TAI"E ( ungi That (nvade Mair, 6/in andPor Kails

Fungus (air S/in )ails

$pidermophyton floccosum > Q Q

Microsporum spp( Q Q >

!richophyton spp( Q Q Q

Malassezia furfur > Q >

$%ophiala &ernec'ii > Q >

Candida spp(/ > Q Q

Q R invades > R does not invade @ R may cause lesions of scalp

TAI"E (( Type of Mair (nvasion by 6ome ungi

$ctothri invasion $ndothri invasion

Microsporum audouinii !richophyton schoenleinii

M( canis !( tonsurans M( ferrugineum !( violaceum

M( gypseum !( rubrum

!richophyton mentagrophytes !( soudanense

!( verrucosum !( yaoundei

!( megninii !( gourvillii

!( e,uinum

!-



$pidermophyton floccosum Microsporum audouinii

Microsporum canis Microsporum fulvum

#



Microsporum gypseum Microsporum nanum

Microsporum vanbreusighemii Microsporum gallinae

#1



Jee/ ! <

!richophyton a+elloi !richophyton concentricum

!richophyton verrucosum !richophyton megninii

#!



!richophyton mentagrophytes !richophyton rubrum

!richophyton schoenleinii !richophyton terrestre

##



!richophyton tonsurans !richophyton violaceum

!richophyton e,uinum !richophyton simii

#$



Laboratory . - Subcutaneous !ycoses

The subcutaneous mycoses are caused by fungi that probably exist as saprophytes in nature andinfect the s/in and subcutaneous tissue, rarely involving internal organs in the body. The threediseases of this type are chromoblastomycosis, mycetoma, and sporotrichosis. =sually a person

acquires infection accidentally as a result of some traumatic implantation.

7bBectives;

1. To become familiar with the subcutaneous mycoses.!. To become familiar with the cultural and microscopic characteristics of the fungiassociated with each of the subcutaneous mycoses.#. To become familiar with the diagnostic procedures for each of the associated diseases.

7rganisms; %slant culture for each $ students&

Jee/ 1 >> ungi associated with chromoblastomycosis Fonsecaea pedrosoi

Fonsecaea compacta

Phialophora verrucosa Phialophora gougerotii

Phialophora richardsiae

$%ophiala spinifera

Jee/ ! >> ungi associated with mycotic mycetomaScedosporium apiospermum 0 %anamorph Pseudallescheria boydii.

$%ophiala +eanselmeiungus causing sporotrichosis

Sporothri% schenc'ii

You are responsible for the species listed

Equipment, edia, and 8eagents;6lides C coverslips6terile glass petri dishes for slide cultures

6terile water 34"5 with aniline blue5elatin tubes6tarch agar plates

#'



3rocedure;Jee/ 1 >> or chromoblastomycotic fungi

1. 3repare slide cultures for each species and incubate at room temperature. Examine aftersufficient growth has occurred %may ta/e ! to # wee/s as these fungi grow relatively slowly&.

Jee/ ! >> or mycetoma fungi

!. 3repare slide cultures for each species and incubate at room temperature. Examine aftersufficient growth has occurred.#. (noculate a gelatin medium tube and a starch medium plate with each organism. (noculate plates at room temperature. Ie sure to include control organisms.$. After fungus growth occurs on starch plate, flood with iodine solution. A clear 2one aroundcolony indicates an area of hydrolysis.

(nterpretation >>

3roteolytic Activity Amylolytic Activity

Scedosporium apiospermum 0 0

$%ophiala +eanselmei 1 1

edia >>5elatin medium

Irain heart infusion broth !'g5elatin 1! g

9istilled water 1 mlAdBust pM to *.! > *.$9ispense ' ml per tube and sterili2e

#)



$%ophiala dermatitidis Phialophora verrucosa

Phialophora gougerotii Phialophora richardsiae

#*



$%ophiala spinifera $%ophiala +eanselmei

Scedosporium apiospermum Sporothri% schenc'ii

#+



2ee/ + 3 Y$4S&S Kote; certain species may not be available. ?hec/ board.

1. 3repare slides for slide box.

!. (ndia (n/Cryptococcus neoformans

#. 5erm tube test > rabbit serumCandida albicans

Candida tropicalis

$. ?hlamydospore production %1 platePpair&Candida albicans

Candida tropicalis

'. Ascospore production %# tubesPpair& 1ansenula anomala

Saccharomyces cerevisiaeSchizosaccharomyces octosporus

). 8apid nitrate %$'?& %! swabsPpair&Cryptococcus terreus

Cryptococcus neoformans

*. ?A? dis/sCandida albicans

Cryptococcus neoformans

1. Add water to slide plate!. 3lace ! ?A? discs on slide#. oisten disc with sterile water $. (noculate discs with test organisms. 7ne with Cryptococcus neoformans, one withCandida albicans

'. (ncubate at #* °? and examine each hour for the production of brown pigment.

+. =rease testCryptococcus terreus

Candida glabrata

-. Candida albicans screenCandida albicansCryptococcus laurentii

1. ?hromAgar Candida 1 plate for each $ people

#-



Candida albicans* Candida 'rusei* Candida tropicalis* Cryptococcus neoformans

$



2ee/ , Y$4S&S - %se %n/nown provided to you

1. 9elman plate %?>Tween +&

!. =rease slant

#. ?arbohydrate assimilation plates

$. A3( !? Aux. system if available

'. K7# assimilation > next wee/ if necessary

). 5erm tube > next wee/ if necessary

*. ?hromAgar Candida 1 plate for each $ people

+. Cryptococcus latex test

$1



Laboratory .I - Pathogenic Yeasts

ycologically, the yeasts encompass a relatively large group of fungi. (n a broad sense, the termyeast refers to those fungi that possess a predominantly unicellular thallus, which reproduceasexually by budding or fission. (f sexual reproduction occurs they typically produce ascospores

in a na/ed ascus. (n growth, yeast colonies typically resemble bacterial colonies and notmycelioid fungal colonies.

(n the diagnostic medical microbiology laboratory, the isolation of yeasts from clinical materialis an everyday occurrence. Mowever, their identification as well as their significance often poses problems for the technologist and the physician. Jith the constant use of antibiotics, steroids,antimitotic and immunosuppressive drugs, yeasts have become a maBor group of opportunistic pathogens. 6everal species of yeasts, including Candida albicans and Cryptococcus neoformans,are now recogni2ed as etiologic agents of disseminated fungal disease. This is especially true in patients whose resistance has been altered by an initial disease %such as A(96&, chemotherapy orcertain surgical procedures. 6ome Rhodotorula species have been isolated from a number of

patients receiving intravenous medication. Candida glabrata is frequently isolated from urinespecimens. Candida parapsilosis, C( guilliermondii and C( tropicalis are seen in increasinginstances as etiologic agents of endocarditis in drug addicts and in patients undergoing someform of cardiovascular surgery. Cryptococcus albidus and Cryptococcus laurentii have beenshown to cause respiratory disease as well as s/in lesions. The incidence of meningitis caused by species other than Cryptococcus neoformans is on the increase. any laboratories now aredemonstrating Cryptococcus neoformans as a frequent causative agent in pneumonitis.

(dentification of yeasts isolated from blood cultures, cerebral spinal fluid, a closed lesion or asurgical specimen should be made. 6uch yeasts are unli/ely to be contaminants. Yeasts from theupper respiratory tract, lower urinary tract or the intestinal tract may represent normal flora, but,

in some instances, may contribute significantly to a patientNs disease process. The yeasts isolatedfrom any of these sources should be reported so that the physician can interpret theirsignificance.

The number of species of yeasts that the diagnostic laboratory should find necessary to identifyis not large. 6even Candida species, two !orulopsis species %usually included in Candida&, two!richosporon species and four species of Cryptococcus represent the maBority of the yeastsisolated. To successfully identify a yeast, a combination of several tests must be employed.These tests are feasible for even the smallest of laboratories and include;

1. ?hec/ing yeast for purity!. ?olony characteristics

#. ?ell si2e and morphology$. ?hlamydospore production'. 5erm tube test). ermentation > assimilation studies*. Kitrate assimilation test+. =rease production-. (ndia (n/ preparation or nigrosin1. Ascospore production

$!



7bBectives;1. To become familiar with the human diseases caused by the yeasts.!. To become familiar with colony characteristics and morphology of various pathogenicyeasts.#. To become familiar with the laboratory diagnostic procedures in identifying the

various pathogenic yeasts.

7rganisms; %1 slant culture for each $ students&Candida albicans

Candida guilliermondii

Candida 'ruseiCandida parapsilosis

Candida 'efyr

Candida stellatoideaCandida tropicalis

Cryptococcus albidus

Cryptococcus neoformansCryptococcus terreus

1ansenula anomala

Rhodotorula rubraSaccharomyces cerevisiae

Candida glabrata

!richosporon beigelii

You are responsible for learning the methods used to distinguish the species and the /inds

of characters that are used5 2e don6t epect you to memori7e the physiological test results8

but you should be able to interpret them5


6lides C coverslips34"5 with aniline blue(ndia in/ or nigrosin6terile pipettes %1 ml x ' ml&, 9isposable pipettes6terile petri dishes5erm sera tubes %.' ml of pooled human or bovine serum in each tube&6terile .+'O salineermentation broth tubes %dextrose, galactose, lactose, sucrose, maltose, C raffinose&Assimilation discs %dextrose, galactose, lactose, sucrose, maltose, raffinose, Trehalose&Ilan/ discsYeast nitrogen base, Yeast carbon base

!O agar deeps6aturated LK7# solution?hlamydospore formation agar %corn meal > Tween > +&=rea agar slantsalt extract agar slants?arbolfuchsin stain-'O ethyl alcohol containing 1O M?lAqueous methylene blue, edia and supplies for special tests

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3rocedure;

+5 !ethod to chec/ yeast for purity5a. Emulsify a loop full of material from the yeast colony in ' ml of sterile distilled water andstrea/ a blood agar plate and a 6abouraud dextrose agar plate with the suspension.

b. (ncubate the blood agar plate at #* °? and the 6abouraud agar plate at room temperature. (f

after $+ hours no bacteria appear on the blood agar plate, the culture may be considered free of bacteria.c. (f bacteria are present, purify as follows; 1. To a tube containing 1 ml of 6abouraud dextrose broth, add 1 drop of 1K: to a second tubeof broth add ! drops of 1K M?l, # drops to a third tube, and $ drops to a fourth tube. !. Add a drop of yeast suspension to each of the four tubes and incuate atroom temperature for !$ hrs. #. 6ubculture from each of the broth tubes to blood agar plates and incubate at #*

°? for $+ hrs. 7ne or more of the plates should be free of bacteria: if not, further attempts to

purify the culture is unli/ely to succeed.d. The initial 6abouraud agar strea/ plate should be incubated for *!>-) hrs. (f all colonies on

the plate are identical, the yeast may be considered pure. (f there is more than one type of colonyon the plate, each different colony type should be isolated, chec/ed for purity and identified.

,5 India in/ preparation - to chec/ for capsulesa. ix a loopful of (ndia in/ %3eli/an& or nigrosin and a loopful of sterile distilled water on aclean slide. b. Transfer a small amount of yeast to the drop, emulsify, add a cover slip and examine forcapsules. The capsule is a clear area around the cell. Cryptococcus and Rhodotorula both produce capsules. Rhodoturola species have reddish pigmented colonies. 8emember that C(

neoformans tends to lose its capsule the longer it has been in culture.

95 Germ tube testa. Touch the yeast colony with the tip of a disposable capillary pipette and insert pipette into.' ml of pooled human or bovine serum. %9o not use heat coagulated serum& %6erum and pipettes need not be sterili2ed.&

b. (ncubate with pipette in serum at #* °? for ! hrs. and $' min., then examine a drop of the

mixture for germ tube production.

Approximately -+O of Candida albicans strains will produce germ tubes. After # hrs.incubation, C( stellatoidea will produce germ tubes, therefore incubation time is very critical. C(albicans and C( stellatoidea can be differentiated by a sucrose assimilation test. or all germtube negative yeasts, additional tests for identification must be conducted.

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:5 Chlamydospore productionA chlamydospore is a thic/>walled, intercalary or terminal cell. (t is a characteristic structure ofCandida albicans.

a. Jith an inoculating needle, ma/e two strea/ inoculations the entire length of a

chlamydospore agar plate: use Bust enough pressure to scarify the agar >> do not cut the agar. b. Jith a loop, strea/ bac/ and forth across the scarified area.c. 3lace a sterile cover glass over a portion of the strea/s.

Kote; A strain of Candida albicans should also be inoculated on the plate as a control.

d. (ncubate the plate at room temperature for 1!>!$ hours.e. Examine the plate by removing the cover and placing the plate on the microscope substage.5et the edge of the coverslip in focus under low power. Jhen present, chlamydospores willusually be found underneath the cover glass near the edge.

; 4scospore production

a. Jith a loop, inoculate a malt agar slant or a 4+ Buice agar slant with the yeast culture. b. (ncubate at room temperature.c. Jith a wet preparation, examine culture every $+>*! hrs. for !># wee/s. 6ometimesascospores may not appear for ) wee/s.

An ascospore stain can be used !. lood slide with carbolfuchsin and steam gently for !># min.#. Jash and destain with -'O ethyl alcohol containing 1O M?l for a few seconds.

$. Jash and counterstain with aqueous methylene blue for 1>! min. Jash, dry, andexamine. Ascospores will be red and vegetative cells will be blue.

<5 %rease test

a. Jith a loop, inoculate a ?hristensenNs urea agar slant. b. (ncubate at room temperature and examine daily for #>$ days.

(f urease is positive, the medium will turn cherry red. Cryptococcus and Rhodotorula species areurease positive. Also, an occasional isolate of Candida 'rusei* Candida humicola* and some!richosporon species may produce urease.

=5 Fermentation &ests

ermentation tests are not needed routinely to identify Candida species, particularly ifthe technologist is familiar with the characteristic morphology of each Candida

species when examined on a chlamydospore agar plate.

a. 3repare a cell suspension of the yeast in .+'O saline to a turbidity of a carland Ko. 1$'



standard. A suspension giving *O transmittancy at #)' nm on a IC" 6pectromic !spectrophotometer is also satisfactory.

b. (noculate each fermentation tube with .# ml of the prepared suspension. Tighten capssecurely.

c. (ncubate at room temperature for 1$ days. 3ositive fermentation is recorded for the production of gas only.

>5 Carbohydrate assimilation testThis test chec/s which sugars can be assimilated by different species

a. 3repare a suspension of the yeast to be tested equivalent to a carland 6tandard number $.

b. lood the surface of the agar in a plate containing yeast nitrogen base and brom cresol purplewith the yeast suspension. =se about 1 ml. 8emove the excess inoculum with a pipette and

leave the lid aBar for 1>1' minutes to allow the surface to dry.

c. 3lace three sugar impregnated discs %different sugars& equidistant apart on the agar surface ofone plate and three different sugar impregnated discs on the other plate.

d. (ncubate at # °? for !$>$+ hours and read for evidence of carbohydrate utili2ation. A color

change from purple to yellow around the carbohydrate dis/ is indicative of the utili2ation of thesubstrate. ?arbohydrate dis/s may be surrounded by growth without a color change and this alsoindicates utili2ation of the substance.

?5 )itrate assimilation test we will not do this test. (t has largely been supplanted by the

rapid nitrate test.

a. 3repare a cell suspension as for the carbohydrate assimilation test. %The yeast cells should bestarved in a yeast carbon base broth&. b. Add .# ml of yeast suspension and 1.' ml of yeast carbon base to the bottom of a sterile petridish.c. Add 1#.' ml of liquid !O water agar, thoroughly mix, and allow to harden.d. 6oa/ a sterile disc in a saturated solution of LK7# and place on the agar in the center of the petri dish.e. (ncubate at room temperature for *! hrs. Assimilation of nitrate is recorded as growth aroundthe disc.

A /nown positive %Cryptococcus albidus& and a /nown negative %Cryptococcus neoformans& arerun in conBunction with the tested yeast as controls.

@@ Kote; Kitrate assimilation tests need be run only on yeasts that are urease positive withnegative fermentation after !$ hrs.

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Some !ethods for 'apid Yeast Identification Kote; we will not do all these tests

1. 8apid urease test

a. 8econstitute a vial of 9ifco 8 broth with # ml of sterile distilled water on the day that

it is to be used. b. 9ispense $>' drops into each well to be used in a microtiter plate.c. Transfer a heavy inoculum of each yeast colony to a well containing urea broth.%(solation of the yeast will be necessary if the culture is contaminated with bacteria.&?olonies to be tested should not be older than * days.d. (n a laboratory situation, include a positive and negative control such as Cryptococcus

neoformans and Candida albicans respectively. Also include one well with uninoculated broth only.

e. 6eal wells with scotch tape and incubate for $ hours at #*° ?.

f. (f urea broth shows any indication of having a pin/ color, it is positive for urease production.

g. (t is not necessary to test Rhodotorula species for urease production.

!. 8apid nitrate assimilation test

a. 6weep the tip of the swab impregnated with nitrate reduction medium across two tothree colonies of the yeast on a plate. b. 6wirl the inoculated swab against the bottom of an empty test tube to imbed the yeastcells within the swab.

c. (ncubate the tube and swab for 1 minutes at $' °?.

d. 8emove swab from tube and add two drops each of a>naphthylamine and sulfanilicacid reagents to the tube, then replace swab.

e. A change in color to red is positive for nitrate assimilation. %A /nown nitrate positiveand negative Cryptococcus should be used as controls.&

Kitrate 8eduction edium >>3otassium nitrate 'g6odium phosphate, monobasic 11.* g6odium phosphate, dibasic 1.1$ gSepharin chloride ! mg9istilled water ! ml

6tandard, medium si2ed tipped, cotton swabs are saturated in the solution. 6wabs are fro2en,lyophili2ed, and autoclaved. An alternative is to dry the swabs by vacuum for !$ hours and

autoclave. 6wabs are stored in containers.

Kitrate 8eduction Test 8eagents >> (t may ta/e overnight for reagents to go into solution.requent agitation helps

8eagent A 8eagent I5lacial acetic acid ' ml 5lacial acetic acid ' mlJater 1!' ml Jater 1!' ml6ulfanilic acid 1.$ g 9imethyl alpha napthylamine 1 g

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#. Irown pigment production by Cryptococcus neoformans

Cryptococcus neoformans produces a brown pigment %melanin& when grown on certainspecific substrates. 8ecent studies have shown that -'>-+O of C( neoformans isolates are positive for pigment production. Also, an occasional isolate of !richosporon cutaneum

and Cryptococcus luteolus produces the pigment. C( neoformans can produce the brown pigment %melanin& from various substrates containing certain ortho> and para>diphenolsand ortho>triphenols. The production of melanin from o>diphenol is due to #, $dihydroxyphenylalanine phenoloxidase, which is an en2yme present in the cell wall of C(neoformans(

elanin pigment production is a fairly reliable test but false positives and falsenegatives do occur, therefore, pigment production should only be used in combinationwith other tests to provide a definitive identification of C( neoformans(

a. ?affeic acid>ferric citrate %?A?& impregnated dis/s

1. or each organism to be tested, moisten one control dis/ %1 cm& and one ?A?dis/ with sterile distilled water %!># small drops&. 9o not over saturate dis/s.3lace dis/s in a petri dish.

!. Transfer a loopful of Cryptococcus neoformans cells from a 6ab agar slant toeach of the dis/s. 8epeat with additional dis/s if other yeast are to be tested.

#. (ncubate within an enclosed container at #* °? for !># hrs. and chec/ for the

production of brown pigment. (f no pigment is seen, then chec/ after ) hrs. and!$ hrs. Ko brown coloration after !$ hrs. is interpreted as a negative reaction.

To prepare ?A? dis/s %note; prepared dis/s may also be purchased&6toc/ solutions >

?affeic acid > 1 mg of caffeic acid per ml in .' 6orensen buffer %pM *.&

erric citrate > .' mg of ferric citrate per ml distilled water.

To each dis/ %1 cm& add >>

1 ml of caffeic acid stoc/ solution

.' ml of ferric citrate stoc/ solution#.' ml of 6orensen phosphate buffer

%?ontrol dis/s are saturated with 6orensen buffer only.&

Allow dis/s to dry overnight in sterile petri dishes at #* °? and store in dar/ bottles at room

temperature for no more than ! months.

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$. A3( !? A= clinical yeast systemJithin recent years several companies have prepared yeast diagnostic /its for a rapididentification of clinically important yeast. The A3( !? clinical yeast system is one method,and has proven to be highly accurate and also has a shelf life of 1! months. The A3( !? A=is a ready to use microtube system which consists of 1- assimilation tests and when combined

with computer assisted interpretation, allows for the identification of a wide range of yeastspecies isolated in a clinical laboratory. The A3( !? system consists of microtubes containingdehydrated substrates for the assimilation reactions. Each microtube is inoculated with a basal

medium yeast cell suspension, incubated at #°? and read after !$, $+, and *! hours. 6ee the

sheets provided with the /it and included here.

#. ?hromAgar http;PPwww.chromagar.comP

A medium for the detection and differentiation of ?andida, allowing easy detection of coloniesfrom different species. ?M87agar Candida > will not only allow the growth and detection of yeasts > li/e traditional

media %6abouraud& but in addition, Bust by the color of the colony, will instantly allow todifferentiate various Candida species.

?M87agar Candida helps to recogni2e the maBor population of Candida infecting the patientas well as for the first time > it offers a panoramic view on a mixed population with ability torecogni2e the presence of a minor population within a patient.

Candida albicans> greenCandida tropicalis > steel blueCandida 'rusei > rose, fu22y

(noculate plates as directed by your instructor.

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http://www.chromagar.com/

http://www.chromagar.com/



Laboratory .II- Systemic Pathogenic Fungi

The systemic mycoses may involve all or any of the internal organs of the body. 6/in,subcutaneous tissue, and bone may also be involved. 9isease symptoms vary from mild to quitesevere. 9eath may also occur if the patient does not respond to therapies.

The fungi involved in systemic mycoses are dimorphic organisms. (n tissue and usually when

cultured at #*°? they exhibit one phase %usually a yeast& and when cultured at room temperature

they produce a different phase. %mycelial&. ungi causing histoplasmosis, blastomycosis,coccidioidomycosis and paracoccidioidomycosis are typical examples of this phenomenon.

7bBectives1. To become familiar with the systemic mycoses

!. To become familiar with the morphological characteristics of the true pathogenic fungi#. To become aware of the ha2ards of wor/ing with the true pathogenic fungi$. To become familiar with the proper laboratory techniques in handling the systemic fungi.

7rganisms; 1istoplasma capsulatum

#lastomyces dermatitidis

Coccidioides immitis

Paracoccidioides brasiliensisSepedonium chryospermum

You will be responsible for identifying all the organisms here to species

Equipment6lides Qcoverslipsultitude of demonstration slides.

These fungi are too pathogenic for us to wor/ with with the equipment we have available.Mowever, we are luc/y to have many demonstration slides of these organisms, both mounts ofthe cultures and in histopathological section. 7bserve all materials available.

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Mistoplasma capsulatum rt° Mistoplasma capsulatum #*°?

#lastomyces dermatitidis rt° #lastomyces dermatitidis #*°?

'1



Coccidioides immitis rt° Coccidioides immitis #*°?

Paracoccidioides brasiliensis rt° Paracoccidioides brasiliensis #*°?

'!



Aspergillus

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Lab Manual Volk-nelson 2011

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Transcript of Lab Manual Volk-nelson 2011