YEAST IDENTIFICATION
I. PRINCIPLE: A. Yeasts are a heterogenous heterogenous group of fungi that that superfic superficially ially appear appear to be homogeneous. homogeneous. Yeasts grow in a conspicuous unicellular form that reproduces by fission, budding, or a combination of both. True yeasts reproduce sexually, developing ascospores or basidiospores under favorable conditions. The majority of ascomycetous and basidiomycetous yeasts isolated by the lab go unrecognized because most of them are heterothallic. In most instances, only one of the mating types is isolated and therefore no asci or basidia are produced. B. Yeast-like Yeast-like fungi fungi (imperfe (imperfect ct yeasts) yeasts) reproduce reproduce only by asexual asexual means. The identific identification ation of these fungi is based upon a combination of morphological and biochemical criteria. Morphology is primarily used to establish the genera, whereas biochemical assimilations are used to differentiate the various species. C.
Princip Principal al Criter Criteria ia and Tests Tests for for Identif Identifying ying Yeasts Yeasts 1. Culture Culture charac character teristi istics cs - Colony Colony color, color, shape, shape, texture texture 2. Asex Asexua uall struc structu ture ress a. Shap Shapee and and siz sizee of cell cellss b. Bipolar Bipolar,, fission fission,, multipol multipolar ar or unipol unipolar ar "buddi "budding" ng" c. Absence or presence presence of arthroconi arthroconidia, dia, ballistoc ballistoconidia, onidia, blastoconidia, blastoconidia, clamp connections, endoconidia, germ tubes, hyphae, pseudohyphae, or sporangia and sporgangiospores. 3. Sexual structures structures - Arrangement, Arrangement, cell cell wall wall ornamentation ornamentation,, number, number, shape and size of ascospores or basidiospores 4. Phys Physio iolo logi gica call studi studies es a. b. c. d. e. f.
Ass Assimil milation Cyclo Cyclohe hexi ximi mide de res resis ista tanc ncee Ferm Fermen enta tati tion on Nitr Nitrog ogen en uti utili liza zati tion on Urea Urea hyd hydrrolys olysiis Temp Temper erat atur uree stu studi dies es
II. ISOLATION TECHNIQUES FOR MIXED CULTURES : All initially isolated yeasts may be contaminated or in mixed culture. Direct mounts and subsequent streaking for colony isolation are necessary to confirm the purity of each yeast isolate. Pure cultures are essential for assimilation procedures. Streaking subcultures for spatial isolation is adequate in most cases. The following additional techniques can be used to purify yeasts: A. Bacte Bacteri rial al conta contamin minat ation ion 1. Colony Colony isola isolati tion on on SAB SAB agar agar a. Suspend a small portion portion of the yeast yeast to be decontaminated decontaminated in sterile sterile distilled distilled water. water. b. Streak a loopful loopful of the the suspension suspension for for colony isolation isolation onto a plate of SAB agar.
YEAST IDENTIFICATION II. ISOLATION TECHNIQUES FOR MIXED CULTURES : (cont'd) c. Incu Incuba bate te at 30oC for for 48 hour hours. s. d. Examine for isolated isolated colonies. colonies. Verify Verify purity purity with a direct direct exam. e. If colonie coloniess are not pure, pure, further further steps steps are neces necessary sary (Step (Step 2). 2. Colony isolati isolation on on SAB agar plus plus chloramphe chloramphenicol nicol SAB SAB plus penicil penicillin lin and streptomycin, streptomycin, or BHI plus 10% blood plus gentamicin and chloramphenicol. a. Suspend a small portion portion of the yeast yeast to be decontaminated decontaminated in sterile sterile distilled distilled water. water. b. Streak a loopful loopful of the the suspension suspension for colony isolatio isolation n onto one of the above media. media. o c. Incubate Incubate at 30 C for for 48 hours. hours. Check Check for purity. purity. If colon colonies ies are not pure, pure, further further steps are necessary (Step 3). 3. Acidi Acidifi fica cati tion on of SAB brot broth h a. Suspend a small portion portion of the yeast yeast to be decontaminated decontaminated in sterile sterile distilled distilled water. water. b. To each each of 4 tubes conta containin ining g 10 ml of SAB broth, broth, add add 1 drop of 1 N HC1 to the the first first tube, 2 drops to the second tube, 3 drops to the third tube, and 4 drops in the fourth tube. c. Add 0.5 ml ml of the conta contamina minated ted yeast yeast suspe suspensio nsion n to each each tube. tube. o d. Incu Incuba bate te at 30 C for for 24 hour hours. s. e. Subculture a loopful loopful of of each each broth broth to to SAB agar plates. Streak for isolation. f. Incuba Incubate te the the SAB SAB pla plates tes at 30o C for for 48 hours. hours. g. Check Check purity. purity. If still still not pure, pure, consult consult the the super supervisor visor.. B. Mix Mixed Yea Yeasts sts 1. 2. 3. 4.
Suspend a portion of each suspected suspected colony colony type in a tube of of sterile sterile distilled distilled water. Streak Streak a loopf loopful ul of the the suspensi suspension on onto onto a SAB agar agar plate. plate. o Incub Incubat atee the the SAB plate plate at 30 C for for 48 hour hours. s. Check for purity. purity. If the yeast yeast is not pure, pure, it must be restreaked. restreaked. Note that that some strains have both rough and smooth colony types in pure culture.
C. Moul Mould d Cont Contam amin inat atio ion n 1. Colony Colony isola isolation tion on yeast yeast malt (YM) agar. agar. a. b. c. d.
Suspend a small small portion portion of the yeast-mo yeast-mould uld colony colony in sterile sterile water. water. Streak a loopful loopful of the the suspension suspension for for colony colony isolation isolation onto a plate plate of YM agar. o Incuba Incubate te the the YM YM plat platee at 30 C for for 4-6 4-6 days. days. Check for purity. If not pure, pure, further further steps are necessary necessary (Step (Step 2). 2).
2. Colony Colony isolatio isolation n in in yeast yeast malt malt broth broth.. a. Transfer Transfer a small small portion portion of the yeast-mould yeast-mould isolate isolate to a tube containing containing 10 ml of YM broth. b. Incubate Incubate the YM broth broth at 30 30o C for 48 hours hours.. Remove Remove a small small portion portion of of the the sedimen sedimentt with a sterile capillary pipette by slipping the pipette along the edge of the tube to the bottom without disturbing the mycelial pellicle.
YEAST IDENTIFICATION II. ISOLATION TECHNIQUES FOR MIXED CULTURES : (cont'd) c. Streak the sediment sediment for colony colony isolation isolation onto a plate of YM agar. agar. o d. Incubate Incubate at 30 C for 4-7 days days and and then then prepare prepare a direct direct mount. mount. If the the yeast yeast is is not pure, pure, further steps are necessary (Step 3). 3. Colony Colony isolati isolation on in shake shake cultur culture. e. a. Transfer Transfer a small small portion portion of the yeast-mould yeast-mould isolate isolate to a 250 ml Erlenmeyer Erlenmeyer flask containing approximately 100 ml of YM broth. b. Place Place the flask flask on a rotary rotary shake shakerr and incubate incubate at 30o C for 4-6 days. days. c. Carefully Carefully remove remove a small small amount of the sediment sediment with a sterile sterile capillary capillary pipette. pipette. Ensure that the balls of mycelium are not removed removed by accident. d. Streak the sediment sediment for colony colony isolation isolation onto a plate of YM agar. agar. o e. Incubate Incubate at at 30 C for 4-7 4-7 days and and then prepa prepare re a direct direct mount mount using using a small small portion portion of each yeast colony to be identified. If the yeast is not pure, consult the supervisor. III. IDENTIFICATION SCHEME: To identify yeasts, first examine the colony color, shape and texture. If the colony is black to brown in color or moist mycelial in texture, prepare a direct direct mount. Using the flow chart, determine the genus based on microscopic morphology. If the colony is pink to red, streak the colony out for isolation. After incubation, check for the presence of satellite colonies. Examine microscopically for forcibly discharged conidia. Using the flow flow chart, determine the genus based on morphology. Assimilations Rhodotorula . If the colony is white or cream color, will be necessary to determine the species of Rhodotorula perform a germ tube test. If the germ tube test is positive, the yeast can be identified as Candida albicans. If germ tubes are absent, confirm the purity of the yeast isolate and inoculate an Vitek YBC or an API20C strip and a Dalmau plate. No identifications will be performed on yeast recovered from respiratory sites except if the yeast resembles Cryptococcus neoformans or if the identification is requested by the physician to the director. For patient care, all germ tube negative yeast from respiratory sites will be reported as yeast not Candida albicans or Cryptococcus sp. Most of the commonly recovered yeasts can be identified to the species level using the morphology on corn meal agar and assimilation results. (See Dalmau morphology flow chart). Rarely, it is hydrolysis, cycloheximide resistance or ascospore induction. These procedures are available to aid in yeast identification. Problem identifications should be brought to the attention of the supervisor. Identifications using a commercial method will be carried out by using materials from the Clinical Microbiology Laboratory which has been ecked for QC. Reimbursement of the material used will be made to them by the MMRC. IV. IDENTIFICATION PROCEDURES: A. Direct Direct Mounts - Direct Direct mounts mounts are made in in order to study study yeast morpholo morphology gy microscopica microscopically lly and to determine purity of the isolates. B. Lact Lactop ophe heno noll Moun Mountt 1. Place a small drop of lactophenol lactophenol (LP) (LP) on a clean glass microsco microscope pe slide. slide. 2. Remove a small small portion portion of the yeast colony colony and place place it into into the drop drop of LP and suspend suspend the cells. 3. Place a clean clean cover glass over the the suspension suspension and and observe observe microscopic microscopically. ally. 4. Seal edges edges of the cover glass glass with fingernai fingernaill polish to temporar temporarily ily preserve preserve the mount.
YEAST MORPHOLOGY FLOW CHART
Brown to black colony
Pink to to re red co colony
Direct Mount
Satellite co colonies
Present
Yeast and hyphae
Aureobasidium Exophiala Wangiella Sporobolomyces
Purify Germ Tube Test
Positive
Direct Mount
Rhodotorula Candida albicans
Negative
Moist mycelial colony
Phaeococcomyces
Forcibly di discharged co conidia pr present
Absent White colony
Yeast only
Purify
Dalmau plate YBC-Vitek
Arthroconidia
Geotrichum Trichosporon
Other types of conidia
hypohomycete
* Young Young colon colonies ies of Cryptococcus neoformans are often indistringuishable from young colonies of Candida. Therefore, technologists performing the germ tube test must be alert to yeasts that have a m icroscopic morphology morphology suggestive of Cr. Cr. neoformans.
DALMAU MORPHOLOGY FLOW CHART
Ascospores present
Hansenula Pichia Saccharomyces
Ballistoconidia present
Sporobolomyces and similar yeasts
Basidiospores present
Filobasidiella Filobasidium Leucosporidium
Hyphae, pseudohyphae, or both present
chlamydospores
Present Absent
Cadida albicans
arthroconidia
Present
Absent
blastoconidia
Present
Trichosporon
Absent
Geotrichum Candida
DALMAU MORPHOLOGY FLOW CHART
Ascospores present
Hansenula Pichia Saccharomyces
Ballistoconidia present
Sporobolomyces and similar yeasts
Basidiospores present
Filobasidiella Filobasidium Leucosporidium
Hyphae, pseudohyphae, or both present
chlamydospores
Present Absent
Cadida albicans
arthroconidia
Present
blastoconidia
Absent
Present
Trichosporon
Absent
Geotrichum Candida Saccharomyces
Sporangia present
Prototheca Sarcinosporon Fissuricella or similar fungi
Yeast only
Cryptococcus Rhodotorula Torulopsis Candida guilliermondii or an ascomycetous yeast not producing ascospores, i.e., Saccharomyces
YEAST IDENTIFICATION IV.
IDENTIFICATION PROCEDURES: (cont'd)
C. Germ Tu Tube Tes Test 1. The germ germ tube test test provides provides a simple, reliable reliable and and economical economical procedur proceduree for the presumptive identification of Candida albicans. About 95% of the clinical isolates produce germ tubes tubes when when incubated incubated in serum at at 35o C for 2.5-3 2.5-3 hours. A germ tube represent represent the initiation of a hypha directly from the yeast cell. They have parallel walls at their point of origin. Germ tube formation is influenced by the medium, inoculum size and temperature of incubation. Fresh normal pooled human sera or a commercially commercially available germ tube solution (Remel Lenexa kansa) are to be used as the medium for the test. The inoculum should result in a very faintly turbid serum suspension. Over-inoculation will inhibit the development development of of germ germ tubes. tubes. Incubate Incubate in in at 35o C-37o C for 2.5-3 hours. 2. A germ tube test is to to be set up up daily on all all cultures cultures positive positive for yeast yeast from from sterile sites, results to be reported in the computer as soon as available as either Candida albicans or yeast not Candida albicans or Cryptococcus identification to follow.
YEAST IDENTIFICATION IV.
IDENTIFICATION PROCEDURES: (cont'd)
C. Germ Tu Tube Tes Test 1. The germ germ tube test test provides provides a simple, reliable reliable and and economical economical procedur proceduree for the presumptive identification of Candida albicans. About 95% of the clinical isolates produce germ tubes tubes when when incubated incubated in serum at at 35o C for 2.5-3 2.5-3 hours. A germ tube represent represent the initiation of a hypha directly from the yeast cell. They have parallel walls at their point of origin. Germ tube formation is influenced by the medium, inoculum size and temperature of incubation. Fresh normal pooled human sera or a commercially commercially available germ tube solution (Remel Lenexa kansa) are to be used as the medium for the test. The inoculum should result in a very faintly turbid serum suspension. Over-inoculation will inhibit the development development of of germ germ tubes. tubes. Incubate Incubate in in at 35o C-37o C for 2.5-3 hours. 2. A germ tube test is to to be set up up daily on all all cultures cultures positive positive for yeast yeast from from sterile sites, results to be reported in the computer as soon as available as either Candida albicans or yeast not Candida albicans or Cryptococcus identification to follow. 3. The germ tube test test is also also used as a preliminary preliminary screen on yeast isolated isolated in the laborato laboratory ry to rule out the presence of Cryptococcus neoformans in respratory or sterile site specimens. If a yeast is encountered with the morphology of Cr. neoformans when screening with the germ tube, the supervisor should be notified immediately, a note should be made in the worksheets and computer, and if proper, physicians should be notified. 4. Since the the time needed needed for the final final identificat identification ion of a yeast yeast after after the germ germ tube test test may take 2-4 days for the germ tube negative yeast and because on occasions, the physician will need a presumptive identification for proper therapy (i.e. urinary infections by T. glabrata versus Candida sp.), a presumptive identification is to be given from the morphology of the yeast in the germ tube test and or colony morphology. 5. Procedure a. Labe Labell 12 12 x 75 75 mm test test tub tubes es.. b. Using a Pasteur pipette, dispense 3 drops of fresh fresh pooled human serum serum into the tubes. tubes. Serum can be obtained from the Serology lab. c. With a sterile sterile wooden applicat applicator or stick, stick, lightly lightly touch a yeast yeast colony colony and place place the stick stick into the serum. d. Suspend the yeast yeast in the serum. Discard Discard the stick in a discard discard container. container. o e. Incuba Incubate te the test test at 35 C for for 2.52.5-3 3 hours hours.. f. Place Place a drop drop of the the suspens suspension ion on a clean clean micro microsco scope pe slide. slide. g. Place a clean cover cover glass glass over the the suspension suspension and and then examine examine it with with a microscope microscope using the low power objective. Use the high power objective to confirm confirm the presence or absence of germ tubes.* h. Read Read cont control rolss and rec recor ord d resul results. ts. i. If time is not allowed allowed to read read and record the test test results results add a drop of 10% 10% formalin/formal formalin/formaldehyde dehyde to each tube, tube, seal the top with with parafilm, parafilm, and store at 2-4 in the refrigerator.
YEAST IDENTIFICATION IV.
IDENTIFICATION PROCEDURES: (cont'd)
* Youn Young g col coloni onies es of Cryptococcus neoformans are often indistinguishable from young colonies of Candida sp., therefore technologists performing germ tube tests must be alert to yeasts that have a microscopic morphology suggestive of Cr. neoformans. Mature cells of Cr. neoformans are spherical, 3-4 um in diameter, with a pointed bud scar. Detached buds are smaller, and oval in shape. Yeasts suspected of being Cr. neoformans must be evaluated as soon as possible with an India Ink mount and supervisor notified. D. Commeri Commerical cal Yeas Yeastt Identif Identifica icatio tion n Systems Systems 1. Principle a. Two commerica commerically lly availab available le yeast yeast identif identificati ication on systems systems are are describ described: ed: API 20C 20C Yeast Identification System (API Analytab Products, Plainview, NY) and the Biomerieux Vitek System (Hazelwood, MO). These two yeast identification identification systems are easy to use. The API 20C requires less preparation of reagents. The Vitek system is an automated system. b. The two systems systems are are based based on modificatio modifications ns of the the classic classic auxanographi auxanographicc technique technique of carbohydrate assimilation. When an organism is able to assimilate a particular carbohydrate, in the cupules of reconstituted substrates (API), or, accompanied by a color change, in the wells containing the substrate of Vitek, the systems must be supplemented with morphological studies, and both systems should have germ tube tests done in conjunction with them as a means of obtaining a more-complete profile of the yeast cells being identified. c. Yeast identific identification ation determine determiness the identity identity of an etiologi etiological cal agent agent causing causing disease and provides access to the information published in the literature regarding diagnosis, patient management, and prognosis. Yeast identifications require isolates in pure culture. d. The Vitek Vitek YBC will be used routinely routinely in this this laboratory laboratory because because it it is semi-aut semi-automated omated and technologist time is not involved in the set up or interpretation of the test. The API 20C is used as a back-up for the YBC. 2. Specimen Specimen - A pure culture culture of of 24- to 48-hour-ol 48-hour-old d yeast cells cells growing growing on Sabouraud Sabouraud glucose glucose (SAB) or other nonselective agar is required. 3. Reagents a. API 20C Yeast Yeast Identific Identification ation System System - In In addition addition to the kit, the following supplies are needed. (1) (2) (2) (3) (4) (5) (6)
Ster Steril ilee wood wooden en appli applica cator tor stic sticks ks Ster Steril ilee Past Pasteu eurr pipe pipett ttes es (5 (5 ml) ml) Squee ueeze bot bottle Incubator (3 (30 C) Wat Water bath (48 to 50 C) SAB ag agar pl plates
YEAST IDENTIFICATION IV.
IDENTIFICATION PROCEDURES: (cont'd)
b. Vitek Yeast Yeast Identifica Identification tion System System - In In addition addition to the kit, the the following following supplies supplies or equipment is needed. (1) (2) (3) (4) (5)
Ster Steril ilee wood wooden en appli applica cator tor stic sticks ks Sterile Sterile tubes tubes contai containing ning 1.8 ml of 0.45 to 0.5% 0.5% saline saline Colorim Colorimete eterr (Vite (Vitek k colori colorimete meter, r, produ product ct no. no. 52-121 52-1210) 0) Fillin Filling g sta stand nd (Vit (Vitek ek produc productt 5252-070 0700) 0) Vitek Vitek Syst System em Senio Senior, r, whic which h includ includes es the the foll followi owing: ng: (a) (a) (b) (b) (c) (d) (e)
(6) (7) (8) (8)
Fill Filler er-s -sea eale lerr modu module le Read Reader er incu incuba bato torr uni unitt Printer Computer Computer (with (with a minimu minimum m of an an R4.01, R4.01, 1989 1989 update updated d data data base) base) Data te terminal
Finene-tip mar markers Incubator (3 (30 C) Sabo Sabour urau aud d dext dextro rose se aga agarr plat plates es
4. Quality Quality Contr Control ol - Incl Include ude known known isolate isolatess of Torulopsis glabrata, Candida albicans, and Cryptococcus laurentii. For specific details refer to the QC Section. 5. Procedure a. API 20C Yeast Yeast Identi Identific ficati ation on System System (1)
(2) (3) (4)
(5)
(6)
Melt Melt the basal basal medium medium in in the ampoul ampoules es by placi placing ng them them in an autocl autoclave ave for for 2 minutes or in a boiling water bath (do not prolong boiling; ampoule may explode). Plac Placee the ampoul ampoules es in in a water water bat bath h at 48 to to 50 C, and and all allowt owthe hem m to coo cool. l. Prepar Preparee an incuba incubation tion tray. tray. Use a squee squeeze ze bottle bottle to to dispense dispense 20 20 ml of water water into into the tray, and then place the strip into the incubation tray. Open the ampoules according according to the the manufact manufacturer's urer's instructions, instructions, and inoculat inoculatee the molten medium with an applicator stickthat has touched one or two colonies (>2 mm diameter). Adjust to a density just below below 1+ on a Wickerham card. Inocula Inoculate te the strip strip (20 (20 cupules; cupules; approx approximat imately ely 0.2 0.2 ml each) each) by using using a Pateur Pateur pipette and following the manufacturer's directions, and then place thelid on the tray. Incubate Incubate the trays trays at at 28 to 30 30 C for for 72 hours hours.. Read and record record the results results after after 24, 48, and 72 hours of incubation.
b. Vitek Vitek Yeas Yeastt Ident Identifi ificat cation ion System System (1)
(2) (3) (4)
Use one one to three three colonie coloniess to prepar preparee the yeast yeast suspen suspension sion in in the 1.8 ml ml saline saline tubes. Adjust the suspension to a McFarland no. 2 standard by using the Vitek colorimeter (46 to 56% transmittance, 450 nm filter). After labeling labeling the yeast cards with a marker, place the card card in the filling filling stand with a transfer tube that is in the yeast suspension. Inocul Inoculate ate the cards cards via via the fillin filling g modul module. e. Seal Seal the the cards cards via the sealer sealer module module and and incub incubate ate at 30 30 C for either either 24 24 or 58 58 hours, depending on the readings provided by the instrument.
YEAST IDENTIFICATION IV.
IDENTIFICATION PROCEDURES: (cont'd)
6. Results a. API 20C Yeast Yeast Identi Identific ficati ation on System System (1) (2) (3) (4)
Although Although API is a widely used commerical commerical yeast identifica identification tion system, system, it does does not include rhamnose (for C.lusitaniae) and urea. Germ Germ tube tests tests andmor andmorphol phologic ogical al studie studiess should should be inclu included. ded. API yeast yeast profiles profiles sometimes sometimes give one one to three different different yeast yeast identificatio identifications ns for an individual isolate. Supplemental tests may then be required. It takes takes 3 days days to to obtai obtain n fina finall resu results lts..
b. Vitek Vitek Yeast Yeast Identi Identific ficati ation on System System (1) (2)
(3)
Heavy encapsulate encapsulated d yeasts yeasts and and isolates isolates with extensive extensive mycelia myceliall growth growth are are sometimes difficult to suspend. Morpholo Morphologic gical al studies studies using using a Dalmau Dalmau plate plate or additiona additionall tests tests are require required d in order to confirm the identification of some isolates or when some strains react similarly in the test system. Most of of the resul results ts are are obtained obtained afte afterr 24 hours hours and and a few isola isolates tes (26%) (26%) may may require additional incubation.
E. Nitr Nitrat atee Uti Utili liza zati tion on Yeasts have the ability to use ammonium sulfate, asparagine, peptone, and urea aerobically as sole sources of nitrogen if adequate vitamins are provided. In contrast, aliphatic amines, potassium nitrate, sodium nitrate, and some amino acids are utilized selectively by different yeasts. In general, if a yeast can utilize nitrate, it can also use nitrate as a nitrogen source. In the auxanographic method, yeast carbon base medium is seeded with a yeast suspension. Potassium nitrate impregnated discs are applied to the surface. Growth around the disc is a positive reaction. 1. Procedure a. Prepare Prepare a yeast suspension suspension from from a 24- to to 48-hour 48-hour old colony in sterile sterile distilled distilled water water equal to a MacFarland number 1 standard. b. Melt a tube tube containing containing 15 15 ml of yeast yeast carbon carbon base medium for for each each yeast to be tested. tested. o Melted tubes tubes can be cooled cooled in a 50 C water bath. c. Add 1.0 ml of the yeast yeast inoculum to the tube of of yeast yeast carbon carbon base base medium. medium. Mix. d. Pour the medium in a 15 x 100 mm sterile sterile Petri Petri dish dish and allow allow the agar agar to harden at room temperature. e. Aseptically Aseptically place discs containing containing KNO3 and peptone on the the medium surface. surface. Label the plates if the discs look alike. f. Incubate Incubate the plate plate with with its inoculat inoculated ed surfa surface ce up up at 30o C. g. Check for growth growth around around the peptone peptone disc. If growth is absent, absent, the test test is is not valid and must be repeated. h. Record Record result resultss of grow growth th around around the KNO3 disc. disc. Growth Growth is a posit positive ive resul result. t.
YEAST IDENTIFICATION IV.
IDENTIFICATION PROCEDURES: (cont'd)
2. Quality Control Control - Control isolates will be used each each time a set of nitrate nitrate utilization utilization tests are are run. a. Posit Positive ive (gro (growth wth aroun around d KNO KNO3 disc) disc) - Rhodotorula glutinis b. Negative - Candida albicans NOTE: This procedure is not routinely performed and is only used for rare situations when the API or Vitex cannot identify a yeast.
F. Urea Urea Hydr Hydrol olysi ysiss - micr microt otit iter er The rapid urea hydrolysis test in microtiter wells is used to screen isolates for Cryptococcus neoformans . Under these conditions, C. neoformans will rapidly hydrolyze urea, which results in a pink to red color. 1. Procedure a. Reconstitute Reconstitute each each vial of Difco Urea Urea R broth with with 3 ml of sterile sterile distilled distilled water water on the day that it is to be used. b. Dispense Dispense 3-4 3-4 drops drops into into each well well to be used used in a micromicro- titer titer plate plate.. c. Transfer Transfer a heavy heavy inoculum inoculum of each each yeast yeast colony colony to a well contain containing ing urea urea broth. broth. A subculture or reincubation of the isolation plate may be necessary if there is insufficient growth of the colony. Isolation of the yeast will be necessary if the culture is contaminated with bacteria. Colonies to be tested should be no older than 7 days. d. Seal Seal wells wells with scotch scotch tape tape and and incubat incubatee for for 4 hours hours at 37o C. 2. Qual Qualit ity y Cont Contrrol a. Posit Positive ive (pink (pink to to red red color color)) - Cryptococcus neoformans b. Negative - Cryptococcus albidus c. Unin Uninoc ocul ulaated ted G. Ascospor Ascosporee Inducti Induction on and Detect Detection ion One step in indentifying a yeast involves determining whether or not the isolate has the ability to form ascospores. Some yeasts will readily form form ascospores on primary isolation medium, whereas others require special media. The ability to form ascospores varies from isolate to isolate and may be lost in old laboratory strains. If only one mating type of a heterothallic yeast is present, no ascospores will be formed. Ascospore media contain small amounts of carbohydrates; this restricts vegetative growth while enhancing ascospore formation. 1. Procedure a. Inoculate Inoculate the yeast to to yeast malt agar agar for enrichment. enrichment. Incubate Incubate 2-3 days. b. Inoculate Inoculate the yeast from the the yeast yeast malt agar to to a V-8 V-8 juice agar slant. Incubate Incubate o aerobica aerobically lly at 20-25 20-25 C.
YEAST IDENTIFICATION IV.
IDENTIFICATION PROCEDURES: (cont'd)
c. Most freshly freshly isolated strains begin forming ascospores ascospores in 1-2 days. Older stock cultures usually require a longer period of time. d. Examine the cultur culturee in 3-5 days days and weekly thereafter thereafter for 3 weeks. Prepare Prepare wet wet mounts of the yeast in distilled water. e. Examine the the wet mounts mounts using the the oil immersio immersion n lense. lense. Ascospore Ascospore form, form, surface surface topography, size, color, brims, number of ascospores per ascus, and the presence or absence of inclusion bodies are characteristics used in part to identify the various species. f. If ascospores ascospores cannot be readily readily seen seen in a wet mount, perform perform an acid-fa acid-fast st stain. stain. The Kinyoun stain is recommended. Ascopsores are acid-fast. 2. Qual Qualit ity y Cont Contrrol Each time a yeast is inoculated to a V-8 juice agar slant, Saccharomyces Saccharomyces cerevisiae will be inoculated concurrently as a control. When performing the Kinyoun stain, use the same S. cerevisiae as a positive control for the staining procedure. V. REFERENCES: 1. Bowman, PI, Ahearn, Ahearn, DG: Evaluation Evaluation of of the the Uni-Yeas Uni-Yeast-Tek t-Tek Kit for the identificatio identification n of medically important yeasts. J. Clin. Microbiol. 2:354-357, 2:354-357 , 1975. 2. Haley, LD: Identifica Identification tion of yeasts in clinical clinical microbiology microbiology laborat laboratories ories.. Am J. Med. Technol. Technol. 37:125-131, 1971. 3. Hupert, M, Harper, Harper, G, Sun, SH, Delanerol Delanerolle, le, V: Rapid methods for identification identification of yeasts. yeasts. J. Clin. Microbiol. 2:21-34, 1975. 4. Land, GA, Harrison, Harrison, BA, Hulme, Hulme, KL, KL, Cooper, Cooper, BH, Byrd, Byrd, JC: JC: Evaluation Evaluation of of the New API 20C strip for for yeast identification against a conventional method. J. Clin. Microbiol. 10:357-364, 10:357-36 4, 1979. 5. Salkin, Salkin, IF, Land, GA, Hurd, NJ, NJ, Goldson, Goldson, PR, McGinnis, MR: Evaluation Evaluation of YeastIdent YeastIdent and and Uni-Yeast-Tek yeast identification systems. J. Clin. Microbiol. 25:624-627, 25:624-62 7, 1987. 6. The Yeasts. Yeasts. A Taxonomic Taxonomic Study, Kreger-van Kreger-van Rij, NJW (ed), (ed), 3rd Edition, Edition, Amsterdam, Amsterdam, Elsevier Elsevier Publishers, 1984. 7. Larone, Larone, DH: Medically Medically Important Important Fungi. A Guide Guide to Identificat Identification. ion. 2nd Edition, Edition, New York, Elsevier Publishers, 1987. 8. McGinnis, McGinnis, MR: Laboratory Laboratory Handbook Handbook of Medical Medical Mycology Mycology,, New York, Academic Academic Press, Press, 1980. 9. Pincus, Pincus, DH, Salkin, Salkin, IF, IF, McGinni McGinnis, s, MR: MR: Rapid methods methods in medical medical mycology. mycology. Lab. Med. 19:315-320, 1988. 10. El-Zaatari, El-Zaatari, M, Pasarell, Pasarell, L, McGinnis, MR, Buckner, Buckner, J, J, Land, GA, Salkin, Salkin, IF: IF: Evaluation Evaluation of the updated Vitek yeast identification identification data base. J. Clin. Microbiol. 28:1938-1941, 28:1938-1941, 1990.
