Teknis Identifikasi Jamur Biakan Identifikasi

IDENTIFIKASI JAMUR SIGIT SULISTYA BALAI LABORATORIUM KESEHATAN YOGYAKARTA 2010

IDENTIFIKASI JAMUR

Untuk mengidentifikasi jamur lebih diutamakan pengujian sifat-sifat morfologinya pengujian sifat-sifat fisiologi

Metode pemeriksaan laboratorium:Pemeriksaan Mikroskopis : Lansung dan Tak LansungKultur Biakan IdentifikasiAPI Medium : API C20 ( Untuk Yeast)Vitek 2 ( Yeast)

IDENTIFIKASI JAMUR

pengujian sifat-sifat morfologinya

Pemeriksaan makroskopis

Pemeriksaan mikroskopis lansung menggunakan Larutan KOH 10 %

Slide Kultur menggunakan teknik biakan identifikasi dengan media Saboroud Agar

Test Fermentasi dan Test Asimilasi

Uji biokimia

MEDIA JAMUR

NOMIKROORGANISMEMEDIA ISOLASIMEDIA DIFERENSIALTEST KONFIRMASI1AspergillusMucorRhizhopusYM brothSaboroud agarPotato AgarKOH 10 %2CandidaS. Ceriviceae YM brothSaboroud agarPotato AgarCandida Elektif Agar WL Nutrien AgarCorn Meal AgarEMB AgarGlukosa Pepton 0,5 %KOH 10 %Cat Gram: A,B,C dan D3TrichosporonTrichophyton Microsporum YM brothSaboroud agarPotato AgarWL Nutrien AgarKOH 10 %

4CryptococcusYM brothSaboroud agarPotato AgarWL Nutrien AgarIndia INKCat Gram: A,B,C dan DUrea Agar

Aspergillus Differential AgarIntended UseAspergillus Differential Agar is used in the differentiation of Aspergillus species based on pigmentation

Summary and ExplanationBothast and Fennel developed Aspergillus Differential Agar as a screening medium to detect pigment produced under colonies of Aspergillus flavus (flavus group).1 The yelloworange pigment differentiates A. flavus from most other Aspergillus species and from organisms of other genera.1-3 Some other Aspergillus species may also produce a yellow-orange pigment indistinguishable from the pigment produced by A. flavus

Aspergillus Differential AgarProcedureThe isolate to be differentiated should be stained with lactophenol cotton blue or an appropriate fungal stain and examined to confirm that morphology is appropriate for Aspergillus species. Using a sterile inoculating loop or needle, pick several isolated colonies and streak the surface of the slant Incubate the tubes at 25C for up to 10 days to allow sufficient time for pigmentation to develop

Expected ResultsExamine the medium for typical growth and pigmentationA. flavus produces a yellow-orange pigment under colonies

Limitation of the ProcedureA. parasiticus, another species associated with aspergillosis,4 as well as some other aspergilli (i.e., A. sulphureus, A. sclerotiorum and A. thomii) may also produce a yelloworange pigment that is indistinguishable from the pigment produced by A. Flavus.

Czapek-Dox Broth Czapek Solution AgarIntended UseCzapek-Dox Broth and Czapek Solution Agar are used forcultivating fungi and bacteria capable of using inorganic nitrogen. Czapek Solution Agar is recommended in Standard Methods for the Examination of Water and Wastewater5 for the isolation of Aspergillus, Penicillium, Paecilomyces and related fungi

ProcedureRefer to appropriate references for specific procedures for thecultivation of fungi and bacteria capable of utilizing inorganicnitrogenExpected ResultsRefer to appropriate references and procedures for results

BiGGY AgarIntended UseBiGGY (Bismuth Sulfite Glucose Glycine Yeast) is a selectiveand differential medium used in the detection, isolation andpresumptive identification of Candida species.

Summary and ExplanationBiGGY Agar is based on the formulation of Nickerson.1Nickerson developed the medium in 1953 following a studyof sulfite reduction by Candida species.Differentiation of Candida is based on growth patterns andpigmentation of isolated colonies. The bismuth sulfite actsas an inhibitory agent to suppress bacterial growth, whichenables the recovery of isolated colonies of Candida. Candidaspecies reduce the bismuth sulfite, resulting in pigmentationof colonies and, with some species, pigmentation in thesurrounding medium.

BiGGY AgarProcedureConsult appropriate references for information about the processing and inoculation of specimens such as tissues, skin scrapings, hair, nail clippings, etc.2-5 The streak plate technique is used primarily to obtain isolated colonies from specimens containing mixed flora. When using slants, streak the surface of the slant with a sterile inoculating loop needle using two to three isolated colonies.Incubate plates in an inverted position (agar side up) for upto 5 days at 25 2C.Expected ResultsWithin 5 days of incubation, the plates should show isolated colonies in streaked areas and confluent growth in areas of heavy inoculation. Slants should show evidence of growth. Examine plates and slants for colonies showing characteristic growth patterns and morphology. The following table summarizes typical Candida colonial morphology.

Candida BCG Agar BaseCandida Bromcresol Green AgarIntended UseCandida Bromcresol Green (BCG) Agar is a differential andselective medium used for primary isolation and detection ofCandida species from clinical specimens

Summary and ExplanationCandida BCG medium employs the formula devised by Harold and Snyder.1 They demonstrated that the triphenyltetrazolium chloride (TTC) being used as an indicator in Pagano Levin medium retarded the growth of some species of Candida and completely inhibited the growth of others. To overcome this, they replaced TTC with bromcresol green, a non-toxic indicator, to develop Candida BCG Agar. Neomycin is incorporated to inhibit gram-negative and some gram-positive bacteria.

Candida BCG Agar BaseCandida Bromcresol Green AgarProcedureUse standard procedures to obtain isolated colonies fromspecimens. Incubate the plates in an inverted position (agar side up) at 30 2C for up to 72 hours

Expected ResultsCandida species produce convex to cone-shaped, smooth to rough colonies. The color of the medium around the colonies becomes yellow, usually within 72 hours. Gram staining, biochemical tests and serological procedures should be performed to confirm findings

Candida Isolation AgarIntended UseCandida Isolation Agar is used for isolating and differentiatingCandida albicans. Candida Isolation Agar is a nutritionally rich medium that supports growth of many yeasts and molds and is differential for Candida albicans. Candida Isolationn Agar was developed using modification of YM Agar as described by Fung and Liang.1 Goldschmidt demonstrated that YM Agar with aniline blue WS could be used to identify C. albicans in clinical samples with high accuracy and predictability.2 Aniline blue is metabolized by C. albicans to produce a fluorescent moiety that can be detected under long-wave UV light.2

Candida Isolation AgarProcedureProcess each specimen as appropriate for that specimen and inoculate directly onto the surface of the medium. Streak for isolationIncubate plates aerobically at 30C for 18-72 hoursExamine plates for growth after 18-72 hours of incubation.Expected ResultsColonies of C. albicans fluoresce yellow-green under long-wave UV light following incubation at 30C for 18-24 hours. Non- C. albicans isolates do not fluoresceLimitations of the ProcedureStrains of Candida albicans have been reported that are false negative for fluorescence on this mediumStrains of C. parapsilosis, C. krusei and C. Pulcherrima that fluoresce on this medium may be encountered.2 These strains may be distinguished from C. albicans based on germ tube formation in serum

ProcedureUse standard procedures to obtain isolated colonies from specimens. Incubate the plates in an inverted position (agar side up) at 30 2C for up to 72 hours. Expected ResultsCandida species produce convex to cone-shaped, smooth to rough colonies. The color of the medium around the colonies becomes yellow, usually within 72 hours. Gram staining, biochemical tests and serological procedures should be performed to confirm findings

Corn Meal Agar Corn Meal Agar withPolysorbate 80 Corn Meal Agar with 1% DextroseIntended UseCorn Meal Agar is a general-purpose medium for the cultivation of fungi. With the addition of polysorbate 80, it is utilized primarily for the testing of Candida species for their ability to produce chlamydospores. BBL prepared plates of Corn Meal Agar with Polysorbate 80 are deep-filled to reduce the effects of drying during prolonged incubation. Corn Meal Agar with 1% Dextrose enhances pigment production.

Summary and ExplanationCorn Meal Agar has been used for many years to cultivate fungi. Pollack and Benham reported on its usefulness for studying the morphology of Candida.1 In 1960, Walker and Huppertmodified the basic formulation of Corn Meal Agar by adding polysorbate 80, which stimulated rapid and abundant chlamydospore formation.2 This modified formulation is recommended for the production and viualization of chlamydospores

Corn Meal Agar Corn Meal Agar withPolysorbate 80 Corn Meal Agar with 1% DextroseProcedureTo prepare plated media from agar deeps, place the agar deeps in a boiling water bath until the medium becomes liquefied (clear). Pour the molten medium into a sterile Petri dish and allow to solidify before use. Organisms to be cultivated for identification must first be isolated in pure culture on an appropriate medium. Using an inoculating needle, streak the medium with growth from a pure culture and incubate at 25 2C. Examine at intervals for up to 28 days for growth and pigmentation. Corn Meal Agar with 1% Dextrose should be incubated for up to 4 weeks to allow sufficient time fo pigmentation to develop. Test for the production of chlamydospores on medium containing polysorbate 80 using the Dalmau plate method.6 With a sterile inoculating needle, lightly touch the yeast colony, and then make two separate streaks approximately 1.5 cm long each and 1.0 cm apart. Do not dig into the agar. Flame the needle, allow to cool. Then lightly make an S-shaped streak back and forth across the two original streak lines. Flame a coverslip and, after it cools, place it over the central area of the stab marks to provide slightly reduced oxygen tension.3 Incubate the plates at room temperature (25 2C) for 24-48 hours. If the test is negative, reincubate plates an additional 48-72 hours and examine again.

Corn Meal Agar Corn Meal Agar withPolysorbate 80 Corn Meal Agar with 1% DextroseThe addition of dextrose enhances fungal growth and pigmentproduction.4 Corn Meal Agar with Dextrose is commonly usedin the differentiation of Trichophyton species based on chromogenesis

Cooke Rose Bengal AgarAntimicrobic Vial AIntended UseCooke Rose Bengal Agar is used with or without AntimicrobicVial A in isolating fungi from environmental and food specimens.Antimicrobic Vial A is used in preparing microbiological culturemedia.ProcedureRefer to appropriate references for specific procedures on theisolation and cultivation of fungi.Expected ResultsRefer to appropriate references and procedures for results.Limitations of the ProcedureAlthough this medium is selective primarily for fungi,microscopic examination is recommended for presumptiveidentification. Biochemical testing using pure cultures isrequired for complete identification.Due to the selective properties of this medium and the type of specimen being cultured, some strains of fungi may be encountered that fail to grow or grow poorly on the complete medium; similarly, some strains of bacteria may be encountered that are not inhibited or only partiallyinhibited. Care should be taken not to expose this medium to light, since photo-degradation of rose bengal yields compounds that are toxic to fungi.

Dermatophyte Test Medium Base DermatophyteTest Medium, Modified with ChloramphenicolIntended UseDermatophyte Test Medium (DTM) is a selective and differential medium used for the detection and presumptive identification of dermatophytes from clinical and veterinary specimens.1 Because of the unavailability of one of the inhibitory agents, chlortetracycline, Dermatophyte Test Medium (DTM), Modified with Chloramphenicol is recommended as a substitute for the original DTM formation Dermatophytes cause cutaneous fungal infections of the hair, skin and nails generally referred to as tinea or ringworm.2-4 Members of the genera Trichophyton, Microsporum and Epidermophyton are the most common etiologic agents of these infections.

Dermatophyte Test Medium Base DermatophyteTest Medium, Modified with ChloramphenicolProcedureInoculate the specimen as soon as possible after it is received in the laboratory. Implant cutaneous specimens by gently pressing the samples into the agar surface.For isolation of fungi from potentially contaminated specimens, a nonselective medium should be inoculated along with the selective medium. Incubate plates at 22-25C in an inverted position (agar side up) with increased humidity and tubes with caps loosened to allow air to circulate.Expected ResultsDermatophytes produce typical morphology and a pink to red color in the medium around the colony within 10-14 days of incubation. Disregard color changes after the fourteenth day of incubation because they may be caused by contaminating fungi.5 Certain strains of Candida albicans are capable of converting the indicator to red, but the yeast can be recognized by their white bacteria-like colonial appearance. Certain nondermatophyte fungi rarely can produce alkaline products (false positives).

Eosin Methylene Blue Agar, Levine

M-Green Yeast and Mold BrothIntended UseM-Green Yeast and Mold Broth is used for the detection offungi in the routine analysis of beverages.

Summary and ExplanationM-Green Yeast and Mold Broth is an improved modificationof the liquid medium, M-Yeast and Mold Broth, which wasdeveloped to improve the efficiency of detection and enumerationof fungi in sugar and other materials by the membranefilter method. The revised formula contains the indicator dyebromcresol green. It is a relatively more complex formula thanmany of the other media exclusively used for the recovery ofyeasts and molds

Procedure1. Saturate a sterile membrane filter pad in a sterile Petri dishwith 2.0-2.5 mL of M-Green Yeast and Mold Broth.2. Roll a membrane filter, which has been used to filter thetest sample, onto the surface of the moistened pad so as toavoid the trapping of air bubbles between the filter and thepad.3. Incubate the plates at 30-35C for 48 hours and up to 5days in an aerobic atmosphere with increased humidity.

Malt AgarIntended UseMalt Agar is used for isolating and cultivating yeasts and molds from food and for cultivating yeast and mold stock cultures.Summary and ExplanationMalt media for yeasts and molds have been widely used for many years. In 1919, Reddish1 prepared a satisfactory substitute for beer wort from malt extract. Thom and Church2 used Reddishs medium for their studies of the aspergilli. Malt Agar was also employed by Fullmer and Grimes3 for their studies of the growth of yeasts on synthetic media. Malt Agar is included in Official Methods of Analysis of AOAC International.

Limitation of the ProcedureDo not heat the medium after addition of acid, as this will hydrolyze the agar and reduce its solidifying properties.

Inhibitory Mold AgarInhibitory Mold Agar with GentamicinIntended UseInhibitory Mold Agar, which contains chloramphenicol, is a moderately selective medium used for the isolation of pathogenic fungi. BBL prepared plates of Inhibitory Mold Agar and Inhibitory Mold Agar with Gentamicin are deep filled to reduce the effects of drying during prolonged incubation.

Summary and ExplanationInhibitory Mold Agar was formulated by Ulrich as a general medium for the selective isolation and cultivation of the majority of pathogenic fungi.

Inhibitory Mold AgarInhibitory Mold Agar with GentamicinProcedureConsult appropriate references for information about the processing and inoculation of specimens.2For isolation of fungi from potentially contaminated specimens, a nonselective medium should be inoculated along with theselective medium. Incubate the plates at 25-30C in an invertedposition (agar side up) with increased humidity. Thetubed slants also should be incubated at 25-30C. For isolation of fungi causing systemic mycoses, two sets of media should be inoculated, with one set incubated at 25-30C and a duplicate set at 35 2C. All cultures should be examined at least weekly for fungal growth and should be held for 4-6 weeks before being reported as negative. Expected ResultsExamine plates for fungal colonies exhibiting typical color andmorphology. Biochemical tests and serological proceduresshould be performed to confirm findings.Limitation of the ProcedureSome fungi may be inhibited by the antibiotics in InhibitoryMold Agar and Inhibitory Mold Agar with Gentamicin

Malt Extract Agar Malt Extract BrothIntended UseMalt Extract Agar is used for isolating, cultivating and enumerating yeasts and molds. Malt Extract Broth is used for cultivating yeasts and molds.

Summary and ExplanationThe use of malt and malt extracts for the propagation of yeasts and molds is quite common. Reddish1 described a culture medium prepared from malt extract that was a satisfactory substitute for wort. Thom and Church,2 following the formula of Reddish, used malt extract as a base from which they prepared the complete media. Malt Extract Broth is recommended for the examination of yeasts and molds in the U.S. Food and Drug Administrations Bacteriological Analytical Manual

OGYE Agar BaseAntimicrobic Vial OxytetracyclineIntended UseOGYE Agar Base is for use with Antimicrobic Vial Oxytetracycline in isolating and enumerating yeasts and molds in foods.Summary and ExplanationAcidified agar may be used for enumerating yeasts and moldsin foods and dairy products. However, in some cases,antimicrobics better suppress bacterial growth and improverecovery of yeasts and molds.1,2 Mossel et al.3,4 described Oxytetracycline-Glucose Yeast Extract (OGYE or OGY) Agar for selectively isolating and enumerating yeasts and molds in foods. Mossel et al. Demonstrated improved recovery compared to acidified agar media. OGYE Agar is specified as a standard methods medium for use with dairy products

Yeast Extract AgarIntended UsePhytone Yeast Extract Agar is used for the selective isolationof dermatophytes, particularly Trichophyton verrucosum, and other pathogenic fungi from routine clinical specimens.Summary and ExplanationCarmichael and Kraus modified the classical formula of Sabouraud medium in order to selectively recover Trichophyton verrucosum, one of the species associated with ringworm, from clinical specimens.1,2Phytone Yeast Extract Agar is used in Petri dishes for earlydetection of dermatophytes. Skin scrapings or hairs are rubbed over the surface of the agar. Blood agar plates should be inoculated in parallel to permit isolation of pyogenic cocci which may also be present. The medium is of value for increasing the yield of isolation of ringworm organisms and for early identification, especially of T. verrucosum. Inoculate skin scrapings, hair or other materials directly on the agar surface of Petri plates. Incubate plates in an aerobic atmosphere at 25-30C or at 30-37C if T. verrucosum is suspected. Expected ResultsAfter the plates have been incubated for 2-3 days, examine them directly under the microscope. If microcolonies are observed, they should be transferred to fresh plates before the original plates become overgrown.

Yeast Extract AgarIntended UsePotato Dextrose Agar conforms with specifications of TheUnited States Pharmacopeia (USP).Potato Dextrose Agar is used for the cultivation and enumerationof yeasts and molds.Potato Dextrose Broth is used for cultivating yeasts and molds.Summary and ExplanationPotato Dextrose Agar is recommended by the AmericanPublic Health Association for plate counts of yeasts andmolds in the examination of foods and dairy products.1,2 It isrecommended in the USP for use in the performance ofMicrobial Limit Tests.3 It is also used for the stimulation ofsporulation (slide preparations), maintenance of stock culturesof certain dermatophytes and for differentiation of atypicalvarieties of dermatophytes by pigment production.4Potato Dextrose Broth is a general-purpose broth medium foryeasts and molds (Potato Dextrose Agar without the agar

Yeast Extract AgarProcedureConsult appropriate references for information concerning the processing and inoculation of specimens.1-3,5,6 Liquefy the medium in pour tubes by heating in boiling water. Cool to 45-50C and pour into sterile Petri dishes. Allow to solidify for a minimum of 30 minutes.Streak the specimen onto prepared media with a sterile inoculating loop to obtain isolated colonies. When used for determining yeast and mold counts, the medium should be adjusted to a Ph of approximately 3.5 with sterile tartaric aid and used in the standard pour plate technique. Incubate the plates at 25-30C in an inverted position (agar side up) with increased humidity. Tubed slants are used primarily for the cultivation and maintenance of pure cultures. They should be inoculated with an inoculating loop and incubated under the same conditions as the plated medium. For isolation of fungi from potentially contaminated specimens, a selective medium should be inoculated along with the nonselective medium. For isolation of fungi causing systemic mycoses, two sets of media should be inoculated, with one set incubated at 25-30C Limitations of the ProcedureHeating Potato Dextrose Agar after acidifying hydrolyzes the agar and may destroy the solidifying properties. Potato Dextrose Agar is not a differential medium. Perform microscopic examination and biochemical tests to identify isolates to genus and species if necessary.

Potato Flakes Agar Potato Flakes CC AgarPotato Flakes Agar with Chloramphenicol andGentamicinIntended UseThese media are used in qualitative procedures for the cultivation of pathogenic and opportunistic fungi encountered in clinical mycology.Summary and ExplanationPotato Flakes Agar induces sporulation, enhancing the production of morphological structures required for the identification of many pathogenic and opportunistic fungi.1The addition of chloramphenicol and cycloheximide (CC) or gentamicin provides selectivity for more effective isolationand identification of medically significant fungi. fungi, while permitting the growth of pathogenic species. Gentamicinis an aminoglycoside antibiotic that inhibits growth of gram-negative bacteria.ProcedureConsult appropriate references for information about the processing and inoculation of specimens such as tissues, skinscrapings, hair, nail clippings, etc.3-5 For isolation of fungi causing cutaneous mycoses, a nonselective medium should be inoculated along with a selective medium.Incubate the plates at 25-30C in an inverted position (agar side up) with increased humidity. For isolation of fungi causing systemic mycoses, two sets of media should be inoculated with one set incubated at 25-30CExpected ResultsExamine the media for growth. Microscopic examination ofthe colony aids in identification.

Sabouraud Brain Heart Infusion Agar BaseSabouraud Brain Heart Infusion Agar SabouraudBrain Heart Infusion Agar with AntimicrobicsIntended UseSabouraud Brain Heart Infusion Agar is used in qualitativeprocedures for cultivation of dermatophytes and other pathogenicand nonpathogenic fungi from clinical and nonclinicalspecimens. The medium is rendered selective by the additionof antimicrobial agents.Summary and ExplanationSabouraud Brain Heart Infusion Agar is based on the formulationof Gorman.1 The combination of Brain Heart InfusionAgar and Sabouraud Dextrose Agar in this medium improvesthe recovery of fungi compared with the recovery on eithermedium individually. The addition of defibrinated sheep bloodis recommended to increase the recovery of fastidious, dimorphicfungi.2The antimicrobial agents chloramphenicol, cycloheximide andgentamicin are incorporated in various combinations toimprove the recovery of pathogenic fungi from specimensheavily contaminated with bacteria and saprophytic fungiProcedureUse standard procedures to obtain isolated colonies from specimens.For isolation of fungi from potentially contaminated specimens,both a nonselective and a selective medium should be inoculated.Incubate the plates at 25-30C in an inverted position (agar sideup) with increased humidity. For isolation of fungi causingsystemic mycoses, two sets of media should be inoculated, withone set incubated at 25-30C Expected ResultsAfter sufficient incubation, the plates should show isolatedcolonies in streaked areas and confluent growth in areas ofheavy inoculation.Examine the plates for fungal colonies exhibiting typical colorand morphology. Biochemical tests and serological proceduresshould be performed to confirm findings

Sabouraud Media (Low pH)Sabouraud Dextrose Agar Sabouraud DextroseAgar with Antimicrobics Sabouraud Dextrose Agarwith Lecithin and Polysorbate 80 SabouraudDextrose Broth Sabouraud Maltose AgarSabouraud Maltose BrothIntended UseSabouraud Dextrose Agar conforms with specifications of TheUnited States Pharmacopeia (USP).Sabouraud Dextrose Agar is used in qualitative procedures forcultivation of pathogenic and nonpathogenic fungi, particularlydermatophytes. The medium is rendered more selective for fungiby the addition of antimicrobics. Sabouraud Dextrose Brothand Sabouraud Maltose Agar and Broth are also used forculturing yeasts, molds and aciduric microorganisms.Fluid Sabouraud Medium is used for cultivating yeasts, moldsand aciduric microorganisms and for detecting yeasts and moldsin normally sterile materials.Summary and ExplanationSabouraud Dextrose Agar is a general-purpose mediumdevised by Sabouraud for the cultivation of dermatophytes.1The low pH of approximately 5.6 is favorable for the growthof fungi, especially dermatophytes, and slightly inhibitory tocontaminating bacteria in clinical specimens.2-4 This mediumis recommended in the USP for use in performing totalcombined mold and yeast counts (Microbial Limit Tests).5The addition of antimicrobics is a modification designed toincrease bacterial inhibition.

an agar medium can be over-filled, producing a meniscus ordome-shaped surface that can be pressed onto a surface forsampling its microbial burden. These plates are used in avariety of programs to establish and monitor cleaning techniquesand schedules.6-10 After touching the surface to besampled with the medium, the environmental sampling dish iscovered and incubated at an appropriate temperature. Thepresence and number of microorganisms is determined by theappearance of colonies on the surface of the agar medium.11Collection of samples from the same area before and aftercleaning and treatment with a disinfectant permits the evaluationof the efficacy of sanitary procedures.Sabouraud Maltose Agar is a modification of SabouraudDextrose Agar with maltose substituted for the dextrose. It isa selective medium due to the acid pH. Davidson et al.reported that Sabouraud Maltose Agar was a satisfactorymedium in their studies of infections caused by Microsporumaudouini, M. lanosum and Trichophyton gypseum.12 Davidsonand Dowding also used this medium in isolating T. gypseumfrom a case of tinea barbae.13Sabouraud Maltose Broth is a modification of SabouraudDextrose Broth in which maltose is substituted for dextrose. It isselective due to its acid pH and is used for the detection of fungi.Fluid Sabouraud Medium is employed in sterility testprocedures for determining the presence of molds, yeasts andaciduric microorganisms. The acid reaction of the finalmedium is inhibitive to a large number of bacteria and makesthe medium particularly well suited for cultivating fungi andacidophilic microorganisms.

ProcedureFor isolation of fungi from potentially contaminated specimens,a selective medium should be inoculated along with the nonselectivemedium. Incubate the containers at 25-30C withincreased humidity. All cultures should be examined at leastweekly for fungal growth and should be held for 4-6 weeksbefore being reported as negative.Liquefy the medium in pour tubes by heating in boiling water.Cool to 45-50C and pour into sterile Petri dishes. Allow tosolidify for a minimum of 30 minutes.Prepared tubed slants primarily are intended for use with purecultures for maintenance or other purposes. With preparedplates and Mycoflask bottles, streak the specimen as soonas possible after it is received in the laboratory, using a sterileinoculating loop to obtain isolated colonies. Consultappropriate references for information about the processingand inoculation of specimens.3,4For the Sterile Pack media, sample selected surfaces by firmlypressing the agar medium against the test area. Hold the platewith thumb and second finger and use index finger to pressplate bottom firmly against surface. Pressure should be thesame for every sample. Do not move plate laterally as thisspreads contaminants over the agar surface making resolutionof colonies difficult. Slightly curved surfaces may be sampledwith a rolling motion.Areas (walls, floors, etc.) to be assayed may be divided intosections or grids and samples taken from specific points withinthe grid.Incubate exposed plates at 35-37C for 48 hours, and 25Cfor 7 days or as required.Expected ResultsAfter sufficient incubation, the containers should show isolatedcolonies in streaked areas and confluent growth in areas ofheavy inoculation. Transfer of growth from slants to platedmedia may be required in order to obtain pure cultures of fungi.Examine containers for fungal colonies exhibiting typical colorand morphology.20 Biochemical tests and serological proceduresshould be performed to confirm findings.In the RODAC procedure, colonies are counted (fewer than200 colonies for accurate counts) and expressed as eitherthe number of colonies per RODAC plate or the number ofcolonies per cm.2,21,22 Criteria for cleanliness of equipment andenvironment (surfaces) can be developed by using a databasederived from repeated routine sampling of specific sites.23Subculture colonies of interest so that positive identificationcan be made by means of biochemical testing and/or microscopicexamination of organism smears.Limitation of the ProcedureSome fungi may be inhibited by the acidic pH of the mediumand by the antimicrobics in the selective media.

Sabouraud Agar, Modified Sabouraud DextroseAgar, Emmons Sabouraud Dextrose Agar, Emmons,with Antimicrobics

Intended UseSabouraud Agar, Modified (Emmons) and Sabouraud DextroseAgar, Emmons are used in qualitative procedures for cultivationof dermatophytes and other pathogenic and nonpathogenic fungifrom clinical and nonclinical specimens.Sabouraud Dextrose Agar, Emmons is rendered selective bythe addition of antimicrobial agents.Summary and ExplanationSabouraud Dextrose Agar was devised by Sabouraud for thecultivation of dermatophytes.1 The low pH of approximately5.6 is favorable for the growth of fungi, especially dermatophytes,and inhibitory to contaminating bacteria in clinicalspecimens.2 The acidic pH, however, also may inhibit somefungal species.2-4 Emmons modified the original formulationby adjusting the pH close to neutral to increase the recovery offungi and by reducing the dextrose content from 40 to 20 g/L.4The two base formulations offered differ in peptone contentand amount of agar. The addition of antimicrobics furtherincreases the selectivity of the medium.3

ProcedureConsult appropriate references for information about theprocessing and inoculation of specimens.2,3Prepared tubed slants primarily are intended for use with purecultures for maintenance or other purposes.For isolating fungi from potentially contaminated specimens,a selective medium should be inoculated along with the nonselectivemedium. Incubate the plates at 25-30C in an invertedposition (agar side up) with increased humidity. For isolationof fungi causing systemic mycoses, two sets of media shouldbe inoculated, with one set incubated at 25-30C and a duplicateset at 35

WL Nutrient Medium and WL Nutrient Broth are used forcultivating yeasts, molds and bacteria encountered in brewingand industrial fermentation processes.WL Differential Medium is used for isolating bacteria encounteredin brewing and industrial fermentation processes.Summary and ExplanationWL (Wallerstein Laboratory) nutrient media were developedby Green and Gray1,2 in their study of various fermentationprocesses. An exhaustive study examining the methods offermentation control procedures in worts, beers, liquid yeastsand similar fermentation products led to the development ofthese media.At a pH of 5.5, counts of viable bakers yeast may be made onthe WL Nutrient Medium. By adjusting the pH to 6.5, themedium is suitable for obtaining counts of bakers and distillersyeast. The medium can support the growth of bacteria, butunless the number of yeast cells is small the bacteria may notbe detected. Due to this limitation, Green and Gray developedWL Differential Medium that inhibits the growth of yeastswithout inhibiting the growth of bacteria present in beersWL Nutrient Medium and WL Differential Medium are usedsimultaneously as a set of three plates. One plate is preparedfrom WL Nutrient Medium and two plates from WL DifferentialMedium.3 The WL Nutrient Medium plate is incubatedaerobically to obtain a total count of mainly yeast colonies. Adifferential agar plate is incubated aerobically for growth ofacetic acid bacteria, Flavobacterium, Proteus and thermophilicbacteria. Another differential agar plate is incubated anaerobicallyfor growth of lactic acid bacteria and Pediococcus.

YM Agar YM BrothIntended UseYM Agar and YM Broth are used for cultivating yeasts, moldsand other aciduric microorganisms.Summary and ExplanationYM Agar and YM Broth (Yeast Mold Agar and Broth) areprepared according to the formulae published by Wickerham.1-3Wickerham suggested that YM Broth acidified to pH 3.0-4.0be used as an enrichment medium for yeasts from populationsalso containing bacteria and molds.Media selectivity may be enhanced through acidification orthrough addition of selective agents. YM Broth may be acidifiedprior to sterilization. YM Agar should be sterilized withoutpH adjustment and sterile acid added to the sterile moltenmedium cooled to 45-50C. Acidified YM Agar should not beheated. Antibiotics may be aseptically added to the sterile media.Other fungistatic materials, such as sodium propionateand diphenyl may be added to YM Agar to eliminate moldsand permit the enumeration of yeasts in mixed populationsProcedureInoculate YM Agar plates or YM Broth tubes with sampleto evaluate for the presence of yeasts, molds, or aciduricmicroorganisms. Incubate at 30 To favor isolation of fermentative species, add a layer ofsterile paraffin oil 1 cm deep on the surface of the inoculatedbroth. Incubate the culture until growth appears and then streakonto YM Agar to obtain isolated yeast colonies. To isolatefermentative and oxidative strains, place acidified inoculatedYM Broth on a rotary shaker for 1 or 2 days. This favors yeastrecovery while preventing the sporulation of molds.Expected ResultsExamine the plates or tubes for growth. Record YM Agarresults as colony-forming units (CFU) per volume of sample.Record YM Broth results as growth or no growth.

Yeast Extract Glucose Chloramphenicol AgarIntended UseYeast Extract Glucose Chloramphenicol Agar is a selective agarrecommended by the International Dairy Federation1,2 for enumeratingyeasts and molds in milk and milk products.Summary and ExplanationThe antibiotic method for enumerating yeasts and molds indairy products has become the method of choice, replacingthe traditional acidified method.2 The use of antibiotics forsuppressing bacteria results in better recovery of injuredfungal cells, which are sensitive to an acid environment, andin less interference from precipitated food particles during thecounting.3-7Yeast Extract Glucose Chloramphenicol Agar is a nutrientmedium that inhibits the growth of organisms other than yeastsand molds due to the presence of chloramphenicol. When asample contains predominantly yeasts and/or injured yeasts,the use of Yeast Extract Glucose Chloramphenicol Agar mayoffer some advantage.2 After incubation at 25C, colonies arecounted and yeast colonies are distinguished from molds bycolony morphology.

Procedure1. Prepare initial sample dilutions using 10 g or 10 mL of samplein 90 mL of diluentAdd 10 mL from the initial dilution prepared above (#1) to90 mL of 1/4-strength Ringers solution. One milliliter(1 mL) of this dilution corresponds to 0.01 g/mL of sample.3. Prepare further dilutions by adding 10 mL of the 0.01 g/mLdilution above (#2) to 90 mL of diluent.Pipette 1 mL of each dilution into two Petri dishes.5. Pour 10 mL of sterile molten agar (cooled to 45C) intoeach dish. Mix thoroughly.6. Incubate at 25C for 4 days.Expected Results1. Select plates containing 10-300 colonies and count the colonies.Distinguish yeasts from molds by colony morphology.2. Express results as yeasts and molds per gram or permilliliter.

Yeast Extract Phosphate (YEP) AgarIntended UseYeast Extract Phosphate (YEP) Agar is used in qualitative proceduresfor the isolation of dimorphic pathogenic fungi fromclinical specimens. The plates are deep-filled to reduce theeffects of drying during prolonged incubation.Summary and ExplanationSmith and Goodman developed YEP Agar for the primary recoveryof Blastomyces dermatitidis and Histoplasmacapsulatum from contaminated specimens.1 The medium isdesigned to be used with ammonium hydroxide, a selectiveagent that improves the recovery of dimorphic pathogens byinhibiting bacteria, yeasts and saprophytic fungi.2,3ProcedureUse standard procedures to obtain isolated colonies from specimens.Add one drop of concentrated NH4OH (ammonia) atthe edge of the inoculated medium and allow the medium tosit for 20 minutes before inverting.Incubate the plates in an inverted position (agar side up) at22-25C.Expected ResultsAll cultures should be examined for growth at least weekly.Cultures should be held for 4-6 weeks before reporting asnegative.

Yeast Fermentation Broth Base with Durham TubeYeast Fermentation Broth with Carbohydrates andDurham TubeIntended UseYeast Fermentation Broth media are used for identification ofyeasts based on the fermentation of specific carbohydrates;e.g., dextrose, galactose, lactose, maltose, sucrose, trehaloseand xylose.1 A Durham tube is provided to detect the gasproduced during fermentation.Summary and ExplanationYeast Fermentation Broth is a modification of a medium developedby Wickerham for the determination of carbohydratefermentation by yeasts.2 In this test, tubes of media, each containinga specific carbohydrate, are inoculated with a yeastisolate. If the carbohydrate is fermented by the yeast, the colorof the medium changes from purple to yellow, due to the formationof acids, and gas is produced.In this modification of the Wickerham formula, bromcresolpurple is substituted for bromthymol blue.ProcedureSubculture the isolate to be identified onto a Sabouraud DextroseAgar slant or Mycophil Agar slant.Air bubbles should be removed from the Durham tube priorto inoculation by inverting the broth tube and gently tappingthe side to dislodge the bubble. Return the broth tube to theupright position, taking care to avoid reintroducing air intothe Durham tube.Using a sterile cotton swab, remove growth from the subcultureand suspend it in sterile water to a density approximatelyequal to that of a McFarland no. 1 standard. Inoculate themedium with one drop of the standardized culture using a sterile1 mL pipette.Incubate the tubes at 25C and examine at 5, 7, 10 and 14days for growth and fermentation (gas production).Expected ResultsGrowth is indicated by turbidity in the broth medium. Fermentationof the carbohydrate is indicated by accumulationof gas in the Durham tube and the change of the indicator toyellow.

IDENTIFIKASI JAMUR

Untuk mengidentifikasi jamur lebih diutamakan pengujian sifat-sifat morfologinya pengujian sifat-sifat fisiologi

Metode pemeriksaan laboratorium:Pemeriksaan Mikroskopis : Lansung dan Tak LansungKultur Biakan IdentifikasiMERANCANG MENYUSUN DAN MEMBUAT TEKNIS PEMERIKSAAN JAMUR

MEDIA JAMUR

NOMIKROORGANISMEMEDIA ISOLASIMEDIA DIFERENSIALTEST KONFIRMASI1234