IDENTIFIKASI JAMUR

SIGIT SULISTYA
BALAI LABORATORIUM
KESEHATAN YOGYAKARTA 2010


IDENTIFIKASI JAMUR

1. Untuk mengidentifikasi jamur lebih diutamakan
pengujian sifat-sifat morfologinya

2. pengujian sifat-sifat fisiologi

3. Metode pemeriksaan laboratorium:
1. Pemeriksaan Mikroskopis : Lansung dan Tak
Lansung
2. Kultur Biakan Identifikasi
3. API Medium : API C20 ( Untuk Yeast)
4. Vitek 2 ( Yeast)


IDENTIFIKASI JAMUR

pengujian sifat-sifat morfologinya

1. Pemeriksaan makroskopis

2. Pemeriksaan mikroskopis lansung menggunakan
Larutan KOH 10 %

3. Slide Kultur menggunakan teknik biakan
identifikasi dengan media Saboroud Agar

4. Test Fermentasi dan Test Asimilasi

5. Uji biokimia



MEDIA JAMUR
NO MIKROORGANISME MEDIA
ISOLASI
MEDIA
DIFERENSIAL
TEST
KONFIRMASI
1 Aspergillus
Mucor
Rhizhopus
YM broth
Saboroud agar
Potato Agar
KOH 10 %
2 Candida
S. Ceriviceae
YM broth
Saboroud agar
Potato Agar
Candida Elektif
Agar
WL Nutrien Agar
Corn Meal Agar
EMB Agar
Glukosa Pepton
0,5 %
KOH 10 %
Cat Gram: A,B,C
dan D
3 Trichosporon
Trichophyton
Microsporum
YM broth
Saboroud agar
Potato Agar
WL Nutrien Agar

KOH 10 %

4 Cryptococcus YM broth
Saboroud agar
Potato Agar
WL Nutrien Agar

India INK
Cat Gram: A,B,C
dan D
Urea Agar
Aspergillus Differential Agar
Intended Use
Aspergillus Differential Agar is used in the differentiation of Aspergillus
species based on pigmentation

Summary and Explanation
Bothast 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 Agar
Procedure
The 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 Results
Examine the medium for typical growth and pigmentation
A. flavus produces a yellow-orange pigment under colonies

Limitation of the Procedure
A. 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 Agar
Intended Use
Czapek-Dox Broth and Czapek Solution Agar are used for
cultivating 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

Procedure
Refer to appropriate references for specific procedures for the
cultivation of fungi and bacteria capable of utilizing inorganic
nitrogen
Expected Results
Refer to appropriate references and procedures for results
BiGGY Agar
Intended Use
BiGGY (Bismuth Sulfite Glucose Glycine Yeast) is a selective
and differential medium used in the detection, isolation and
presumptive identification
of Candida species.

Summary and Explanation
BiGGY Agar is based on the formulation of Nickerson.1
Nickerson developed the medium in 1953 following a study
of sulfite reduction by Candida species.
Differentiation of Candida is based on growth patterns and
pigmentation of isolated colonies. The bismuth sulfite acts
as an inhibitory agent to suppress bacterial growth, which
enables the recovery of isolated colonies of Candida. Candida
species reduce the bismuth sulfite, resulting in pigmentation
of colonies and, with some species, pigmentation in the
surrounding medium.
BiGGY Agar
Procedure
1. Consult 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.
2. When using slants, streak the surface of the slant with a sterile
inoculating loop needle using two to three isolated colonies.
3. Incubate plates in an inverted position (agar side up) for up
to 5 days at 25 2C.
Expected Results
Within 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 Base
Candida Bromcresol Green Agar
Intended Use
Candida Bromcresol Green (BCG) Agar is a
differential and
selective medium used for primary isolation
and detection of
Candida species from clinical specimens

Summary and Explanation
Candida 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 Base
Candida Bromcresol Green Agar
Procedure
Use 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 Results
Candida 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 Agar
Intended Use
Candida Isolation Agar is used for isolating
and differentiating Candida 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 Agar
Procedure
1. Process each specimen as appropriate for that specimen and inoculate directly
onto the surface of the medium. Streak for isolation
2. Incubate plates aerobically at 30C for 18-72 hours
3. Examine plates for growth after 18-72 hours of incubation.
Expected Results
Colonies 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
fluoresce
Limitations of the Procedure
1. Strains of Candida albicans have been reported that are false negative for
fluorescence on this medium
2. Strains 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

Procedure
Use 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 Results
Candida 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 with
Polysorbate 80 Corn Meal Agar with 1% Dextrose
Intended Use
Corn 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 Explanation
Corn 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 Huppert
modified 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 with
Polysorbate 80 Corn Meal Agar with 1% Dextrose
Procedure
1. To prepare plated media from agar deeps, place the agar deeps in a boiling water bath until the
medium becomes liquefied (clear).
2. Pour the molten medium into a sterile Petri dish and allow to solidify before use.
3. 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.
4. Corn Meal Agar with 1% Dextrose should be incubated for up to 4 weeks to allow sufficient time
fo pigmentation to develop.
5. 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.
6. Flame the needle, allow to cool. Then lightly make an S-shaped streak back and forth across the
two original streak lines.
7. 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.
8. If the test is negative, reincubate plates an additional 48-72 hours and examine again.
Corn Meal Agar Corn Meal Agar with
Polysorbate 80 Corn Meal Agar with 1% Dextrose
The addition of dextrose enhances
fungal growth and pigment
production.4 Corn Meal Agar with
Dextrose is commonly used
in the differentiation of Trichophyton
species based on chromogenesis
Cooke Rose Bengal Agar
Antimicrobic Vial A
Intended Use
Cooke Rose Bengal Agar is used with or without Antimicrobic
Vial A in isolating fungi from environmental and food specimens.
Antimicrobic Vial A is used in preparing microbiological culture
media.
Procedure
Refer to appropriate references for specific procedures on the
isolation and cultivation of fungi.
Expected Results
Refer to appropriate references and procedures for results.
Limitations of the Procedure
1. Although this medium is selective primarily for fungi,
microscopic examination is recommended for presumptive
identification. Biochemical testing using pure cultures is
required for complete identification.
2. 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 partially
inhibited.
3. 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 Dermatophyte
Test Medium, Modified with Chloramphenicol
Intended Use
Dermatophyte 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 Dermatophyte
Test Medium, Modified with Chloramphenicol
Procedure
1. Inoculate 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.
2. 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 Results
Dermatophytes 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 Broth
Intended Use
M-Green Yeast and Mold Broth is used for the detection of
fungi in the routine analysis of beverages.

Summary and Explanation
M-Green Yeast and Mold Broth is an improved modification
of the liquid medium, M-Yeast and Mold Broth, which was
developed to improve the efficiency of detection and enumeration
of fungi in sugar and other materials by the membrane
filter method. The revised formula contains the indicator dyebromcresol green. It is a relatively more complex formula than
many of the other media exclusively used for the recovery of
yeasts and molds

Procedure
1. Saturate a sterile membrane filter pad in a sterile Petri dish
with 2.0-2.5 mL of M-Green Yeast and Mold Broth.
2. Roll a membrane filter, which has been used to filter the
test sample, onto the surface of the moistened pad so as to
avoid the trapping of air bubbles between the filter and the
pad.
3. Incubate the plates at 30-35C for 48 hours and up to 5
days in an aerobic atmosphere with increased humidity.
Malt Agar
Intended Use
Malt Agar is used for isolating and cultivating
yeasts and molds from food and for cultivating
yeast and mold stock cultures.
Summary and Explanation
Malt 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 Procedure
Do not heat the medium after addition of acid, as
this will hydrolyze the agar and reduce its
solidifying properties.
Inhibitory Mold Agar
Inhibitory Mold Agar with Gentamicin
Intended Use
Inhibitory 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 Explanation
Inhibitory Mold Agar was formulated by Ulrich as a
general medium for the selective isolation and cultivation
of the majority of pathogenic fungi.


Inhibitory Mold Agar
Inhibitory Mold Agar with Gentamicin
Procedure
1. Consult appropriate references for information about the processing and inoculation of specimens.2
2. For isolation of fungi from potentially contaminated specimens, a nonselective medium should be inoculated
along with the
selective medium. Incubate the plates at 25-30C in an inverted
position (agar side up) with increased humidity. The
tubed slants also should be incubated at 25-30C.
1. 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 Results
Examine plates for fungal colonies exhibiting typical color and
morphology. Biochemical tests and serological procedures
should be performed to confirm findings.
Limitation of the Procedure
Some fungi may be inhibited by the antibiotics in Inhibitory
Mold Agar and Inhibitory Mold Agar with Gentamicin
Malt Extract Agar Malt Extract Broth
Intended Use
Malt Extract Agar is used for isolating, cultivating and enumerating
yeasts and molds. Malt Extract Broth is used for cultivating yeasts
and molds.

Summary and Explanation
The 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 Base
Antimicrobic Vial Oxytetracycline
Intended Use
OGYE Agar Base is for use with Antimicrobic Vial
Oxytetracycline in isolating and enumerating yeasts and molds in
foods.
Summary and Explanation
Acidified agar may be used for enumerating yeasts and molds
in foods and dairy products. However, in some cases,
antimicrobics better suppress bacterial growth and improve
recovery 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 Agar
Intended Use
Phytone Yeast Extract Agar is used for the selective isolation
of dermatophytes, particularly Trichophyton verrucosum, and other pathogenic fungi from
routine clinical specimens.
Summary and Explanation
Carmichael 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.1,2
Phytone Yeast Extract Agar is used in Petri dishes for early
detection 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 Results
After 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 Agar
Intended Use
Potato Dextrose Agar conforms with specifications of The
United States Pharmacopeia (USP).
Potato Dextrose Agar is used for the cultivation and enumeration
of yeasts and molds.
Potato Dextrose Broth is used for cultivating yeasts and molds.
Summary and Explanation
Potato Dextrose Agar is recommended by the American
Public Health Association for plate counts of yeasts and
molds in the examination of foods and dairy products.1,2 It is
recommended in the USP for use in the performance of
Microbial Limit Tests.3 It is also used for the stimulation of
sporulation (slide preparations), maintenance of stock cultures
of certain dermatophytes and for differentiation of atypical
varieties of dermatophytes by pigment production.4
Potato Dextrose Broth is a general-purpose broth medium for
yeasts and molds (Potato Dextrose Agar without the agar

Yeast Extract Agar
Procedure
Consult 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 Procedure
1. Heating Potato Dextrose Agar after acidifying hydrolyzes the agar and may destroy the
solidifying properties.
2. 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 Agar
Potato Flakes Agar with Chloramphenicol and
Gentamicin
Intended Use
These media are used in qualitative procedures for the cultivation of pathogenic and opportunistic fungi encountered in
clinical mycology.
Summary and Explanation
Potato Flakes Agar induces sporulation, enhancing the production of morphological structures required for the identification
of many pathogenic and opportunistic fungi.1
The addition of chloramphenicol and cycloheximide (CC) or gentamicin provides selectivity for more effective isolation
and identification of medically significant fungi. fungi, while permitting the growth of pathogenic species. Gentamicin
is an aminoglycoside antibiotic that inhibits growth of gram-negative bacteria.
Procedure
1. Consult appropriate references for information about the processing and inoculation of specimens such as tissues, skin
scrapings, hair, nail clippings, etc.3-5 For isolation of fungi causing cutaneous mycoses, a nonselective medium should be
inoculated along with a selective medium.
1. 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 25-30C
Expected Results
Examine the media for growth. Microscopic examination of
the colony aids in identification.
Sabouraud Brain Heart Infusion Agar Base
Sabouraud Brain Heart Infusion Agar Sabouraud
Brain Heart Infusion Agar with Antimicrobics
Intended Use
Sabouraud Brain Heart Infusion Agar is used in qualitative
procedures for cultivation of dermatophytes and other pathogenic
and nonpathogenic fungi from clinical and nonclinical
specimens. The medium is rendered selective by the addition
of antimicrobial agents.
Summary and Explanation
Sabouraud Brain Heart Infusion Agar is based on the formulation
of Gorman.1 The combination of Brain Heart Infusion
Agar and Sabouraud Dextrose Agar in this medium improves
the recovery of fungi compared with the recovery on either
medium individually. The addition of defibrinated sheep blood
is recommended to increase the recovery of fastidious, dimorphic
fungi.2
The antimicrobial agents chloramphenicol, cycloheximide and
gentamicin are incorporated in various combinations to
improve the recovery of pathogenic fungi from specimens
heavily contaminated with bacteria and saprophytic fungi
Procedure
Use 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 side
up) with increased humidity. For isolation of fungi causing
systemic mycoses, two sets of media should be inoculated, with
one set incubated at 25-30C
Expected Results
After sufficient incubation, the plates should show isolated
colonies in streaked areas and confluent growth in areas of
heavy inoculation.
Examine the plates for fungal colonies exhibiting typical color
and morphology. Biochemical tests and serological procedures
should be performed to confirm findings
Sabouraud Media (Low pH)
Sabouraud Dextrose Agar Sabouraud Dextrose
Agar with Antimicrobics Sabouraud Dextrose Agar
with Lecithin and Polysorbate 80 Sabouraud
Dextrose Broth Sabouraud Maltose Agar
Sabouraud Maltose Broth
Intended Use
Sabouraud Dextrose Agar conforms with specifications of The
United States Pharmacopeia (USP).
Sabouraud Dextrose Agar is used in qualitative procedures for
cultivation of pathogenic and nonpathogenic fungi, particularly
dermatophytes. The medium is rendered more selective for fungi
by the addition of antimicrobics. Sabouraud Dextrose Broth
and Sabouraud Maltose Agar and Broth are also used for
culturing yeasts, molds and aciduric microorganisms.
Fluid Sabouraud Medium is used for cultivating yeasts, molds
and aciduric microorganisms and for detecting yeasts and molds
in normally sterile materials.
Summary and Explanation
Sabouraud Dextrose Agar is a general-purpose medium
devised by Sabouraud for the cultivation of dermatophytes.1
The low pH of approximately 5.6 is favorable for the growth
of fungi, especially dermatophytes, and slightly inhibitory to
contaminating bacteria in clinical specimens.2-4 This medium
is recommended in the USP for use in performing total
combined mold and yeast counts (Microbial Limit Tests).5
The addition of antimicrobics is a modification designed to
increase bacterial inhibition.
an agar medium can be over-filled, producing a meniscus or
dome-shaped surface that can be pressed onto a surface for
sampling its microbial burden. These plates are used in a
variety of programs to establish and monitor cleaning techniques
and schedules.6-10 After touching the surface to be
sampled with the medium, the environmental sampling dish is
covered and incubated at an appropriate temperature. The
presence and number of microorganisms is determined by the
appearance of colonies on the surface of the agar medium.11
Collection of samples from the same area before and after
cleaning and treatment with a disinfectant permits the evaluation
of the efficacy of sanitary procedures.
Sabouraud Maltose Agar is a modification of Sabouraud
Dextrose Agar with maltose substituted for the dextrose. It is
a selective medium due to the acid pH. Davidson et al.
reported that Sabouraud Maltose Agar was a satisfactory
medium in their studies of infections caused by Microsporum
audouini, M. lanosum and Trichophyton gypseum.12 Davidson
and Dowding also used this medium in isolating T. gypseum
from a case of tinea barbae.13
Sabouraud Maltose Broth is a modification of Sabouraud
Dextrose Broth in which maltose is substituted for dextrose. It is
selective due to its acid pH and is used for the detection of fungi.
Fluid Sabouraud Medium is employed in sterility test
procedures for determining the presence of molds, yeasts and
aciduric microorganisms. The acid reaction of the final
medium is inhibitive to a large number of bacteria and makes
the medium particularly well suited for cultivating fungi and
acidophilic microorganisms.
Procedure
For isolation of fungi from potentially contaminated specimens,
a selective medium should be inoculated along with the nonselective
medium. Incubate the containers at 25-30C with
increased humidity. All cultures should be examined at least
weekly for fungal growth and should be held for 4-6 weeks
before 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 to
solidify for a minimum of 30 minutes.
Prepared tubed slants primarily are intended for use with pure
cultures for maintenance or other purposes. With prepared
plates and Mycoflask bottles, streak the specimen as soon
as possible after it is received in the laboratory, using a sterile
inoculating loop to obtain isolated colonies. Consult
appropriate references for information about the processing
and inoculation of specimens.3,4
For the Sterile Pack media, sample selected surfaces by firmly
pressing the agar medium against the test area. Hold the plate
with thumb and second finger and use index finger to press
plate bottom firmly against surface. Pressure should be the
same for every sample. Do not move plate laterally as this
spreads contaminants over the agar surface making resolution
of colonies difficult. Slightly curved surfaces may be sampled
with a rolling motion.
Areas (walls, floors, etc.) to be assayed may be divided into
sections or grids and samples taken from specific points within
the grid.
Incubate exposed plates at 35-37C for 48 hours, and 25C
for 7 days or as required.
Expected Results
After sufficient incubation, the containers should show isolated
colonies in streaked areas and confluent growth in areas of
heavy inoculation. Transfer of growth from slants to plated
media may be required in order to obtain pure cultures of fungi.
Examine containers for fungal colonies exhibiting typical color
and morphology.20 Biochemical tests and serological procedures
should be performed to confirm findings.
In the RODAC procedure, colonies are counted (fewer than
200 colonies for accurate counts) and expressed as either
the number of colonies per RODAC plate or the number of
colonies per cm.2,21,22 Criteria for cleanliness of equipment and
environment (surfaces) can be developed by using a database
derived from repeated routine sampling of specific sites.23
Subculture colonies of interest so that positive identification
can be made by means of biochemical testing and/or microscopic
examination of organism smears.
Limitation of the Procedure
Some fungi may be inhibited by the acidic pH of the medium
and by the antimicrobics in the selective media.

Sabouraud Agar, Modified
Sabouraud Dextrose
Agar, Emmons Sabouraud Dextrose
Agar, Emmons,
with Antimicrobics
Intended Use
Sabouraud Agar, Modified (Emmons) and Sabouraud Dextrose
Agar, Emmons are used in qualitative procedures for cultivation
of dermatophytes and other pathogenic and nonpathogenic fungi
from clinical and nonclinical specimens.
Sabouraud Dextrose Agar, Emmons is rendered selective by
the addition of antimicrobial agents.
Summary and Explanation
Sabouraud Dextrose Agar was devised by Sabouraud for the
cultivation of dermatophytes.1 The low pH of approximately
5.6 is favorable for the growth of fungi, especially dermatophytes,
and inhibitory to contaminating bacteria in clinical
specimens.2 The acidic pH, however, also may inhibit some
fungal species.2-4 Emmons modified the original formulation
by adjusting the pH close to neutral to increase the recovery of
fungi and by reducing the dextrose content from 40 to 20 g/L.4
The two base formulations offered differ in peptone content
and amount of agar. The addition of antimicrobics further
increases the selectivity of the medium.3
Procedure
Consult appropriate references for information about the
processing and inoculation of specimens.2,3
Prepared tubed slants primarily are intended for use with pure
cultures for maintenance or other purposes.
For isolating fungi from potentially contaminated specimens,
a selective medium should be inoculated along with the nonselective
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 25-30C and a duplicate
set at 35

WL Nutrient Medium and WL Nutrient Broth are used for
cultivating yeasts, molds and bacteria encountered in brewing
and industrial fermentation processes.
WL Differential Medium is used for isolating bacteria encountered
in brewing and industrial fermentation processes.
Summary and Explanation
WL (Wallerstein Laboratory) nutrient media were developed
by Green and Gray1,2 in their study of various fermentation
processes. An exhaustive study examining the methods of
fermentation control procedures in worts, beers, liquid yeasts
and similar fermentation products led to the development of
these media.
At a pH of 5.5, counts of viable bakers yeast may be made on
the WL Nutrient Medium. By adjusting the pH to 6.5, the
medium is suitable for obtaining counts of bakers and distillers
yeast. The medium can support the growth of bacteria, but
unless the number of yeast cells is small the bacteria may not
be detected. Due to this limitation, Green and Gray developed
WL Differential Medium that inhibits the growth of yeasts
without inhibiting the growth of bacteria present in beers
WL Nutrient Medium and WL Differential Medium are used
simultaneously as a set of three plates. One plate is prepared
from WL Nutrient Medium and two plates from WL Differential
Medium.3 The WL Nutrient Medium plate is incubated
aerobically to obtain a total count of mainly yeast colonies. A
differential agar plate is incubated aerobically for growth of
acetic acid bacteria, Flavobacterium, Proteus and thermophilic
bacteria. Another differential agar plate is incubated anaerobically
for growth of lactic acid bacteria and Pediococcus.
YM Agar YM Broth
Intended Use
YM Agar and YM Broth are used for cultivating yeasts, molds
and other aciduric microorganisms.
Summary and Explanation
YM Agar and YM Broth (Yeast Mold Agar and Broth) are
prepared according to the formulae published by Wickerham.1-3
Wickerham suggested that YM Broth acidified to pH 3.0-4.0
be used as an enrichment medium for yeasts from populations
also containing bacteria and molds.
Media selectivity may be enhanced through acidification or
through addition of selective agents. YM Broth may be acidified
prior to sterilization. YM Agar should be sterilized without
pH adjustment and sterile acid added to the sterile molten
medium cooled to 45-50C. Acidified YM Agar should not be
heated. Antibiotics may be aseptically added to the sterile media.
Other fungistatic materials, such as sodium propionate
and diphenyl may be added to YM Agar to eliminate molds
and permit the enumeration of yeasts in mixed populations
Procedure
Inoculate YM Agar plates or YM Broth tubes with sample
to evaluate for the presence of yeasts, molds, or aciduric
microorganisms. Incubate at 30
To favor isolation of fermentative species, add a layer of
sterile paraffin oil 1 cm deep on the surface of the inoculated
broth. Incubate the culture until growth appears and then streak
onto YM Agar to obtain isolated yeast colonies. To isolate
fermentative and oxidative strains, place acidified inoculated
YM Broth on a rotary shaker for 1 or 2 days. This favors yeast
recovery while preventing the sporulation of molds.
Expected Results
Examine the plates or tubes for growth. Record YM Agar
results as colony-forming units (CFU) per volume of sample.
Record YM Broth results as growth or no growth.

Yeast Extract Glucose
Chloramphenicol Agar
Intended Use
Yeast Extract Glucose Chloramphenicol Agar is a selective agar
recommended by the International Dairy Federation1,2 for enumerating
yeasts and molds in milk and milk products.
Summary and Explanation
The antibiotic method for enumerating yeasts and molds in
dairy products has become the method of choice, replacing
the traditional acidified method.2 The use of antibiotics for
suppressing bacteria results in better recovery of injured
fungal cells, which are sensitive to an acid environment, and
in less interference from precipitated food particles during the
counting.3-7
Yeast Extract Glucose Chloramphenicol Agar is a nutrient
medium that inhibits the growth of organisms other than yeasts
and molds due to the presence of chloramphenicol. When a
sample contains predominantly yeasts and/or injured yeasts,
the use of Yeast Extract Glucose Chloramphenicol Agar may
offer some advantage.2 After incubation at 25C, colonies are
counted and yeast colonies are distinguished from molds by
colony morphology.

Procedure
1. Prepare initial sample dilutions using 10 g or 10 mL of sample
in 90 mL of diluent
Add 10 mL from the initial dilution prepared above (#1) to
90 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/mL
dilution 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) into
each dish. Mix thoroughly.
6. Incubate at 25C for 4 days.
Expected Results
1. 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 per
milliliter.
Yeast Extract Phosphate (YEP)
Agar
Intended Use
Yeast Extract Phosphate (YEP) Agar is used in qualitative procedures
for the isolation of dimorphic pathogenic fungi from
clinical specimens. The plates are deep-filled to reduce the
effects of drying during prolonged incubation.
Summary and Explanation
Smith and Goodman developed YEP Agar for the primary recovery
of Blastomyces dermatitidis and Histoplasma
capsulatum from contaminated specimens.1 The medium is
designed to be used with ammonium hydroxide, a selective
agent that improves the recovery of dimorphic pathogens by
inhibiting bacteria, yeasts and saprophytic fungi.2,3
Procedure
Use standard procedures to obtain isolated colonies from specimens.
Add one drop of concentrated NH4OH (ammonia) at
the edge of the inoculated medium and allow the medium to
sit for 20 minutes before inverting.
Incubate the plates in an inverted position (agar side up) at
22-25C.
Expected Results
All cultures should be examined for growth at least weekly.
Cultures should be held for 4-6 weeks before reporting as
negative.
Yeast Fermentation Broth Base with Durham Tube
Yeast Fermentation Broth with Carbohydrates and
Durham Tube
Intended Use
Yeast Fermentation Broth media are used for identification of
yeasts based on the fermentation of specific carbohydrates;
e.g., dextrose, galactose, lactose, maltose, sucrose, trehalose
and xylose.1 A Durham tube is provided to detect the gas
produced during fermentation.
Summary and Explanation
Yeast Fermentation Broth is a modification of a medium developed
by Wickerham for the determination of carbohydrate
fermentation by yeasts.2 In this test, tubes of media, each containing
a specific carbohydrate, are inoculated with a yeast
isolate. If the carbohydrate is fermented by the yeast, the color
of the medium changes from purple to yellow, due to the formation
of acids, and gas is produced.
In this modification of the Wickerham formula, bromcresol
purple is substituted for bromthymol blue.
Procedure
Subculture the isolate to be identified onto a Sabouraud Dextrose
Agar slant or Mycophil Agar slant.
Air bubbles should be removed from the Durham tube prior
to inoculation by inverting the broth tube and gently tapping
the side to dislodge the bubble. Return the broth tube to the
upright position, taking care to avoid reintroducing air into
the Durham tube.
Using a sterile cotton swab, remove growth from the subculture
and suspend it in sterile water to a density approximately
equal to that of a McFarland no. 1 standard. Inoculate the
medium with one drop of the standardized culture using a sterile
1 mL pipette.
Incubate the tubes at 25C and examine at 5, 7, 10 and 14
days for growth and fermentation (gas production).
Expected Results
Growth is indicated by turbidity in the broth medium. Fermentation
of the carbohydrate is indicated by accumulation
of gas in the Durham tube and the change of the indicator to
yellow.

IDENTIFIKASI JAMUR

1. Untuk mengidentifikasi jamur lebih diutamakan
pengujian sifat-sifat morfologinya

2. pengujian sifat-sifat fisiologi

3. Metode pemeriksaan laboratorium:
1. Pemeriksaan Mikroskopis : Lansung dan Tak
Lansung
2. Kultur Biakan Identifikasi
4. MERANCANG MENYUSUN DAN MEMBUAT TEKNIS
PEMERIKSAAN JAMUR

MEDIA JAMUR
NO MIKROORGANISME MEDIA
ISOLASI
MEDIA
DIFERENSIAL
TEST
KONFIRMASI
1


2

3

4