LABORATORY DIAGNOSIS OF FUNGI 1
Practical No. 2
Practi cal No. 2
(L a b o r a to r y e xa m i n a ti o n o f fu n g i )
A. Fi l am en t ou s f u n gi
Ob j e c t i v e s :
1 - To learn how to collect various specimens of fungal diseases.
2 - To learn various methods to identify the fungi.
3 - Describe the general structures of filamentous fungi.
4 - To be familiar with the most common method to identify filamentous fungi.
Lab o r at o r y a p p r o ach t o t he d i a g n o s i s o f f ung al i nf ect i o ns :
1. Sp e ci m e n co l l e ct i o n a nd t ra nsp o rt :
Specimens for fungal microscopy and culture may be:
1 - Scrapings of scale, best taken from the leading edge of the rash after the skin
has been cleaned with alcohol.
2 - Skin stripped off with adhesive tape, which is then stuck on a glass slide.
3 - Hair, which has been pulled out from the roots.
4 - Brushings from an area of scaly scalp.
5 - Nail clippings.
6 - Skin biopsy.
7 - Moist swab from a mucosal surface (inside the mouth or vagina) in a special
transport medium.
8 - A swab should be taken from pustules in case of secondary bacterial infection.
3rd
Dr. Nessrin AL-abdallat
year Laboratory Medicine, 1434-1435H
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LABORATORY DIAGNOSIS OF FUNGI 1
Practical No. 2
Sealed, sterile transport containers should be used for all liquid or moist specimens.
However, skin scrapings, nail fragments, and hair can be transported in a black
envelope or petri dish.
Because many specimens contain contaminating bacteria that may compromise their
quality, 50.000U of penicillin, 100.000
µg of streptomycin, or 0.2 mg of
chloramphenicol can be added per milliliter of specimen if it is anticipated that transit will
be prolonged (such as transport through the mail).
2. Di r ec t ex am i n at i on :
It is highly recommended that a direct microscopic examination be made on most
specimens submitted for fungal culture.
A phase-contrast microscope is a valuable adjunct in the direct examination of
specimens. The advantages include:
1. Mounts can be made and examined quickly.
2. There is no need for direct staining.
3. The objects can be clearly visualized.
The material is examined by microscope by one or more of these methods:
1- Wet preparation with potassium hydroxide (KOH) preparation.
2- Lacto-phenol cotton blue (LPCB) (tease mount).
3- Scotch tape technique.
4- Slide culture technique.
5- Histopathology of biopsy with special stains.
6- India ink test.
7- Germ tube test.
3rd
Dr. Nessrin AL-abdallat
year Laboratory Medicine, 1434-1435H
LABORATORY DIAGNOSIS OF FUNGI 1
Practical No. 2
Not e: Fungal elements are sometimes difficult to find, especially if the tissue is very
inflamed, so a negative result does not rule out fungal infection.
A negative culture may arise because:
1 - The condition is not due to fungal infection.
2 - The specimen was not collected properly.
3 - Antifungal treatment had been used prior to collection of the specimen.
4 - There was a delay before the specimen reached the laboratory.
5 - The laboratory procedures were incorrect.
6 - The organism grows very slowly.
3. Se l e ct i o n a nd i no cul a t i o n o f cul t ure m e d i a :
Two types of culture media are essential to ensure the primary recovery of all clinically
significant fungi from clinical specimens. One medium should be non-selective and a
second medium, more selective for the recovery of fungi, should also be used.
For the recovery of the more fastidious dimorphic fungi, an enriched agar base, such as
brain-heart infusion, must be used. Antibiotic combinations may be added because the
incubation of the plate may require one month or more.
It is currently recommended that all fungal cultures be incubated at a controlled 30oC.
Incubation of a second set of plates at 35oC for the recovery of the yeast forms of
dimorphic fungi is not cost-effective. All fungal cultures should be incubated for a
minimum of 30 days before discarding as negative.
4. Se ro l o g i ca l t e st s:
Serological tests are not useful for the diagnosis of superficial fungal infections. But in
subcutaneous and systemic infection, several tests may be useful.
3rd
Dr. Nessrin AL-abdallat
year Laboratory Medicine, 1434-1435H
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LABORATORY DIAGNOSIS OF FUNGI 1
Practical No. 2
In i ti a l o b s e r v a ti o n s i n th e s tu d y o f f u n g u s i s o l a te s :
1. Appearance of the growth.
2. Rate of growth.
3. Colony pigmentation.
4. Growth on media containing antifungal agents.
5. Dimorphic growth.
Ex a m i nat i o n o f f i l a m ent o us f ung i ( M o l d s ) :
I. M a c r o sco p i c e xa m i na t i o n o f fi l a m e nt o us fung i :
The identification of filamentous fungi is based upon an evaluation of their colony
characteristics as well as their microscopic morphology.
Visual examination of the colony will reveal important data concerning texture,
topography and pigments. Some of the textures commonly seen include:
1- Cottony/woolly (resembling cotton candy).
2- Suede (velvety, like pigskin).
3- Powdery (soft, like flour or talc).
4- Granular (gritty and more coarse than powdery).
5- Glabrous (smooth leathery, skin-like hairless).
Fungal colonies exhibit distinctive pigmentation on the surface and/or the reverse.
Brightly colored surface pigments include blue, green, yellow, and red. Dull-colored
pigments seen are brown and gray to black. Dematiaceous pigments are brown to black
in color and impart a dark color to microscopic structures. Pigmentation of the reverse
varies in color with some colors being characteristic of specific organisms. Water-soluble
pigments are those that diffuse into the medium. Discrete pigments are those that do
not diffuse into the medium.
3rd
Dr. Nessrin AL-abdallat
year Laboratory Medicine, 1434-1435H
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LABORATORY DIAGNOSIS OF FUNGI 1
Practical No. 2
II. M i c r o s c o p i c e x a m i n a ti o n o f f i l a m e n to u s f u n g i :
One or more of these methods can examine the specimens of filamentous fungi
microscopically:
1. Lacto-phenol cotton blue (LPCB) “tease mount”.
2. Slide culture technique.
3. Scotch tape technique.
Microscopy can identify filamentous fungi by the presence of:
1 - Fungal hyphae (branched filaments) making up a mycelium.
2 - Asexual spores ''conidia''.
3rd
Dr. Nessrin AL-abdallat
year Laboratory Medicine, 1434-1435H
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LABORATORY DIAGNOSIS OF FUNGI 1
Practical No. 2
St u d y o f t w o e x a m p l e s o f f i l a m e n t o us f ung i
1. As p er g i l l us ni g e r
Ma c r o s c o p i c e x a mi n a t i o n :
1 - Ob s e r v e c o l o r : black.
2 - Re v e r s e pi gme n t : beige.
3 - Tex t u r e: wooly, cottony or velvet.
4 - Topogr aph y : heaped.
Mi c r o s c o p i c e x a mi n a t i o n :
1 - Hy ph ae : septate, hyaline.
2 - Ves i cl e: round.
3 - Ph i al i des : single row.
4 - Large dark brown co ni d i al h ead that became
radiate.
5 - Con i di a: brown to black and rough-walled.
6 - Con i di oph or e: smooth-walled, hyaline or turning dark towards the vesicle.
Dr. Nessrin AL-abdallat
3rd year Laboratory
Medicine, 1434-1435H
6
LABORATORY DIAGNOSIS OF FUNGI 1
Practical No. 2
2 . pen i c i l l i u m s pec i es
Ma c r o s c o p i c e x a mi n a t i o n :
1 - Ob s e r v e c o l o r : centrally grayish-turquoise with
white periphery.
2 - Re v e r s e pi gme n t : white.
3 - Tex t u r e: velvety.
4 - Topogr aph y : folded.
Mi c r o s c o p i c e x a mi n a t i o n :
1 - Hy ph ae : septate, hyaline.
2 - P hi a l i d e s: Biserated.
3 - Con i di a: smooth or slightly roughened.
4 - Ves i cl e: absence.
5 - Con i di oph or e: hyaline.
3rd
Dr. Nessrin AL-abdallat
year Laboratory Medicine, 1434-1435H
7
LABORATORY DIAGNOSIS OF FUNGI 1
Practical No. 2
Worksheet
Ma t e r i a l s :
1 - SDA plate from the previous practical.
2 - Culture of molds.
3 - Lacto-phenol cotton blue stain (LPCB).
4 - Pasteur pipette.
5 - Microscopic slide.
6 - Coverslip.
7 - Bent dissecting needles.
8 - Microscope.
Exe rci se 1 :
Examine the SDA plate from the previous practical macroscopically.
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3rd
Dr. Nessrin AL-abdallat
year Laboratory Medicine, 1434-1435H
8
LABORATORY DIAGNOSIS OF FUNGI 1
Practical No. 2
Exe rci se 2 :
Perform tease mount method from given plate and record your result by drawing with
labeling.
Method:
1- With a Pasteur pipette, place a drop of LPCB to a sterile microscopic slide.
2- With pair of sterile dissecting needle, pick-up a bit of the mycelium and put it
directly to the drop of stain.
3- Gently tease apart the mycelium.
4- Cover it with a cover slip and examine under the microscope.
Result:
3rd
Dr. Nessrin AL-abdallat
year Laboratory Medicine, 1434-1435H
9