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MICROBIOLOGY PRAX REVIEWER:
Exercise 10:
KOH MOUNT
 
KOH (potassium hydroxide) Mount 
-
 
is a rapid method for the demonstration of fungal forms in clinical material-
 
facilitates the clearing of specimen forenhanced microscopic observation without altering the fungal elements.-
 
when a specimen if placed in KOH, thespecimen will dissolve faster than the fungisince the chitinous cell walls of the fungi willprotect it from disintegration
 A.
 
Collection of Specimen
1.
 
Clean the area with 70% alcohol andallow to air dry2.
 
With a sharp scalpel, scrape theepidermal scales at the active edgeof the skin or nail lesion.
B.
 
Microscopic Examination
1.
 
Place specimen on the glass slide2.
 
Add 1
–
2 drops of 10% KOH forskin scrapings and 20% KOH fornails and hair specimen3.
 
Put a cover slip and let the mount set for 10
–
30mins. If material ishard (nail or tissue), heat the slidegently by passing it over the flameonce of twice. Do not overheat orKOH will crystallize.4.
 
Examine the mount under themicroscope. Note the presence of granules, hypal elements, spores oryeast cells.
Demo Slide:Two advantages of KOH preparation
:1.
 
Facilitates clearing of specimens for enhancedmicroscopic observation without alteringfungal elements2.
 
Simple, cheap and rapid
Two disadvantages of KOH preparation:
1.
 
Gives an idea about the presence of hypalelements, but cannot distinguish different fungi2.
 
Preparation cannot be kept for too long
Three factors that may affect the results of aKOH preparation:
1.
 
Pressure application2.
 
KOH droplet size3.
 
KOH indicationExercise 11:
Microscopic Morphology of Fungal Culture
 
Identification of fungi is often accomplishedby isolation organisms on appropriate culturemedia and observing their morphologicalcharacters
 
Accurate Identification of filamentous fungi isbased on the microscopic examination of sporulating parts of a colony
 
, since eachspecie has a characteristic morphology andarrangement of its spores and fruiting bodies
Laboratory Techniques
:
Tease Mount Preparation
-
 
technique
 
traditionally used by most laboratories-
 
primary purpose is to demonstrate conidiaor other reproductive structures ormorphological forms which might giveinformation toward the identification of theorganism
Procedure:
1.
 
Using an inoculating needle, pick a smallportion of each fungus growth and place onseparate slides containing a drop of LPCB(Lacto-phenol cotton blue)2.
 
Tease with inoculating needle. Emulsify thegrowth of the yeast and yeast-like fungi3.
 
Put a cover slip and examine under low andhigh power objectives.
 
4.
 
Observe record and draw the followingimportant morphological features of thevarious fungi.a.
 
Nature and type of mycelium(presence or absence of crosswalls)b.
 
Characteristic arrangement, size,shape, and type of spores produced(blastospores, arthrospores,chlamydospores, sporangiospores,macro/microconidia, etc) and somespecial characteristicc.
 
Nature of growth (fluffy, velvety,powdery, dry, moist)d.
 
Color of growth on surface andreverse side.5.
 
Label all drawings accordingly.6.
 
Observe and characterize the different fungalcolonies
Demo Slides:I. Trichophyton MentagrophyteMicrosporum GypseumE. FloccosumCandida AlbicansSlide Culture or van Tieghem Cell
-
 
Considered the best method for preservingand observing the actual structure of afungus.-
 
in an undisturbed state, important microscopic structures and morphologicdetails are demonstrated
Procedure:
1.
 
Inoculate the microculture plates separatelywith each of the given fungus.2.
 
Hold a microscope coverslip with a pair of forceps and put it on top of the agar inside theplates.
 
3.
 
Check the culture periodically for growth andsporulation. When the fungus has grown ontothe square of the
Sabouraud’s dextrose agar
 and out onto the small part of the slide andcoverslip, proceed with the next step.Incubation time varies with each fungus,usually 5
–
10 days for contaminants and 1
–
3weeks for pathogens.4.
 
Remove the slide from the petri dish. Take anew clean slide, and place a drop of LPCB on it near the center and put the coverslip that hasfungus growth from the other slide. Label theslide.5.
 
Examine the slide for reproductive structuresand notice the undisturbed morphology.
Demo Slides:Penicillium sp.
a. Small, ovoid structures dividing by transverse fissionb. Flat, powdery to velvety, tan to reddish yellow coloniesc. Conidiosphore bearing short broad metullaed. At 37
C colonies are soft and yeast-like, round or ovoidcells with central septum are seen
 Aspergillus sp
a. Myceliab. Fruiting headsc. Septate, monomorphic fungid. Forms branches at acute angles
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