KOH mount preparation involves mixing specimens like skin or nails with 10-20% potassium hydroxide solution to clear tissues and cellular debris, allowing visualization of fungal hyphae and spores under a microscope. Papanicolaou stain uses acidic and basic dyes to stain cell nuclei blue and cytoplasm colors, aiding identification of candida species. India ink preparation detects encapsulated yeasts like Cryptococcus neoformans in cerebrospinal fluid by staining the background ink black and leaving the capsule clear.

1. DIRECT MICROSCOPIC EXAMINATION
by-Sanju sah
st. xaviers college, maitighar
department of microbiology

2. KOH mount preparation
PRINCIPLE
KOH is strong alkali, when specimen such as skin, hair, nails or sputum is mixed with 10-20% w/v
KOH, it softens, digests and clears the tissues (eg- keratin present in the skin) and cellular debris
surrounding the fungi without damaging the fungal cells so that the hyphae and conidia of fungi can be
seen under the microscope.
COMPOSITION
100ml of KOH 20% w/v preparation:
Ingrediants gm/ml
Potassium hydroxide (KOH) - 10gm
Glycerol - 10ml
Distilled water - 80ml

3. PROCEDURE
Specimens (tissues, nail )
Place on a slide containing a drop of KOH (10-20%)
Cover by coverslip and leave for 5-10 mins
Observe under microscope

4. CONTD…
ADVANTAGES
• Rapid detection
• Readily visible
• Useful for dermatophytes, candida spp
• DISADVANTAGE
• Reaction of KOH with specimens produce background artifacts
• Clearing may take extended time

5. Wet mount of candidial vulvovaginitis

6. PAPANICOLAOU STAIN
PRINCIPLE
papanicolaou stain includes both acidic and basic dyes. Acidic dye stains the basic components of the
cell and basic dye stain the acidic component of the cell. The polychromatic PAP stain involves five
dyes in three solutions.
1. Hematoxylin - stains cell nuclei blue
2. Orange green 6 -stains mature and keratined cells orange
3. Eosin azure -mixture of eosin Y, light green SF snd Bismarack brown.
Eosin Y - stains cytoplasm of metabolically active cells to blue
Bismarck brown Y - stains nothing and sometimes it is often ommitted

7. procedure
slide prepared is stained in a solution of haematoxylin
for 20 to 40 minutes
washed in tap water for about 3 minutes until it turns blue,
differentiated in 70percent ethanol that contains 1 percent of HCL
for about 5 seconds to remove excess dye and allow the nuclear to emerge,
This is then washed in tap water
Stain with eosin for 10 minutes
Then wash for about 1 to 5 minutes in tap water and dehydrated

8. Contd…
ADVANTAGES
• Used in staining benign urine cytology sample, squamous cell carcinoma, liver biospsy
• Useful for candida spp
• Good for initial differentiation of dimorphic fungi. Works well on sputum smears.

9. India ink preparation
PRINCIPLE
This stain is used for capsulated organism. Capsules are non-ionic , so neither acid nor basic stains will adhere to
their surfaces. Therefore the best way to visualize them is to stain the background using an acidic stain.

10. procedure
Specimen (CSF) is centrifuged
Drop of india ink is mixed with a drop of centrifuged deposit
and then examined under the microscope
Budding yeast surrounded by a large clear area
against dark background

11. Contd…
ADVANTAGES
• Detects encapsulated yeast C. neoformans in CSF
• Positive in CSF is diagnostic of fungal meningitis
• For the detection of coccidioides immitis in spinal fluids
DISADVANTAGES
• White blood cells and other artifacts may resemble encapsulated organism.

12. Hematoxylin and eosin stain
PRINCIPLE
Hematoxylin and Eosin(H&E) staining is one of the basic stains used in many of the diagnostic setting .
It is used to stain the nuclei by oxidized hematoxylin (hematin) through mordant (chelate) bonds of
metals such as aluminium followed by counterstaining by the xanthene dye-eosin , which colors
different tissues fibres and cytoplasm.
COMPOSITION
Hematoxylin -5gm Eosin Y -1.0gm
Ethyl alcohol - 50ml d/w -80ml
Potassium alum - 100gm 95% alcohol -320ml
d/w - 950ml glacial acetic acid - 0.4ml
Glacial acetic acid - 40ml

13. procedure
flame the slide on burner and place in the xylene
Hydrate the tissue section by passing through decreasing concentration
of alcohol baths and water. (100%, 90%, 80%, 70%)
Stain in hematoxylin for 3-5 minutes
Wash in running tap water until sections “blue” for 5 minutes or less
Differentiate in 1% acid alcohol (1% HCl in 70% alcohol) for 5 minutes
Wash in running tap water until the sections are again blue

14. procedure
Stain in 1% Eosin Y for 10 minutes
Wash in tap water for 1-5 minutes
Dehydrate in increasing concentration of alcohols and clear in xylene
Mount in mounting media
Observe under microscope

15. Contd…
Results:
Keratohyalin, nuclei, cytoplasmic RNA, some calcium salts, bacteria - Blue
Muscle, keratin coarse elastic fibers, fibrin, fibrinoid - Bright red
Collagen, reticulin, myelinated nerve fibers, amyloid - Pink
Red blood cells - orange
ADVANTAGES
• Routinely used in the pathological laboratory
• Useful for Aspergillosis, Candidiasis, and Trichosporonosis.
DISADVANTAGE
• Many fungi stain poorly and some fungi donot stain at all with H&E

16. Haematoxylene and eosin stainining of Aspergillus

17. Giemsa staining
PRINCIPLE
This is a compound stain formed by the interaction of methylene blue and eosin. On exposure to acids,
alkali and ultraviolet light, a number of oxidation products (methylene azures) are formed from methylene blue
which give contrast staining.
COMPOSITION
Azur II eosin -3.000gm
Azur II -0.800gm
Glycerine -125.0gm
Methyl alcohol, absolute - 375.0gm

18. procedure
Fix the smears in methanol for 5 to 10 minutes.
Stain in dilute M-G solution for 10 minutes
Rinse in pH 6.8 buffer.
Stain in Giemsa solution for 30 minutes.
Wash and differentiate in pH 6.8 buffer for 5-20 minutes
Air dry and see under a microscope.

19. Contd…
Results
Nuclei: purple
Cell cytoplasm : blue to mauve
Red blood cells : pink
ADVANTAGES
• used for blood and bone marrow specimens.
• Useful for basidiomycetesss

20. Contd…

21. Fungal culture
• For some fungal infections, the fungal load may be low. Inadequate amounts of specimen may yield
false-negative results. In order to maximize the chance of recovering organisms in sterile fluids, it is
recommended that as much specimen as possible be sent for culture.
• When possible, to increase the chance of recovery of a fungal organism, the specimen should be
collected before an antifungal agent is administered