Direct microscopic examination

Direct microscopic examination presentation

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. Hematoxylin - stains cell nuclei blue Orange green 6 -stains mature and keratined cells orange 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 B udding yeast surrounded by a large clear area against dark background

11. Contd … ADVANTAGES Detects encapsulated yeast C. n eoformans 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 A spergillus

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, a bsolute - 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