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Direct microscopic examination

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St. Xavier's college, maitighar,Kathmandu
St. Xavier's college, maitighar,Kathmandu

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.

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DIRECT MICROSCOPIC EXAMINATION by-Sanju sah st. xaviers college, maitighar department of microbiology
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
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
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
Wet mount of candidial vulvovaginitis
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
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
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.
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.
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
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.
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
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
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
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
Haematoxylene and eosin stainining of Aspergillus
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
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.
Contd… Results Nuclei: purple Cell cytoplasm : blue to mauve Red blood cells : pink ADVANTAGES • used for blood and bone marrow specimens. • Useful for basidiomycetesss
Contd…
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

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Direct microscopic examination

  • 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
  • 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