Staining

Sandiford gram stain Acridine Orange (Trichomonas) Calcofluor stain (Microsporidia, fungus) Auramine phenol (AFB) Auramine phenol (Cryptosporidium) Modified ZN/Kinyoun AFB/Crypto India Ink (Cryptococcus) Lugols iodine (Parasite) Modified Trichrome (Microsporidium) Fields stain (malaria) Vincents stain Spore stain ZN stain

Sandiford gram stain

Air dry Crystal violet 2 min Wash Lugols iodine 2 min Wash Blot dry Acetone alcohol 10-15 sec Wash Blot dry Sandiform malachite green 3 min Wash Air dry Acridine orange for Trichomonas Specimen vaginal smear Air dry Acridine Orange 5-10 Sec Wash Decolourise with alcohol saline 5-10 sec Rinse with physiological saline Dry on a rack Add a drop of saline and cover with a cover slip

Positive Result Gram positive organisms stain deep blue/purple. Negative Result Gram negative or Gram variable organisms stain pink against a blue green background. Good for intracellular gram neg diplococci

Fluorescent microscopy BG 12 excitor filter and No 44, no 53 barrier filter Trichomonas appear brick red pear shaped with green nucleus. Yeasts stain red with a bright green nucleus. Calcofluor stain for microsporidia, fungus Thin smear air dry. Methanol 5 min Calcofluor (0.5% w/v) 1-2 drops 2-3 min Rinse with water Counterstain Evans blue (0.1%) 1min Rinse with water Air dry 1-2 drops of mounting fluid cover slip Fluorescence microscope (395-415 nm)

Auramine phenol for AFB Process in class 1 cabinet in Cat 3 room Dont use diamond marker, use pencil to mark Hotfix the smear Flood with auramine phenol (1:10 v/v) 10 min. Rinse with filtered water (Tap water if not available say assuming it is free of AFB) Put 1% acid alcohol 3-5 min. Rinse with filtered water. Repeat acid alcohol step until no stain seeps from the slide. Rinse with filtered water. Counterstain with thiazine red (or 0.1% pot permanganate) 15 sec Rinse with water. Air dry UV epi-fluorescence microscope 25X and 40X (if it is a non-cover glass objective 40X then dont put a cover slip) AFB appears bright yellow green against a black background. Auramine phenol for cryptosporidium Prepare medium to thick slide Air dry Methanol 3 min Auramine phenol 10 min Rinse with tap water Decolourise with 3% acid methanol 5 min Rinse Counterstain 0.1% Pot permanganate 30 sec Rinse air dry Fluorescent microscope incident light, blue/FITC filter, excitation(690nm), emission (510nm) 20X objective examine 50 fields Modified ZN/Kinyouns method AFB heat fix 80deg -15min/60-70deg 2 hrs Flood with Kinyoun 3% curbol fuchsin 5 min Rinse with deionised water Decolourise with acid alcohol 3 min Rinse with deionised water Repeat acid alcohol step 1-2 min (till no red colour seeping from slide) Rinse with deionised water Flood with methylene blue 4 min Rinse with water air dry Examine high dry 40X, and oil 100X

For cryptosporidium - Fix in methanol for 3 min and air dry Kinyoun curbol fuchsin 15 min Rinse water Decolourise 1% acid methanol 15-20 sec Rinse Counterstain 0.4% malachite green 30 sec

India ink stain for Cryptococcus Place a drop of India ink on a slide Put one drop of specimen or rub the sample next to the ink. Mix it Cover with cover slip. Press it through a sheet of blotting paper. Lugols iodine for parasites Put 1 drop of 0.85% NaCl and 1 drop of Lugols iodine on 2 sides of slide. Mix small amount of stool with each drop wet mount and iodine mount. Put cover slip and see under 10X and 40X

Modified trichrome for microsporidia Make a thin smear unconcentrated stool: 10% formalin 1:3 Air dry Flood with Chromotrope based stain 90 min Rinse with tap water 1 min Acid alcohol (0.45% glacial acetic acid in ethyl alcohol) 10 sec Rinse 95% alcohol Place the slide in 95% alcohol 5 min Place it in 100% alcohol 10 min Place in Hemo-de 10min Air dry High power microscope

Field stain for falciparum thin film

Prepare thin film. Air dry Methanol 1 min Cover the slide with 1 mL of diluted Fields stain B (1 in 4 in buffered water pH 7.2). Immediately add an equal volume of Fields stain A and mix. Leave for 1 min. Rinse with water and dry

For thick film

Hold the slide film facing downward Dip in Fild A 3 sec Drain the excess stain by touching the slide at the corner of the container. Wash in water 3 sec, drain Dip in field B 3 sec Drain excess stain Wash in water Dry in air. If the slide appears to brown, blue/pink restain use 1 sec instead of 3. Other stain for malaria Giemsa stain

Positive Result Chromatin of parasite - Dark red Cytoplasm of parasite Blue Schffners dots/Jamess dots- Red Maurers dots (clefts) - Red-mauve Malaria pigment in white cells - Brown-black Negative Result Red cells Grey to pale mauve-pink Reticulocytes Grey-blue Nuclei of neutrophils Dark purple Cytoplasm of mononuclear cells Blue-grey Granules of eosinophils Red

Vincents stain
Presence of B vincenti with fusiform bacilli and GNR = vincents angina Follow gram stain step but counterstain with 1% carbol fuschin - 30 s.

Spore stain- Schaeffer and Fultons method

Smear, heat fix Place the slide on the rim of a beaker of boiling water (smear up) When large droplet of water appears on the underside, flood with malachite green and leave for 1 min (on beaker). Wash with cold water Safranin 30sec Wash and dry

ZN stain for AFB

Flood the slide with strong carbol fuschin. Heat gently, and once slide is just steaming leave for 3-5 min. Rinse well with water. Decolourise for 2-3 min with a (3% v/v) acid-alcohol solution, rinse with water, then replace with fresh acid-alcohol for 3-4 min until the slide remains a faint pink colour. Rinse well with water. Counter stain with (1% w/v) methylene blue or malachite green for 30 s. Rinse with water and allow to dry. Apply immersion oil and read with a transmitted light microscope