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Staing of Organisms

graph LR
    Y[staining ] ==>|organism absorbs color| X[Positive Stain]
    Y ==>|environment absorbs the Color| Z[negative staining]
    Z ==> z1[India ink, nigrosin stain]
    X ==>x1[Simple] & x2[Differentiate organisms] & x3[special staining ];
    x1 ==> x1a[monochrome dyes];
    x1a-.-> x1a1[Methylene blue,crystal violet];
    x2-.-> x2a[Gram stain] & x2b[acid fast stain];
    x2b ==>|Types| x2b1[Heat fixation method ] & x2b2[Cold method ];
    x2b1-.-> x2b1a[ziehl neelsan];
    x2b2 -.-> x2b2a[kingyoun];
    x3 ==> x3a[Capsule] & x3b[DNA/Nuclecaspid] & x3c[granules in cell] & x3d[spores] & x3e[flagella ] & x3f[cell wall] & x3g[fluroscent stains]
    x3f-.-> x3f1[chan stain]
    x3a-.-> x3a1[welch];
    x3b -.-> x3b1[feulgen];
    x3d-.-> x3d1[fultan stain];
    x3e ==> x3e1[silver impregnation stain];
    x3e1 -.Syphilis tissue samples .-> x3e1a[levaditi stain]
    x3e1 -.Syphilis bodily fluids.-> x3e1b[fontana];
    x3e1 -.other organisms flagella .->x3e1c[ashley/schaffer fultan stain ]
    x3c==> x3c1[lipid granules of M.tuberculosis ] & x3c2[volutin granules or metachromatic granules];
    x3c1-.-> x3c1a[sudan Black];
    x3c2-.-> x3c2a[albert stain /ponder/babes];
    x3g ==>x3g1[plasmodium species] & x3g2[TB] & x3g3[ fungal ]
    x3g1 -.-> x3g1a[Acridine orange];
    x3g2 -.-> x3g2a[Auramine Rhodamine stain];
    x3g3 -.-> x3g3a[calcof:octicons-arrow-down-24:er white];

Steps on gram staining

  1. heat/methanol fixation to smear
  2. crystal violet/methylene viotel pigment added , gram posotive organism absorb
  3. adding mordant - fixes the stain to gram positive organisms
  4. decolorizing - washing the blue pigment with Alcohol/Acetone/Acetone Alcohol
  5. adding counter stain(saffron/neutral red pigment)- all other organisms that bind to primary stain binds to red pigment

Steps in Acid fast staining

  1. Heat fixation in hot method (or) no heat in cold method
  2. then primary staining
  3. washed with H2SO4
  4. counter staining

Percentage H2SO4 washing based on organisms

  1. M TB /Atypical = 20-25 % H2SO4
  2. partial acid fast organisms= 5% > M.leprae,Oocytes of Cyclosporia,cryptosporidium,Cyclosporia
  3. 0.5 -1% = nocardia,leginella micaedia
  4. 0.25 - 0.5% = bacterial spores and head of sperm.