ROMANOWSKY STAIN

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ROMANOWSKY STAIN
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ROMANOWSKY TECHNIQUES

• Ehrlich (1879) described neutral dye
  (mixtures of acidic and basic dyes)
• Romanowsky Malakowsky (1891) developed method
  which used mixtures of Eosin Y and ripened
  Methylene Blue
• Jenner (1899) May, Grunwald (1902) introduced
  methanol as a solvent for dye precipitate

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• Leishman (1901), Wright (1902), Giemsa (1902)
  improved the technique
• controlled methods of oxidation
• adding glycerol to increase solubility
  stability

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ROMANOWSKY STAIN

• Polychrome stain ?????????????????
  ??????
• Compound dye consist of
• Basic dye
• Acid dye

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Basic dye

• Cationic dye
• Methylene blue, oxidative product of methylene
  blue (Azure)
• Methylene blue chloride (salt)
• MbCl Mb Cl-
• DNA (nucleus), Basophil granules

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Acid dye

• Anionic dye
• Eosin
• Sodium eosinate (salt)
• Na2E 2Na E-
• Hemoglobin, Eosinophil granules

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2MbCl Na2E Mb2E NaCl Mb2E 2Mb E-
(Metalic sheen)
Buffer
pH 6.4
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Romanowsky Stain

• Wright-Giemsa stain
• Leishman stain
• May-Granwald Giemsa stain (MGG)
• Jenners stain

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Staining characteristics
Nuclei Purple Cytoplasm Erythrocytes Deep
pink Reticulocytes Grey-blue Neutrophil Orang
e-pink Lymphocytes Blue Monocytes Grey-blue
Basophils Blue
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Staining characteristics
Granules Neutrophils Fine purple Eosinophils
Red-orange Basophils Purple-black Monocytes
Fine reddish (azurophil) Platelets Purple
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Metachromatic Stain

• ?????????????????????????????????????????????????
  ???????????????? ?????????????????????????????????
  ??
• ???????????????????????????????????? dimer,
  trimer, polymer ??????????????????????????????????
  ????
• Azure A, Azure B
• ???? ????????? ????????????? ??? ????????

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Metachromatic Stain

• ???????????-??????? buffer
• ???????????????????????????
• Solvent polar solvent
• ????????????
• ????????????????????????????????

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???????????!!!!!
1. ?????????????????????????? 6 ????? 2.
??????????????????????????????? 3. ?????????
polyethylene, ????????? 4. ???????????????????????
???????? ??????? pH Buffer ???????
?????????????????????????????, ??????? Buffer
???????? ???????????????????????????????????? 5.
Metachromatic effect ??????????????????????????
?
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WORK
1. ?????? Wright-Giemsa stain 1000 ml 2. ??????
Leishman stain 500 ml
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Wright-Giemsa stain
Dye solution Powdered Wright stain 3
g Powdered Giemsa stain 0.33
g Glycerol 40 ml Absolute methyl alcohol
to 1000 ml
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Wright stain 3 g
Giemsa stain 0.33 g
???? methyl alcohol glycerol
??????????????
?????????????????????
Dye solution
40oC, 1 wk. , ???????????
???????? Whatman No.1 ?????????????????
????????????
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Leishman stain
Dye solution Powdered Leishman stain 1
g Absolute methyl alcohol to 500
ml Phosphate Buffer pH 6.4 Monopotassium
phosphate 13.26 g Disodium phosphate 5.12
g Distilled water to 2000 ml
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Leishman stain methyl alcohol 500 ml
??????????
50oC waterbath, 15 min
???????? Whatman No.1 ?????????????????
????????????
?????????????????? ?????!!! ???????
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STAINING
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Dye solution ?? Min
Buffer ?? min
wash
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Wright-Giemsa stain Leishman stain Tray Couplin
jar Tray Couplin jar
Fix(min) stain(min) 1 1 2 1
2 2 2 3 3 3
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Tray
Fix
?? min
stain
?? min
wash
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Couplin Jar
W-G stain 5 ml buffer 45 ml
buffer 45 ml
W-G stain
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?????????????
1. ?????????????????????? central pallor 2.
???????????????????????????? 3.
??????????????????????????? granule, cytoplasm
??? nucleus
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STAINING FAULTS
Too blue Smear too thick Inadequate time
in buffer Staining time too long Dye
solution incorrectly made or made from
impure dye Dye solution left exposed to
bright daylight
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STAINING FAULTS
Too pink Excessive time in buffer Buffer
pH too low Dye solution incorrectly made or
made from impure dye Too faint Staining
time too short Excessive washing after
staining
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STAINING FAULTS
Stain deposit Stain solution left in
uncovered jar or tray Stain solution
not filtered Dirty slides Blue
background Inadequate fixation Prolonged
storage before fixation Blood anticoagulated
with heparin
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ROMANOWSKY TECHNIQUES

• Are produced by oxidation of Methylene Blue in
  aqueous solution using heat and alkali
• Solution contains a mixture of Azure A , Azure
  B, Methylene Blue
• Eosin Y is added to produce a neutral dye
• The precipitate formed is dissolved in methanol
  or mixture of methanol glycerol

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ROMANOWSKY TECHNIQUES

• Working solution are prepared by diluting with
  distilled water or aqueous buffer to allow
  ionisation of the dyes