Romanowky Stains

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THE ROMANOWSKY STAINS
Universally used routine stains for blood films.
These are NEUTRAL STAINS.
It contains both positively & negatively charged particles
which imparts different colors to different components.
Eg : Leishman’s stain
wright stain
Giemsa stain etc.
• Making subtle distinctions in shades of staining and
differential granular stains.
• Stain cell & parasites within the cell.
 cell
ACIDIC COMPONENTS
 Nucleus (DNA)
 Some cytoplasmic components (RNA, Granules)
 BASOPHILIC
 Imparts BLUE SHADES
BASIC COMPONENTS
 Cytoplasm (Hb)
 Mitochondria
 Secretory granules
 ACIDOPHILIC
 Imparts ORANGE-RED SHADES
 PRINCIPLE
 The main components of a Romanowsky stains are
BASIC DYE (cationic)
 Binds to anionic/acid components in the cell ( Nuecleus)
 Gives Blue grey colour
 Eg : Nucleus, basophil granules, neutrophil granules etc.
ACIDIC DYE (anionic)
 Binds to cationic/basic components in the cell
 Gives Orange Red colour
 Eg : hemoglobin, eosinophil granules,etc

• pH value of phosphate buffer is very important.

CONT..
 These stains contain eosin Y which is an acidic anionic dye and
azure B and other thiazine dyes (derived from the oxidation, or
polychroming, of methylene blue) which are basic cationic dyes.
 When diluted in buffered water, ionization occurs.
 Eosin stains the basic components of blood cells
 haemoglobin stains pink-red,
 granules of eosinophils stain orange-red.
 Azure B and other methylene blue derived dyes, stain the acidic
components of cells.
 Nucleic acids and nucleoprotein stain various shades of mauve-purple and
violet
 granules of basophils stain dark blue-violet,
 cytoplasm of monocytes and lymphocytes stains blue or blue grey.
• The staining reactions of Romanowsky stains are pH dependent
which is why the stains are diluted in buffered water of specific pH.
COLOR RESPONSES OF BLOOD
CELLS
Nuclei – chromatin purple
Nucleoli – light blue
Erythrobalst – dark blue
Erythrocyte – dark pink
Reticulocyte - grey blue
 ROMANOWSKY STAINS
1. Leishman
2. Wright
3. Giemsa
4. Jenner
5. Field
6. May-Grunwald Giemsa
7. Wright - Giemsa
8. Jenner - Giemsa
9. Azure B - Eosin Y
 APPLICATION
Stain blood smear for differential count and
RBC count
Stain Bone marrow
Blood parasites studies (eg ; Malaria)
LEISHMAN STAIN
LEISHMAN STAIN
It is named after Sir William Boog Leishman from
Sotland.
It is a polychromatic stain
PRINCIPLE
LEISHMAN STAIN is a polychromatic stain
Components
 Methanol : fixes cell
 Methylene blue : stains RNA , DNA (blue)
 Eosin : stains Hb, Eosin granules
• Eosin + methyelene blue = Thiazine complex.
• This complex will not stain untila buffer is added
- 0.05 M sodium phosphate (pH 6.4)
- Aged distilled water (pH 6.8)
COMPONENTS
Polychrome methylene blue (basic)
Eosin azure (acidic)
Acetone free methanol
Phosphate buffer ( pH 6.8)
PREPARATION OF STAIN
Dissolve 0.15 g of Leishman powder in 100 ml of
acetone free methanol.
The stain is then filtered to a stock bottle.
Place 50 ֯C in water bath for 15 minutes.
Again filter into a clean brown borosilicate bottle and
store at room temperature away from light.
POINTS TO BE NOTED
● Leishman stain
 For daily use, store the stain in an amber container, e.g. TK dropper bottle,
which can be closed in between use to prevent moisture entering the stain.
 Make sure the stain is kept in a cool place (not refrigerated) and never left
in direct sunlight.
 Bright light and heat will cause the stain to deteriorate rapidly.
 Keep the stock stain in a tightly stoppered light opaque (e.g. amber)
container in a cool dark place.
 Renew every 3 months or more often if indicated.
 Allow 3–5 days before using freshly made stain (to obtain optimum colour
reaction).
● pH 6.8 buffered water
- Renew if the water becomes cloudy.
- When refilling the dispensing container, always check that the pH is
correct, e.g. by using narrow range pH papers
REQUIREMENTS
Stain
Buffer
Tweet/ pasteur pipette
Distilled water
Filter paper
Tissue paper
Timer
 BUFFER
Disodium hydrogen phosphate- 49.6 ml
Pottassium dihydrogen phosphate-50.4 ml
Check the and make it pH 6.8
Use Ph paper
PROCEDURE
1) Cover the blood film with undiluted Leishman stain
- FIXATION STEP
 Methanol act as a fixative
 Do not flood the slide
 If using a dropper bottle count the number of drops
required to cover the film.
 Note: The undiluted stain not only acts as a fixative
but also partially stains the smear.
 This stage is required to obtain the best possible
staining results.
PROCEDURE
2) Add twice the volume of pH 6.8 buffered water (i.e. twice
the number of drops as stain).
- STAINING STEP
 The diluted stain should not overflow.
 Ensure the water is well mixed with the stain by blowing on
the diluted stain or mixing the stain and water using a plastic
bulb pipette.
 Allow to stain for 10 minutes (time may require adjusting).
 Note: Diluting the stain in buffered water brings about full
staining of the blood cells.
 The exact staining time to use should be decided when a
new batch of stain is prepared
3)Wash off the stain with tap water (filtered if not clean).
 Do not tip off the stain, because this will leave a fine
deposit covering the film.
 Wipe the back of the slide clean and stand it in a draining
rack for the smear to dry.
 The blood film should appear neither too pink nor too blue
(check results microscopically).
 Tap water: If the tap water is highly acidic, resulting in too
pink a blood film or highly alkaline, resulting in too blue a
blood film, try using boiled cooled water or filtered rain
water. If neither of these are suitable, wash the film with pH
6.8 buffered water.
RESULT
 Red cells . . . . . . . . . . . . Pink-red
 Nucleus of cells . . . . . . .Purple-violet

Cytoplasm
 Neutrophils, eosinophils . . . . . . . . . . . . . . Pale pink
 Large lymphocytes . . . . . . . . . . . . . . . . . . Clear blue
 Small lymphocytes . . . . . . . . . . . . Darker clear blue
 Monocytes . . . . . . . . . . . . . . . . . . . . . . . . . Grey-blue

Granules
 Eosinophils . . . . . . . . . . . . . . . . . . . . . . . Orange-red
 Neutrophils . . . . . . . . . . . . . . . . . . . . . Mauve-purple
 Toxic granules. . . . . . . . . . . . . . . . . . . . . . Dark violet
 Basophils. . . . . . . . . . . . . . . . . . . . . . Dark blue-violet
 Platelets . . . . . . . . . . . . . . . . . . . . . . . . . . Purple-blue

Inclusions
 Malaria pigment (in monocytes) . . . . . . Brown-black
 Howell-Jolly body. . . . . . . . . . . . . . . . . . Purple-violet
 Auer body (in myeloblast) . . . . . . . . . . . . Purple-red
QUALITY CONTROL
 ●When a new batch of stain is prepared, decide the best staining
time to use.
 e.g. stain films made from the same blood at different times, e.g. 5,
7, 10, 12, 15 minutes. Compare the results with a stained control
blood film.
 ● Check the pH of newly prepared buffered water and re-check it at
weekly intervals. The pH of the buffered water used to dilute the
stain must be correct. It is mainly responsible for the staining
reactions.
 ● Maintain consistency in the staining procedure by following exactly
a standard operating procedure (SOP). If the quality of staining
changes, always report this to the person in charge of the laboratory
and ensure the fault is rectified. The staining procedure should be
checked at the beginning of each week.
FALSE POSITIVE REACTON
THANK YOU
GIEMSA STAIN
…………………………
 GIEMSA STAIN
It is named after Dr.Gustav Geimsa from Germany.
It is a nucleic acid stain used in cytogenetics and
parasite examination.
This consist of number of compound made by mixing
different proportion of methylene blue and eosin.
These are Azure I, Azure II, and Azure II-Eosin.
It is an advatage over Leishman stain that fixation is
by alcohol instead of buffer.
Rapid and slow methods can be used for parasite
detection.
GIEMSA STAIN
 Stain preparation
CONTENTS
• Giemsa powder - 1 g
• Methanol - 250 m
PREPERATION
1. Add 1g Geimsa powder to 250 ml methanol in a conical flask.
2. Warm the mixure to 50֯ C and keep for 15 minute with occasional
shaking.
3. Filter the solution and use after few hours ripening.

Buffer
- M/15 disodium phosphate – 61.1 ml
- M/15 potasssium dihydrogen phosphate – 38.9 ml
- Check the pH
 Procedure
1. Prepare smear and air dry it.
2. Fix in methanol for 3-5 minutes.
3. Take out and dry it.
4. Dilute 1 volume of stain with 9 volume of buffer
(1:10).
5. Flood the slide with the stain and keep it for 10-
15 minutes.
6. Wash with the tap water and dry at RT.
 result
It is a classical blood film stain for
peripheral blood smeares and bone marrow
specimens.
RBC stains Pink ,
Platelet show light pale pink
Lymphocyte cytoplasm stains sky blue
Monocyte cytoplasm stains pale blue
luekocyte & chromatin stains Magenta
 USES
Used to in cytogenetics
It stain Trichomonas vaginalis.
It is differential stain when combined with wright stain
to form Wright- Giemsa stain.
Used as a bacteriological stain (spirochetes)
Used in histopathology for malarial parasite.
TYPES
Jenner Giemsa
- Wright Giemsa
- May Grunwald Giemsa
Other stains
Wright s stain
 Composition of Wright stain powder:
Azure A Eosinate:24%
Azure B :35%
Azure C :6%
Methylene blue :35%

PREPARATION
 Wright stain powder -0.25gm
 Acetone free methanol-100ml
Dissolve powder in methanol.Keep for few days {for
ripening}before using.
SIGNIFICANCE
Wright's stain is a hematologic stain that facilitates
the differentiation of blood cell types
It is named for James Homer Wright, who devised the
stain, a modification of the Romanowsky stain, in
1902.
Because it distinguishes easily between blood cells, it
became widely used for performing differential
white blood cell counts, which are routinely ordered
when conditions such as infection or leukemia are
suspected.
Used to differentiate nuclear/ cytoplasmic morphology
of platelets , RBC, WBC and parasites.
 The related stains are known as the buffered Wright stain,
the Wright-Giemsa stain (a combination of Wright and
Giemsa stains)buffered Wright-Giemsa stain,
 Made by specific instructions depend on the solutions being
used, which may include eosin Y, azure B, and methylene
blue (some commercial preparations combine solutions to
simplify staining).
 The May–Grünwald stain, which produces a more intense
coloration, also takes a longer time to perform.
 Urine samples stained with Wright's stain will identify
eosinophils, which can indicate interstitial nephritis or
urinary tract infection.[2]
WRIGHT STAIN
Field stain
Field stain is a histological method for staining of
blood smears.
 It is used for staining thick blood film in order to
discover malarial parasites.
 Field stain is a version of a Romanowsky stain,used
for rapid processing of specimens.
 Field stain is named after physician
John William Field, who developed it in 1941.
COMPOSITION
Field's stain consists of two parts - Field's stain A is
methylene blue and Azure 1 dissolved in phosphate
buffer solution;
 Field's stain B is Eosin Y in buffer solution
MGG stain
Most commonly used for bone marrow.
Used for differential count
 Jenners stain
Jenner's Stain (methylene blue eosinate) is used in
microscopy for staining blood smears. The stain is
dark blue and results in very observable clearly stained
nuclei.
Most commonly used for DLC [differential leucocyte
count]
JSB stain
- It is a method of rapid staininig of malaria parasites by
a water soluble dye/stain.
- Jaswant Singh–Bhattacharji stain, commonly
referred to as JSB stain, is a rapid staining method for
detection of malaria.
- It is useful for the diagnosis of malaria in thick smear
samples of blood.
- The JSB stain is commonly used throughout India, but
rarely used in other countries.
composition
The JSB stain consists of two solutions which are used
in sequence to stain various parts of the sample.
 The first solution consists of methylene blue,
potassium dichromate, and sulfuric acid diluted in
water.
solution is heated for several hours to oxidize the
methylene blue. The second solution is eosin dissolved
in water.
THANK YOU