RAPID STAIN KITS & REAGENTS
                                      ------->RAPID- MALARIA STAIN
                                                                             [JSB I & II] (KIT FOR STANING MALARIA PARSITES)
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PRINCIPLE
Polychromated Methylene Blue and Eosin stains specifically to basophilic and acidophilic cellular elements to demonstrate blood cells and hemoparasites.

STAINS SUPPLIED
Cat. No. HE 807
Composition
  1. RAPID-MALARIAJSBI     500 mL
    Methylene blue     4.3mMol/L
    SulphuricAcid     1%
    Potassium Dichromate    0.5 gms
    Phosphate Buffer           0.1mMol
    Contains polychromating materials, preservatives and stabilizers.
  2. RAPID-MALARIA JSB II  500 mL
    Eosin             4.3mMol/L
    Phosphate    0.1 mMol/L
    Contains preservatives and stabilizers.
  3. EASYFIX         50 mL
    Peripheral Smear Fixative

Preparation of Working Reagent
All Reagents are ready to use.

STABILITY
All reagents are stable between 25 to 300C for 60 months. RAPID-MALARIA FIXATIVE must be stored tight capped away from heat and fire. Rapid-MALARIA JSB STAIN may be exhausted on prolonged use over 150 smears. It is advised to discard Rapid-MALARIA JSB Stain as and when the smear gets lighter stain.

SAMPLES
Peripheral blood samples on clean glass slides may be collected upon febrile episodes from susceptible patients. Avoid anticoagulated samples.

THIN SMEAR
A drop of blood can be spread with a fine edge spreader. Dry the smear at air and fix it with FIXATIVE or rectified spirit within 4 hours. Fixed smear can be stored for longer period without much variation in staining result, however, it is preferable to stain all smears as early as possible.

THICK SMEARS
In mass or conclusive screening it is preferable to prepare thick smears by collecting 1-2 drop of blood on a clean glass slide and spread it to the shape of a coin. Dry at air, add few drops of distilled water to the slide to cover the smears and wait for 1-2 minutes to dehaemoglobinize the smear. Carefully remove water and dry at air. Fix the smear

LIMITATIONS
The performance of stain must be periodically checked by known positive and known negative samples. The accuracy of reporting Malarial parasite is subject to the professional experience of each person as well as the use of a good optical system that could make clear magnification of the parasite from the smear.

 PROCEDURE

Fill uptwoCoplinjarsorwide mouth bottles with Rapid-MALARIASTAIN -I and II

A FOR THIN SMEARS
  1. Spray RAPID-MALARIA JSB STAIN FIXATIVE to thin smears and dry at air.
  2. Dip fixed smear to RAPID-MALARIA JSB STAIN - II for 10-20 seconds and wash in running tap water.
  3. Dip smear in RAPID-MALARIA JSB STAIN -I for 25-35 seconds. Wash in running tap water. Dry at air and see under oil immersion objective for malarial parasites.

B. FOR THICK SMEARSDEHAWMO GLOBINIZING
  1. Arrange air dried unfixed thick smears in a slide rack keeping the smear side up.
  2. Add few drops of distilled water to the slide to cover the smears and wait for 1-2 minutes to dehaemoglobinize the smear. Carefully remove water and dry at air and fix smear by spraying fixative dry at air.
  3. Dip fixed smear to RAPID-MALARIA JSB STAIN - II for 10-20 seconds and wash in running tap water.
  4. Dip smear in RAPID-MALARIA JSB STAIN - I for 25-35 seconds. Wash in running tap water. Dry at air and see under oil immersion objective for malarial parasites.
RESULTS

Neutrophils = Segmented polymorph with purple granules.
Eosinophils =  Polymorph with large orange granules.
Basophils = Polymorph with large blue granules.
Lymphocytes = Dark blue nuclei with clear blue cytoplasm.
Monocytes = Kidneyshapednucleuswithsmokyblue
cytoplasm = Small pale bluish cells.
Malaria Parasites = Pale bluish cytoplasam and Red Scifner's Dot(Trophozoit).

BULKSTAINING PROCEDURE
Using staining tanks number of slides can be stained with relatively less time.

A) THIN SMEAR
  1. Label all smears with appropriate patient identification marks. Fix thin smear by immersing or spraying with Rapid-MALARIA JSB STAIN Fixative and arrange in a slide holder.
  2. Fill Rapid-MALARIA JSB STAIN II in a suitable staining jar and dip the smears in the stain for 10-20 seconds.
  3. Remove and dip in another jar filled with water for 10-20 seconds. Agitate the slide holder to remove excessive stain from the smear.
  4. Remove the water by keeping the slides holder over a filter paper.
  5. Fill Rapid-MALARIA JSB STAIN - I in a suitable staining jar and dip the smears in the stain for 25-35 seconds.
  6. Remove and dip in another jar filled with water for a minute. Agitate the slide holder to remove excessive stain from the smear. Dry the smears at room temperature or in an oven thermostatically controlled to 50-60°C.


B. THICK SMEAR
  1. Label all smears with appropriate patient identification marks and arrange in a slide holder
  2. Dehaemoglobinize by dipping in a jar containing distilled waterfor 1-2 minutes. Carefully remove from water.
    Note : Unfixed thick smears may run out from the slides therefore, all steps of Dehemoglobinization and washing must be without much agitation.
  3. Proceed to Bulk staining procedure thin smear step No.2.


BIBLIOGRAPHY
  1. Mac. Neal,J.A.M.Med.Assoc. 78,1112(1922)
  2. Biological Stain 9th Ed. The Williams & Wilkins Co.BaltiMore,MD1977P.424.
  3. J.MedRes.7,138(1902)
  4. "Histopathological technic and pract histochem The Blackston Co., New York, N.Y. 1954.

BIOLAB DIAGNOSTICS (I) PVT. LTD.
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BOISAR-401 501, MAHARASHTRA.
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