DIFFERENTIAL STAIN KIT

differential stain kit microorganism stain

(use: Hematological Staining & Special Stain for Helicobacter pylori)

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Part # 9112B500ml kit $68.50H

 

DIFFERENTIAL STAIN KIT INCLUDES: 

    500 ml Kit
Solution A: Xanthene Stain Solution 500 ml
Solution B: Thiazine Stain Solution 500 ml
Solution C: Fixative 500 ml

     

Differential Stain Kit bottles are not available to be purchased individually. "Component" bottles may be available under separate catalog numbers.  All Solutions manufactured by Newcomer Supply.

For storage temperatures refer to individual bottle labels.

 

DIFFERENTIAL STAIN KIT APPLICATION:

Fresh uncoagulated venous or arterial blood should be used.  If the smear is not prepared immediately, Davidsohn and Henry recommend EDTA in a concentration of 1.0 to 2.0 mg/ml of blood as the anticoagulant of choice.

If the smear is not to be made immediately, thoroughly mix the blood when it is drawn and again before it is examined.  This is best done by rotating the specimen for not less than 2 minutes on a mechanical rotator.  During the interval, the blood should be kept in the refrigerator at 2-8° C.  Do not freeze sample.

This procedure is for the rapid, differential staining of hematological smears and yields qualitative results similar to a Wright-Giemsa stain. 

The Differential Stain Set is a modification of the Wright Stain technique which essentially converts the classical 4 minute, 3-step process into a 15 second staining operation.  The modification was achieved by Bernard Willing in 1970.  He produced a water soluble Wright Stain and separated the stain into two aqueous solutions.

Blood smears are fixed using the methanolic fixative to stabilize cellular components.  Solutions A and B are then applied individually to the fixed smear to differentially stain specific cellular components.                                                                                                      

DIFFERENTIAL STAIN KIT STAINING PROCEDURE:  Standard

Blood smears and cytology smears are prepared on microscope slides in the same way that they are prepared for a Wright or Wright-Giemsa Stain. 

  1. Dip slide in Solution C, Fixative 5-10 times, one second each time.  Allow excess to drain.
  2. Dip slide in Solution A, Xanthene Stain Solution 5-10 times, one second each time. Allow excess to drain.
  3. Quickly rinse slide with distilled or deionized water.
  4. Dip slide in Solution B, Thiazine Stain Solution 5-10 times, one second each time.  Allow excess to drain.
  5. Quickly rinse slide with distilled or deionized water.
  6. Allow to dry.  Examine under oil immersion lens.
  7. If coverslip is preferred, allow slide to completely air dry.  Dip slide in xylene or substitute and coverslip with a compatible mounting media.

 

OUR PREFERRED PROCEDURE METHOD FOR HEMATOLOGICAL SMEARS:

  1. Dip slide in Solution C, Fixative 5 times, one second each time.  Allow excess to drain.
  2. Dip slide in Solution A, Xanthene Stain Solution 8 dips of 1 second each.  Blot on paper towel. 
  3. Dip slide in Solution B, Thiazine Stain Solution 3 dips of 1 second each.  Allow excess to drain.
  4. Rinse quickly in distilled or deionized water.
  5. Allow to air dry.  Examine under oil immersion lens.
  6. If coverslip is preferred, allow slide to completely air dry.  Dip slide in xylene or substitute and coverslip with a compatible mounting media.

 

DIFFERENTIAL STAIN KIT PROCEDURE NOTES:

  1. If more intense overall stain is desired, increase the number of dips in Solutions A and B.
  2. If a paler stain is desired, decrease dips in Solutions A and B.
  3. To increase eosinophilic staining, increase the number of dips in Solution A.  To increase basophilic staining, increase the number of dips in Solution B.
  4. Coplin jars should be kept tightly covered when not in use.

 

RESULTS:

Erythrocytes     Pink or yellowish-red
Platelets     Violet or purple granules
       
Granular Leukocytes  
  Polymorphonuclear Neutrophils  
    Nucleus Dark blue
    Cytoplasm Pale blue
    Granules Reddish-blue
  Eosinophils    
    Nucleus Blue
    Cytoplasm Blue
    Granules Red to red-orange
  Basophils    
    Nucleus Purple of dark blue
    Granules Dark purple (almost black)
       
Non-Granular Leukocytes                                        
  Monocytes    
    Nucleus Violet
    Cytoplasm Sky blue
  Lymphocytes    
    Nucleus Violet
    Cytoplasm Dark blue
  Bacteria   Blue
  Area between cells   Clear

    

LIMITATIONS OF PROCEDURE:

Good technique in preparing the blood smear should be used at all times.  The unique division of stains in the Differential Stain gives the user the advantage of varying dips in Solutions A and B to produce different degrees of shading and intensity.  However, one must never use fewer than three dips of one full second each.  The Differential Stain Kit set is an aqueous stain, and the water soluble portions of cell material may take the stain differently than the classical alcohol-based Wright Stain.  This phenomenon is most noticeable when observing basophils.  The granules will appear “washed out” or partially dissolved.  If basophils are suspected, the specimen should be stained using Wright Stain.

 

REFERENCES:

  1. "Selected Histochemical and Histopathological Methods", Thompson, S.W., and Hunt, R.D., Second Edition, Charles Thomas, Springfield, IL, 1966, pp. 756-762.
  2. "Clinical Diagnosis by Laboratory Methods", Davidsohn and Henry, Fourteenth Edition, W.B. Saunders Co., Philadelphia, PA, 1969.