SPECIMEN CONSERVATION AND STAINING METHODS

Contents of This Section

(All links are to subsections within this file.)

Specimen Conservation

Several solutions are possible for the conservation of interesting specimens. The simplest consists in adding an equal amount of glycerol or propylene glycol to the sediment volume. The resulting solution is kept at -20C. This mixture preserves the elements for a certain time, but cellular degradation is rapid.

Conservative media—Sediments can be kept refrigerated in Hank's solution, with added antibiotics. The antibiotics used can be the same as those used for cell culture. We are using streptomycin with a 1g/L concentration. Nystatin should be added to prevent yeast growth.

Method

• 10 mL of Hank's solution is added to the sediment.
  
  
• The mix is centrifuged.
  
  
• The added volume is aspirated.

Conservation times vary with the element and the type of examination. Cast's general picture is preserved for weeks. Cells are preserved only a few days. Cell degradation is seen as many types of vacuolisation, rendering identification almost impossible. For PAP staining, cell characteristics not noticeable on a wet preparation are preserved for only a few hours.

Staining Methods

Toluidine Blue Staining Procedure

Reagent

• 150 mg of Toluidine blue dissolve in PBS buffer.
  
  
• Hank's solution (Difco)
  
  
• PBS buffer: phosphate buffered saline solution (Sigma)

Technique

1. In a test tube, add 3 drops of sediment.
   
   
2. Add 1 ml of PBS buffer or Hank's solution (Difco).
   
   
3. Add 1 or 2 drops of stain depending on cellularity.
   
   
4. Centrifuge.
   
   
5. Aspirate the added fluid volume and read.

Results

Hansel Eosinophils Stain

Reagents

• Hansel stain (This reagent is obtained from a scientific vendor.)

Procedure

Prepare a smear or a cytospin of the sediment. Nature or dilute 50:50.

1. Let the smear dry at room temperature.
   
   
2. Fix 5 sec with methanol 95%.
   
   
3. Add to the slide 25 drops of Hansel stain, 45 sec.
   
   
4. Add to the slide 25 drops of H₂O, 30 sec.
   
   
5. Rinse with water, clean the bottom of the slide with methanol.
   
   
6. Let dry.
   
   
7. Mount the slide with Eukitt (facultative).
   
   
8. Read with the 100x objective.

Naphtyl AS-D Chloroacetate Esterase

Principle

The aim of this stain is to demonstrate the presence of granulocytes. Granulocyte lysozomes contain a rather specific hydrolase that can use the Naphtol AS-D Chloroacetate as substrate. The liberated naphtol reacts with the diazonium salt "Fast Red Violet LB", forming red depots.

Reagents

• Sigma: Kit num. 91C
  
  
• Naphtol AS-D Chloroacetate Esterase

Working reagent preparation

1. Mix
0,1 ml of the sodium nitrite solution and
0,1 ml of the Fast Red Violet LB solution.


2. Let stand for 2 minutes (diazo preparation).
   
   
3. Add 4 ml H₂O at 37°C. Mix.
   
   
4. Add 0,5 ml of Trizmal buffer pH 6,3. Mix.
   
   
5. Add 0,1 ml of Naphtol AS-D Chloroacetate reagent. Mix.
   
   
6. Solution will become slightly red. If the solution forms a precipitate, filter or centrifuge.

Staining procedure

1. In a test tube, add 100 to 200 ul of sediment.
   
   
2. Add to this tube 2 ml of working reagent.
   
   
3. Incubate at 37°C for 15 minutes.
   
   
4. Centrifuge and read the results.

The nucleus can be stained by adding the methylene blue.

Results

Polynuclear cells stain deep red, while the rest of cells remain unstained, or take a light pink to orange red color due to non-specific adsorbtion. Mastocytes and some macrophages are stained with this method.

Acetate Filter PAP Stain for the Urinary Cytodiagnostic

Reagents

• Filter (Gelman GN6 13 mm) or (Millipore RAW 013)
  
  
• Filter holder sweeny 13 mm
  
  
• Hank's solution
  
  
• Hématoxiline acide free
  
  
• Orange G (Ortho)
  
  
• Ea 65 ou Ea 50 (Ortho)
  
  
• EtOH 95%, 1-propanol, tolučne
  
  

Acetate filter procedure

1. Centrifuge 10 ml of urine
   
   
2. Decant a maximum of supernatant
   
   
3. Resuspend the pellet to 1 ml with Hank's solution. Verify the cellularity. Dilute if necessary.
   
   
4. Bring to volume to its initial value using Hank. Mix.
   
   
5. Place the acetate filter in ethanol 95% for 10-15 sec, mount on the sweeny filter holder.
   
   
6. Rinse the filter by injecting 2 ml of Hank.
   
   
7. Inject with a seringe, 1ml or less of the sediment suspension, depending on the cellularity.
   
   
8. Rinse the filter by injecting 2 to 5 ml of Hank.
   
   
9. Un-mount the holder and retrieve the filter.
   
   
10. Fix immediately with EtOH 95%. Never let the filter dry out.
    
    
11. Stain and mount.

PAP stain for acetate filter

From: H. Barwick, McGill University (personnal communication)

1. EtOH 95% (10 min.)
   
   
2. H₂O (10 min.)
   
   
3. Hematoxiline** (2 min.)
   
   
4. H₂O tap (10 min.)
   
   
5. HCl 0.05% (7 dips; destain as needed.)
   
   
6. H₂O tap 37°C (6 min.)
   
   
7. EtOH 95% (2 min.)
   
   
8. Orange G** (2 min.)
   
   
9. EtOH 95% (2 min.)
   
   
10. EtOH 95% (2 min.)
    
    
11. EtOH 95% (2 min.)
    
    
12. EA 65** (2 min.)
    
    
13. EtOH 95% (2 min.)
    
    
14. EtOH 95% (2 min.)
    
    
15. EtOH 95% (2 min.)
    
    
16. 1-propanol 100% (2 min.)
    
    
17. 1-propanol 100% (2 min.)
    
    
18. 1-propanol /Tolučne (1:1) (2 min.)
    
    
19. Tolučne (6 min.)

Mount

1. Slide
   
   
2. 1 drop of Eukitt
   
   
3. Put the filter on the drop.
   
   
4. 2 to 4 drops of Eukitt on the filter.
   
   
5. Add the coverslip.

Put on a heating plate adjusted at 50°C, and let the resin disperse.

Results

This technique is used for collection purposes.