Stains and Solutions Used in Hematology and Cytology

Stains and Solutions Used in Hematology and Cytology A APPENDIX Acid-Fast Stain Commercially prepared acid-fast stains are available 1. Ziehl Neelsen carbolfuchsin: Dissolve 3.0 g basic fuchsin in 100 ml 95% ethyl alcohol. Prepare a 5% phenol solution by dissolving 5.0 g phenol in 100 ml distilled water. Prepare the Ziehl Neelsen carbolfuchsin by mixing 10 ml alcoholic basic fuchsin and 90 ml 5% phenol and allowing the mixture to stand for 24 hours. Filter the solution prior to use. 2. Acid alcohol: Mix 2.0 ml concentrated hydrochloric acid and 98.0 ml 95% ethyl alcohol. 3. Methylene blue: Prepare a saturated solution of methylene blue by adding 1.5 g powdered methylene blue to 100 ml 95% ethyl alcohol. Slowly add the alcohol to dissolve the powder. Add 30 ml saturated alcoholic solution of methylene blue to 100.0 ml distilled water and 0.1 ml 10% potassium hydroxide. Filter this solution and dilute it 1:20 with distilled water to prepare the final methylene blue counterstain. 1. Allow the film to air-dry and then gently heat-fix it (see Gram s stain procedure below). 2. Cover the slide with Ziehl Neelsen carbolfuchsin and steam it gently using a water bath for 3 5 minutes. 3. Rinse the slide with tap water. 4. Decolorize the slide with acid alcohol until little red color remains visible to the unaided eye. 5. Rinse again with tap water. 6. Counterstain the slide with aqueous methylene blue solution for 5 20 seconds, depending upon the thickness of the sample. 7. Rinse the slide with tap water and allow to air-dry. Acid-fast positive organisms, such as Mycobacterium spp. and Cryptosporidium spp., appear distinctly red, whereas other bacteria, leukocytes, and debris appear blue. Gram s Stain Commercially prepared Gram s stains are available 1. Crystal violet stain: Dissolve 2.0 g powdered crystal violet into 20 ml 95% ethyl alcohol and mix with 80 ml 1.0% aqueous ammonium oxalate. The solution is stable and can be stored for months. 2. Gram s iodine: Dissolve 1.0 g iodine (I 2 ) and 2.0 g potassium iodide (KI) into 300 ml distilled water. Prepare a fresh solution every 3 weeks. 3. 95% ethyl alcohol 4. Safranin stain: Dissolve 0.25 g Safranin O into 10 ml 95% ethyl alcohol. Add this solution to 100 ml distilled water. 1. Heat-fix an air-dried slide by passing the slide (film side up) five or six times through a low flame (i.e., Bunsen burner or lighter). Allow the slide to cool. 2. Flood the heat-fixed film with crystal violet for 1 minute and gently wash the slide with water for 1 5 3. Flood the slide with Gram s iodine solution for 1 minute and gently wash it with water. 4. Decolorize the film with 95% ethyl alcohol until stain no longer elutes from the film (15 30 seconds). Wash the slide with water. Exotic Animal Hematology and Cytology, Fourth Edition. Terry W. Campbell. 2015 John Wiley & Sons, Inc. Published 2015 by John Wiley & Sons, Inc. 377

378 EXOTIC ANIMAL HEMATOLOGY AND CYTOLOGY 5. Counterstain the slide with Safranin stain solution for 1 2 minutes. Wash the slide with water for 1 5 seconds and allow to air-dry. A satisfactorily stained smear should show grampositive organisms as deep violet and gram-negative organisms as red. The Gram stain may be affected by the nature of the material on the smear. Smears often vary in thickness on the slide and excessive decolorization may occur in very thin areas, causing the gram-positive organisms to appear gram-negative. Likewise, thicker areas may be poorly decolorized, causing gram-negative organisms to appear gram-positive. Because Gram staining has a varied technique, the procedure requires practice before the cytologist gains confidence in the stain. Macchiavello s Stain 1. Basic fuchsin stain: Prepare a 0.1 M phosphate buffer (ph 7.3 7.4) by adding 80 ml 0.1 M anhydrous dibasic sodium phosphate (Na 2 HPO 4 )to20ml0.1m monobasic sodium phosphate (NaH 2 PO 4 H 2 O). Dissolve 0.25 g basic fuchsin chloride into 100 ml 0.1 M phosphate buffer to make the stain. Prepare the 0.25% stain solution fresh each day of use and filter the stain prior to staining the slide. 2. Citric acid solution: Dissolve 0.5 g citric acid in 100 ml distilled water. Make fresh 0.5% citric acid solution when mold growth occurs. 3. Methylene blue stain: Prepare a stock solution by adding 1.0 g methylene blue chloride to 10 ml 95% ethyl alcohol and slowly adding 100 ml distilled water and 5 ml phenol (melted crystals). Make a working solution by diluting the stock solution 1:10 with distilled water. 1. Heat-fix the air-dried film (see Gram s stain procedure). 2. Flood the slide with the basic fuchsin stain and allow it to stain for 5 minutes. 3. Quickly wash the slide in tap water and dip it one to three times in the citric acid solution (1 3 seconds). 4. Rinse the slide in tap water. 5. Counterstain the slide with the methylene blue stain for 20 30 6. Wash the slide in tap water and allow to air-dry. In satisfactorily stained smears, the elementary bodies (0.2 0.3 μm) of the Chlamydophila (Chlamydia) spp. organism stain red and the larger initial bodies (0.9 1.0 μm) stain blue. Some films contain nonchlamydophial particles that stain red, making the interpretation difficult. Heterophil and eosinophil granules frequently stain red. Mycoplasma colonies may resemble Chlamydophila (Chlamydia). Excessive decolorization with citric acid may decolorize the elementary bodies, making them appear blue; therefore, the citric acid decolorization step may be omitted or shortened in some films. Modified Giménez Stain s 1. Carbol basic fuchsin a. Stock solution: Dissolve 10 g basic fuchsin in 100 ml ethyl alcohol. Add 250 ml 4.0% phenol solution to 100 ml basic fuchsin ethanol solution. Finally, add 650 ml distilled water and incubate the solution at 37 C for 48 hours. b. Buffer solution: Dissolve 27.60 g NaH 2 PO 4 H 2 O (dibasic sodium phosphate) into 100 ml distilled water. Prepare the buffer solutions by adding 3.5 ml monobasic sodium phosphate solution (0.2 M) and 15.5 ml dibasic sodium phosphate solution (0.2 M) to 10.0 ml distilled water. c. Working carbol basic fuchsin solution: Mix 4.0 ml carbol fuchsin stock solution with 10 ml buffer solution and filter twice before using. Filtration is required to minimize the amount of red stain precipitate that may interfere with staining results. 2. Malachite green: Dissolve 0.8 g powdered malachite green into 100 ml distilled water. 1. Allow the film to air-dry and then gently heat-fix the slide (see Gram s stain procedure above). 2. Cover the slide with a working solution of carbol fuchsin and allow to stand for 1 2 minutes. 3. Rinse the slide with tap water. 4. Cover the slide with malachite green solution for 6 9 5. Rinse the slide in tap water and recover the slide with malachite green solution for an additional 6 9 6. Wash the slide with tap water and allow it to air-dry. Chlamydophila (Chlamydia) inclusions are circular and stain red against a blue-green cellular background. Chlamydophila (Chlamydia) organisms may be seen as intracellular and extracellular red organisms. New Methylene Blue Stain New methylene blue stain is used for reticulocyte staining and counting procedures. It can also be used to examine cytology specimens.

APPENDIX A / STAINS AND SOLUTIONS USED IN HEMATOLOGY AND CYTOLOGY 379 1. Dissolve 0.5 g powdered new methylene blue into a solution containing 99.0 ml 0.85% saline and 1.0 ml 40% formalin. 2. Filter the stain solution and store it in a brown bottle. 1. Reticulocyte stain: Mix equal parts of whole blood and stain in a test tube and allow it to stand for 15 20 minutes. Prepare a standard blood film and allow it to air-dry. 2. Cytology stain: Use new methylene blue stain as a wet-mount on a dried film. Apply a small drop of the stain to an air-dried film and place a coverslip over the film for microscopic examination. New methylene blue does not stain hemoglobin; therefore, erythrocytes have a colorless cytoplasm, distinct cytoplasmic borders, and, in the case of lower vertebrates, a purple nucleus. Erythrocytes of many of the lower vertebrates contain a variable amount of reticulum, which appears as blue cytoplasmic precipitate or clumps. Granulocytes have purple nuclei and pale blue cytoplasm. The cytoplasmic granules of heterophils and eosinophils do not stain with new methylene blue. New methylene blue stain provides a more distinctive chromatin and nucleolar appearance to nuclei than do alcohol-based stains, such as Wright s stain. Since new methylene blue stain is water soluble, it can be used to demonstrate fibrin, lipid droplets, and fungal hyphae, which either dissolve or do not stain well with alcoholbased stains. Standard Natt and Herrick s Solution and Stain Add the following ingredients together and bring them to a total volume of 1000 ml with distilled water, using a volumetric flask: Sodium chloride NaCl 3.88 g Sodium sulfate NaSO 4 2.50 g Sodium phosphate Na 2 HPO 4 1.74 g Potassium phosphate KH 2 PO 4 0.25 g Formalin (37%) 7.50 ml Methyl violet 0.10 g Allow the solution to stand overnight and filter through Whatman No. 10 medium filter paper before use. and Draw whole blood to the 0.5 mark of the red blood cell-diluting pipette, and draw the Natt and Herrick s solution to the 101 mark to dilute the blood for obtaining total cell counts, using a hemacytometer. The diluted blood is discharged onto the hemacytometer counting chamber and allowed to settle for a minimum of 5 minutes before counting. Nonmammalian erythrocytes have a small, dark blue nucleus surrounded by a colorless to faint pink cytoplasm with this stain. The total number of erythrocytes in the four corner and central red blood cell squares of the central large square of a Neubauer-ruled hemacytometer chamber is counted using 400 (40, high-dry objective) magnification. Both sides of the hemacytometer are counted in order to obtain duplicate counts. The duplicate counts are averaged if they are in at least a 15% agreement between the two sides. The TRBC (total red blood cell count per microliter) is then calculated by multiplying the number of erythrocytes by 10 000. For the total leukocyte determination, the same charged Neubauer-ruled hemacytometer used for obtaining a TRBC from blood diluted 1:200 in Natt and Herrick s diluent is used. The small, dark blue staining leukocytes are counted in nine large squares in the Neubauer-ruled hemacytometer chamber using 400 (40, high-dry objective) magnification. Both sides of the hemacytometer should be counted in order to obtain duplicate counts. The duplicates are averaged if they are within at least a 15% agreement between the two sides. If the two sides do not agree within 15% of each other, then the process should be repeated using a freshly charged hemacytometer. The TWBC per microliter is then calculated using the following formula: TWBC μl + (total cells in nine large squares + 10%) 200 It may be difficult to distinguish small mature lymphocytes from thrombocytes if counts are made using a 100 magnification (10 objective). In general, thrombocytes typically stain lighter than lymphocytes; however, staining the sample for 60 minutes in the Natt Herrick s solution may improve the differentiation between small lymphocytes and thrombocytes. Elasmobranch-Modified Natt and Herrick s Solution and Stain applications. In: Smith M, Warmolts D, Thoney D, Hueter R (eds), The Elasmobranch Husbandry

380 EXOTIC ANIMAL HEMATOLOGY AND CYTOLOGY Manual: Captive Care of Sharks, Rays, and Their Relatives. Add the following ingredients together and bring them to a total volume of 100 ml with distilled water, using a volumetric flask: NaCl Na 2 SO 4 NaH 2 PO 4 KH 2 PO 4 Formalin (37% formaldehyde) Methyl violet 2B 2.28 g 0.25 g 0.29 g 0.025 g 750 ml 0.01 g Allow the solution to stand overnight and filter through Whatman No. 10 medium filter paper before use. Store at room temperature. and Same as described for the standard Natt and Herrick s solution. This solution prevents osmotic effects of the stain on elasmobranch cells. Hemolysis of elasmobranch blood can also be decreased using a modified heparin EDTA or citric acid (ACD) solution as an anticoagulant when collecting blood samples as follows. Elasmobranch-Modified Heparin EDTA applications. In: Smith M, Warmolts D, Thoney D, Hueter R (eds), The Elasmobranch Husbandry Manual: Captive Care of Sharks, Rays, and Their Relatives. 1. Prepare an Elasmobranch-modified phosphate buffered saline (E-PBS) in distilled water as follows: g/100 ml g/500 ml g/1000 ml NaCl 2.63 13.15 26.3 NaH 2 PO 4 0.12 0.6 1.2 Adjust the ph to 7.0 with 1N HCl. Filter through 0.2-mm sterile filter and store at 4 F. The final osmolarity is approximately 920 mosm. 2. Prepare a stock solution of the anticoagulant by adding 200 mg EDTA and 2000 units heparin in 10 ml E-PBS. 3. Filter through 0.2-mm sterile filter and use the following volumes for specified amounts of blood: 0.5 ml for 10 ml blood; 0.25 ml for 5 ml blood; 0.15 ml for 3 ml blood. Store at 4 C, or premeasured aliquots can be frozen and thawed when needed. Elasmobranch-Modified ACD Solution applications. In: Smith M, Warmolts D, Thoney D, Hueter R (eds), The Elasmobranch Husbandry Manual: Captive Care of Sharks, Rays, and Their Relatives. 100 ml 200 ml 500 ml Citric acid (anhydrous) 0.73 g 1.46 g 3.65 g or (monohydrate) 0.795 g 1.59 g 3.98 g Sodium citrate (hydrous) 2.2 g 4.4 g 11 g Dextrose (hydrous) 2.45 g 4.9 g 12.25 g For 100 ml, dissolve above ingredients in approximately 67 ml E-PBS and adjust to a final volume of 100 ml with distilled water. Filter through 0.2-mm sterile filter and store at 41 F. Use this anticoagulant in amounts equal to the ratio of 7 ml ACD to 40 ml whole blood. For 5 ml samples, add 875 ml per tube. Quick or Stat Stains s These commercially prepared stains are designed to provide the staining characteristics of Wright Giemsa stains Examples of two commonly used quick stains are Diff Quik (American Scientific Products, Division of American Hospital Supply Corporation, McGraw Park, IL) and Hema-Tek (Ames Division, Miles Laboratories, Inc., Elkhart, IN). 1. Dip the air-dried slide into the methanol fixative solution for five 1-second dips and allow the excess to drain. 2. Dip the alcohol-fixed slide into Solution I (Diff Quik s buffered Eosin Y solution) for five 1-second dips and drain off the excess. 3. Dip the slide into Solution II (Diff Quik s buffered solution of methylene blue and azure A dyes) for five 1-second dips and drain off the excess. 4. Rinse the slide with distilled or deionized water for five 1-second dips and allow it to air-dry. The staining procedure can be modified according to the desired staining effects or thickness of the

APPENDIX A / STAINS AND SOLUTIONS USED IN HEMATOLOGY AND CYTOLOGY 381 film. Increasing the number of dips in Solution I or II will intensify the overall staining of the film. A paler stain is obtained by fewer dips (a minimum of three dips is required). Eosinophilic staining is enhanced by increasing the number of dips in Solution I, and basophilic staining is increased by additional dips in Solution II. Overall, the staining qualities for the films are similar to those of Wright Giemsa stain. Sudan III and Sudan IV Stains s Because of the difficulty of preparing stain solutions, it is advised that commercially prepared Sudan stains be used. Use Sudan stains as wet-mount stains. Fat droplets or globules stain a red-orange color. Wright s Stain Commercially prepared Wright s stains are available 1. Wright s stain: Dissolve 0.1 g Wright s stain into 60 ml absolute methanol. Allow the solution to stand in a tightly sealed brown bottle for 1 2 weeks. Filter the solution before using. 2. Wright s buffer: Dissolve 3.80 g Na 2 HPO 4 (dibasic sodium phosphate) and 5.47 g KH 2 PO 4 (monobasic potassium phosphate) into 500 ml distilled water. Bring the total volume to 1000 ml with distilled water. 1. Flood an air-dried film with Wright s stain and allow to stand for 1 3 minutes. 2. Add an equal amount of Wright s buffer and mix by gently blowing on the slide until a metallic green sheen forms on the surface. Allow it to stand for 2 6 minutes (the exact time must be determined for each batch of stain). 3. Gently rinse the stain from the slide using tap water or distilled water and a wash bottle or beaker. 4. Prop up the slide and allow it to air-dry. A satisfactorily stained blood film will reveal erythrocytes with a yellowish red cytoplasm. The erythrocytes of lower vertebrates and leukocytes will have dark violet nuclei. Heterophils of lower vertebrates will exhibit red-orange, rod-shaped cytoplasmic granules. Eosinophils will exhibit a pale blue cytoplasm and redorange round granules in many species. Basophils will exhibit dark purple cytoplasmic granules. The cytoplasm of thrombocytes of lower vertebrates and the platelets of mammals will stain colorless to light blue with red granules. Wright Giemsa Stain Commercially prepared Wright Giemsa stains are available and make the staining procedure relatively simple. 1. Dissolve 300 mg powdered Wright s stain and 30 g powdered Giemsa stain into 100 ml absolute methanol. Allow the solution to stand for 1 2 days in a tightly sealed brown bottle. 2. Filter the solution and use as indicated for the Wright s staining procedure. The results are similar to Wright s stain alone except the cell nuclei become reddish purple instead of violet.