Blood smear staining
5.1 Preparation of stock solution of Gimsa stain Gimsa powder 5 g, methanol 250 mL, glycerol 250 mL.
Put 50 solid glass beads in a dark glass bottle and pour measured Gimsa powder and methanol. Gently cover the lid, wait until the powder is fully soaked in methanol, and shake the glass bottle in the same direction for 2-3 minutes. Add the measured glycerol and shake the glass bottle in the same direction for 2-3 minutes. Repeat the same shake process every half an hour for at least six times. Leave the glass bottle for 2 or 3 days, shake the glass bottle in the same direction 3 to 4 times a day for 2 or 3 minutes each time, until the stock solution is fully prepared.
Note: if there is no dark glass bottle, use a chemical grade clean, dry transparent glass bottle or polyethylene plastic bottle in suitable size, but keep it away from light. For each newly prepared Gimsa stock solution it must be labelled the date of preparation. In addition, the best dye dilution ratio and dying time must be tested before use.
5.2 Preparation of Gimsa working solution
In order to dye the blood film better, the phosphate buffer of pH 7.2 should be used to prepare the dye solution and rinse the blood slides. According to the laboratory and field conditions, bottled pure water and mineral water with a pH about 7.2 can also be used as rinse water for blood smears after staining.
5.2.1 Preparation of pH 7.2 phosphate buffer
Stock solution I: weigh 9.50 g disodium hydrogen phosphate and dissolve in 1,000 mL distilled water. Stock solution Ⅱ: weigh 9.07 g potassium dihydrogen phosphate and dissolve in 1,000 mL distilled water. When preparing 1 L pH 7.2 phosphate buffer, take 105 mL stock solution Ⅰ and 77.20 mL stock solution Ⅱ respectively, fully mix the two solutions, dilute them to 1,000 mL with distilled water, or buy commercial buffer tablets directly followed the instruction.
5.2.2 3% Gimsa solution preparation
Take 3 mL Gimsa stock solution, add 97 mL pH 7.2 phosphate buffer and mix well.5.2.3 10% Gimsa solution preparation
Take 10 mL Gimsa stock solution, add 90 mL pH 7.2 phosphate buffer and mix well.
5.3 Dyeing method
5.3.1 Batch dyeing (for 20 or more slides)
Insert the blood slides into the dyeing tank in one direction, or each pair of slides with films facing outward, pour into the newly prepared 3% Gimsa work solution, immerse the thick and thin blood films, dye for 30 minutes. 2 or 3 blood smears are taken from different parts of the dyeing tank to check the dyeing effect. After the dyeing is proper, pour the buffer or water into the dyeing tank until spilling. Remove the scum on the surface of the dye solution. Pour out the residual dye solution from the dyeing tank, then add clean water, rinse repeatedly for 2 to 3 times, take out the slides, put the blood slides downwards in a slide rack to drain and dry naturally.
5.3.2 Single blood slide staining (3% Gimsa work solution dye)
Drop 3% Gimsa work solution about 3 mL on the thick and thin blood films, completely cover the films, dye for 20 to 30 minutes, then rinse the films with slow and flowing water along the upper edge of the slide for about 1 min to make sure the slide clean and then dry naturally.
5.3.3 Single blood slide staining (10% Gimsa work solution, quickly stain)
Drop 10% Gimsa work solution about 3 mL on the thick and thin blood films, completely cover the films, dry for 8 minutes to 10 minutes, then rinse the films with slow and flowing water along the upper edge of the slide for about 1 min to make sure the slide clean and then let it dry naturally.(https://www.daowen.com)
5.3.4 Re-staining method
When the color fades or the dyeing effect is not good, wash the oil on the blood slide with xylene firstly, drop 75% alcohol on the films, and repeatedly rinse the slide with clean water until no blue on the thick film. Then add 1 drop of Gimsa stock solution into 1 mL pH 7.2 phosphate buffer, mix them well and drop on thick and thin blood films, dye for 1 to 4 hours (depending on the storage time of blood slides). Rinse with clean water for 2 to 3 minutes, add 1 to 2 drops of 75% alcohol on the blood films, rinse with clean water after 1 to 2 seconds and let them dry naturally.
5.4 Factors affecting the quality of blood film staining
The quality of blood staining is not only related to the cleanliness of slides, the quality of blood film making and dyeing technique, but also related to the factors as follows:
5.4.1 Quality of dyes and solvents
Analytically pure methanol and neutral glycerol must be used. In addition, the utensils used in preparation must be clean and waterless. The newly prepared Gimasa stock solution should be tried first to observe whether the dyeing is ideal, otherwise it is necessary to be reprepared. The stock solution should be filtered with qualitative filter paper according to the amount before use.
5.4.2 Newness and oldness of the dye solution
When the dye solution is placed for a longer time, the dyeing power increases gradually. As a consequence, the dye solution should be prepared 1-2 weeks before use. Bottles with dye solution should be sealed and kept away from light in a cool and dry place avoid to affect the quality of the dye solution.
5.4.3 Dilution concentration of dye solution
The dye concentration is high, the staining is fast, all kinds of cells are darkly stained, and Schuffner’s dots and Maurer’s dots are significantly fine. However, the dye concentration is low, the staining is slow. the inner structure of all kinds of cells is uniform and clear, such as trophozoite, merozoite and pigment, but Schuffner’s dots and Maurer’s dots are not clear enough. When the dye solution is diluted, it should be used within half an hour, too long time will also affect the dyeing effect. The dye solution should be prepared when used consequently.
5.4.4 Dyeing time
The longer the dyeing time, the better the dyeing effect; while the shorter, the worse. The dyeing time is related to the temperature: the dyeing is fast when the temperature is high, so the dyeing time can be shortened appropriately accordingly; when the temperature is low, so the dyeing is slow, So the dyeing time should be prolonged properly accordingly.
5.4.5 Dilution and rinse water
In order to stain the thick and thin blood films well, the diluted dye solution should be neutral or slightly alkaline. Therefore, it should be diluted with pH 7.0-7.2 water. When dyeing color is too blue, it can be rinsed with lower pH value buffer; if dyeing color is too red, rinse with higher pH value buffer; if dyeing color is too dark, rinse time can be prolonged to correct.
5.4.6 Rinse method
In order to avoid dye particles stained with blood films, do not pour out the dye solution directly after dyeing, but add water along the end side of thin blood film or the edge of the dyeing tank to overflow the dye surface and then rinse gently.