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PREPARATION OF THE BLOOD SMEAR
MATERIALS
- sterilized lancet or needle
- 20 clean microscope slides and coverslips
- Canada balsam or other medium for permanent preparations
- 95% ethyl or methyl alcohol
- distilled water
- Giemsa stain
- low containers (you can make them with aluminum sheet also)
- microscope which magnifies 200 times at least
TAKING THE BLOOD
Cleanse a finger. With a sterile lancet, make a puncture on a fingertip. If you have difficulties in doing this, you can wait until you have a casual wound. In the meantime, keep all the materials needed ready and protected from dust, particularly the clean microscope slides.
MAKING THE SMEAR
Place a small drop of blood near an end of a slide. According to figure 7, bring the edge of another slide in contact with the drop and allow the drop to bank evenly behind the spreader. The angle between the two slides has to be 30-40 degrees. Now, push to the left in a smooth, quick motion. The smear should cover about half the slide.
It is important that the quantity of blood is not excessive, otherwise the red cells could hide the leukocytes. So, if you succeed in making a gradual transition from thick to thin in your smear, you should get a zone with a satisfactory distribution of cells.
With a single drop of blood, you can make several smears. In fact, to make a smear, it is enough to leave a spot of blood of 3 mm about in diameter on the slide. It is useful to perform many smears. In fact, not always they are successful, and with some attempts, it is easier to get one well prepared. To avoid producing clots, you must make each smear with fresh blood and straight after having deposited it. To this purpose, it is useful to be helped by another person where one deposits the blood, and the other makes the smears. With the microscope, you should observe the smears to check that some of them are properly made. The red cells must not overlap each other, nor be so scarce as to be too spread out.
FIXING
If you apply the stain to a smear without having fixed it beforehand, the cells will explode because of the so-called osmotic or hypotonic shock. This happens because the saline concentration inside the cells is much higher than that of staining fluid which is diluted in distilled water. In the attempt to equal the internal saline concentration to the values of the external one, the cells undergo swelling by osmosis. To attain the same saline concentration of the external liquid, the cells should swell more than their membrane allows, in fact they explode. The cell contents are released, and the preparation becomes unusable. To avoid this, before staining, you have to fix the smear. This operation hinders the inflation of the cells which keep sound when they are stained.
A simple and effective fixing technique consists of dipping the smear in a vessel containing 95% ethyl or methyl alcohol for 3-5 minutes. In order to put alcohol on the smear, you can also use a dropper or a bottle dispenser.
STAINING
If you observe the smear as it is after fixing, you will see very little because all cells are very transparent. The erythrocytes are slightly visible, but the leukocytes are too pale, almost invisible and you will not see anything inside them. To be able to observe and recognize the different kinds of leukocyte, you must stain them. For this purpose, normally Giemsa stain is used. It is a mixture of stains, based on methylene blue and eosin. It is cheaply available commercially in volumes of 100 cc. It consists of a concentrated solution which you have to dilute in the proportion1/10, that is one part of Giemsa in nine of distilled water, or buffer solution (pH = 6,8-7,2). You can buy the stain in a store of chemicals and laboratory equipment.
To stain a smear, take a slide with a fixed and dry smear. Put on the slide a drop of stain until it is fully covered. Stain for about 16 minutes, renewing the stain about four times. Then rinse the slide with distilled water at room temperature. Drain off the water and leave the slide to dry.
CHECKING
With the microscope, verify that the cells are well stained. If necessary, apply the stain for a few more minutes. If you were planning to mount the slide with Canada balsam, the staining has to be stronger.
COVER-SLIPPING
At this point, your smear is ready to be observed, but if you want to keep it for a long time, you should make the preparation permanent. To this purpose, after drying the slide, place a drop of Canada balsam or another medium mountant on the smear, then mount the coverslip. If the balsam is too viscous, you may heat a few of the slides (but not over 40 degrees C) to help the balsam flow between the slide and coverslip.
OBSERVATION
A magnification of 200 times is enough to allow you to observe and identify the different types of cells. If you use a higher power, you can also see the cells details better. You can examine either with dry objectives or with the oil immersion technique. In this last case, if you have put on a coverslip, you must wait a day to allow the balsam to set, otherwise, when you move the slide, oil will displace the coverslip.
-- ERYTHROCYTES
The red cells are very numerous in the blood. Usually, they measure 6,6-7,5 µm in diameter. However, cells with a diameter higher than 9 µm (macrocytes) or lower than 6 µm (microcytes) have been observed. In the observation field of the microscope, you will see a lot of erythrocytes and, sometimes, some isolated leukocytes. Erythrocytes are without nucleus (among vertebrates, only the red cells of mammalians are lacking a nucleus). Their typical shape is that of a cake depressed in the center (fig. 1). Under the microscope, they look like pink discs clearer in the middle (fig. 2-6: pink cells around the leukocytes). Sometimes, they are piled up like coins. As we saw, the red cells can also have different shapes from those we described. Sometimes, this is normal, other times, this is due to diseases or to defective process of preparation and staining of the smear.
-- PLATELETS
Platelets are not true cells. They gemmate from big leukocytes called megakaryocytes. They are small sized diskettes about 3µm in diameter. They appear a purple color and are more intense than red cells (you can see some platelets in figures 5 and 6).
LEUKOCYTES
Unlike red cells, leukocytes have a nucleus. It is easily visible under the microscope, but only after having stained the smear. The nucleus of these cells can show multiple lobes, or be indented or kidney-shaped (reniform). Usually, the shape of the nucleus of various kind of leukocytes is different. Together with the different colors of granules, the shape of nucleus helps us to recognize these cells. Leukocytes are divided into granulocytes and lymphoid cells. In the drawings which follow, besides nuclei and granules, you can see even mitochondria, Golgi apparatus, endoplasmic reticula and ribosomes.
- GRANULOCYTES
They come from the marrow bone. Their cytoplasm is rich in granules which take typical colors which help their recognition. The nucleus is condensed in a little masses or lobes. In the blood, there are immature cells as well. They distinguish themselves by having a less segmented nucleus. As we have said, there are three types of granulocyte: neutrophil, eosinophil, basophil.
-- NEUTROPHIL Granulocytes
The neutrophil are the more common leukocytes. They have a diameter of 12-15 µm. You can recognize them as their nucleus is divided into 2 - 5 lobes connected by a fine nuclear strand or filament (fig. 8). The cytoplasm is transparent because its granules are small and faintly pink colored. Immature neutrophils have a band-shaped or horseshoe-shaped nucleus and are known as band cells. In the nucleus of the neutrophil of cells from females, you may see an appendage like a little drumstick (Barr body). It is the second X chromosome, inactivated.
-- EOSINOPHIL Granulocytes
The eosinophils are quite rare in the blood. They have the same size as the neutrophils. Generally their nucleus is bi-lobed. But even nuclei with three or four lobes have been observed. The cytoplasm is full of granules which assume a characteristic pink-orange color (fig. 9). As for the neutrophil, the nucleus is still easily visible.
-- BASOPHIL Granulocytes
Basophils are the rarest leukocytes: less than 1 %. They are quite small: 9-10 µm in diameter. Cytoplasm is very rich in granules which take a dark purple color. The nucleus is bi- or tri-lobed, but it is hard to see because of the number of granules which hide it (fig. 10).
- LYMPHOID CELLS (or agranulocytes)
Because usually these cells appear lacking in granules, they are also named agranulocytes. They have a compact nucleus and a transparent cytoplasm. There are two types of lymphoid cells: lymphocytes and monocytes. Their look is similar, but their origin is different. In fact, whereas lymphocytes spring from lymphatic organs, mono[IMG]cytes have the same origin as the granulocytes.
-- LYMPHOCYTEShttp://funsci.com/fun3_en/blood/blood_05.jpg[/IMG]
Lymphocytes are quite common in the blood: 20-40%, 8-10 µm in diameter and generally they are smaller than the other leukocytes but they are still a few larger than red cells (fig. 11). The cytoplasm is transparent. The nucleus is round and large in comparison to the cell and it occupies most of it. In any case, some of the cytoplasm remains visible, generally in a lateral position. According to the quantity of cytoplasm, lymphocytes are divided into small, medium and large. With Giemsa stain, we cannot distinguish the different types of lymphocyte (B, T, NK), either in the blood because they are not activated, or because it would be necessary to perform special immunochemical staining.
-- MONOCYTES
Monocytes are the biggest leukocytes: 16-20 µm. They have a great reniform or horseshoe-shaped nucleus, in some cases even bi-lobed. The cytoplasm is transparent, but with an appearance of "ground glass" (fig. 12
).
MATERIALS
- sterilized lancet or needle
- 20 clean microscope slides and coverslips
- Canada balsam or other medium for permanent preparations
- 95% ethyl or methyl alcohol
- distilled water
- Giemsa stain
- low containers (you can make them with aluminum sheet also)
- microscope which magnifies 200 times at least
TAKING THE BLOOD
Cleanse a finger. With a sterile lancet, make a puncture on a fingertip. If you have difficulties in doing this, you can wait until you have a casual wound. In the meantime, keep all the materials needed ready and protected from dust, particularly the clean microscope slides.
MAKING THE SMEAR
Place a small drop of blood near an end of a slide. According to figure 7, bring the edge of another slide in contact with the drop and allow the drop to bank evenly behind the spreader. The angle between the two slides has to be 30-40 degrees. Now, push to the left in a smooth, quick motion. The smear should cover about half the slide.
It is important that the quantity of blood is not excessive, otherwise the red cells could hide the leukocytes. So, if you succeed in making a gradual transition from thick to thin in your smear, you should get a zone with a satisfactory distribution of cells.
With a single drop of blood, you can make several smears. In fact, to make a smear, it is enough to leave a spot of blood of 3 mm about in diameter on the slide. It is useful to perform many smears. In fact, not always they are successful, and with some attempts, it is easier to get one well prepared. To avoid producing clots, you must make each smear with fresh blood and straight after having deposited it. To this purpose, it is useful to be helped by another person where one deposits the blood, and the other makes the smears. With the microscope, you should observe the smears to check that some of them are properly made. The red cells must not overlap each other, nor be so scarce as to be too spread out.
FIXING
If you apply the stain to a smear without having fixed it beforehand, the cells will explode because of the so-called osmotic or hypotonic shock. This happens because the saline concentration inside the cells is much higher than that of staining fluid which is diluted in distilled water. In the attempt to equal the internal saline concentration to the values of the external one, the cells undergo swelling by osmosis. To attain the same saline concentration of the external liquid, the cells should swell more than their membrane allows, in fact they explode. The cell contents are released, and the preparation becomes unusable. To avoid this, before staining, you have to fix the smear. This operation hinders the inflation of the cells which keep sound when they are stained.
A simple and effective fixing technique consists of dipping the smear in a vessel containing 95% ethyl or methyl alcohol for 3-5 minutes. In order to put alcohol on the smear, you can also use a dropper or a bottle dispenser.
STAINING
If you observe the smear as it is after fixing, you will see very little because all cells are very transparent. The erythrocytes are slightly visible, but the leukocytes are too pale, almost invisible and you will not see anything inside them. To be able to observe and recognize the different kinds of leukocyte, you must stain them. For this purpose, normally Giemsa stain is used. It is a mixture of stains, based on methylene blue and eosin. It is cheaply available commercially in volumes of 100 cc. It consists of a concentrated solution which you have to dilute in the proportion1/10, that is one part of Giemsa in nine of distilled water, or buffer solution (pH = 6,8-7,2). You can buy the stain in a store of chemicals and laboratory equipment.
To stain a smear, take a slide with a fixed and dry smear. Put on the slide a drop of stain until it is fully covered. Stain for about 16 minutes, renewing the stain about four times. Then rinse the slide with distilled water at room temperature. Drain off the water and leave the slide to dry.
CHECKING
With the microscope, verify that the cells are well stained. If necessary, apply the stain for a few more minutes. If you were planning to mount the slide with Canada balsam, the staining has to be stronger.
COVER-SLIPPING
At this point, your smear is ready to be observed, but if you want to keep it for a long time, you should make the preparation permanent. To this purpose, after drying the slide, place a drop of Canada balsam or another medium mountant on the smear, then mount the coverslip. If the balsam is too viscous, you may heat a few of the slides (but not over 40 degrees C) to help the balsam flow between the slide and coverslip.
OBSERVATION
A magnification of 200 times is enough to allow you to observe and identify the different types of cells. If you use a higher power, you can also see the cells details better. You can examine either with dry objectives or with the oil immersion technique. In this last case, if you have put on a coverslip, you must wait a day to allow the balsam to set, otherwise, when you move the slide, oil will displace the coverslip.
-- ERYTHROCYTES
The red cells are very numerous in the blood. Usually, they measure 6,6-7,5 µm in diameter. However, cells with a diameter higher than 9 µm (macrocytes) or lower than 6 µm (microcytes) have been observed. In the observation field of the microscope, you will see a lot of erythrocytes and, sometimes, some isolated leukocytes. Erythrocytes are without nucleus (among vertebrates, only the red cells of mammalians are lacking a nucleus). Their typical shape is that of a cake depressed in the center (fig. 1). Under the microscope, they look like pink discs clearer in the middle (fig. 2-6: pink cells around the leukocytes). Sometimes, they are piled up like coins. As we saw, the red cells can also have different shapes from those we described. Sometimes, this is normal, other times, this is due to diseases or to defective process of preparation and staining of the smear.
-- PLATELETS
Platelets are not true cells. They gemmate from big leukocytes called megakaryocytes. They are small sized diskettes about 3µm in diameter. They appear a purple color and are more intense than red cells (you can see some platelets in figures 5 and 6).
LEUKOCYTES
Unlike red cells, leukocytes have a nucleus. It is easily visible under the microscope, but only after having stained the smear. The nucleus of these cells can show multiple lobes, or be indented or kidney-shaped (reniform). Usually, the shape of the nucleus of various kind of leukocytes is different. Together with the different colors of granules, the shape of nucleus helps us to recognize these cells. Leukocytes are divided into granulocytes and lymphoid cells. In the drawings which follow, besides nuclei and granules, you can see even mitochondria, Golgi apparatus, endoplasmic reticula and ribosomes.
- GRANULOCYTES
They come from the marrow bone. Their cytoplasm is rich in granules which take typical colors which help their recognition. The nucleus is condensed in a little masses or lobes. In the blood, there are immature cells as well. They distinguish themselves by having a less segmented nucleus. As we have said, there are three types of granulocyte: neutrophil, eosinophil, basophil.
-- NEUTROPHIL Granulocytes
The neutrophil are the more common leukocytes. They have a diameter of 12-15 µm. You can recognize them as their nucleus is divided into 2 - 5 lobes connected by a fine nuclear strand or filament (fig. 8). The cytoplasm is transparent because its granules are small and faintly pink colored. Immature neutrophils have a band-shaped or horseshoe-shaped nucleus and are known as band cells. In the nucleus of the neutrophil of cells from females, you may see an appendage like a little drumstick (Barr body). It is the second X chromosome, inactivated.
-- EOSINOPHIL Granulocytes
The eosinophils are quite rare in the blood. They have the same size as the neutrophils. Generally their nucleus is bi-lobed. But even nuclei with three or four lobes have been observed. The cytoplasm is full of granules which assume a characteristic pink-orange color (fig. 9). As for the neutrophil, the nucleus is still easily visible.
-- BASOPHIL Granulocytes
Basophils are the rarest leukocytes: less than 1 %. They are quite small: 9-10 µm in diameter. Cytoplasm is very rich in granules which take a dark purple color. The nucleus is bi- or tri-lobed, but it is hard to see because of the number of granules which hide it (fig. 10).
- LYMPHOID CELLS (or agranulocytes)
Because usually these cells appear lacking in granules, they are also named agranulocytes. They have a compact nucleus and a transparent cytoplasm. There are two types of lymphoid cells: lymphocytes and monocytes. Their look is similar, but their origin is different. In fact, whereas lymphocytes spring from lymphatic organs, mono[IMG]cytes have the same origin as the granulocytes.
-- LYMPHOCYTEShttp://funsci.com/fun3_en/blood/blood_05.jpg[/IMG]
Lymphocytes are quite common in the blood: 20-40%, 8-10 µm in diameter and generally they are smaller than the other leukocytes but they are still a few larger than red cells (fig. 11). The cytoplasm is transparent. The nucleus is round and large in comparison to the cell and it occupies most of it. In any case, some of the cytoplasm remains visible, generally in a lateral position. According to the quantity of cytoplasm, lymphocytes are divided into small, medium and large. With Giemsa stain, we cannot distinguish the different types of lymphocyte (B, T, NK), either in the blood because they are not activated, or because it would be necessary to perform special immunochemical staining.
-- MONOCYTES
Monocytes are the biggest leukocytes: 16-20 µm. They have a great reniform or horseshoe-shaped nucleus, in some cases even bi-lobed. The cytoplasm is transparent, but with an appearance of "ground glass" (fig. 12
).
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