Intro Lab - Eritrosit Summon and Osmotic

fragility

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Intro Lab - Eritrosit Summon and

Osmotic Fragility Test
Bu Lelly
Blood smear preparation
Specimen

EDTA anticoagulated blood/capillary blood

Equipment

Glass slide

Blood lancet

Reagents

Methanol

Giemsa Solution

Cotton wall soaked with alcohol 70%

Procedure
1. Identify samples ID (Name, Medical Record, Age, Sex).
2. Wash your hand thoroughly by using antiseptic hand soap, put on gloves.
3. Put a small drop of blood on an object glass, about 2 cm from the end of
the slide.
4. Place the slide on the table and immediately place the end of another slide
(spreader slide) against the surface of the first slide, holding it at an angle
450.
5. Draw the spreader slide back against the drop of blood which will spread
across the surface between the two slides.
6. Push the spreader slide slowly and steadily across the first slide and the
blood will follow making an even film.
7. The thickness of the film can be varied by the rapidity of the spreader. The
slower the motion, the thinner the smear.
8. Dry the slide in the air.
9. Fix the smear with methanol for 5 minutes
10.Pour Giemsa solution on the slide.
11.Let stand for 30 min.
12.Remove Giemsa stain
13.Clean the slide from the excess stain with tap water.
14.Dry the slide in the air.

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EDTA anticoagulated blood)

1 drops capillary blood on the glass slide

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1. Put the slide on the rack

2. 2. Fix the slide with methanol for 5 minutes
3. Stain the slide with Giemsa solution for 30 minutes

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How to Use the Microscope

General Procedure
1. Make sure all backpacks and junk are out of the aisles.
2. Plug your microscope in to the extension cords. Each row of desks uses the
same cord.
3. Always start and end with the Scanning Objective. Do not remove slides
with the high power objective into place - this will scratch the lens!
4. Always wrap electric cords and cover microscopes before returning them
to the cabinet. Microscopes should be stored with the Scanning Objective
clicked into place.
5. Always carry microscopes by the arm and set them flat on your desk.
6. Set your microscope on a tabletop or other flat sturdy surface where you
will have plenty of room to work.
7. Plug the microscopes power cord into an outlet.
8. Note: some compound microscopes do not use electric lighting, but have a
mirror to focus natural light instead.
9. Switch on your microscope's light source and then adjust the disc
diaphragm to the largest hole diameter, allowing the greatest amount of
light through.
10.If you have an iris diaphragm, slide the lever till the most light comes
through.
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11.Rotate the nosepiece to the lowest-power objective (usually 4x for 40x

magnification).
12.It is easiest to scan a slide at a low setting, as you have a wider field of
view at low power.
13.Place a microscope slide on the stage, either under the stage clips or
clipped onto the mechanical stage if your microscope has one.
14.A prepared slide works best when you do this for the first time. (If you do
not have a prepared slide, place a strand of colored yarn or thread on a
blank slide and place a coverslip over it.)
15.Move the slide until the specimen is under the objective lens.
16.Adjust the large coarse focus knob until the specimen is in focus
17.Slowly move the slide to center the specimen under the lens, if necessary.
18.Do this by nudging it gently with your fingers or by turning the slide
control knobs if you have a mechanical stage.
19.Adjust the small fine focus knob until the specimen is clearly in focus
20.Then adjust the diaphragm to get the best lighting.
21.Start with the most light and gradually lessen it until the specimen image
has clear, sharp contrast.
22.Scan the slide (right to left and top to bottom) at low power to get an
overview of the specimen. Then center the part of the specimen you want
to view at higher power
23.Rotate the nosepiece to the 10x for 100x magnification.
24.Refocus and view your specimen carefully. Adjust the lighting again until
the image is most clear (you will need more light for higher powers).
25.Repeat with the 40x objective for 400x magnification, which will enable
you to see all of the specimen detail that's necessary for high school
biology lab work.
26.If your microscope has a 100x
27.you'll need to put 1-2 drops of immersion oil over the slide cover slip
28.(the piece of glass over the middle of the slide) before viewing it at
highest power.
29.Move the 100x objective lens into position, and then slowly move the
stage up until the lens makes contact with the oil.
30.Continue focusing with the coarse focus knob until the color or blurred
outline of the specimen appears.
31.Finish focusing with the fine focus knob.
32.With the 100x lens, you will be able to see additional cell detail, but you
will need to take extra care with focus and contrast for a clear image.
33.When you are done using the slide, clean the oil off of the slide and the
lens with lens cleaning paper and solution.

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Peripheral blood smear from a patient with a microangiopathic hemolytic anemia

with marked red cell fragmentation. The smear shows multiple helmet cells
(small black arrows), other fragmented red cells (large black arrow);
microspherocytes are also seen (blue arrows). The platelet number is reduced;
the large platelet in the center (red arrow) suggests that the thrombocytopenia is
due to enhanced destruction. Courtesy of Carola von Kapff, SH (ASCP).

High power view of a normal peripheral blood smear. Several platelets (black
arrows) and a normal lymphocyte (blue arrow) can also be seen. The red cells are
of relatively uniform size and shape. The diameter of the normal red cell should
approximate that of the nucleus of the small lymphocyte; central pallor (red
arrow) should equal one-third of its diameter. Courtesy of Carola von Kapff, SH
(ASCP).

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Normal Blood Cell

Progress stage in the discocyte to echinocyte

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Two stages of stomatocyte - spherocyte transformation

a knizocyte ("pinched" cell) and the lower one is a codocyte with a very deep
depression

Leptocytes (flattened cells). The lower photograph also contains a

microstomatocyte

spherocytes are almost spherical in shape.

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microspherocytes hereditary spherocytosis

Diffusion
Diffusion is an important process where substances are moved without use of
energy.
It is the movement of particles (or molecules; or ions) from a region where they
are in a higher concentration to a region of lower concentration
Thus the movement is down a concentration gradient.

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Osmosis
Osmosis is the movement of water molecules from a region of their higher
concentration to a region of their lower concentration, through a partially
permeable membrane.

6. Erythrocytes

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