SERUM

ClottingTime

2 1. Capillary Method. Sterilize thoroughly the tip of your left fourth finger with 70% alcohol.
Puncture with a sterile lancet. Dip the tip of a capillary tube in the drop of blood formed and
maintain the position until blood has filled the capillary tube. Note the time.
After 2 minutes, break off a small piece of the end of the capillary tube. Observe for the
presence of a thread-like fibrin. If there is none, continue breaking off a small piece of the end at a
Blood time every 30 seconds. Note the time when a fibrin thread does form. This is your clotting time. Is
your clotting time within normal limits (normal limits is about 4-8 minutes)?
Physiology 2. Drop Method. Put several drops of blood from the puncture made above on a clean slide. Note the
time. Draw a needle through the drops at 30-second intervals. Note the formation of shreds of fibrin
clinging to the needle. The time it takes for the first fibrin threads to appear is your clotting time.
3. Factors Affecting Coagulation. Prepare 7 test tubes as follows:
a. Test tube 1 – with a piece of cotton
b. Test tube 2 – plain
c. Test tube 3 – inner surface lined with paraffin
d. Test tube 4 – immersed in ice
e. Test tube 5 – with 5 drops of 5% potassium oxalate
f. Test tube 6 – with 20 drops of 2% sodium citrate
g. Test tube 7 – with 15 drops of 25% MgSO4 sol’n
Place about 3 cc of fresh blood to each tube. Note the time required for the blood to clot in each
tube. Explain your results.

Bleeding Time
1. Puncture another finger. Allow the blood to flow freely and note the first time the blood appears.
Remove the drops as they appear by blotting with filter paper, but without touching the skin.
Note the time the bleeding finally stops. This is your bleeding time. How does it compare with
the normal population values (normal bleeding time by this method is from 2 – 5 minutes)?

BLOOD CELLS

1. Blood Smear. Get two clean microscope slides. Cleanse the inner surface of your third fingertip
with an alcohol prep and allow to dry. Prick the finger with a sterile lancet deep enough to give a
free flow, and place one drop of blood on one end of a clean slide. Position the other clean slide at a
45 angle to the first slide so that this second slide touches the drop of blood on the first slide.
Quickly move the second slide across the top of the first slide, dragging the drop of blood into a thin,
uniform layer. Allow the blood smear to dry thoroughly (about 10 minutes).
Stain the film by adding 5 drops of Wright’s blood stain. Allow to stand for 30 seconds. Add 5
drops of water and let stand for 5 minutes, then wash gently in water for 30 seconds. Dry thoroughly
then examine under oil immersion. Identify the red blood cells and all types of white blood cells.
How do you distinguish the types of white blood cells from each other?

2. Red Blood Cell Count.

Get a hemacytometer. In the center of the counting slide are three grooves arranged in the form of a
letter H. The two polished platforms within the H lie exactly 0.1 mm below the surface of the slide.
The pipette with the red plastic float is used for counting red blood cells (RBC). This pipette is
graduated to dilute blood 200x when the sample and diluting solutions are mixed correctly.
Cleanse the inner surface of your third fingertip with an alcohol prep and allow to dry. Prick the
finger with a sterile lancet deep enough to give a free flow of blood. Wipe off the first drop
and allow a good-sized drop to form.
 Dip the tip of the red cell pipette into the drop and fill to the 0.5 mark exactly. Be careful to
keep the tip constantly in the blood, otherwise air will enter the column and the sample will be
inaccurate. If this happens, clean and dry the pipette and start again.
Immediately wipe the excess blood from the outside of the tip. Quickly insert the pipette in the
Hayem’s solution and fill it to the 101 mark exactly. Close the tip of the pipette with your thumb and
forefinger and shake the contents (by rotating the pipette) for about 2 minutes. It is very important
not to shake in the direction of the longitudinal axis.
With the hemacytometer and coverslip in place on the microscope stage, discharge 2 drops of the
dilution on a paper napkin and place the next small drop at the edge between the counting chamber
and the coverslip. The counting chamber will fill by capillary action. Do not attempt to adjust the
coverslip after it has been filled.
Allow about 3 minutes for the blood cells to settle on the ruled squares. Immediately after
discharging blood into the hemacyto-meter, rinse the pipette with distilled water and then with
acetone.
Count using the high power objective. For RBC counting, focus on the middle 5x5 small square
grid. Each small square is divided into 16 smaller squares. Use the four corner small squares plus
the middle square for counting RBC. To avoid counting the same RBC twice, include in counting all
cells that touch the line at the left and top borders. To calculate the number of RBC per cubic mm of
blood, use the following formula:

RBC /mm3 = No. of cells counted X dilution X 4000

No. of small squares counted

Or, no. of cells counted in 5 squares X 10,000

3. White Blood Cell Count

Follow the same procedure as in the RBC count, but this time use
the pipette with a white plastic float. This pipette is graduated to dilute blood 20x when the sample
and diluting fluid are mixed correctly.
Fill the pipette with blood to the 0.5 mark, then follow with Turk’s solution and fill to the 11.0
mark exactly. Observe the same precautions as the RBC procedure.
Charge the hemacytometer with the solution and count after 3 minutes.
This time use the four large corner squares, each one divided into 4x4 small squares. The WBC
count is done using the low power objective. Count all WBC in each of the 4x4 corner squares, also
including the left and top borders.
To obtain the number of WBC per cubic mm of blood, use the following formula:

No. of cells counted X dilution X 10

No. of large squares counted

Blood Typing (ABO)

Obtain the anti-A and anti-B sera and place a drop each on two ends of a clean glass slide. Mark
these with a marking wax pencil.
Puncture your finger with a sterile lancet and place a drop of blood each into the two antisera.
Mix gently with a glass probe and tilt the slide from side to side, being careful not to mix the two
antisera together.
Observe for clumping (agglutination) in the two mixtures. This should occur within a minute
after mixing. Do not read after two minutes.
Agglutination in anti-A but not in anti-B suggests blood type A, while the reverse suggests blood
type B. Agglutination in both suggests type AB and no agglutination indicates type O. Can you
explain the mechanisms involved?
Report: Blood Physiology

Name ____________________________________ Date _______________

Serum

1. What information is suggested by the clotting time? the bleeding time? How will you get an abnormal
clotting time but a normal bleeding time and vice versa?

2. Draw a diagram of the stages of blood coagulation with the clotting factors involved.

3. What is hemophilia? Thrombocytopenia?

4. The following tests are often requested by doctors from the hospital laboratory to analyze levels of
certain chemicals found in serum. What are their general uses / significance?

a. fasting blood glucose

b. serum creatinine
c. SGPT (ALT)

d. cholesterol

Blood Cells and Blood Typing

1. Enumerate and describe the functions of different cells found in the blood, including the subtypes of
white blood cells.

2. Why do you need to wipe off the first drop in the procedure?

3. What is the mechanism for the ABO blood typing reaction? Based on this mechanism, why is type O
considered the “universal donor” and type AB the “universal recipient”? How is blood type inherited?