EXERCISE NO.

1
PIPETTING TECHNIQUES

I. INTRODUCTION:

Pipettes are volumetric instruments used extensively in the laboratory. It is a cylindrical glass
tube used in measuring fluids and calibrated to deliver or transfer a specified volume of liquids
and solutions from one vessel to another. Care and discretion should be used in selecting pipettes
for clinical laboratory use, for this is one of the determining factors in the accuracy of a laboratory
procedure.

II. OBJECTIVES:
At the end of the activity, the students must be able to:
1) be acquainted with the different techniques of correct pipetting.
2) develop skills acquired from this activity.
3) Identify different kinds of pipettes and their uses

III. MATERIALS:
1) Serological pipettes (10 ml, 5 ml, 1 ml, 0. 1 ml)
2) Beaker with liquid solution
3) Clean receptacles

IV. PROCEDURE:
1) The pipette is grasped by the thumb and middle finger. Place the index finger over
the upper opening that will be used to control the flow of liquid.

2) Lower the pipette into the solution. Allow sufficient depth to fill the pipette above
the calibration mark.

3) Apply suction using the pipette bulb and load the pipette to a point above the
calibration mark. In cases of a critically low volume of the solution, fill the pipette
slowly and watch carefully to avoid aspiration of air. “DO NOT PIPETTE BY
MOUTH”

4) Remove the pipette from the solution. Wipe the tip with a tissue or gauze.

5) Hold the pipette in a vertical solution. Empty the pipette slowly until the lower
meniscus touches the calibration mark.

6) Touch the tip to a clean, dry receptacle to remove any pendant drop.

7) Drain the pipette freely up to the desired calibration in a vertical position. The pipette
has been calibrated to deliver its specified volume in a vertical position with a
constant rate of delivery. Changing the angle of the pipette changes the rate of
 delivery. For the same reason, do not attempt to force the liquid from the pipette at a
faster rate than free drainage permits.

8) When the liquid enters the stem just below the bulb, touch the tip to the side of
receiving vessel, but not into the liquid. Allow several seconds for pipette to drain.

8.1 If the pipette has a continuous ring near at the top of the pipette, it is called a
blowout pipette. This means that the last drop of liquid shall be expelled into
the receiving vessel.

8.2 When the markings are absent from a pipette, it is self-draining, and contents
should be allowed to drain by gravity.

V. ACTIVITY:
1) Make sure that all serological pipettes are scrupulously clean.
2) Pipette the following volumes of solution observing correct pipetting techniques
a. 9 ml
b. 8 ml
c. 6 ml
d. 4 ml
e. 4.5 ml
f. 3 ml
g. 2 ml
h. 1 ml
i. 0.9 ml
j. 0.5 ml
k. 0.2 ml
 EXERCISE NO. 1
PIPETTING TECHNIQUES
Performance Assessment Sheet

Name : __________________________________________ Date : _____________________

PROCEDURE :
You Must Rating Comments
1. Wear a laboratory gown.
2. Wash hands and wear gloves.
3. The pipette is grasped by the thumb and middle
finger. place the index finger over the upper opening
that will be used to control the flow of liquid
4. Lower the pipette into the solution. Allow sufficient
depth to fill the pipette above the calibration mark.
5. Apply suction using the pipette bulb and load the
pipette to a point above the calibration mark.
6. Remove the pipette from the solution. Wipe the tip
with a tissue or gauze.
7. Hold the pipette in a vertical solution. Empty the
pipette slowly until the lower meniscus touches the
calibration mark.
8. Touch the tip to a clean, dry receptacle to remove
any pendant drop.

9. Drain the pipette freely up to the desired calibration

in a vertical position.
10. Touch the tip to the side of receiving vessel, but not
into the liquid. Allow several seconds for pipette to
drain.
11. Allow to drain by gravity or blow the last drop of the
fluid depending on the type of pipette.

RATING 4: EXCELLENT
3: VERY SATISFACTORY
2: SATISFACTORY
1: POOR
0: NOT DONE

LEVEL OF COMPETENCY:

Instructor Date:
 EXERCISE NO. 1
PIPETTING TECHNIQUES
REPORT SHEET

Name: ___________________________ Score: ________

Date:______________

ILLUSTRATIONS:

1. Different kinds of pipettes

2. Step-wise procedure of proper pipetting
REVIEW QUESTIONS:

1. Enumerate the Ten Commandments of Pipetting.

2. Describe the following:

A. Graduation on Pipettes:
a. To contain (TC)

b. To deliver (TD)

c. Between two marks

B. Types of Pipettes:
a. Volumetric pipettes

b. Serological pipettes

c. Micropipettes
i. Lang Levy pipettes

ii. Piston operated pipettes

iii. Positive displacement pipettes

 EXERCISE NO. 2
VENIPUNCTURE

I. INTRODUCTION:
Many types of laboratory tests are performed using blood specimens. The process of collecting
blood is called phlebotomy, translated literally as “to cut a vein”. Venipuncture is a procedure of
blood collection that involves the insertion of a collection needle directly into a vein.

II. OBJECTIVES:

At the end of the activity, the students must be able to:

1) perform the proper collection of blood correctly through venipuncture.
2) observe patient care and safety during the process of venipuncture
3) properly dispose of sharp objects after the procedure
4) identify the different complications of venipuncture

III. MATERIALS:
1) Cotton with and without alcohol
2) 5 ml or 3 ml syringe
3) Hypodermic needle (G 21)
4) Tourniquet
5) Sample containers

IV. PROCEDURE:

A. Preparation:

1) Examine the requisition form; verify the order, tests, and tubes.

2) Greet the client. Identify yourself. Explain what you intend to do. Reassure the patient
that the procedure is safe.

3) Ask the patient for his or her full name, address and or date of birth, if possible ask
for any Identification Card to verify the information given.

4) Verify compliance with dietary restrictions, if applicable. Note any medication which
the client is taking. Inquire about latex allergies.

5) Assemble all supplies.

a. Gloves
b. Laboratory gown
c. Vacuum tubes or special collection tubes, with an extra supply whenever the
procedure is to be repeated.
 [Note: Verify that the tubes have not surpassed the expiration date.
d. Needle
e. Adapter for vacuum system, or syringe
f. Alcohol prep pads
g. 2x2 gauze pads
h. Tourniquet
i. Adhesive bandage or paper tape
j. Permanent marker or pen
k. Approved Sharps Biohazard Container

6) Assemble and position equipment. Assemble the needle and tube holder or syringe.

7) Position tubes in the correct order of draw. Position supplies comfortably within
reach of the dominant hand.

8) Wash hands and apply gloves.

9) Comfortably position the client so that his/her arm is straight and pointed in a slightly
downward direction.

B. VENIPUNCTURE PROPER

10) Apply the tourniquet; make it sure that it should not be applied for more than 1
minute.

11) Select the vein to be punctured.

12) Clean the site by rubbing the alcohol prep pad in concentric circles. Release the
tourniquet if necessary. Take note of the following:
• Do not puncture a site which is wet with alcohol
• Do not wipe the alcohol with an unsterile gauze
• Do not touch the site after cleaning.

13) Reapply the tourniquet if necessary. Avoid contaminating the site of puncture.

14) Anchor the vein

15) Insert the needle at a 15° angle with the bevel up, following the direction of the vein.
Securely rest your hand which is holding the vacuum assembly on the client’s arm.
Be sure that the needle does not move.

16) Fill the vacuum tubes. Follow the correct order of draw. Gently invert additive tubes,
then insert the next tube into the holder and repeat the process.
 17) Once there is evidence that blood was obtained, release the tourniquet. (This should
be within 1 minute.) Pull the looped free end downward. Continue filling, changing
and mixing (if an additive tube) until complete. Remove the last filled tube from the
tube holder.

18) Withdraw the needle. Immediately apply pressure with a clean gauze pad to the
puncture site for the required time. Ask the client to continue applying pressure and
elevate his or her arm overhead.

C. AFTER CARE:

19) Immediately dispose of needle and adapter or syringe in a sharps biohazard container.

20) Label the tubes.

• Label with the client’s name, laboratory number, date, time of collection and your
initials.

21) Follow special handling procedures, if applicable.

22) Examine the client’s arm for bleeding. Apply a pressure bandage, if needed.

23) Remove all collection materials. Remove and discard gloves and other contaminated
items into a biohazard container.

24) Wash your hands.

25) Recheck the site of puncture again.

26) Thank the client

 EXERCISE NO. 2
VENIPUNCTURE
Performance Assessment Sheet

Name : _____________________________________ Date : ______________________

PROCEDURE
You Must Rating Comments
1. Wear a laboratory gown.

2. Examine requisition; verify the order, tests, and

tubes.

3. Greet the client. Identify yourself. Explain what

you intend to do. Reassure the patient that the
procedure is safe.

4. Ask the patient for his or her full name, address

and or date of birth, if possible, ask for any
Identification Card to verify the information given

5. Verify compliance with dietary restrictions, if

applicable.

6. Assemble all supplies.

7. Wash hands and apply gloves.

8. Position the client.

9. Apply the tourniquet. Make it sure that it should

not be applied for more than 1 minute.
10. Select the vein.

11. Cleanse the site

12. Reapply the tourniquet if it necessary. Avoid

contaminating the site of puncture

13. Anchor the vein

14. Insert the needle.

15. Fill the vacuum tubes.

16. Release the tourniquet.

17. Withdraw the needle.

18. Properly dispose the needles.

19. Label the tubes.

20. Follow special handling procedures, if applicable.

21. Examine the client’s arm for bleeding.

22. Remove all collection materials. Wash your hands.

23. Recheck the site of puncture.

24. Thank the client

RATING 4: EXCELLENT
3: VERY SATISFACTORY
2: SATISFACTORY
1: POOR
0: NOT DONE

LEVEL OF COMPETENCY:

Date:
Instructor
 EXERCISE NO. 2
VENIPUNCTURE
REPORT SHEET

Name: ___________________________ Score: ________

Date:______________

ILLUSTRATION:

1. Illustrate the stepwise procedure of venipuncture

REVIEW QUESTIONS:

1. What is the most important aspect of any phlebotomy procedure?

2. Describe the proper protocol for the identification of

a. an Out patient

b. an In patient
3. What techniques can you use to help locate a vein?

4. What is hemoconcentration? How will you prevent hemoconcentration.

5. List down the common errors committed in venipuncture.

 EXCERCISE NO. 3
BLOOD SPECIMEN PREPARATION

I. INTRODUCTION:

Collection, handling, and processing of specimen represents a critical step in laboratory

analysis, for even the most sophisticated laboratory equipment cannot deliver valid results if
specimen integrity is compromised. Samples commonly prepared for clinical chemistry analysis
includes whole blood, plasma and serum.

II. OBJECTIVES:

At the end of the activity, the students must be able to:

1) enumerate the different anticoagulants employed in the preparation of samples
2) be acquainted with the different processes of sample preparation for chemistry
analysis
3) differentiate plasma from serum

III. MATERIALS:

1) Test tube and evacuated tubes with anticoagulant

2) Centrifuge
3) Venipuncture set
4) Applicator sticks

IV. PROCEDURE:

1) Have the following containers ready

a. Evacuated tubes with anticoagulant
b. Plain test tube

2) Obtain 5 ml of blood employing good venipuncture technique.

3) Remove and dispose the needle in a puncture free or biohazard container

4) Dispense

a. The required amount of blood in an evacuated tube with anticoagulant

b. The remaining blood in a plain test tube

5) Mix contents of the former tube by inverting several times. DO NOT SHAKE

6) Whole blood preparation

 Mix the sample placed in an evacuated tube with an anticoagulant. This will
now represent a whole blood sample that includes both the solid and liquid
portions of blood.

7) Plasma Preparation

a. Centrifuge the tube containing the blood sample mixed with an anticoagulant
at 2500 rpm for 5 to 10 minutes
b. Separate the clear supernatant fluid using a pipette or a dropper.
c. The supernatant portion is the PLASMA specimen.

8) Serum preparation

a. Allow the plain tube containing the blood sample to stand at room temperature
for about 30 minutes to allow the sample to clot.
b. After the sample has clotted, rim the sides of the container with applicator
stick to dislodge the clot from the sides of the tube.
c. Centrifuge the sample at 2500 rpm for 5-10 minutes
d. Separate the clear supernatant fluid using a pipette or a dropper
e. This supernatant portion is the serum specimen.
 EXERCISE NO. 3
BLOOD SPECIMEN PREPARATION

Performance Assessment Sheet

Name : ________________________________ Date: ________________

PROCEDURE
Rating Comments
You Must

1. Wear a laboratory gown.

2. Wash hands and wear gloves

3. Have the following containers ready

a. Evacuated tubes with anticoagulant
b. Plain test tube
4. Obtain 5 ml of blood employing good venipuncture
technique

5. Remove and dispose the needle in a puncture free

or biohazard container
6. Dispense

a. The required amount of blood in an

evacuated tube with an anticoagulant
b. The remaining blood in a plain test tube

7. Mix contents of the former tube by inverting it

several times. DO NOT SHAKE
WHOLE BLOOD PREPARATION
8. Mix the sample placed in an evacuated tube with an
anticoagulant
Plasma Preparation
9. Centrifuge the tube with an anticoagulant at 2500
rpm for 5 to 10 minutes
10. Separate the clear supernatant

SERUM PREPARATION
11. Allow the tube without an anticoagulant to stand at
room temperature for about 30 minutes
12. Rim the sides of the container with applicator stick
to dislodge the clot from the sides of the tube.
13. Centrifuge the sample at 2500 rpm for 5-10
minutes
14. Separate the clear supernatant

RATING 4: EXCELLENT
3: VERY SATISFACTORY
2: SATISFACTORY
1: POOR
0: NOT DONE

LEVEL OF COMPETENCY:

Date:
Instructor
 EXERCISE NO. 3
BLOOD SPECIMEN PREPARATION
REPORT SHEET

Name: ___________________________ Score: ________

Date:______________

ILLUSTRATION:

1. Illustrate the preparation of:

a. whole blood
b. plasma
c. serum
REVIEW QUESTIONS:

1. In a tabulated form enumerate the anticoagulants for blood chemistry analysis, its action, the
correct proportion to the amount of blood, and the different laboratory tests that could be
done for each anticoagulant.

2. What is the significance of the order of draw? Is it still applicable to date?

 EXERCISE NO. 4
DERMAL PUNCTURE

I. INTRODUCTION:

Dermal puncture is an alternative collection procedure when minute amount of blood is

needed for testing or for patients in whom venipuncture is not possible. This is also the usual
collection procedure for infants, obese patients and for point of care testing.

II. OBJECTIVES:

At the end of the activity, the students must be able to:

1) perform the procedure of dermal puncture
2) explain the significance of dermal puncture
3) identify the limitations of dermal puncture

III. MATERIALS AND EQUIPMENTS:

1) Alcohol wipes
2) Skin puncture device
3) Collection tubes
4) Sealing caps or clay
5) Bandage

IV. PROCEDURE:

1) Select an appropriate puncture site.

a. For infants younger than 12 months old, the site is usually the lateral or the
medial plantar heel surface.
b. For infants older than 12 months, children, and adults, the site is usually the
fleshy area located near the center of the third and fourth fingers on the palmar
side of the nondominant hand.
c. The site of puncture must not be edematous and never collect on previously
punctured site.
d. Do not use the thumb and the index finger.

2) Position and hold the finger and make the puncture on an appropriate area.

3) Wipe away the first drop of blood.

4) Collect the specimen using appropriate containers avoiding formation of air bubbles.
5) Seal the specimen containers.

6) Mix specimens with anticoagulant to prevent clot formation.

7) Apply pressure to the puncture site with gauze.

8) Bandage the puncture site appropriately.

9) Label the specimen with the client’s name, laboratory number, date, time of
collection and your initials.

10) Thank the patient

 EXERCISE NO. 4
DERMAL PUNCTURE
Performance Assessment Sheet

Name : _________________________________ Date : ___________________________

PROCEDURE
You Must Rating Comments
1. Wear a laboratory gown

2. Examine requisition to verify order, tests, and tubes

3. Greet the client. Identify yourself. Explain what

you intend to do. Reassure the patient that the
procedure is safe.

4. Ask the patient for his or her full name, address

and or date of birth, if possible ask for any
Identification Card to verify the information given.

5. Verify compliance with dietary restrictions, if

applicable

6. Assemble all supplies.

7. Wash your hands and wear personal protective

equipment.

8. Position and hold the finger and make the puncture

on an appropriate area and position.

9. Wipe away the first drop of blood.

10. Collect the specimen using appropriate containers

avoiding formation of air bubbles.

11. Seal the specimen containers.

12. Mix specimens with anticoagulant to prevent clot

formation.

13. Apply pressure to the puncture site with gauze.

14. Bandage the puncture site appropriately. Label the
specimens and thank the patient.
RATING 4: EXCELLENT
3: VERY SATISFACTORY
2: SATISFACTORY
1: POOR
0: NOT DONE

LEVEL OF COMPETENCY:

Date:
Instructor
 EXERCISE NO. 4
DERMAL PUNCTURE
REPORT SHEET

Name: ___________________________ Score: ________

Date:________________

ILLUSTRATIONS:

1. Illustrate the proper collection sites for dermal puncture

2. Illustrate the stepwise procedure of dermal puncture
REVIEW QUESTIONS:

1. Explain why it is best to perform a dermal puncture rather than a venipuncture on children.

2. In what types of patients is dermal puncture applicable and why?

3. Discuss the reason why the first drop of blood in dermal puncture is discarded.