A Laboratory Manual forHuman Physiology

Salman Bin Abdulaziz University

COLLEGE OF PHARMACY

2014-15 / 1435-36

S. NO. TABLE OF CONTENTS PAGE

GENERAL LABORATORY ORIENTATION 2-7

1 INTRODUCTION TO HUMAN PHYSIOLOGY

LABORATORY AND WORK

8-9

2 INTRODUCTION TO MICROSCOPE 10-11

3 MEMBRANE TRANSPORT & OSMOSIS 12-14

4 BLOOD GROUP 15-16

5 RBC COUNT 17-22

6 WBC COUNT 23-29

7 BLEEDING TIME 30

8 CLOTTING TIME 31-32

9 HEMOGLOBIN ESTIMATION 33-34

10 ERYTHROCYTE SEDIMENTATION RATE (ESR) 35

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LABORATORY ORIENTATIONScope of Physiology

1. Physiology: Physiology is the Science of body functions, that is, how the body parts work.

2. To inquire into fascinating complexity of human body.

3. As gateway to careers in health related fields. Mass therapy and Athletics training.

4. As a foundation to advanced scientific studies.

5. To know the structure and function of human body.

6. For understanding pathology of disease and pathological changes.

7. For determining techniques of surgeries.

8. To know parameters of normal health.

9. Factors affecting various physiological processes and its effects.

10. Overall effective maintenance of individual and community health

11. The Principles of Physiology to meet the existing requirements of introductory anatomy

and Physiology courses.

12. It also gives values, simplicity, direction and sort of power to the learners.

13. Human Physiology is formidable body of knowledge to present in an introductory course

and mastering subject.

14. It also highlights the practical application of anatomical and physiological concepts to

students.

15. The dynamic physiological constancy known as Homeostasis is the cardinal theme in

principles of Anatomy and Physiology.

16. By studying concepts of Physiology, we know, how the various feedback mechanisms

work to maintain physiological processes within a narrow range that is compatible with

life.

17. It is needed to understand how individual structures are related to the composition of

entire body.

18. Therefore anatomical nomenclature such as regional names, directional terms and planes

to sections that enable the learners to precisely describe the relationship of one body

structure to another.

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LINK / BLOCK DIAGRAM SHOWING INTER RELATIONSHIP OF SUBJECT AREAS, CURRICULUM

OBJECTIVES AND JOB PROFILE

CORE TECHNOLOGY1. Pharmaceutics-I2. Pharmaceutical Chemistry-I3. Pharmacognosy4. Biochemistry and Clinical Pathology5. Human Anatomy and Physiology6. Health Education and CommunityPharmacy

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TECHNOLOGY SUBJECT1. Pharmaceutics-II2. Pharmaceutical Chemistry-II3. Pharmacology and Toxicology4. Pharmaceutical Jurisprudence5. Drug Stores and BusinessManagement6. Hospital and Clinical Pharmacy7. Practical Training

JOB PROFILE1. Entrepreneur (wholesaler, Distributor,Chemist and Druggist)2. Industry Skilled personnel (Bulk drugformulations, cosmetics)Medical representative3. Hospital Pharmacist (dispensing,Manufacturing)Supervisor4. Community Pharmacist5. Academic Institution(Laboratory technician)6. Repackaging of Drugs other thanthose specified in Schedule C andC1 of Drugs and Cosmetics Act1940 and Rules 1945.

CURRICULUM OBJECTIVES1. Develop attitude for personalDevelopment.2. Develop social skills for socialDevelopment.3. Develop continued learning skills forlifelong learning4. Gain basic knowledge of humanbody and various illnesses,disorders5. Counsel for use of various drugs,their formulations and theirAppropriate use.6. Develop communication skills.7. Develop technical skills for industryAnd hospital activities.

DEVELOPMENT OF SKILLS

Following is the broad perspective of acquisition of intellectual and motor skills. Due care is to be taken, that a student systematically studying the subject will acquire the skills enlisted below.

A) Intellectual skills

1. To understand the concepts. 2. To understand the procedure for performance of experiments.3. To interpret results of experiments.4. To investigate and discriminate the various situations. 5. To acquire ability to plan and design the experiment. 6. To co-ordinate the role of each organ in functioning of a system.

B) Motor skills:

1. Sketching and labeling proportionate diagrams and wherever necessary. 2. Handling and using correctly the instruments, microscopes, models and equipments.3. Measuring and recording accurately with the help of instruments/equipments4. To follow systematic, hygienic and safe procedure of working.5. To prepare and stain the sample slide and observe. 6. To collect sample of blood.

GUIDELINES FOR TEACHERS

Teachers shall discuss the following points with students before start of practicals of the subject

1. Learning Overview: To Develop better understanding of importance of the subject. To know related skills to be developed such as intellectual skills and Motor Skills.

2. Link / Block Diagram: Context of the subject in the form of link diagram showing interrelationship of various subject areas, curriculum objectives and job profile.

3. Graphical structure: In this topics and sub topics are organized in systematic way so that ultimate purpose of learning the subject is achieved. This is arranged in the form of fact, concept, principle, procedure, application / problem.

4. Know your Laboratory work: To understand the layout of laboratory specifications of Equipments/ Instruments / Chemicals, procedures, working in groups, planning time etc. Also to know total amount of work to be done in the laboratory. Teacher shall ensure that required equipments are in working condition before start of experiment, Also keep operating instruction manual and safety manual available. Explain prior concepts to the students before starting of each experiment.

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5. Involve student’s activity at the time of conduct of each experiment.6. While taking reading / observation each student shall be given a chance to perform/observe

the experiment.7. List of questions is given at the end of each experiment. Teacher shall instruct the students

to attempt all questions given at the end each experiment / exercise. Teacher shall ensure that each student writes the answers to the allotted questions in the laboratory manual after performance is over.

8. If the experimental setup has variations in the specifications of the equipments, the teachers are advised to make the necessary changes, wherever needed.

9. Teacher shall assess the performance of students continuously as per norms prescribed by the university.

10. Teacher should ensure that the respective skills and competencies are developed in the students after the completion of the practical exercise.

11. Teacher is expected to share the skills and competencies are developed in the students.12. Teacher may provide additional knowledge and skills to the students even though not

covered in the manual but are expected from the students by the industries.13. Teacher may suggest the students to refer additional related literature of the technical

papers/ reference books/ Seminar Proceedings, etc.14. During assessment teacher is expected to ask questions to the students to tap their

achievements regarding related knowledge and skills so that students can prepare while submitting record of the practical. Focus should be given on development of enlisted skills rather than theoretical /codified knowledge.

15. Teacher should organized group discussions / brain storming sessions /Seminars to facilitate the exchange of knowledge amongst the students.

16. Teacher should give more focus on hands on skills and should actually share the same.17. Teacher shall also refer to the ____________dated _______________ for additional

guidelines.

INSTRUCTIONS FOR STUDENTS

Students shall read the points given below for understanding the theoretical concepts &

practical applications.

1. Listen carefully to the lecture given by teacher about importance of subject, curriculum philosophy, graphical structure, and skills to be developed, information about equipment, instruments, procedure, method of continuous assessment, tentative plan of work in laboratory and total amount of work to be done in a year.

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2. Students shall undergo study visit of the laboratory for types of equipment, instruments, material to be used, before performing experiments.

3. Read the write up of each experiment to be performed, a day in advance.4. Organize the work in the group and make a record of all observations.5. Understand the purpose of experiment and its practical implications.6. Write the answers of the questions allotted by the teacher during practical hours if possible

or afterwards, but immediately.7. Student should not hesitate to ask any difficulty faced during conduct of practical / exercise.8. The student shall study all the questions given in the laboratory manual and practice to

write the answers to these questions.9. Student should develop the habit of pocket discussion / group discussion related to the

experiments/ exercises so that exchanges of knowledge /skills could take place.10. Student shall attempt to develop related hands-on-skills and gain confidence.11. Student shall focus on development of skills rather than theoretical or codified knowledge.12. Student shall visit the nearby workshops, workstation, industries, laboratories, technical

exhibitions, trade fair etc. even not included in the Lab Manual. In short, students should have exposure to the area of work right in the student hood.

13. Student shall insist for the completion of recommended Laboratory Work, industrial visits, answers to the given questions, etc.

14. Student shall develop the habit of evolving more ideas, innovations, skills etc. than included in the scope of the manual.

15. Student shall refer technical magazines, proceedings of the Seminars, refer websites related to the scope of the subjects and update their knowledge and skills.

16. Student should develop the habit of not to depend totally on teachers but to develop self-learning techniques.

17. Student should develop the habit to react with the teacher without hesitation with respect to the academics involved.

18. Student should develop habit to submit the practical exercise continuously and progressively on the scheduled dates and should get the assessment done.

19. Student should be well prepared while submitting the write up of the exercise. This will develop the continuity of the studies and he will not be over loaded at the end of the term.

20. Read the appropriate part of the text and experiment carefully, assure work before you come to the Laboratory.

21. Bring the necessary instruments rough notebook/manual with you.22. Come in time, preferably a few minute before the schedule.23. Take down the relevant notes while the lecturer gives the instructions.24. Never begin to work until you are perfectly clear about what you intend to do.

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25. Do not abuse the apparatus instructed to you. It is valuable and you will have to pay heavy fine in case of breakage/damage.

26. While working keep your table neat and clean. Do not take any excessive material.27. When your work is over detached the apparatus, clean it and keep it is in safe place or

locker.28. Do not be afraid of asking any difficulty for which you cannot find logical answer.29. Learn to rely upon your own observation.30. Before you leave the laboratory find out what work has been assigned for the next

experiment.31. Enter all your observations directly in a tabular form.32. Observation, diagrams should preferably be entered in journal.

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LAB 1: INTRODUCTION TO HUMAN PHYSIOLOGY LABORATORY AND

WORK

AIM: To discuss the basic information about the Human Physiology Laboratory and Work

Physiology is an experimental science. Because of this and because doing experiments or seeing

them done facilitates learning, laboratory exercises are integral to the physiology course. The

objectives of the laboratory in this course are:

1. To permit the student to have direct "hands-on" experience with many physiological

phenomena.

2. To familiarize the student with laboratory equipment and techniques essential for the

study of physiological phenomena.

3. To train the student to acquire data and make accurate and thorough observation, and

4. To present the student with the opportunity for analyzing and explaining the

observations critically.

During the semester you will have an opportunity to do several animal and human studies as

well as computer simulations. How much you gain from the laboratories depends upon your

attitude, interest, and common sense. Frequently, observations of a neighbor's experiment may

aid your understanding of the phenomena being examined; this is all the more true if your own

experiment is not altogether successful, as may occasionally occur.

In order to facilitate your work as well as that of the instructor, certain suggestions and

regulations should be followed:

1. Read each experiment before you come to the laboratory and read the pertinent

sections of your textbook and lecture notes.

2. Keep accurate records in a data book (notebook, etc.) of all laboratory observations and

results. These notes may then be used in the preparation of your laboratory reports. In

some cases the class data will be distributed or presented on the board. Each student is

responsible for providing their own graph paper (including logarithmic).

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3. Since you will be working in groups of four or five, you must make sure that each

member of your group has an opportunity to see the experimental data. It is advisable

that each group find some time when they can discuss the results of each experiment

preparatory to writing their own reports. Each person must write their own laboratory

report.

4. Keep your apparatus neat looking and clean. Report any breakage immediately. Be sure

to keep the computer, interface and software dry and clean.

5. When using solutions take only as much as you need from the stock bottles and never

pour solutions back into them.

6. Keep all stock bottles where they belong and return all equipment in clean condition to

its proper place at the end of each laboratory period.

7. Should you have any questions always ask the instructor - but only after you have given

some thought to answer them yourself.

If you are uncertain how to use any apparatus, how to perform a technique or how to proceed

with an experiment, do not waste time and risk damaging an expensive apparatus or ruining an

experiment, but ask the instructor for assistance.

8. Computer software will be distributed by the instructor. If you would like to use the

computers during hours other than the labs please make arrangements.

9. The execution of a laboratory experiment and the preparation of a report of results

involves (a) some prior understanding of the nature and purpose of the work, (b)

understanding the methods used and some awareness of their limitation, (c) collection

of valid and significant data and expressing them in proper, understandable form, (d)

discussion and critical evaluation of the results, and, finally (e) drawing logical and

unbiased conclusion or generalizations from the results. The last step you will recognize

as a restatement of one of the prime objectives of laboratory work mentioned above.

Your performance in achieving this objective is the best test of your development in

laboratory science. Conclusions must be stated in a general way. They are more than a

mere recital of the data or a chronological description of what you did. The reports will

be due as instructed and you should make sure your name is on each sheet.

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LAB 2: INTRODUCTION TO MICROSCOPE

AIM: To study the microscope and its use

THE MICROSCOPE:

1. Familiarize yourself with the different parts of the scope labeled in Figure below.Illustration provided separately of the microscope used in this lab, with essential components labeled.

2. Before inserting a slide into your microscope make sure that… The lowest power objective lens has been rotated into the locked position over

the slide stage. The stage has been lowered all the way down using the coarse focus knob. Lock the slide into the slide holder on the stage.

3. Using the slide position controls, position the slide so that the sample is immediately above the

4. Turn on the light source for the scope and look through the eyepiece. If the 4x objective lens is properly locked in place, you should see light (although probably not in focus).

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5. Using the coarse focus knob, focus the image you see in the eyepiece. You will be observing the cells at 40x magnification (the product of the 10x eyepiece lens and the 4x objective lens).

6. Once the image is in focus with the 4x objective lens, rotate the objective lenses to bring the 10x objective lens into place. Refocus using the FINE FOCUS KNOB ONLY. Once focused, you will be observing the cells at 100x magnification.

7. Rotate the objective lenses once again to bring the 40x objective lens into place, which will give you a 400x magnification of the cells. Again, refocus using the FINE FOCUS KNOB ONLY— this is critical now—since the stage has now lifted the slide so close to the objective lens it would be very easy to smash the objective lens through the slide!

8. Make the necessary observations under the microscope and describe on your data sheet. When you are done with the slide, rotate the 4x objective lens back into place, lower the stage all of the way down with the coarse focus knob, and remove the slide. UNDER NO CIRCUMSTANCES ARE YOU TO REMOVE A SLIDE WITHOUT SETTING THE OBJECTIVE LENS BACKTO 4X AND LOWERING THE STAGE.

9. Wipe off any fluid from the stage.10. Repeat, if needed as well.11. When you are finished, be sure that:

The lowest power objective lens has been rotated into the locked position over the slide stage.

The stage has been lowered all the way down using the coarse focus knob. There is no blood on the stage of the scope (or anywhere else) That the power has been turned off The slides have been properly disposed. Summon your lab instructor to inspect your scope to be sure that it has been properly

shut down.

CONCLUSION:

RESULT:

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LAB 3: MEMBRANE TRANSPORT & OSMOSIS

AIM: To study the phenomenon of osmosis using living RBC’s

BACKGROUND INFORMATION:

1. With your lab group, discuss and identify which solutions for this experiment are hypotonic,

hypertonic, or isotonic to the RBCs. The isotonic solution is also called physiologic saline.

• 0.9% saline solution ____________________________________

• 5% saline solution______________________________________

• Distilled water_________________________________________

2. In Figure, identify the shape of the RBC (normal, swollen, or shrunk) and the type of solution

the RBC is in (isotonic, hypertonic, or hypotonic).

Hypertonic_______________ Isotonic_________________ Hypotonic_______________

3. With your lab group, predict the net movement of the water in all three types of solutions.

Circle your answer for all three situations:

• In the isotonic solution, the RBCs will swell, shrink, or not change shape:

• In the hypotonic solution, the RBCs will swell, shrink, or not change shape:

• In the hypertonic solution the RBCs will swell, shrink, or not change shape:

MATERIALS:

Blood.

Gloves, Microscopes, microscope slides, cover slips

Isotonic solution (0.9% saline solution)

Hypertonic solution (5 % saline solution)

Hypotonic solution (Distilled water)

Ethanol 70%

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PROCEDURE:

Decide who will set up and initially observe each slide. Every member of your group should

observe each slide.

Wash hands and put on gloves.

Assemble equipment and materials.

Label the microscope slides as Isotonic, Hypertonic or Hypotonic.

SLIDE # 1: 0.9% saline solution

• Place a drop of blood on a slide with a dropper and cover with a coverslip. With a different

dropper, add a drop of 0.9% saline solution to the blood. Tilt the slide to intermix the two

solutions, and cover with a coverslip.

• Using high power, observe the slide for changes in cell shape. Blood cells are very tiny and

difficult to see under low power.

• Record shape of RBCs and type of solution in which they were placed.

Shape__________________________ Type of solution________________________

SLIDE # 2: 5% Saline solution

• Place a drop of blood on a second slide and cover with a coverslip. Observe the RBCs using

high power. On one side of the coverslip, add one drop of 5% saline with a clean medicine

dropper. Place a small piece of paper towel on the opposite side of coverslip to absorb the

liquid and pull the 5% saline solution into the RBCs

• Immediately observe the second slide under high power and watch for changes in cell

shape. You may have to wait a few minutes for cell changes to occur.

• Record shape of RBCs and type of the solution in which they were placed.

Shape ________________________ Type of solution _____________________

SLIDE #3: distilled water

• Place a drop of blood on a second slide and cover with a coverslip. Observe the RBCs using

high power. On one slide of the coverslip, add one drop of distilled water with a clean

dropper. Place a small piece of paper towel on the opposite side of coverslip to absorb the

liquid and pull the distilled water into the RBCs

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• Immediately observe the second slide under high power and watch for changes in cell

shape. You may have to wait a few minutes for cell changes to occur.

• Record shape of RBCs and type of solution in which they were placed.

Shape________________________ Type of solution______________________

Clean up your lab area:

• Place blood-stained items (slides, coverslips, and droppers) in a disinfectant solution of as

directed by your instructor.

• Place gloves in a bag or location indicated by your instructor.

• Wash down the lab counters with disinfectant solution and wipe with paper towels wet

with disinfectant solution. Allow to air dry.

• Wash your hands with soap and water before leaving the lab area.

Complete the Experimental Report with your lab group:

EXPERIMENTAL REPORT:

RESULTS: Describe how the RBC shape changes when placed in each solution.

• 0.9% Saline solution ___________________________________

• 5 % Saline solution ____________________________________

• Distilled water _______________________________________

DISCUSSION:

1. Describe how solute concentration affects osmosis.

2. Describe how water movement affects the cell shape.

CONCLUSION:

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LAB 4: BLOOD GROUP

AIM: To determine blood group of blood sample by ABO system (Use of own blood sample is

suggested)

REAGENTS & APPARATUS:

High Titer Antisera-A, Antisera-B, and Antisera-D,

70% alcohol,

Microscopic slides,

Marking pencil, Pricking needle, Cotton, etc.

DESCRIPTION:

PROCEDURE:

1. Prick a finger and place one drop of blood in each of the compartments A, B & D. these

are clearly labeled on the microscopic slides provided.

2. Quickly add a drop of Antisera-A, Antisera-B, and antisera-D, to compartments A, B &

D respectively.

3. Mix the serum with the drop of blood by moving the slides gently to and fro for a

minute. Now examine the mixtures for sign of red blood cell agglutination. When red

blood cell clumps together they have a speckled or peppered appearance. If you are

not sure, examine the slide using the low power of a microscope.

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EXPERIMENTAL REPORT:

OBSERVATIONS: Agglutination = Y; No Agglutination = N

Compartment A Compartment B Compartment D

Anti A

Anti B

Anti D

RESULT: The blood group of blood sample was found to be __________

DISCUSSION:

1. Explain Antigen-Antibody reaction?

2. Describe blood transfusion?

CONCLUSION:

The blood group of blood sample is ___________________________ (Universal

donor/universal recipient)

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LAB 5: RBC COUNT

AIM: To determine the total RBC count of the blood sample (Use of own blood sample is

suggested)

GENERAL REQUIRMENT

1. Rubber glove

2. Lab Coat

3. Mask

4. Tissue paper

SPECIFIC EQUIPMENT

1. RBC pipette

2. Rubber pipe

3. Plastic syringe

4. Hemacytometer & cover slip (Counting chamber)

5. Microscope

6. Counter (hand tally)

7. Report form

REAGENTS:

RBC diluting fluid, either of the following fluids can be used:

Hayam’s solution

Sodium Sulphate

Sodium Chloride

Mercuric Chloride

Distilled Water

Gower’s solution

Sodium Sulphate

Glacial acetic acid

Distilled water

Citrate-formalin solution

Tri-sodium Citrate, Formalin

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[PLEASE NOTE: The Haemocytometer is made of glass, so do not drop it!].

DESCRIPTION:

The haemocytometer has a central area, slightly lower (0.1 mm) than the rest of surface of the

slide. This depression forms the counting area/ chamber (Figure 1a; below).

Looking at the surface of the slide; finely ruled areas that form grids can be seen (Figure 1b;

above). These define an area on the slide of a known size. The area measured from the grid and

the height between the slide and the base of the depression of the counting area defines a

known volume. By counting the cells or other objects in this volume the no. of cells etc. per unit

volume can be calculated and this value used to give the number in the original volume.

INSTRUCTIONS FOR USE

1. Ensure both the haemocytometer and it’s cover slip are clean. If they are dirty wash them in

distilled water and then wipe them over with alcohol. When dry breath on the surface of

the haemocytometer and quickly place the cover slip in position so that it is centred over

the counting chamber.

2. Gently, but firmly press down on the cover slip, using a thumb placed either side, holding

the haemocytometer firmly between both hands, until Newton's rings (refraction rings of

light of a rainbow/oil slick coloration) are seen. If no rings are seen, try again. If problems

are experienced obtaining rings, e.g. on a hot day, then cool the haemocytometer under

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running cold water for few minutes before drying and then trying to the mount the cover

slip again.

3. Dilute the blood using an RBC diluting pipette with a RBC diluting fluid and mix well. Discard

the first 3-4 drops and then gently touch the pipette against the side of the cover slip where

it touches the base of the depression (Figure 3, below). A drop of liquid should be drawn

out of the pipette and into the chamber. If it is not then gently and slowly squeeze out one

drop. This will fill one counting chamber. If both chambers are to be used then repeat for

the other chamber.

4. Place the haemocytometer under a microscope. Turn the microscope on and under low

power (*5/*10) focus on the grid area of the counting chamber. PLEASE NOTE: do not take

too long to count as the liquid will start to evaporate from the sides of the chamber, and the

chamber dry out, with the heat of the lamp.

5. Under the microscope the grids area can be seen to be formed of 9 large squares in a 3*3

grid (Figure 4; below). Each of these squares covers an area of 1 mm 2 which, with the depth

of the chamber being 0.1 mm, means that the volume of each large square is 0.1 mm3.

The central area of the grid is made up of triple ruled lines that in the centre form a 5*5 grid

of 25 squares with a total volume of 0.1 mm3 (Figure 4, above, and Figure 5a, below ). Each

of these 25 squares has an area of 0.04 mm2 and a volume of 0.004 mm3. Each one of these

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25 squares is further divided up into 16 smaller ones in a 4x4 grid (Figure 5b, below). Each

of these 16 smaller squares has a surface area of 1/400 mm2 (0.0024 mm2) and a volume of

0.00025 mm3.

COUNTING RBC’s (A HIGH-DENSITY CELL POPULATION).

When counting high densities of cells squares use the 40x objective and count the four corner

and one centre 0.04 mm2 square (Figure below, left). When counting a 0.004 mm2 square scan

from the top left of one of 16 1/400 mm2 squares and move to the bottom right as shown in

Figure below, right.

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Cells falling on the bordering triple lines should only be counted if they are on either the top or

on the left lines only. Exclude those touching the bottom and the right hand side lines. When

performing cell counting, the most accurate count is achieved when 120-200 cells can be

counted. If you have more than this in the five squares then you should dilute your sample and

re-count.

Calculations: The concentration (Number) of RBC’s as below and completes the Experimental

Report with your lab group.

Volume of 1 red square = 0.2 x 0.2 x 0.1 cu.mm. = 0.004 cu.mm.

Volume of 5 red square = 0.004 x 5 cu.mm. = 0.02 cu.mm.

In 0.02 cu.mm. the RBC count = N (counted No.)

In 1 cu.mm. the RBC count = N x 1 / 0.02 = N x 50

The dilution for RBC = 100/0.5 = 200

The final RBC count = N x 50 x 200 = 10,000 N (/cu.mm.)

Red cell count = Number of cells counted (N) x Volume factor (50) x Dilution factor(200)

= N x 10,000

Normal range = 3.8 - 6.0 x 106 / cu.mm.

Errors in hemocytometry most frequently arise as a result of:

1. Apparatus

2. Personal technique

3. Inherent error

(1) Errors caused by apparatus:

1. Chipped pipette tips

2. Obscure markings on pipettes

3. Non-optically plane cover glasses

4. Dirty glassware

5. Inaccurate rulings on chamber

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(2) Errors caused by personal technique:

1. Not thoroughly mixing blood

2. Inadequate shaking

3. Failure to discard first 4 drops

4. Not loading chamber properly (overfilling, trapped air bubbles)

5. Counting cells inaccurately (skipping cells, counting cells twice, counting on wrong

borders)

6. Calculation error

"EXPERIMENTAL REPORT"

OBSERVATIONS:

CALCULATIONS:

Normal Range of RBC's:

RESULTS and CONCLUSION:

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Cells counted in each square

R1 = R2 = R3 = R4 = R5 = N =

LAB 6: WBC COUNT

AIM: To determine the total WBC count of the blood sample (Use of own blood sample is

suggested)

GENERAL REQUIRMENT

Rubber glove, Lab Coat, Mask, Tissue paper

SPECIFIC EQUIPMENT

1. Hemacytometer (Counting chamber, WBC pipette &cover slip)

2. Microscope

3. Counter (hand tally)

4. Report form

REAGENTS: WBC diluting fluid, either of the following fluids can be used:

1. Turk’s solution:

Glacial acetic acid

Gential violet

Distilled water

2. Acetic acid (2%)

3. Hydrochloric acid (1%)

[PLEASE NOTE: The Haemocytometer is made of glass, so do not drop it!].

DESCRIPTION:

The haemocytometer has a central area, slightly lower (0.1 mm) than the rest of surface of the

slide. This depression forms the counting area/ chamber (Figure 1a; below).

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Looking at the surface of the slide; finely ruled areas that form grids can be seen (Figure 1b;

above). These define an area on the slide of a known size. The area measured from the grid and

the height between the slide and the base of the depression of the counting area defines a

known volume. By counting the cells or other objects in this volume the no. of cells etc. per unit

volume can be calculated and this value used to give the number in the original volume.

INSTRUCTIONS FOR USE

1. Ensure both the haemocytometer and it’s cover slip are clean. If they are dirty wash them in

distilled water and then wipe them over with alcohol. When dry breath on the surface of

the haemocytometer and

quickly place the cover slip in

position so that it is centred

over the counting chamber.

2. Gently, but firmly press down

on the cover slip, using a thumb placed either side, holding the haemocytometer firmly

between both hands, until Newton's rings (refraction rings of light of a rainbow/oil slick

coloration) are seen. If no rings are seen, try again. If problems are experienced obtaining

rings, e.g. on a hot day, then cool the haemocytometer under running cold water for few

minutes before drying and then trying to the mount the cover slip again.

3. Dilute the blood using a WBC diluting pipette with a WBC diluting fluid and mix well. Discard

the first 3-4 drops and then gently touch the pipette against the side of the cover slip where

it touches the base of the depression (Figure 3, below). A drop of liquid should be drawn

out of the pipette and into the chamber. If it is not then gently and slowly squeeze out one

drop. This will fill one counting chamber. If both chambers are to be used then repeat for

the other chamber.

4. Place the haemocytometer under a

microscope. Turn the microscope on and

under low power focus on the grid area of

the counting chamber. PLEASE NOTE: do

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not take too long to count as the liquid will start to evaporate from the sides of the

chamber, and the chamber dry out, with the heat of the lamp.

5. Under the microscope the grids area can be seen to be formed of 9 large squares in a 3*3

grid (Figure 4; below). Each of these squares covers an area of 1 mm 2 which, with the depth

of the chamber being 0.1 mm, means that the volume of each large square is 0.1 mm3.

PROCEDURE:

1. Draw well-mixed capillary or venous blood exactly to the 0.5 mark in a white blood cell

diluting pipet. This blood column must be free of air bubbles.

2. Wipe the excess blood from the outside of the pipet to avoid transfer of cells to the

diluting fluid. Take care not to touch the tip of the pipet with the gauze.

3. Immediately draw diluting fluid to the "11" mark while rotating the pipet between the

thumb and forefinger to mix the specimen and diluent. Hold the pipet upright to prevent

air bubbles in the bulb.

4. Mix the contents of the pipet for 3-5 minutes to ensure even distribution of cells. Expel

unmixed and relatively cell-free fluid from the capillary portion of the pipet (usually 4

drops).

5. Place the forefinger over the top (short end) of the pipet, hold the pipet at a 450 angle,

and touch the pipet tip to the junction of the cover glass and the counting chamber.

6. Allow the mixture to flow under the cover glass until the chamber is completely charged.

Similarly, fill the opposite chamber of the hemocytometer.

NOTE: If the mixture overflows into the moat or air bubbles occur, clean and dry the

chambers, remix the contents of the pipet, and refill both chambers.

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7. Allow the cells to settle for about 3 minutes. Under low-power magnification and reduced

light, focus on the ruled area and observe for even distribution of cells.

8. Count the white cells in the four 1 sq mm corner areas corresponding to those marked A,

B, C, and D of Figure given below in each of two chambers.

9. Count all the white cells lying within the square and those touching the upper and right-

hand center lines. The white cells that touch the left-hand and bottom lines are not to be

counted. In each of the four areas, conduct the count as indicated by the "snake-like" line

in figure 5-1. A variation of more than 10 cells between any of the four areas counted or a

variation of more than 20 cells between sides of the hemocytometer indicate uneven

distribution and require that the procedure be repeated.

Hemacytometer counting chamber (WBCs). Areas marked A, B, C, and D are used to count

white blood cells.

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COUNTING WBC’s

When counting WBC squares use the 10x objective and count the four corner 1 mm2 square

(Figure below, left). When counting a 1 mm2 square scan from the top left of one of 16 squares

and move to the bottom right as shown

in Figure below, right. Cells falling on the

bordering lines should only be counted if

they are on either the top or on the left

lines only. Exclude those touching the

bottom and the right hand side lines.

Calculations: The concentration (Number) of WBC’s as below and completes the Experimental

Report with your lab group.

Volume of 1 white square = 1 x 1 x 0.1 cu.mm. = 0.1 cu.mm.

Volume of 4 white square = 0.1 x 4 cu.mm. = 0.4 cu.mm.

In 0.4 cu.mm. the WBC count = N (counted No.)

In 1 cu.mm. the WBC count = N x 1 / 0.4 = N x 2.5

The dilution for WBC = 10/0.5 = 20

The final WBC count = N x 2.5 x 20 = 50 N (/cu.mm.)

White cell count = Number of cells counted (N) x Volume factor (2.5) x Dilution factor (20)

= N x 50

Normal range = 5000 - 10000/ cu.mm.

Errors in hemocytometry most frequently arise as a result of:

(1) Errors caused by apparatus:

1. Chipped pipette tips

2. Obscure markings on pipettes

3. Non-optically plane cover glasses

4. Dirty glassware

5. Inaccurate rulings on chamber

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(2) Errors caused by personal technique:

1. Not thoroughly mixing blood

2. Inadequate shaking

3. Failure to discard first 4 drops

4. Not loading chamber properly (overfilling, trapped air bubbles)

5. Counting cells inaccurately (skipping cells, counting cells twice, counting on wrong

borders)

6. Calculation error

"EXPERIMENTAL REPORT"

OBSERVATIONS:

CALCULATIONS:

Normal Range of WBC's:

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Cells counted in each square

W1 = W2 = W3 = W4 = N =

1. Adults (both sexes): 4,500-11,500 WBC’s per cu mm.

2. Childhood: 6,000-14,000 WBC’s per cu mm.

3. Birth: 9,000-30,000 WBC’s per cu mm.

RESULTS and CONCLUSION:

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LAB 7: BLEEDING TIME AIM: To find out the bleeding time of blood sample (Use of own blood sample is suggested)

REAGENTS & APPARATUS:

Filter Paper

70% alcohol

Pricking needle

Cotton, etc.

PROCEDURE:

1. Tip of ring finger is sterilized with 70% alcohol & a bold prick is given so that blood flows.

2. Stop watch is started immediately.

3. Blood is soaked on a filter paper. While soaking, the paper should not touch the skin of

the finger. This is repeated every 10 sec till no blood appears on filter paper.

4. Time of first appearance of blood to cessation of bleeding is bleeding time.

EXPERIMENTAL REPORT:

RESULT:

The bleeding time of own blood is ………………………... (Normal bleeding time is 1 to 3 min)

DISCUSSION:

Describe the mechanisms of hemostasis?

CONCLUSION:

The bleeding time is …………………………………. (Normal / Prolonged / Reduced)

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LAB 8: CLOTTING TIME

AIM: To determine clotting time of blood sample by capillary glass method. (Use of own blood

sample is suggested)

DESCRIPTION: Coagulation of blood

When the blood vessel ruptures, in a few minutes blood loses its fluidity and sets into a

semisolid mass called clot.

This process is called blood coagulation.

1. In vitro - blood clots outside the body on cuts and injuries.

2. In Vivo - Blood clots inside the blood vessels.

Hemophilia: A genetic disorder where longer clotting time due to absence of some clotting

factors.

Requirements:

1. Apparatus:

1. Sterile disposable pricking needle or lancet.

2. Stop watch

3. Dry glass capillary tube (narrow diameter 1 top 2 mm, minimum 10 cm long.)

4. Cotton Swab of absorbent cotton.

5. Spirit wetted, cotton swab.

2. Chemicals:

70 % v/v ethyl alcohol or 70 % v/v denatured spirit.

PROCEDURE:

1. Apply alcoholic 70 % v/v to the clean finger with cotton swab. Allow it to dry naturally.

2. Prick the finger with usual aseptic precautions. Immediately stop watch is started.

3. Dip one end of the capillary into blood drop gently without pressure.

4. Allow to fill the capillary with blood by lowering the end of fitted capillary. (Do not suck

the blood) around ¾th of its length undipped.

5. After every 30 seconds, using stopwatch, break a small piece of capillary.

6. Repeat breaking at regular time intervals, till fibrin thread appears at the broken end of

capillary tube. Do not pull away the cut pieces ling apart and bristly.

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7. Record time interval between pricking finger and first appearance of fibrin thread at the

broken ends of capillary tube. That is clotting time of blood.

DIAGRAM: Clotting time of

blood

Breaking of capillary tube

OBSERVATION:

The fibrin thread is formed at the breaking point after clot is formed. Note the time……

RESULT:

The Clotting time of own blood is ……………….. (normal clotting time is 4 to 9 min)

CONCLUSION:

The clotting time is …………. (normal/prolonged/reduced)

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LAB 9: HEMOGLOBIN ESTIMATION

AIM: To estimate the hemoglobin content and oxygen carrying capacity of blood sample (Use of

own blood sample is suggested)

REAGENTS & APPARATUS:

Sahli’s Hemoglobinometer

Spirit Swab

70% alcohol,

Pricking needle,

Diluting Pipette,

N/10 HCl

Distilled water etc.

PROCEDURE:

1. The graduated diluting tubes and micropipette are cleaned thoroughly and dried.

2. Fill the Sahli’s measuring tube with N/10 HCl unto mark 4 of scale.

3. Wash the pipette with 1:1000 heparin solution in order to prevent coagulation of

blood.

4. Tip of ring finger is sterilized with 70% alcohol & a bold prick is given with usual

precautions so that blood flows.

5. Suck the blood into the Hemoglobinometer pipette unto the mark 20 c.m.

6. Clean the tip of pipette with filter paper and blow the blood out into the

Hemoglobinometer tube dipping the tip of pipette below the HCl level. By sucking

and blowing out the solution a few times the pipette will be completely freed of

blood which mixes with HCl.

7. Wait for one minute when the solution will turn brown, as a result of reaction

hemoglobin with HCl.

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8. Add distilled water drop by drop (stirring after each addition) until the color of blood

acid solution matches with the color of standard brown glass in the

Hemoglobinometer.

9. Read the hemoglobin level directly in grams percent of blood, on the scale given on

Sahli’s measuring tube.

CALCULATION:

1 gm Hb carries 1.34 c.c. of O2

Y gm Hb will carry Y x 1.34 c.c. of O2

RESULT:

My own hemoglobin level is__________________________ gms%

Oxygen carrying capacity____________________________ c.c.

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LAB 10: ERYTHROCYTE SEDIMENTATION RATE (ESR)

AIM: To estimate the Erythrocyte Sedimentation Rate (ESR) of the blood sample.

REAGENTS & APPARATUS: Westergren’s sedimentation apparatusAnticoagulant (EDTA)70% alcohol, Disposable sterile syringe and needle, Cotton etc.

PROCEDURE: 1. Using a sterile syringe remove 1.6 ml of blood from a suitable vein. Transfer it to a test

tube containing EDTA and then draw up blood into a Westergren’s tube exactly to the zero mark.

2. Place the tube upright in the stand and leave undisturbed. The height of the column of clear plasma at the top of tube is noted at the end of an hour and again at the end of 2 hrs.

RESULTS:ESR after 1 hour = _________ mm (Normal 3-5 mm - male)ESR after 2 hour = _________ mm (Normal 7-15 mm - male)

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