VISION COLLEGE OF

PHARMACY
DEOLI - 304804

DISTRICT - TONK

SUBJECT: HUMAN ANATOMY AND

PHYSIOLOGY

PRACTICAL MANUAL BOOK

( I - B. Pharm and I - D. Pharm)
 EXPERIMENT – I base of this, stage is fixed. On the top
of the arm body tube of the microscope
is fixed and two knobs are fitted. One is
COMPOUND MICROSCOPE for the coarse adjustment and the other
for the fine adjustment. These are used
Objects which are ordinarily not visibleby naked for focussing the bodytube.
eye are seen with microscope. Generally an (c) Body Tube : This is attached to the knob of
object smaller than 0.1 mm cannot be seen by our the arm. It has one lens on the upper
eyes. Therefore, to observe an object smaller than end known as eye piece. This lens can
this, compoundmicroscope is very helpful. be changed according to the required
Hand lens (magnifying lens) is also a type of magnificaton. On the bottom of this
microscopebut its magnifying capacity is very
tube there is a nose piece. Two to four
low. Dissecting microscope is also used to
lenses can be fitted in this nose piece.
visualizetiny things, but it has only one lens.
Because the lenses are fitted on the
Compoundmicroscope is generally used in the
objective, these are known as
laboratories. Therefore, description, use and
maintenance of ordinary compound objective lenses. These are fitted in
microscope are mentioned here. the body tube, known as objective
lens body. The objective lens body is
1.1 Parts of a Compound fitted into the noise piece.
Microscope (d) Stage : It is a platform having a circular hole
Take out the microscope from the box in the centre to allow the passage for
holding the arm by one hand and supporting light from below. It is fixed to the base
the base by another hand. Place carefully on
by the stand. One mirror is fixed to the
the table and study the names and functions of
the parts as mentioned in the figure. The parts stand. It is known as reflecting mirror.
of a compound microscope can been divided Below the stage is a condenser through
into 4 main parts: which concentrated beamof light passes.
(a) Base : This is U-shaped lower portion of the Iris diaphragm is also attached to the
microscope on which the other parts of condenser. The reflecting mirror reflects
the microscope lie. Above the U-shaped the light upward through the iris and
portion, there is a perpendicular portion diaphragm. This beam of light passes
known as the pillar. On the top of this,
through the hole in the stage and provide
another arm is fixed. This is known as
inclination joint. This can be used to tilt light to the object kept on the slide.
the microscope at a desired angle. There are two clips for holding
(b) Arm : It supports the body tube and base of
the microscope. This portion is used to
hold or carry the microscope. On the

1
the slide above the hole on the stage. are also available. The multiplication of
Operation (Use) : Keep a clean prepared slide magnification of eye piece and nose piece
in the centre of the stage. Use clips to denotes the size of the object under
fix the slide on the stage so that it does observation.
not move. Now move the body tube Maintenance of Microscopes:
by the help of coarse adjustment
Microscope is costly equipment.
knobs. Bring the slide in focus under
Therefore, it should be handled carefully.
the objective lens. Focusing should
Always keep the microscope in an upright
be made sharp by the use of fine
position while taking it from one place to
adjustment knobs. When the focus is
another. As far as possible don’t tilt the arm.
sharp then study the slide. The
Clean the lenses of the microscope with the
specimen is viewed by keeping one
lens paper or muslin cloth, never with the filter
eye on the eye piece and the second
or any other kind of paper. If you are using the
eye should be kept open. This type of
high power objective lens then after the
compound microscope is known as
observation is over, turn the nose piece and
monocular compound microscope.
bring low power objective lens in line with the
Some compound microscopes have two hole in the stage. Objective lens should be kept
body tubes. So there are 2 eye pieces and at least 1 cm above the stage. After using the
specimen can be viewed by both the eyes. Such microscope always keep it in the box. Take
type of compound microscope is known as care to see that the stages of microscope, the
binocular compound microscope. In the eye piece, the objective lens are dry and
research work generally binocular compound clean. No chemical should stick to these.
microscope is used. Adjustment knobs and joints should be
1.2 How to use a Compound protected from rusting by applying Vaseline.
Microscope
To use the microscope first of all rotate
the nose piece until the low power objectives
is in line with the body tube and clicks into
position. Open the iris diaphragm. Look
through the eye piece, adjust the mirror and
diaphragm to set a complete field of vision.
Place the slide you want to examine on the
stage of the microscope and by the help of
the clips fix it. Move the slide till the object
comes roughly to the centre of the hole or the
stage. Bring the object into focus using the
coarse adjustment knob. Turn the fine
adjustment knob to bring the object into
sharp focus.
How much magnification the object
needs will be learnt through experience.
Eye lenses of 5x, 10x or 15x are available.
Some way objective lenses of 4x, 10x & 40x Fig. 1.1 A compound microscope
 EXPERIMENT – II

HISTOLOGY
1. Ciliated Epithelium tissue :

Ciliated columnar
epithelial cells are
rectangular in shape
and have between 200
to 300 hair-like
protrusions called cilia.
The mitochondria are
found toward the
apical region of the cell
while the cell nuceliare
found towards thebase
and are often
elongated. Cells are
interconnected via
desmosomses and tight
junction, creating a
semipermeable
membrane that is more
selective that
membrane found in
other types of cell.

2. Cuboidal Epithelium :

Cuboidal epithelia are epithelial cells having a cube-like shape; that is, their width is
approximately equal to their height. They may exist in single layers (simple cuboidal epithelium) or
multiple layers (stratified cuboidal epithelium) depending on their location (and thus function) in the
body.
 3. Stratified Columnar Epithelium :

Stratified columnar epithelia are found in the ocular conjunctiva of the eye, in parts of the
pharynx and anus, the female's uterus, the male urethra and vas deferens. Also found in Lobar
ducts in salivary glands. The cells function in secretion and protection. In simple terms, we can
say that the upper and lowermost layer of cells are columnar in shape. The middle layer contains
cuboidal cells. It forms the lining of respiratory tract, ureter, ovi duct ,etc.

4. Stratified Cuboidal Epithelium :

Stratified cuboidal epithelium is a type of epithelial tissue composed of multiple layers

of cube-shaped cells.

Only the most superficial layer is made up of cuboidal cells, and the other layers can be cells of
other types. This is because, conventionally, naming of stratified epithelium is based on the type
of cell in the most superficial layer.
 4. Areolar connective tissue :

Loose connective tissue is the most common type of connective tissue in vertebrates. It holds organs
in place and attaches epithelial tissue to other underlying tissues. It also surrounds the blood vessels and
nerves. Cells called fibroblasts are widely dispersed in this tissue; they are irregular branching cells that
secrete strong fibrous proteins and proteoglycans as an extracellular matrix. The cells of this type of tissue
are generally separated by quite some distance by a gelatinous substance primarily made up of
collagenous and elastic fibers.

5. Hyaline cartilage :

Hyaline cartilage is covered externally by a fibrous membrane, called the perichondrium,

except at the articular ends of bones and also where it is found directly under the skin, i.e. ears
and nose. This membrane contains vessels that provide the cartilage with nutrition.

Hyaline cartilage matrix is mostly made up of type II collagen and chondroitin sulfate, both of
which are also found in elastic cartilage.

Hyaline cartilage exists on the ventral ends of ribs; in the larynx, trachea, and bronchi; and on the
articular surface of bones
6. Tendon :

A tendon (or sinew) is a tough band of fibrous connective tissue that usually connects muscle to
bone and is capable of withstanding tension. Tendons are similar to ligaments and fasciae; all
three are made of collagen. Ligaments join one bone to another bone; fasciae connect muscles to
other muscles. Tendons and muscles work together to move bones.

7. Human Vein :

Veins (from the Latin vena) are blood vessels that carry blood toward the heart. Most veins carry
deoxygenated blood from the tissues back to the heart; exceptions are the pulmonary and
umbilical veins, both of which carry oxygenated blood to the heart. In contrast to veins, arteries
carry blood away from the heart. Veins are less muscular than arteries and are often closer to the
skin. There are valves in most veins to prevent backflow.
8. Cardiac Muscle :

Cardiac muscle (heart muscle) is involuntary, striated muscle that is found in the walls and
histological foundation of the heart, specifically the myocardium. Cardiac muscle is one of three
major types of muscle, the others being skeletal and smooth muscle. These three types of muscle
all form in the process of myogenesis. The cells that constitute cardiac muscle, called
cardiomyocytes or myocardiocytes, contain only three nuclei. The myocardium is the muscle
tissue of the heart, and forms a thick middle layer between the outer epicardium layer and the
inner endocardium layer.

9. Smooth Muscle :

Smooth muscle is an involuntary non-striated muscle. It is divided into two

subgroups; the single-unit (unitary) and multiunit smooth muscle. Within single-unit
cells, the whole bundle or sheet contracts as a syncytium (i.e. a multinucleate mass of
cytoplasm that is not separated into cells). Multiunit smooth muscle tissues innervate
individual cells; as such, they allow for fine control and gradual responses, much like
motor unit recruitment in skeletal muscle.

Smooth muscle is found within the walls of blood vessels (such smooth muscle
specifically being termed vascular smooth muscle) such as in the tunica media layer of
large (aorta) and small arteries, arterioles and veins. Smooth muscle is also found in
lymphatic vessels, the urinary bladder, uterus (termed uterine smooth muscle), male and
female reproductive tracts, gastrointestinal tract, respiratory tract, arrector pili of skin, the
ciliary muscle, and iris of the eye. The structure and function is basically the same in
smooth muscle cells in different organs, but the inducing stimuli differ substantially, in
order to perform individual effects in the body at individual times. In addition, the
glomeruli of the kidneys contain smooth muscle-like cells called mesangial cells.
 10. Neuron :

Nervous tissue is the main component of the two parts of the nervous system; the brain and
spinal cord of the central nervous system (CNS), and the branching peripheral nerves of the peripheral
nervous system (PNS), which regulates and controls bodily functions and activity.

11. Adipose tissue :

adipose tissue or body fat or just fat is loose connective tissue composed mostly of
adipocytes. In addition to adipocytes, adipose tissue contains the stromal vascular fraction (SVF) of cells
including preadipocytes, fibroblasts, vascular endothelial cells and a variety of immune cells (i.e., adipose
tissue macrophages [ATMs]). Adipose tissue is derived from preadipocytes. Its main role is to store
energy in the form of lipids, although it also cushions and insulates the body. Far from hormonally inert,
adipose tissue has, in recent years, been recognized as a major endocrine organ,[1] as it produces hormones
such as leptin, estrogen, resistin, and the cytokine TNFα. Moreover, adipose tissue can affect other organ
systems of the body and may lead to disease. The two types of adipose tissue are white adipose tissue
(WAT), which stores energy, and brown adipose tissue (BAT), which generates body heat.
 HEAMATOLOGY
EXPERIMENT - III

ESTIMATION OF HEMOGLOBIN BY SAHALI’S METHOD

AIM: To determine the hemoglobin content in 20µl of blood sample.

PRINCIPLE: A hemoprotein composed of globin and heme that gives red

blood cells their characteristic color; function primarily to transport oxygen from
the lungs to the body tissues. The red blood cells are broken down with
hydrochloric acid to get the hemoglobin into a solution. The free hemoglobin is
exposed for a while to form hemin crystals. The solution is diluted to compare
with a standard colour.

MATERIALS: Hemometer, Single mark pipette, Distilled water, Needle,

Spirit, Cotton, HCl.

PROCEDURE: Take 1/10 HCl in the Hb tube upto the lowest mark ‘2’.

2. Prick the finger with needle and collect 20µl of blood sample with single
mark pipette.

3. Place the Hb tube on working table for five minutes for the formation of
hemin crystals.

4. Place the Hb tube in the compater/hemometer and add drop by drop of

distilled water into it until the colour of the solution in the Hb tube coincides
with the glass plates of the compater.

5. If the colour coincides with the glass plates of the compater, observe the
reading in the Hb tube. The percentage of Hb can be calculated from the
reading.

DATA ANALYSIS: Hb content in grams X 100 / 14.5

NORMAL VALUES: Males = 14 to 18 grams

Females = 13 to 14 grams
Children = 10 to 13 grams

RESULT: The hemoglobin content present in 20µl of blood sample is

 EXPERIMENT – IV

Determination of Bleeding Time

Aim

To determine the bleeding time of a patient.

Theory

The time required for complete stopping of blood flow from the punctured blood vessels called
the bleeding time. Normally it is 1-3 minutes for a normal human's blood. Normal clotting time
and bleeding time values differ because bleeding time is the time for stopping bleeding by the
formation of fibrin network on the surface of punctured skin; that is it is the surface phenomenon.
But the clotting time is the time for clotting the whole blood, collected in the capillary tube;
therefore it is a volume phenomenon. For this reason clotting time is more than the bleeding
time, when determining by conventional methods.

Clinical significance

It plays a significant role

i) to study the haemorrhagic disorders.
ii) to study the coagulation defects
iii)to have an idea about the platelets count of the patient. Bleeding time is prolonged in few
disorders like: vascular lesions, platelet defect, severe liver disease, uremia and anti-coagulant
drug administration.

Materials

Sterilized needle, filter paper, cotton, spirit, and stop watch.

Procedure (Duke's method)

Finger of a subject is sterilized with spirit and pricked with sterilized needle. Time of pricking is
noted. Take the stain of the punctured point on a filter paper on 30 second and keep taking stain
of blood in 20 second intervals until the bleeding stops. The time of no stain has come is noted
properly; it is the bleeding time of the subject.

Precaution
Following precautions should be enforced
i) Needle should be sterilized.
ii) A fain stain of blood should not be avoided.
iii) Time should be noted properly.
 EXPERIMENT – V

Determination of Clotting Time

AIM

To determine the clotting time of a subject.

Requirements:

Fine capillary glass tubes of about 10 mm length, cotton, rectified spirit, lancet, stop
watch.

Procedure:

Capillary tube method: (Wright’s method)

Under sterile precautions make a sufficiently deep prick in the finger tip. Note the time
when bleeding starts (start the stop watch). Touch the blood drop at the finger tip using one end
of the capillary tube kept tilted downwards. The tube gets easily filled by capillary action. After
about two minutes start snapping off small lengths of the tube, at intervals of 15 seconds, each
time noting whether the fibrin thread is formed between the snapped ends. Note the time (stop
the stop watch) when the fibrin thread is first seen.

Discussion:

Clotting time is the interval between the moment when bleeding starts and the moment
when the fibrin thread is first seen.

Normal value is 3to 10 minutes.

Bleeding time and clotting time are not the same. Bleeding time depends on the integrity
of platelets and vessel walls, whereas clotting time depends on the availability of coagulation
factors. In coagulation disorders like haemophilia, clotting time is prolonged but bleeding time
remains normal.

Clotting time is also prolonged in conditions like vitamin K deficiency, liver diseases,
disseminated intravascular coagulation, overdosage of anticoagulants etc.

Other method:

Modified Lee and White method:

Under aseptic precaution venepuncture is done and one ml. of blood is collected in each 3 small
test tubes. Note the time when blood is taken. Keep the test tube in a water bath maintained at
37°c. Tilt the tubes every 30 seconds and see whether the blood is flowing. Repeat this till the
tube can be inverted without the blood flowing out. Nore the time. Average value of the results in
the 3 test tubes gives the clotting time.

Normal value is 2 to 7 minutes

 EXPERIMENT – VI

Identification of Blood Groups

ABO blood group system, the classification of human blood based on the inherited properties of
red blood cells (erythrocytes) as determined by the presence or absence of the antigens A and B,
which are carried on the surface of the red cells. Persons may thus have type A, type B, type O,
or type AB blood. The A, B, and O blood groups were first identified by Austrian immunologist
Karl Landsteiner in 1901.

Blood containing red cells with type A antigen on their surface has in its serum (fluid) antibodies
against type B red cells. If, in transfusion, type B blood is injected into persons with type A
blood, the red cells in the injected blood will be destroyed by the antibodies in the recipient’s
blood. In the same way, type A red cells will be destroyed by anti-A antibodies in type B blood.
Type O blood can be injected into persons with type A, B, or O blood unless there is
incompatibility with respect to some other blood group system also present. Persons with type
AB blood can receive type A, B, or O blood.

The ABO and Rh groups in transfusion

system recipient type donor red cell type donor plasma type
ABO A A* or O A or AB
ABO B B or O B or AB
ABO O O only O, A, B, or AB
ABO AB AB*, A*, B, or O AB
Rh positive positive or negative positive or negative
Rh negative negative or positive**, *** negative or positive**
*Not if the patient’s serum contains anti-A1 (antibody to common type A red cell in subgroup A
patients).
**Not if the patient is a female less than 45 years old (childbearing possible), unless life-
threatening hemorrhage is present and transfusion of Rh-positive blood is lifesaving.
***Not if the patient’s serum contains anti-D (antibody to positive red cells), except underunusual
medical circumstances.

Blood group O is the most common blood type throughout the world, particularly among peoples
of South and Central America. Type B is prevalent in Asia, especially in northern India. Type A
also is common all over the world; the highest frequency is among the Blackfoot Indians of
Montana and in the Sami people of northern Scandinavia.

The ABO antigens are developed well before birth and remain throughout life. Children acquire
ABO antibodies passively from their mother before birth, but by three months of age infants are
making their own; it is believed that the stimulus for such antibody formation is from contact
with ABO-like antigenic substances in nature. ABO incompatibility, in which the antigens of a
mother and her fetus are different enough to cause an immune reaction, occurs in a small number
of pregnancies. Rarely, ABO incompatibility may give rise to erythroblastosis fetalis (hemolytic
disease of the newborn), a type of anemia in which the red blood cells of the fetus are destroyed
by the maternal immune system. This situation occurs most often when a mother is type O and
her fetus is either type A or type B.
 EXPERIMENT – VII

RBC Count
AIM

To find out the number of red blood cells in one cubic millimeter of blood

PRINCIPLE:

The number of RBC in a known volume of diluted blood is counted and the number of
cells in one cmm of undiluted blood is calculated from this.

APPARATUS:

Hemocytometer, RBC diluting fluid, compound microscope, sterile lancet,

watchglass, cotton, rectified spirit

HAEMOCYTOMETER: This includes a counting chamber, a special cover slip,

and RBC diluting pipette and a WBC diluting pipette.

The improved Neu-Bauer’s double counting chamber:

This is a thick rectanguar glass with a polished transverse bar in the centre,
separated from the rest of the slide by two parallel grooves on either side. The
polished bar is divided into two equal platforms by a groove in the middle resulting in
‘H’ shaped depression (moats). The surface of the platforms is 1/10 mm below the
surface of the rest of the slide. So if a cover glass is placed over the surface of the
counting chamber, the under surface of the coverglass remains 1/10 mm above the
polished surface of the platform.

The counting area is in the form of a central ruled area on the polished surface
of each platform. It is a square of 3 mm side, divided into 9 equal squares of 1 mm
side. Of these, the four corner squares are used for WBC counting. Each WBC square
of 1 mm side is again divided into sixteen smaller squares each of 1/4mm side. The
central 1 mm square is divided into 25 equal small squares of 1/5mm side, by means
of triple lines of which the 4 corner ones and the central one are used for RBC
counting. Each of these squares is subdivided into 16 smallest squares each of
1/20mm side.

Other uses of R.B.C. pipette:

1. WBC count in leukemia

2. Platelet counting
Uses of the bead in the bulb

1. For proper mixing

2. To know whether the pipette is dry

3. To identify the pipette

R.B.C diluting fluid: Hayem’s fluid is the commonly used diluting fluid.

Composition:

Sodium chloride 0.5 Gm

Sodium sulphate 2.5 Gm

Mercuric perchloride 0.25 Gm

Distilled water 100 mlSodium chloride and sodium sulphate together keeps the
isotonicity of fluid. Sodium sulphate also prevents clumping of red cells. Mercuric
perchloride fixes the cells and acts as a preservative.

Other diluting fluids:

Gower’s fluid

Toison’s fluid

Formol Citrate solution

Procedure:

Clean and dry the counting chamber and put on the special cover slip provided.
Focus under the high power objective and identify the RBC counting area. Clean the
RBC pipette first with distilled water, then with absolute alcohol and finally with ether and
keep it dry. Take a small quantity of diluting fluid in a watch glass and keep aside. Clean
the finger tip using rectified spirit and make a deep prick with a sterile lancet,so that blood
comes out freely without squeezing. Wipe off the first drop which may contain tissue
fluid also. Allow a good sized blood drop to form hanging drop and keep the pointed tip
of the pipette touching the drop. Suck in blood upto the 0.5 mark carefully, without any
air bubble. Excess blood at the tip of the pipette is removed using a bloating paper or
piece of cotton. Immediately, diluting fluid from the watch glass is sucked in upto the
101 mark with out any air bubble by keeping the pipette in vertical position.

Then thoroughly mix the blood and diluting fluid in the pipette by gently rolling
the pipette held horizontally between the palms and keep aside. Mixing takes place only
in the bulb of the pipette. The column of diluting fluid contained in the stem of the
pipette does not enter into the dilution (i.e. 101-1 = 100). So that the blood sucked upto
0.5 mark will have a dilution of 0.5 in 100 or 1 in 200. Now take out the
counting chamber for charging discard first few drops from the pipette, as the stem
contains only diluting fluid. Bring one small drop of diluted blood at the tip of the
pipette, to the edge of the cover slip on the counting chamber at an angle of about 450
The fluid enters by capillary action under the cover slip and fills the counting chamber.
Both areas are filled.

Focus the RBC counting area under high power. Keep the counting chamber
undisturbed about 3 minutes for the cells to settle down in the counting area, and start
counting. At least 5 squares, each having 16 smallest squares (preferably 4 corner and 1
central) should be counted to obtain a satisfactory average and a better dispersal value.
While counting each small square, cells touching the top and left margin of each square
should be omitted and cells touching bottom and right margin of each square should be
counted. Draw a chart of the counting squares in the record and enter the number of cells
in each square and when counted.

Precautions:

1. Counting chamber and pipette should be clean and dry.

2. Fingertip and pricking lancet should be sterile.

3. Blood should freely come out without squeezing.

4. Be careful to prevent clotting of blood inside the pipette.

5. While filling the pipette and charging the counting chamber, no air bubble should
enter.

6. Blood should be taken only upto the 0.5 mark and diluting fluid sucked only upto
101 mark.

7. Blood should be properly mixed with the diluting fluid.

8. Discard first few drops before charging because it will not contain RBC5.

9. While charging the counting chamber, over filling and spilling should be avoided.

10. Cells should be settled down and more or less evenly distributed before counting.

11 Don’t keep the microscope in tilted position.

12. Count from Left to Right and avoid counting of the same cell.

Calculation:

Let the number of cells counted in (5x16) 80 smallest squares be “N”

Number of cells in 1 smallest square is N/80

Side of 1 square = 1/20mm

Area of 1 square = 1/400mm2Depth of fluid film in counting chamber is 1/10mm
Volume of diluted blood in 1 square=1/400x1/10=1/4000mm3

Number of cells in 1/4000mm3 diluted blood = N

80 Number of cells in 1 mm, 3 of diluted blood N 80x1/4000

= Nx4000 80

The dilution factor is 1 in 200

(Total diluted volume in bulb of the pipette is 100 parts, out of which 0.5 is blood. So
dilution is 0.5 in 100

i.e.1 in 200)

So number of cells in 1 mm3 of undiluted blood = Nx 4000x200 = Nxl0000 80

Discussion:

(Expressed in millions/mm3)

Normal RBC Count Adult male:

5- 5.5 millions/mm3

Adult female: 4.5 - 5 millions/mm3

Variations in Count:

Increase in count Decrease in count

Physiological variations

[Link] -

[Link] -

3. Sex -

4. High altitude -

5. Muscular exercise -

- Polycythemia (Erythrocytosis)

- Anaemia (Erythropenia)

Lessduring night, minimum in early morning, gradually increasesduring day

In newborn, high count is seen

More in males
Count higher due to hypoxia

Count increases

Pathological increase in count:

1. Lung diseases like emphysema, pulmonary tuberculosis

2. Congenital heart disease

3. Carbon monoxide poisoning

4. Primary polycythemia (Polycythemia rubra vera)

Secondary polycythemis is due to hypoxia resulting from any cause, physiological or

pathological.

Pathological decrease in count:

1. Increased destruction of RBC

2. Decreased production of RBC

 EXPERIMENT – VIII
Total Leukocyte Count
WBC Diluting fluid ( Turk’s Fluid )
Composition: Each 100 ml of fluid contains 3ml (to lyse/destroy the Glacial acetic acid membrane of WBC,
RBCs and platelets) 1ml (to stain the nuclei ofGentian violet (1%) WBCs deep violet black so that they can
be identified easily) to make 100ml (solvent)Distilled water.

PRINCIPLE: Since the normal WBC count runs into thousands, the count is made possible by diluting the
sample of blood before counting and subsequently multiplying the count by the dilution factor.
• The dilution employed is 1:20.

PROCEDURE • Take WBC diluting fluid (Turk’s Fluid) in a watch glass • After pricking finger, suck the
second drop of blood into the WBC pipette exactly up to 0.5 mark and dilute it with WBC diluting fluid
by sucking the fluid up to 11mark ( dilution 1 in 20 ) • Gently rotate the pipette at least 3-4 minutes in the
palm of the hand to ensure the proper mixing of the blood an the fluid.

Discard first few drops of WBC fluid in the stem of the pipette, charge the counting chamber ( a small
drop of fluid is allowed to form at the tip of the pipette and gently brought into contact with the edge of
the cover slip that is already placed on the chamber) and allow time for settling of the cells • Under low
power objective, identify and check the distribution of WBCs in the 4 corner squares.

Recharge the chamber if distribution is not uniform ( WBCs are seen as regular nucleated, rounded bodies
with a clear refractivity around them )

Count the number of WBCs in each WBC square preferably under low power objective. • Count the
WBCs in 4 corner WBC squares and enter your observations in the corresponding squares

CALCULATIONS • Calculation of diluting factor Dilution factor:Final volume achieved (10 parts) Original
volume taken (0.5 parts) [Link] 20

Calculation of volume fluid examined :Area of 4 WBC squares = 4 X 1mm X 1mm = 4 sqmm Depth of
the chamber = 0.1mm Therefore, volume of fluid in the 4 WBC squares = [Link] X 0.1mm =
0.4 [Link].

Calculation of Total Leukocyte count or Total WBC count: Let ‘N’ be the total number of WBCs in 4
WBC squares i.e. in 0.4 [Link] of diluted blood Then total number of WBCs in 1 [Link] of undiluted blood
: N x Dilution factor (20) 0.4 = N x 50

PRECAUTIONS • Prick should be bold enough to give free flowing blood. At no stage finger should be
squeezed to take out the blood • Both the chamber and the cover slip should be dry and free from grease •
Use only dry pipette • Never use a broken cover slip • Before charging the chamber the fluid from the stem
of the pipette should be discarded.

The cover slip should be placed symmetrical so as to cover the ruled area completely • There should be
no under or over charging of the chamber ( The count will be low in both the cases) • After charging
the chamber time should be given for the cells to settle down, but counting should be started before the
fluid in the chamber starts drying up. • While counting the cells the stage of the microscope should not
be tilted.

ESTIMATION OF DIFFERENTIAL LEUCOCYTE COUNT ( D LC )

*APPARATUS • • • • • 4-5 glass slides Pricking apparatus Compound microscope Cedar – wood oilLeishman’s stain
[Link]
Leishman’s Stain:It belongs to the Romanowsky group of stains containing an acidic and a basic dye. The
unique property of this stain is •Making clear distinction in the shades of staining •Stains granules
differentially 1. Eosin :- It is an acidic dye ( negatively charged ) and stains positively charged (basic )
particles such as RBCs and granules of Eosinoplhils 2. Methylene blue:- It is a basic dye ( positively
charged ) and stains negatively charged ( acidic ) particles such as cytoplasm, nuclei of WBCs and
granules of basophils.

Acetone free methyl alcohol:- It is a fixative It preserves the cell in whatever chemical and metabolic state
they are at the time of staining The blood smear also gets fixed to the slide ( precipitation of protein in it, by
alcohol ) so that it can not be washed off ( Methyl alcohol should be acetone free as it causes shrinkage of
cells and even lysis. It should be also water free as water may affect appearance of thefilm causing rouleaux
formation)

PROCEDURE A. Preparation of Blood Smear – Take 4-5 grease free slides with smooth edges; 3- 4 to be
covered by the blood film and one as a spreader – Lay the slides on the table, prick the finger, touch the
bleeding point on the centerline of the slide at about 1 to 2 cm from one end of the slide, on eachof the
slides – Place the narrow edge of the spreader on the surface of slide just in front of the drop of the blood at
an angle of 45 deg.

Draw the spreader backwards so as to touch the drop and hold it there till the blood runs along the width
of the spreader at the line of junction – The spreader is then moved to the other end of the slideslowly and
smoothly maintaining the 45 deg angle – Allow the film to dry in air and repeat the procedurewith the
other slides.

Criteria of a good blood smear:1. It should cover almost the entire width and about 3-4 cm lengthof the
slide 2. It is tongue shaped with no tail at the end 3. It is of uniform thickness, not too thick or toothin ( A
thick film appears red when placed on a white back ground ) 4. There should not be longitudinalstriations
or cross striations or air gaps in the smear.

B. Fixing and staining the Blood smear :1. Fixing the blood smear: i) Place the blood smear across2
parallel supports on the glass rods, to make them horizontal ii) Pour 8-12 drops of the Leishman’s stainon
the slides so as to cover the smear, leave it on for 2 mins ( fixation time) 2. Staining the smear: i) At the
end of 2 mins add an equal no. drops of buffered ( pH 6.8) water over the stain so that water is not spilled
over ii) Mix the stain and water evenly by gently blowing air intermittently with the help of a dropper.

act iii) If the dilution of the stain is correct, the fluid will be covered by a thin greenish scum.
Leave the stain to for 10 mins. ( staining time) 3. At the end of 10 mins, pour off the stain hold the slide in
a slanting position below the tap and the water is allowed to flow over the smear 4. Wash the slide ( up to
2 mins) with tap water gently and thoroughly till the film gets a pinkish tinge. Make sure that :i) none of
the greenish scum settles on the surface of the blood film ii) water stream should not strike the blood
stream directly or else it may be washed off. 5. Wipe clean the back of the slide and set upright to dry.

C. Assessment of the quality of the stained smear: A well stained smear has the following characteristics:1.
The smear is single thick, with cells uniformly distributed 2. At least 1 WBC is seen per high power field
(X 100) 3. The RBCs are stained light pink 4. In an over stained smear, RBCs look bluish black and WBCs
will take up more methylene blue, hence look totally purple 5. In an under stainedsmear, RBCs appear very
pale and WBCs look almost colorless.

D. Examination of the stained smear under oil immersion:- Identification of the different types of blood
cells is done only under oil immersion 1. Place a drop of cedar- wood oil on the blood film. Focus the oil
immersion objective such that it just touches the oil drop. Now raise the objective and focus with a
 fine adjustment. 2. Study the distribution and characteristics of different types of cells in the different
parts of the smear.

The following is observed:i) The most abundant cell type foun anywhere in the smear is RBCs, as light
pink non nucleated dics of uniform size. ii) At the head end end of the smear the RBCs are crowded and
superimposed, and the WBCs are poorly stained iii) At the extreme tail end, the cells are wide apart and
the WBCs are distorted. iv) At the upper and lower edges of the smear, the WBCs are found in plenty but
are poorly stained and abnormally rich in granulocytes

E. Identification of different types of Leucocytes: The WBCs can be differentiated from RBCs by the
presence of nucleus and Large size. While identifying the WBCs, keep 4 factors in mind:1. Size of thecells
( compare with the surrounding RBCs which are of uniform size, 7.2µm) 2. Features of the nucleus (colour,
number of lobes ) 3. Features of the cytoplasmic granules ( pink or blue, fine or coarse ) 4. Nuclear /
Cytoplasmic ratio.

F. Counting of the different types of leucocytes: A minimum of 100 WBCs are identified in a systematic
manner and counting is made using the tally- bar method. Ideally all the cells should be counted in a single
strip running the length of the smear, proceeding from the base to the apex. Use the mechanical stage to
traverse the full length of the film. Move the slide along vertically by a distance of 2mm and again traverse
the full length of the film this time in the opposite direction . This method ensuresthat the cells are not
counted more than once.

Alternatively a box containing 100 squares can be made in which entry of each one of identifiedcells can
be made.

precautions [Link] slides should be claen and greese free there fore , these should be cleaned troughly with
soap and water immediately [Link] glass spreader should be carefully selected [Link] about4-5 bllod films
so that at least one is satisfactory.

[Link] smeared surface with glass marking pencil [Link] dry or heat or blot dry smear [Link]
must not be used [Link] smear should be stained not later than 2 hours after it has been prepared.

8. Assess the quality of the smear ,both grossly and microscopically before staining it [Link]
well stained slide should be examined under the oil-immersion lens.
EXPERIMENT – IX

Differential Leucocyte Count

DIFFERENTIAL WHITE BLOOD CELL COUNT

A differential white cell count (leukocyte formula) consists of an examination of blood to determine
the presence and the number of different types of white blood cells. It is obtained examining a blood
film or a peripheral blood smear.
A peripheral blood smear is a microscope slide made from a drop of blood, which allows the cells
to be examined microscopically. Blood films are made by placing a drop of blood on one end of a
slide, and using a spreader slide to disperse the blood over the slide's length. The aim is to get a region
where the cells are spaced far enough apart to be counted and differentiated. The slide is left to air
dry, after which the blood is fixed to the slide by immersing it briefly in methanol. The fixative is
essential for good staining and presentation of cellular detail. After fixation, the slide is stained using
the May-Grünwald-Giemsa method to distinguish the cells from each other: the basophilic structures
(which take up basic dyes) are colored in blue; the acidophilic or eosinophilic structures (which
take up acid dyes) are colored in red whereas neutrophilic structured take up both dyes and are
colored brownish- purple.

There are several different types of white blood cells. A major distinguishing feature of some
leukocytes is the presence of granules in their cytoplasm; white blood cells are often
characterized as granulocytes or agranulocytes.

Granulocytes (polymorphonuclear leukocytes) are subdivided according to the specific coloration

of granules:
► Neutrophils account for the largest percentage of leukocytes found in a normal blood
sample. On a stained blood smear, the cytoplasm of a neutrophil has numerous fine lilac-
colored granules and a dark purple nucleus. The diameter of a neutrophil is
10-15 μm. Neutrophils are subclassified according to their age or maturity, which is
indicated by changes in the nucleus (Figure no. 7):
– metamyelocyte – is the youngest neutrophil generally reported, the nucleus is large,
round or bean-shaped, the cytoplasm is abundant, pale blue;
– neutrophilic band or stab – the nucleus is elongated and curved (horseshoe or S-
shape), cytoplasm is abundant, pink;
– segmented neutrophil – is a mature neutrophil, the nucleus is separated into
2-5 segments or lobes, the cytoplasm is pale red.

Figure: Neutrophil granulocytes with different maturation degree: (from left toright)
metamyelocyte, neutrophilic band and segmented neutrophil.

► Eosinophils have a diameter of 12-14 μm, the nucleus usually large, typically a bilobate
(two-lobed), the cytoplasm is eosinophilic (pale red) with coarse round granules of
uniform size which appear brick-red after staining with eosin (Figure no.
► Basophils have a diameter of 11-13 μm. Their nucleus is usually large, irregular,
 sometimes having three lobes, the cytoplasm is basophilic, containing scattered large, dark-
blue granules which may overlay the nucleus.

Figure: Basophil granulocytes

Agranulocytes (mononuclear leucocytes) are characterized by the apparent absence of granules in

their cytoplasm. Although the name implies a lack of granules these cells do contain non-specific
azurophilic granules.
► Lymphocytes have a diameter of 8-10 μm. The nucleus is large, generally round, oval, or
slightly indented. The cytoplasm of a lymphocyte is basophilic, scanty, (in circle or “half
moon” around the nuclues) (Figure no. 10, left).
Monocytes are the largest of the normal white blood cells (the diameter is 15-25 μm). The
nucleus is large, round, indented or lobulated, the cytoplasm is muddy gray-blue (Figure no.
10, right).

Figure no. Agranulocytes: left: two lymphocytes, right: monocyte.

MATERIALS

– stained blood films

– light microscope with oil immersion objective (90x)
– immersion oil (cedar tree oil)

PROCEDURE

Focus at low power (10x) on a region of a blood smear. Do not alter focus for the following steps.
Partially rotate turret so that 10x and 90x objectives straddle the specimen. Place a small drop of oil
on the slide in the center of the lighted area. Rotate turret so that the 90x oil immersion objective
touches the oil and clicks into place. Focus only with fine focus.
Scan the blood film in crisscross examining both the center and the sides of the smear. Search for 100
successive white blood cells and identify them. Note the cells in Schilling's table (Table 1): mark 10
white blood cells in every column.

cell type total (%)

metamyelocytes
band neutrophils
segmented neutrophils
eosinophils
basophils
lymphocytes
monocytes
total 10 10 10 10 10 10 10 10 10 10 100
Table 1. Schilling's table

Clean up! Cleanse microscope stage and workspace if any oil has spilled on it. Recap the immersion
oil container securely. DO NOT wipe clean the 90x oil immersion objective nor the slide; this will be
done by the assistant!

RESULTS

Sum the number of different cell types by rows in Schilling's table. Report the ratio of different white
blood cells in percents.

DATA INTERPRETATION

Normal distribution of different white blood cell types:

metamyelocytes <1%
band neutrophils 1-2 %
segmented neutrophils 55-65 %
eosinophils 2-4 %
basophils 0-1 %
lymphocytes 23-35 %
monocytes 4-8 %
Alteration of the ratio of different white blood cells:

cell type increase decrease

neutrophil neutrophilia neutropenia
eosinophil eosinophilia eosinopenia
basophil basophilia basopenia
lymphocyte lympocytosis lymphopenia
monocytes monocytosis monocytopenia

Neutrophilia refers to a higher number of neutrophils in the peripheral blood. The most
common cause of neutrophilia is a bacterial infection. Neutrophils are also increased in any
acute inflammation and might also be the result of a malignancy.
A "left shift" refers to the presence of increased proportions of younger, less differentiated
neutrophils in the blood. This generally reflects early or premature release of myeloid cells from
the bone marrow, the site where neutrophils are generated. A "right shift" refers to the presence
of older, hypersegmented neutrophils in the blood, reflecting the inadequate release or
insufficient production of neutrophils.
Neutropenia refers to an abnormally low number of neutrophils. It can be caused by several
conditions, including decreased production in the bone marrow or viral infections.
EXPERIMENT – X

Erythrocyte Sedimentation rate

There are two main methods used to measure the ESR: the Westergren method and the Wintrobe Method. Each
method produces slightly different results. Most laboratories use the Westergren method.

Westergren method:
The Westergren method requires collecting 2 ml of venous blood into a tube containing 0 .5 ml of sodium
citrate. It should be stored no longer than 2 hours at room temperature or 6 hours at 4 °C. The blood is drawn
into a Westergren-Katz tube to the 200 mm mark. The tube is placed in a rack in a strictly vertical position for 1
hour at room temperature, at which time the distance from the lowest point of the surface meniscus to the upper
limit of the red cell sediment is measured. The distance of fall of erythrocytes, expressed as millimeters in 1
hour, is the ESR.

Wintrobe method:
The Wintrobe method is performed similarly except that the Wintrobe tube is smaller in diameter than the
Westergren tube and only 100 mm long. EDTA anticoagulated blood without extra diluent is drawn into the
tube, and the rate of fall of red blood cells is measured in millimeters after 1 hour. The shorter column makes
this method less sensitive than the Westergren method because the maximal possible abnormal value is
lower. However, this method is more practical for demonstration purposes.

Average values in healthy men are: <15mm/hr; in healthy females, they are somewhat higher: <20mm. The
values are slightly higher in old age, in both genders.

Theoretical considerations

The RBCs sediment because their density is greater than

that of plasma; this is particularly so, when there is an
alteration in the distribution of charges on the surface of
the RBC (which normally keeps them separate form each
other) resulting in their coming together to form large
aggregates known as rouleaux.

Rouleaux formation is determined largely by increased

levels of plasma fibrinogen and globulins, and so the
ESR reflects mainly changes in the plasma proteins that
accompany acute and chronic infections, some tumors
and degenerative diseases. In such situations, the ESR
values are much greater than 20mm/hr. Note that the ESR
denotes merely the presence of tissue damage or disease,
but not its severity; it may be used to follow the progress
of the diseased state, or monitor the effectiveness of
treatment.

Some interferences which increase ESR:

 increased level of fibrinogen, gamma globulins.

 technical factors: tilted ESR tube, high room temperature.

Some interferences which decrease ESR:

 abnormally shaped RBC (sickle cells, spherocytosis).

 technical factors: short ESR tubes, low room temperature, delay in test performance (>2 hours), clotted
blood sample, excess anticoagulant, bubbles in tube.
EXPERIMENT – XI

Recording of Pulse Rate

The heart, the blood, and the blood vessels make up the circulatory system. The heart pushes the
blood through the blood vessels to vitals organs and tissues; the blood carries the oxygen they need
to function, removes waste products that are a byproduct of metabolism, and carries them to the
lungs for elimination.

The heart acts like a pump, and each heartbeat has two phases, a resting phase (diastole) and a
pumping phase (systole). During diastole, the chambers of the heart fill with oxygenated blood.
During systole, the walls of these chambers contract and send out a “wave” of blood to the lungs,
brain, kidneys, muscles, etc. Taken together, systole and diastole make up the heartbeat. The pulse
is a measurement of the number of times the heart beats in one minute.

In certain areas of the body, the blood vessels are close to the surface of the skin and this wave
that represents a heartbeat can be felt. The pulse can also be measured by listening to heart with a
stethoscope. Measuring the pulse rate is like measuring the body temperature; it is a quick, reliable,
and easy way to determine someone’s basic state of health or to determine if he/she is sick. The
heart rate speeds up or slows down in response to stress, injury, infection, activity level, changes in
the environment, drugs, etc.

As with the other vital signs there is a range of pulse are that is considered to be normal.

 Babies up to the age of 1: 100-160 beats per minute.

 Children of the ages 1 to 10: 60 to 120 beats per minute
 Children of the ages 11-17: 60 to 100 beats per minute.
 Adults: 60 to 100 beats per minute.
 Athletes: 40-60 beats per minute.

These values are considered to be normal for the given ages. But just as important as the rate,
however, is the rhythm. A normal heartbeat will be regular like the ticking of a clock. The amount
of time between each beats will be the same and if it is not, this is considered abnormal.

Measuring and recording the pulse is simple. It can be done in many places in the body, but the two
most common sites used to check the pulse are a) the chest, directly over the heart, using a
stethoscope, and b) on the side of the wrist using the radial artery.
 Radial artery: The radial artery is located on the wrist (on the side opposite the back of the
hand) just below the base of the thumb. You can find it taking two fingers and placing them
in this area: you should easily feel a rhythmic pulse. Count the pulse for at least 30 seconds
and then multiply times by two; the result will be the heart rate. Do not use your thumb to
count the pulse. Many people have a strong pulse in their thumbs and this can interfere
with accurately feeling someone’s pulse.
 The chest: The heart is located on the left side of the chest, approximately midway between
the waist and the shoulder. Place your stethoscope in that area and listen for the heartbeat.
Count for 30 second and then multiply times two; the result will be the pulse rate.
 The pulse is a measurement of the number of heartbeats in one minute.
 The pulse can be increased or slowed down by illness, injury, infection, drugs, the
environment, or activity level.
 The normal pulse for an adult should be regular and between 60 and 100 beats per minute.
 A pulse rate that is abnormally slow is called bradycardia.
 A pulse rate that is abnormally fast is called tachycardia.
 The two most accurate places to measure the pulse are the radial artery and the chest.
 Notify the R.N. or your supervisor if the patient’s heart rate bradycardic, tachycardic or is
unusually slow or fast for that patient.
EXPERIMENT – XII

Recording of Arterial Blood Pressure

Definition of Blood Pressure
Arterial blood pressure
is the force exerted by the blood on the wall of a blood vessel
as the heart pumps (contracts) and relaxes.
Systolic
blood pressure is the degree of force when the heart
is pumping (contracting). The
diastolic
blood pressure is the degree of force when the hearts relaxed.

Method of Measuring Arterial Blood Pressure

In the measurement procedure a cuff is wrapped around a person’s arm with an inflatable
rubber bag inside the cuff centered over the brachial artery. Enough air pressure is pumped into the cuff
to close the artery. Air pressure is then released by opening the thumb valve. When the pressure in the
cuff is equal to the pressure on the artery, the artery opens and the blood begins to return to the part of
the artery that was closed.
As the blood returns to the artery, pulse sounds begin. These sounds can be heard through
a stethoscope placed over the brachial pulse point. The sounds continue for a time while the cuff is
deflated slowly, eventually becoming too faint to hear.
The cuff is connected by tubing to a manometer, which shows the amount of pressure on
the artery. When the first pulse sounds are heard, the reading on the manometer measures the systolic
blood pressure. The last sound heard is the diastolic blood pressure. In children, the muffling of sound
or fourth sound is often used as the diastolic blood pressure rather than the disappearance of sound.

Reference ranges for blood pressure in children

Stage Approximate age Systolic Diastolic

Infants 1 to 12 months 75–100 50–70

Toddlers and preschoolers 1 to 5 years 80–110 50–80

School age 6 to 12 years 85–120 50–80

Adolescents 13 to 18 years 95–140 60–90

 OSTEOLOGY
EXPERIMENT – XIII

Classification of Bones
EXPERIMENT – XIV

Sense Organs
1) EYE

Lecture notes 1: The human eye

The human eye is not much in use as a professional tool of astronomy. On the
other hand, it is of great interest to understand how it works and by doing so
we may illustrate many of the principles and problems that we will meet later
in the course.
The eye and brain work together, and the brain can correct for many of the
aberrations suffered by the eye. Thus the brain compensates for the fact that
the image on the retina is inverted and for chromatic aberration.

Figure 1: Cross section of the human eye (left), illustration of the eyes receptor
cells; cones, used for color vision with the iodopsin layers arranged to the rigth,
and rods with rhodopsin layers. Light enters these cells from the left before
being absorbed by either iodopsin or rhodopsin.

Light is focussed on the retina, where there are two types of receptors: rods
and cones. Cones for color reception, rods for black and white with higher
sensitivity.
In the rods a pigment known as rhodopsin absorbs radiation. A protein
with a weight of some 40 000 amu, arranged in layers 20 nm thick and 500 nm
wide. Under influence of light a small fragment, a chromophore, will will split
off. The chromophore is a vitamin A derivative called retinal (or retinaldehyde)
with a molecular weight of 286 amu. The portion left behind is a colorless
protein called opsin. The moment of visual excitation occurs during this break
off process as the cell’s electrical potential changes. This change in potential
can then propagate along nerve cells to the brain. The rhodopsin molecule is
then (slowly) regenerated.
The response of cones is similar, but in this case the pigment is known as
iodopsin which also contains the retinaldehyde group. Cone cells come in three
varieties with different spectral sensitivities (see figure 2).
In bright light much of the rhodopsin is broken up into opsin and retinalde-
hyde, and the rod sensitivity is much reduced so that vision is primarly provided
by the cones, even though their light sensitivity is only of order 1% of the rods.
The three varieties of cones combine to give color vision. At low light levels
only rods are triggered by the ambient radiation and vision is then in black and
white.

Figure 2: Absorption curves for the various types of cones and for rhodopsin.

Upon entering the dark from a brightly lit region rhodopsin will build up
over a period of roughly 30 min, thus dark-adaptation takes this long and is
based on rod cells. Somewhere between 1–10 photons are necessary to trigger
an individual rod. However, several rods must be triggered in order to result
in a pulse being sent to the brain as many rods can be connected to a single
nerve fibre. The total number of rods is of order 108, of cones 6 × 106, these
must share some 106 nerve fibres. Thus there are roughly 100 visual receptors
per nerve fibre, note that there can be many cross connections between groups
of receptors. Cones are concentrated towards the fovea centralis which is the
region of most acute vision, while rods are most plentiful towards the periphery
of the field of view. Weak ob jects are thus most easily visible with averted
vision, ie when it is not looked at directly. In sum with all these effects the eye
is usable over a range of illuminations differing by a factor 109 – 1010.
The Rayleigh limit of the eye, roughly given by λ/D where λ is the wave-
length of the observed light and D is the size of the observing aperature, is of
order 20 arcsec when the iris has its maximum diameter of 5–7 mm. However,
for two separate images to be distinguished, they must be separated by at least
one unexcited receptor cell, so even on the fovea centralis resolutin is limited in
practice to between 1 arcmin an 2 arcmin. This is much better than elsewhere
on the retina, since the fovea centralis is populated by small, tightly packed,
singly connected cones. The average resolution of the eye lies between 5 arcmin
and 10 arcmin. The effect of granularity of the retina is countered by rapid
oscillations of the eye through a few 10 arcsec with a frequency of a few Hz, so
that several receptors are involved in the detection when averaged over time.
The response of the eye to changes in illumination is logarithmic; if two
sources A and B are observed to differ by a given amount, and a third source
C is seen to lie midway between them, then the energy from C will differ from
A by the same factor as it differs from B. The faintest stars visible at a good
site (magnitude 6m) corresponds to a detection of approximately 3 × 1015 W.
Sensitivity will vary between indivduals and decreases with age, the retina of
a 60 year old person will receive some 30% of the light seen by a person of 30-
years.
The system used by astronomers to measure the brightness of stars is a very
old one, and is based on the sensitivity of the eye. Hipparchos’ catalogue of
stars divided the stars into six classes from the brightest, of the first rank or
magnitude, to the dimmest of the sixth magnitude. The present day system
is based on this after the work of Norman Pogson put the magnitude scale on
a firm basis in 1856. Pogson suggested a logarithmic scale that approximately
agreed with earlier measurements: the difference between stars of magnitude
m1 and m2 are given by

m1 − m2 = −2.5 log E1
E2
 where E1 and E2 are the energies per unit area at the surface of Earth for the
two stars
Exercises
1. Vega has a magnitude of roughly 0.0, Polaris a magnitude of 2.0. Estimate
the magnitudes of the stars in Orions belt as well as Betelgeuse, Rigel, and
Bellatrix. What is the magnitude of the weakest star you can find in the
sky?
2. Mizar and Alcor in Ursa Ma joris are separated by some 110. Can you
separate them and see both stars? Estimate their magnitudes as well.

2) EAR

Outer ear, middle ear, inner ear

Outer ear: Pinna - external part, Auditory canal

Middle ear: Eardrum - hard membrane, Ossicles

Inner ear: Oval window, Cochlea, Auditory nerve

The Pinna's function is to help in direction finding at high frequencies and helps funnel high
frequencies into the auditory canal. The auditory canal acts as a resonant cavity in the range
of 3,400 Hz, and causes our hearing to be more sensitive in this range.
Middle ear: Eardrum - hard membrane, Ossicles

The eardrum is a little more then 0.5 cm in diameter. Its function, along with the ossicles
(hammer, anvil, stirrup), is to convert the sound waves into mechanical vibrations.
Remember that sound is pressure waves and that pressure is force/area. The mechanical
vibrations carried through the lever action of the ossicles presses on the oval window in the
cochlea. The cochlea is very small, in part, to be able to detect very small vibrations. The
middle ear enhances the sound wave energy two ways. First, through a reduction in size of
the oval window relative to the size of the eardrum. The area of the oval window is 20-25
times smaller then the eardrum, Second, through a level action of the ossicles, which
increases the force imparted on the oval window by 30%. The net effect is to increase the
pressure up to 30 times on the much smaller oval window of the cochlea.

Eardrum - changes pressure fluctuations into mechanical vibrations.

Ossicles - transfer these vibrations to the oval window of the cochlea. hammer, anvil and
stirrup

The function of the inner ear

Cochlea - tapered, coiled tube, snail shaped, size of a pea! Stretched out it would be about 3
cm long. The cochlea is a very complex, very small device that converts the mechanical
vibrations into neural impulses.

How the Cochlea works

Two tubes connected at the end of the cochlea (scala vestubuli and scala timpani) are filled
with fluid called perilymph. Vibrations pass from the oval window to the round window.
When the oval window pushes in the round window pushes out and visa-versa. The basilar
membrane and related structures (cochlear duct, organ of corti), vary along the channel
from thin and stiff to thick and loose. The cochlear duct is filled with viscous fluid called
endolymph that is like spinal fluid. This causes the cochlear duct to be acoustically inactive.
The vibrations of the basilar membrane primarily determine what we hear. The organ of
corti rides along as the basilar membrane vibrates and senses its motion. High frequencies
tend to vibrate the front end of the basilar membrane and low frequencies vibrate the back
end.

Hair cells (approx. 24,000) sense the vibration of the basilar membrane and excite nerve
cells that send nerve impulses down the individual fibers of the auditory nerve. The nerve
cells fire more frequently when the vibrations are large amplitude. In fact, the hairs
themselves vibrate when stimulated causing a positive feedback mechanism (this is a fairly
recent research result). The location of the vibrations and the associated nerve impulses
crudely determines the frequency of the sound. The auditory cortex in the brain does much
more "signal processing," helping us to better distinguish musical sounds.
3) SKIN

The first, topmost, or superficial, layer of the skin the sun's rays hit is called the epidermis.
Again, the epidermis is the outermost layer of the skin. The epidermis is itself made up of
several layers. From outer to innermost, they are the:

 Stratum corneum
 Stratum lucidum
 Stratum granulosum
 Stratum spinosum
 Stratum basale

Do note, however, that the stratum lucidum is typically only found in places like the soles of
your feet or the palms of your hand. Regardless, it's pretty easy to remember the exact order
of the layers of the epidermis. Since we're on the topic of possible sunburns, the coolest
mnemonic to remember the layers of the epidermis from top to bottom, or superficial to
deepest, is:

'Come, Let's Get Sun Burn'.

Each word's first letter represents the first letter of each layer. In case you were wondering,
the epidermis is actually the layer of skin that is primarily affected in most cases of sunburn
and begins to peel off if damaged by the light's dangerous UV rays.

Types of Skin Cells

However, your skin isn't a weakling, and does have a defense mechanism that tries to fight
off dangerous ultraviolet rays found in sunlight. In the deepest layer of the epidermis, the
stratum basale, which is also sometimes called the basal layer, are cells called melanocytes.
These are cells that produce the pigment melanin. It is this substance, melanin, which
determines the skin color of an individual. Those with larger amounts of melanin in their
skin have darker skin, or their skin darkens with more exposure to sunlight.

Melanocytes in the basal layer of the epidermis produce the pigment melanin

Basically, as the sunlight hits your skin, the light rays stimulate the production of melanin
by melanocytes. Since the majority of melanin is called eumelanin, which is a brownish
black color, your skin begins to darken as more melanin is produced. Keep in mind that this
melanin isn't produced to give you a nice tan for aesthetic reasons, but instead, helps protect
you from cancer-causing ultraviolet radiation found in the sunlight that is baking and
peeling your skin off at the beach. At least the pale vampires who come out after twilight
don't have to worry about this.

Pale vampires aside, your epidermis has other cells that are quite important. One of these
cells are called keratinocytes. Keratinocytes are cells that eventually die in order to
comprise the majority of the stratum corneum. The keratinocytes actually originate in the
stratum basale, but as they mature and age, they move from the deepest to the most
superficial layer of the epidermis.

Once the really old keratinocytes reach the stratum corneum they are known as
'corneocytes'. The corneocytes are basically the cells that are shed off your skin and become
part of the dust floating around you. Disgusting, isn't it? When you inhale dust, you also
inhale dead human skin cells.

As yucky as that might sound, the keratinocytes do play a lot of important roles. One of
these roles actually involves the melanin produced by melanocytes. The keratinocytes take
up and store some of the melanin produced by the melanocytes, and this gives your skin an
extra layer of protection from the damaging ultraviolet radiation of the sun's light rays.

Keratinocytes store melanin, giving skin an extra layer of protection from UV rays

In addition to housing young keratinocytes and melanocytes, the basal layer of your skin
also contains other cells, such as Merkel cells, which are cells that are important in the
sensation of touch.

With all of that in mind, I do have an important point to make. The topmost layer of your
skin we are going over, the epidermis, is made up of something called 'squamous' cells,
which are basically a bunch of really flat cells. Bearing those squamous cells in mind, the
'basal' layer of the epidermis where the 'Merkel' cells and 'melanocytes' are located, it
should come as absolutely no shocker that:

 Squamous cell carcinoma

 Basal cell carcinoma
 Merkel cell carcinoma
 Melanoma
are just some of the types of skin cancer you can get due to overexposure to damaging
ultraviolet radiation from the sun's light rays. The next time you're frying at the beach,
remember, your tan may be pretty, but skin cancer looks really nasty.
 HUMAN SYSTEMS
Experiment – XV

1) Cardio Vascular System

2) Respiratory System
3) Digestive System
4) Urinary System

5) Brain