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UNIT II
Fluid and hemodynamic derangements, - edema, normal hemostasis,thrombosis,
disseminated intravascular coagulation, embolism, infarction, shock.
Hematological disorders-Bleeding disorders, Leukaemias, Lymphomas.

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Fluid and hemodynamic derangements:

The cardiovascular disease which were responsible for 35% to 40% of

deaths Included in this category are diseases the primarily affect one of the major
component of cardiovascular system: the heart, the blood vessels, and the blood.

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And they composed of water , salts and a wide variety of proteins, elements that
regulate the clotting mechanisms. Here we focus on disorders of haemodynamic

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(edema,congestion,and shock) and hemostasis (hemorrahage and thrombosis) as
well as various forms of embolism.

Edema:
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The Greek word oidema means swelling. Oedema may be defined as abnormal and
excessive accumulation of “free fluid” in the interstitial tissue spaces and serous
cavities. The presence of abnormal collection of fluid within the cell is sometimes
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called intracellular oedema.
Free fluid in body cavities: Depending upon the body cavity in which the fluid
accumulates, it is correspondingly known as ascites (if in the peritoneal cavity),
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hydrothorax or pleural effusion (if in the pleural cavity), and hydropericardium or

pericardial effusion (if in the pericardial cavity).
Free fluid in interstitial space: The oedema fluid lies free in the interstitial space
between the cells and can be displaced from one place to another. In the case of
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oedema in the subcutaneous tissues, momentary pressure of finger produces a

depression known as pitting oedema. The other variety is non-pitting or solid
oedemain which no pitting is produced on pressure e.g. in myxoedema,
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elephantiasis.
The oedema may be of 2 main types:
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1. Localised when limited to an organ or limb e.g. lymphatic oedema,

inflammatory oedema, allergic oedema.
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2. Generalised (anasarca or dropsy)when it is systemic in distribution, particularly

noticeable in the subcutaneous tissues e.g. renal oedema, cardiac oedema,
nutritional oedema.
Depending upon fluid composition, oedema fluid may be:

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Transudate: It is the fluid pushed through the capillary tube due to high pressure
within the capillary.
Exudate: The fluid that leak around the cells of capillaries caused due to
inflammation.
PATHOGENESIS OF OEDEMA

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Edema is caused by mechanisms that interfere with normal fluid balance of
plasma, interstitial fluid and lymph flow. The following mechanisms may be
operating singly or in combination to produce edema.

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1. Decreased plasma oncotic pressure
2. Increased capillary hydrostatic pressure
3. Lymphatic obstruction
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4. Tissue factors (increased oncotic pressure of interstitial fluid, and decreased
tissue tension)
5. Increased capillary permeability
6. Sodium and water retention
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DECREASED PLASMA ONCOTIC PRESSURE:

The oncotic pressure is a kind of osmotic pressure exerted by the plasma
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protein present in the body which tends to draw fluid into the vessels normally.
A fall in the total plasma protein level (hypoproteinaemia), results in lowering of
plasma oncotic pressure in a way that it can no longer counteract the effect
of hydrostatic pressure of blood. This results in increased outward movement of
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fluid from the capillary wall and decreased inward movement of fluid from the
interstitial space causing oedema.
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INCREASED CAPILLARY HYDROSTATIC PRESSURE:

The hydrostatic pressure of the capillary is the force that normally tends to
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drive fluid through the capillary wall into the interstitial space by counteracting the
force of plasma oncotic pressure. A rise in the hydrostatic pressure at the venular
end of the capillary which is normally low (average 12 mmHg) to a level more
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than the plasma oncotic pressure results in minimal or no reabsorption of fluid at

the venular end, consequently leading to oedema.

LYMPHATIC OBSTRUCTION:

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Normally, the interstitial fluid in the tissue spaces escapes by way of
lymphatics. Obstruction to outflow of these channels causes localised
oedema, known as lymphoedema.

TISSUE FACTORS:

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The two forces acting in the interstitial space—oncotic pressure of the
interstitial space and tissue tension, are normally quite small and insignificant to
counteract the effects of plasma oncotic pressure and capillary hydrostatic pressure

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respectively. However, in some situations, the tissue factors in combination with
other mechanisms play a role in causation of oedema.
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INCREASED CAPILLARY PERMEABILITY:
An intact capillary endothelium is a semipermeable membrane which
permits the free flow of water and crystalloids but allows minimal passage of
plasma proteins normally. However, when the capillary endothelium is injured by
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various ‘capillary poisons’ such as toxins and their products, histamine, anoxia,
venoms, certain drugs and chemicals, the capillary permeability to plasma proteins
is enhanced due to development of gaps between the endothelial cells, leading
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to leakage of plasma proteins into interstitial fluid. This, in turn, causes reduced
plasma oncotic pressure and elevated oncotic pressure of interstitial fluid which
consequently produces oedema.
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SODIUM AND WATER RETENTION:

The possible factors responsible for causation of oedema by excessive
retention of sodium and water in the extravascular compartment via stimulation of
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intrinsic renal and extra-renal mechanisms as well as via release of ADH are as
under:
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i) Reduced glomerular filtration rate in response to hypovolaemia.

ii) Enhanced tubular reabsorption of sodium and consequently its decreased renal
excretion.
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iii) Increased filtration factor i.e. increased filtration of plasma from the
glomerulus.

ORGAN SPECIFIC EDEMA:

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1) Cerebral edema: the interstitial fluid collection in the brain tissues.
2) Pulmonary edema: fluid collection in lungs.
3) Periorbital edema: eye puffiness.
4) Lymph edema: mainly due to obstruction in the lymph channel lead to
edema in the lower extremities.

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THROMBOSIS:
Thrombosis is the process of formation of solid mass in circulation from the

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constituents of flowing blood; the mass itself is called a thrombus. In contrast, a
blood clot is the mass of coagulated blood formed in vitroe.g. in a test tube.
Haematomais the extravascular accumulation of blood clot e.g. into the tissues.
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Haemostatic plugsare the blood clots formed in healthy individuals at the site of
bleeding e.g. in injury to the blood vessel. Haemostatic plugs are useful as they
stop the escape of blood and plasma, whereas thrombi developing in the
unruptured cardiovascular system may be life-threatening by causing one of the
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following harmful effects:
1. Ischaemic injury:Thrombi may decrease or stop the blood supply to part of an
organ or tissue and cause ischaemia which may subsequently result in infarction.
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2. Thromboembolism.The thrombus or its part may get dislodged and be carried

along in the bloodstream as embolus to lodge in a distant vessel.

PATHOPHYSIOLOGY:
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Since the protective haemostatic plug formed as a result of normal

haemostasis is an example of thrombosis. Virchow described three primary events
which predispose to thrombus formation (Virchow’s triad): endothelial injury,
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altered blood flow, and hypercoagulability of blood.

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ENDOTHELIAL INJURY:
pa
The integrity of blood vessel wall is important for maintaining normal blood
flow. Vascular injury exposes the subendothelial connective tissue (e.g. collagen,
elastin, fibronectin, laminin) which are thrombogenic and thus plays important role
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in initiating haemostasis as well as thrombosis. Injury to vessel wall also causes
vasoconstriction of small blood vessels briefly so as to reduce the blood loss.
Endothelial injury is of major significance in the formation of arterial thrombi and
thrombi of the heart, especially of the left ventricle.
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ROLE OF PLATELETS:
Following endothelial cell injury, platelets come to play a central role in
normal haemostasis as well as in thrombosis. The sequence of events is as under
i) Platelet adhesion:

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The platelets in circulation recognize the site of endothelial injury and
adhere to exposed subendothelial collagen (primary aggregation); von
Willebrand’s factor is required for such adhesion between platelets and collagen.
Normal non-activated platelets have open canalicular system with cytoplasmic
organelles (granules, mitochondria, endoplasmic reticulum) dispersed throughout

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the cytoplasm. During the early adhesion process, there is dilatation of canalicular
system with formation of pseudopods and the cytoplasmic organelles shift to the
centre of the cell.

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ii) Platelet release reaction:

The activated platelets then undergo release reaction by which the platelet
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granules are released to the exterior. Two main types of platelet granules are
released:
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a) Alpha granules: containing fibrinogen, fibronectin, plateletderived growth

factor, platelet factor 4 (an antiheparin) and cationic proteins.
b) Dense bodies: containing ADP (adenosine diphosphate), ionic calcium, 5-HT
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(serotonin), histamine and epinephrine. As a sequel to platelet activation and

release reaction, the phospholipid complex-platelet factor 3 gets activated which
plays important role in the intrinsic pathway of coagulation.
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iii)Platelet aggregation:
Following release of ADP, a potent platelet aggregating agent, aggregation
of additional platelets takes place (secondary aggregation). This results in
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formation of temporary haemostatic plug. However, stable haemostatic plug is
formed by the action of fibrin, thrombin and thromboxane A2.
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3. ROLE OF COAGULATION SYSTEM:

Coagulation mechanism is the conversion of the plasma fibrinogen into
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solid mass of fibrin. The coagulation system is involved in both haemostatic

process and thrombus formation is by intrinsic (blood) pathway, the extrinsic
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(tissue) pathway, and the common pathway leading to formation of fibrin

polymers.
i) In the intrinsic pathway,contact with abnormal surface leads to activation of
factor XII and the sequential interactions
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of factors XI, IX, VIII and finally factor X, alongwith calcium ions (factor IV) and
platelet factor 3.
ii) In the extrinsic pathway,tissue damage results in the release of tissue factor or
thromboplastin. Tissue factor on interaction with factor VII activates factor X.

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iii)The common pathwaybegins where both intrinsic and extrinsic pathways
converge to activate factor X which forms a complex with factor Va and platelet
factor 3, in the presence of calcium ions. This complex activates prothrombin
(factor II) to thrombin (factor IIa) which, in turn, converts fibrinogen to fibrin.
Initial monomeric fibrin is polymerised to form insoluble fibrin by activation of

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factor XIII.

4. ALTERATION OF BLOOD FLOW:

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Turbulence means unequal flow while stasis means slowing.
i) Normally, there is axial flow of blood in which the most rapidly-moving central
stream consists of leucocytes and red cells. The platelets are present in the slow-
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moving laminar stream adjacent to the central stream while the peripheral stream
consists of most slow-moving cell-free plasma closeto endothelial layer.
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ii) Turbulence and stasis occur in thrombosis in which the normal axial flow of
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blood is disturbed. When blood slows down, the blood cells including platelets
marginate to the periphery and form a kind of pavement close to endothelium
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(margination and pavementing) . While stasis allows a higher release of oxygen

from the blood, turbulence may actually injure the endothelium resulting in
deposition of platelets and fibrin.
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5. HYPERCOAGULABILITY OF BLOOD:

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The occurrence of thrombosis in some conditions such as in nephritic
syndrome, advanced cancers, extensive trauma, and burns. Hypercoagulability
may occur by the following changes in the composition of blood:

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i) Increase in coagulation factorse.g. fibrinogen, prothrombin, factor VIIa, VIIIa
and Xa.
ii) Increase in platelet countand their adhesiveness.
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iii) Decreased levels of coagulation inhibitorse.g. antithrombin III, fibrin split
products.

DISSEMINATED INTRAVASCULAR COAGULATION (DIC):

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Disseminated intravascular coagulation (DIC), also termed defibrination
syndrome or consumption coagulopathy, is a complex thrombo-haemorrhagic
disorder (intravascular coagulation and haemorrhage) occurring as a secondary
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complication in some systemic diseases.

ETIOLOGY:
Although there are numerous conditions associated with DIC, most frequent
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causes are listed below:

1. Massive tissue injury:in obstetrical syndromes (e.g. abruptio placentae, amniotic
fluid embolism, retained dead foetus), massive trauma, metastatic malignancies,
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surgery.
2. Infections:especially endotoxaemia, gram-negative and meningococcal
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septicaemia, certain viral infections, malaria, aspergillosis.

3. Widespread endothelial damage:in aortic aneurysm, haemolytic-uraemic
syndrome, severe burns, acute glomerulonephritis.
4. Miscellaneous:snake bite, shock, acute intravascularhaemolysis, heat stroke.
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PATHOGENESIS:

1. Activation of coagulation.The etiologic factors listed above initiate widespread

activation of coagulation pathway by release of tissue factor.

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2. Thrombotic phase.Endothelial damage from the various thrombogenic stimuli
causes generalised platelet aggregation and adhesion with resultant deposition of
small thrombi and emboli throughout the microvasculature.
3. Consumption phase.The early thrombotic phase is followed by a phase of
consumption of coagulation factors and platelets.

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4. Secondary fibrinolysis.As a protective mechanism, fibrinolytic system is
secondarily activated at the site of intravascular coagulation. Secondary
fibrinolysis causes breakdown of fibrin resulting in formation of FDPs(fibrin

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degraded products) in the circulation.

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CLINICAL FEATURES:
There are 2 main features of DIC— bleeding as the most common manifestation,
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and organ damage due to ischaemia caused by the effect of widespread

intravascular thrombosis such as in the kidney and brain. Less common
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manifestations include: microangiopathic haemolytic anaemia and thrombosis in

larger arteries and veins.
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PATHOPHYSIOLOGY OF DIC:

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EMBOLISM:
Embolismis the process of partial or complete obstruction of some part of
the cardiovascular system by any mass carried in the circulation; the transported
intravascular mass detached from its site of origin is called an embolus. Most
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usual forms of emboli (90%) are thromboemboli i.e. originating from thrombi or
their parts detached from the vessel wall.
Emboli may be of various types:
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1) Depending upon the matter in the emboli:

i) Solide.g. detached thrombi (thromboemboli), atheromatous material,
tumour cell clumps, tissue fragments, parasites, bacterial clumps, foreign bodies.
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ii) Liquide.g. fat globules, amniotic fluid, bone marrow.

iii) Gaseouse.g. air, other gases.
2) Depending upon whether infected or not:
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i) Bland, when sterile.

ii) Septic, when infected.
3) Depending upon the source of the emboli:
i) Cardiac emboli from left side of the heart e.g. emboli originating from

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atrium and atrial appendages, infarct in the left ventricle, vegetations of
endocarditis.
ii) Arterial embolie.g. in systemic arteries in the brain, spleen, kidney,
intestine.
iii) Venous embolie.g. in pulmonary arteries.

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iv) Lymphatic emboli can also occur.
4) Depending upon the flow of blood,two special types of emboli are mentioned:
i) Paradoxical embolus: An embolus which is carried from the venous side

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of circulation to the arterial side or vice versa is called paradoxical or crossed
embolus.
ii) Retrograde embolus. An embolus which travels against the flow of blood
is called retrograde embolus.
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TYPES OF EMBOLISM:
THROMBOEMBOLISM:
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A detached thrombus or part of thrombus constitutes the most common type
of embolism. These may arise in the arterial or venous circulation :
Arterial (systemic) thromboembolism: Arterial emboli may be derived from the
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following sources:
A.Causes within the heart(80-85%): These are mural thrombi in the left atrium
or left ventricle, vegetations on the mitral or aortic valves, prosthetic heart valves
and cardiomyopathy.
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B. Causes within the arteries: These include emboli developing in relation to

atherosclerotic plaques, aortic aneurysms, pulmonary veins.
Venous thromboembolism: Venous emboli may arise from the following sources:
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i) Thrombi in the veins of the lower legs are the most common cause of
venous emboli.
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ii) Thrombi in the pelvic veins.

iii) Thrombi in the veins of the upper limbs.
iv) Thrombosis in cavernous sinus of the brain.
v) Thrombi in the right side of heart
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PULMONARY THROMBOEMBOLISM:
Pulmonary embolism is the most common and fatal form of venous

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thromboembolism in which there is occlusion of pulmonary arterial tree by
thromboemboli. Pulmonary thrombosis as such is uncommon and may occur
in pulmonary atherosclerosis and pulmonary hypertension.

FAT EMBOLISM:

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Obstruction of arterioles and capillaries by fat globules constitutes fat
embolism. If the obstruction in the circulation is by fragments of adipose tissue, it
is called fat-tissue embolism.

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ETIOLOGY. Following are the important causes of fat embolism:
i) Traumatic causes:
Trauma to bones is the most common cause of fat embolism e.g. in fractures
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of long bones leading to passage of fatty marrow in circulation, concussions of
bones, after orthopaedic surgical procedures etc.
Trauma to soft tissuee.g. laceration of adipose tissue and in puerperium due
to injury to pelvic fatty tissue.
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ii) Non-traumatic causes:
Extensive burns
Diabetes mellitus
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Fatty liver

GAS EMBOLISM:
Air, nitrogen and other gases can produce bubbles within the circulation and
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obstruct the blood vessels causing damage to tissue. Two main forms of gas
embolism—air embolism and decompression sickness.
Air Embolism
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Air embolism occurs when air is introduced into venous or arterial

circulation.
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Venous Air Embolism:

Air may be sucked into systemic veins under the following circumstances:
i) Operations on head and neck, and trauma.
ii) Obstetrical operations and trauma.
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iii)Intravenous infusion of blood and fluid.

iv) Angiography.
Arterial Air Embolism:
Entry of air into pulmonary vein or its tributaries may occur in the following

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conditions:
i) Cardiothoracic surgery and trauma.
ii) Paradoxical air embolism.
iii)Arteriography.
Decompression Sickness

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This is a specialised form of gas embolism known by various names such as
caisson’s disease, divers’ palsy or aeroembolism.
PATHOGENESIS:

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Decompression sickness is produced when the individual decompresses
suddenly, either from high atmospheric pressure to normal level, or from normal
pressure to low atmospheric pressure. In divers, workers in caissons (diving-bells),
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offshore drilling and tunnels, who descendto high atmospheric
pressure, increased amount of atmospheric gases (mainly nitrogen; others are O2,
CO2) are dissolved in blood and tissue fluids. When such an individual ascends too
rapidly i.e. comes to normal level suddenly from high atmospheric pressure, the
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gases come out of the solution as minute bubbles, particularly in fatty tissues
which have affinity for nitrogen. These bubbles may coalesce together to form
large emboli.
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AMNIOTIC FLUID EMBOLISM:

This is the most serious, unpredictable and unpreventable cause of maternal
mortality. During labour and in the immediate postpartum period, the contents of
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amniotic fluid may enter the uterine veins and reach right side of the heart resulting
in fatal complications. The amniotic fluid components which may be found in
uterine veins, pulmonary artery and vessels of other organs.
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TUMOUR EMBOLISM:
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Malignant tumour cells invade the local blood vessels and may form tumour
emboli to be lodged elsewhere, producing metastatic tumour deposits. Notable
examples are clear cell carcinoma of kidney, carcinoma of the lung.
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MISCELLANEOUS EMBOLI:
Various other endogenous and exogenous substances may act as emboli.
These are:
i) Fragments of tissue

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ii) Placental fragments
iii) Red cell aggregates (sludging)
iv) Bacteria
v) Parasites

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INFARCTION:
Infarction is the process of tissue necrosis resulting from some form of
circulatory insufficiency; the localised area of necrosis so developed is called an

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infarct.

ETIOLOGY:
pa
Most commonly, infarcts are caused by interruption in arterial blood supply,
called ischaemic necrosis. Less commonly, venous obstruction can produce
infarcts termed stagnant hypoxia. Generally, sudden, complete, and continuous
occlusion (e.g. thrombosis or embolism) produces infarcts. Infarcts may be
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produced by non occlusive circulatory insufficiencyas well e.g. incomplete
atherosclerotic narrowing of coronary arteries may produce myocardial infarction
due to acute coronary insufficiency.
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TYPES OF INFARCTS:
Infarcts are classified depending upon different features:
1. According to their colour:
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Pale or anaemic,due to arterial occlusion and are seen in compact organs e.g.
in the kidneys, heart, spleen.
Red or haemorrhagic,seen in soft loose tissues and are caused either by
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pulmonary arterial obstruction (e.g. in the lungs) or by arterial or venous occlusion

(e.g. in the intestines).
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2. According to their age:

Recent or fresh
Old or healed
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3. According to presence or absence of infection:

Bland, when free of bacterial contamination
Septic, when infected.
PATHOGENESIS:

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The process of infarction takes place as follows:
i) Localised hyperaemiadue to local anoxaemia occurs immediately after
obstruction of the blood supply.
ii) Within a few hours, the affected part becomes swollen due to oedema and
haemorrhage. The amount of haemorrhage is variable, being more marked in the

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lungs and spleen, and less extensive in the kidneys and heart.
iii) Cellular changessuch as cloudy swelling and degeneration appear early, while
death of the cells (i.e. necrosis) occurs in 12-48 hours.

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iv) There is progressive proteolysisof the necrotic tissue and there is lysis of the
red cells.
v) An acute inflammatory reaction and hyperaemia appear at the same time in the
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surrounding tissues in response to products of proteolysis.
vi) Blood pigments, haematoidin and haemosiderin, liberated by lysis of RBCs are
deposited in the infarct. At this stage, most infarcts become pale-grey due to loss of
red cells.
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vii) Following this, there is progressive ingrowth of granulation tissue from the
margin of the infarct so that eventually the infarct is replaced by a fibrous scar.
Dystrophic calcification may occur sometimes.
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INFARCTS OF DIFFERENT ORGANS:

A few representative examples of infarction of some organs (lungs, kidney,
liver and spleen).
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SHOCK:
Shock is a life-threatening clinical syndrome of cardiovascular collapse
characterised by:
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1) an acute reduction of effective circulating blood volume (hypotension);

2) an inadequate perfusion of cells and tissues (hypoperfusion).
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TRUE SHOCK:
The term“true (or secondary) shock”is a circulatory imbalance between
oxygen supply and oxygen requirements at the cellular level, and is also called as
circulatory shock.
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INITIAL SHOCK:
The term “initial (or primary) shock”is used for transient and usually a
benign vasovagal attack resulting from sudden reduction of venous return to the
heart.

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CLASSIFICATION AND ETIOLOGY:
A simple etiologic classification of shock syndrome divides it into following
3 major types and a few other variants.
1. Hypovolaemic shock:
This form of shock results from inadequate circulatory blood volume by

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various etiologic factors that may be eitherfrom the loss of red cell mass and
plasma from haemorrhage, or from the loss of plasma volume alone.
2. Cardiogenic shock:

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Acute circulatory failure with sudden fall in cardiac output from acute
diseases of the heart without actual reduction of blood volume results
in cardiogenic shock.
3. Septic (Toxaemic) shock:
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Severe bacterial infections or septicaemia induce septic shock. It may be the
result of Gramnegative septicaemia (endotoxic shock) which is more common, or
Gram-positive septicaemia (exotoxic shock).
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4. Other types:
These include following types:
i) Traumatic shock:
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Shock resulting from trauma is initially due to hypovolaemia, but even after
haemorrhage has been controlled, these patients continue to suffer loss of plasma
volume into the interstitium of injured tissue and hence is considered separately in
some descriptions.
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ii) Neurogenic shock:

Neurogenic shock results from causes of interruption of sympathetic
vasomotor supply.
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iii) Hypoadrenal shock:

Hypoadrenal shock occurs from unknown adrenal insufficiency in which the
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patient fails to respond normally to the stress of trauma, surgery or illness.

PATHOGENESIS:
In general, all forms of shock involve following 3 derangements:
1) Reduced effective circulating blood volume.
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2) Reduced supply of oxygen to the cells and tissues with resultant anoxia.
3) Inflammatory mediators and toxins released from shockinduced cellular
injury.
1. Reduced effective circulating blood volume:

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It may result by either of the following mechanisms:
i) by actual loss of blood volume as occurs in hypovolaemic shock; or
ii) by decreased cardiac output without actual loss of blood (normovolaemia)
as occurs in cardiogenic shock and septic shock.
2. Impaired tissue oxygenation:

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Following reduction in the effective circulating blood volume from either of
the above two mechanisms and from any of the etiologic agents, there is decreased
venous return to the heart resulting in decreased

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cardiac output. This consequently causes reduced supply of oxygen to the organs
and tissues and hence tissue anoxia, which sets in cellular injury.
3. Release of inflammatory mediators:
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In response to cellular injury, innate immunity of the body gets activated
as a body defense mechanism and release inflammatory mediators but eventually
these agents themselves become the cause of cell injury.
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PATHOGENESIS OF HYPOVOLAEMIC SHOCK:
Hypovolaemic shock occurs from inadequate circulating blood volume due
to various causes. The major effects of hypovolaemic shock are due to decreased
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cardiac output and low intracardiac pressure. The severity of clinical features
depends upon degree of blood volume lost, haemorrhagic shock is divided into 4
types:
< 1000 ml: Compensated
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1000-1500 ml: Mild

1500-2000 ml: Moderate
>2000 ml: Severe
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Accordingly, clinical features are increased heart rate (tachycardia), low blood
pressure (hypotension).
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PATHOGENESIS OF CARDIOGENIC SHOCK:

Cardiogenic shock results from a severe left ventricular dysfunction from
various causes. The resultant decreased cardiac output has its effects in the form of
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decreased tissue perfusion and movement of fluid from pulmonary vascular bed
into pulmonary interstitial space initially (interstitial pulmonary oedema) and later
into alveolar spaces (alveolar pulmonary oedema).

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PATHOGENESIS OF SEPTIC SHOCK:
Septic shock results most often from Gram-negative bacteria entering the
body from genitourinary tract, alimentary tract, respiratory tract or skin, and less
often from Gram-positive bacteria. In septic shock, there is immune system
activation and severe systemic inflammatory response to infection as follows:

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i) Activation of macrophage-monocytes.
ii) Activation of other inflammatory responses.

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PATHOGENESIS OF CIRCULATORY SHOCK:

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PATHOPHYSIOLOGY (STAGES OF SHOCK):

Shock has been divided arbitrarily into 3 stages
1. Compensated (non-progressive, initial, reversible) shock.
2. Progressive decompensated shock.

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3. Irreversible decompensated shock.
COMPENSATED (NON-PROGRESSIVE, INITIAL, REVERSIBLE)
SHOCK:
In the early stage of shock, an attempt is made to maintain adequate cerebral
and coronary blood supply by redistribution of blood so that the vital organs (brain

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and heart) are adequately perfused and oxygenated. This is achieved by activation
of various neurohormonal mechanisms causing widespread vasoconstriction and
by fluid conservation by the kidney.

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PROGRESSIVE DECOMPENSATED SHOCK:
This is a stage when the patient suffers from some other stress or risk
factors (e.g. pre-existing cardiovascular and lung disease) besides persistence of
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the shock so that there is progressive deterioration.
IRREVERSIBLE DECOMPENSATED SHOCK:
When the shock is so severe that in spite of compensatory mechanisms
and despite therapy and control of etiologic agent which caused the shock, no
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recovery takes place, it is called decompensated or irreversible shock.
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UNIT-III
MICROSCOPE
• A microscope ("to look" or "see") is an instrument used to see objects that are too small
to be seen by the naked eye. Microscopy is the science of investigating small objects and

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structures using such an instrument. Microscopic means invisible to the eye unless aided
by a microscope.
• Total magnification= Magnifying power of the objective lens by that of eye piece lens.

Why do we need microscopes?

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We used to ignore things we couldn't see. But thanks to modern science, we know there's a
whole lot happening on the microscopic scale that can help us to live our lives more
effectively. Scientists have known since the 17th century that the insides of living things are

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made up of tiny functioning factories called cells; understanding how they work helps us to
tackle sickness and disease. More recently, during the 20th century, scientists figured out how
materials are made of atoms and how atoms themselves are built from smaller "subatomic"
particles; understanding atomic structure paved the way for all kinds of amazing inventions, from
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electronic transistors to nuclear power.

Principle of Microscopes
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A general biological microscope mainly consists of an objective lens, eyepiece lens, lens tube,
stage, and reflector. An object placed on the stage is magnified through the objective lens. When
the target is focused, a magnified image can be observed through the eyepiece lens. A
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microscope is designed to emit light onto or through objects and magnify the transmitted or
reflected light with the objective and eyepiece lenses.

Types of Microscope


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Optical / Light Microscope

 Dark Field Microscope
 Phase Contrast Microscope
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 Fluorescent Microscope
 Electron Microscope
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1. Optical/Light microscope

• It is a type of microscope which uses visible light and a system of lenses to magnify
images of small samples.

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• The image from an optical microscope can be captured by normal light-sensitive
cameras to generate a micrograph. Originally images were captured by photographic
film but modern developments in cameras allow the capture of digital images.

Types
There are two basic types of optical microscopes: Simple Microscopes and Compound

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Microscopes. A simple microscope is one which uses a single lens for magnification, such as a
magnifying glass. A compound microscope uses several lenses to enhance the magnification of
an object. The vast majority of modern research microscopes are compound microscopes while
some cheaper commercial digital microscopes are simple single lens microscopes. Compound
microscopes can be further divided into a variety of other types of microscopes which differ in

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their optical configurations, cost, and intended purposes.

Simple microscope

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A simple microscope uses a lens or set of lenses to enlarge an object through angular
magnification alone, giving the viewer an erect enlarged virtual image. The use of a single
convex lens or groups of lenses are still found in simple magnification devices such as
the magnifying glass, loupes, and eyepieces for telescopes and microscopes.
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Ray Diagram of a simple microscope

Compound Microscope
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A compound microscope is an optical instrument consisting of two convex lenses of short focal
lengths which is used for observing the highly magnified images of tiny objects. The compound
microscope can magnify the image of a tiny object up to 1000.

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Principle of Compound Microscope

A compound microscope works on the principle that when a tiny object to be magnified is
placed just beyond the focus of its objective lens, a virtual, inverted and highly magnified image
of the object is formed at the least distance of distinct vision from the eye held close to the eye
piece.

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Construction
The compound microscope is most commonly used in clinical and educational laboratories. It
has a combination of lenses that enhances both magnifying power as well as the resolving power.
The specimen or object, to be examined is usually mounted on a transparent glass slide and
positioned on the specimen stage between the condenser lens and objective lens.

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A beam of visible light from the base is focused by a condenser lens onto the specimen. The
objective lens picks up the light transmitted by the specimen and create a magnified image of the

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specimen called primary image inside the body tube. This image is again magnified by the ocular
lens or eye piece.
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When higher magnification is required, the nose piece is rotated after low power focusing to
bring the objective of higher power (generally 45X) in line with the illuminated part of the slide.
The objective lens comes very near the cover slip but it does not touch the same. Only fine
adjustment it moved for proper focusing. More light may be required. After observation under
high power, the nose piece is rotated to bring back the slide under low power.
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Occasionally very high magnification it required (e.g. for observing bacterial cell). In that case,
oil immersion objective lens (usually 100X) is employed. After focusing under low power a drop
of immersion oil (e.g. cedar oil, olive oil) placed over the illuminated part of the cover-slip.
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The eye piece is rotated to bring the oil immersion lens in line with die specimen. It comes in
contact with the oil. By using fine adjustment only, the specimen is brought under focus.
Immersion oil increases the sharpness of the image. Soon after observation, both the lens and the
slide are cleared of the oil by fine cotton cloth or lens paper.
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Working Principle:
The most commonly used microscope for general purposes is the standard compound
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microscope. It magnifies the size of the object by a complex system of lens arrangement.

It has a series of two lenses; (i) the objective lens close to the object to be observed and (ii) the
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ocular lens or eyepiece lens, through which the image is viewed by eye. Light from a light source
(mirror or electric lamp) passes through a thin transparent object.

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The objective lens produces a magnified ‘real image’ first image) of the object. This image is
again magnified by the ocular lens (eyepiece) to obtain a magnified ‘virtual image’ (final image),

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which can be seen by eye through the eyepiece. As light passes directly from the source to the
eye through the two lenses, the field of vision is brightly illuminated. That is why; it is a bright-

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field microscope.

Applications:
-> Blood analysis pa
-> Human cells examination
-> Plant cells
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Sl. No Simple Microscope Compound Microscope
1 There is single lens in simple There are 3 to 5 objective lenses in a
microscope. compound which helps in magnifying
algae, fungi and bacterium.
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2 Has only one lens for Has two sets of lenses for magnifying
magnifying objects. objects: eyepiece lens and objective
lenses

3 Condenser lens is absent. Condenser lens is present which is

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used to adjust the intensity of light for

magnification of object.

4 Light source is natural Illuminator is a source of light which

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is helpful when small, minutest pieces

needed to be seen.
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5 Total magnification is 10x Total magnification is (400-1000)x

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Dark field microscope:
A microscope that has a special condenser and objective with a diaphragm or stop that scatters
light from the objectobserved, with the result that the object appears bright on a dark backgroud.
It is arranged so that the light source is blocked off, causing light to scatter as it hits the
specimen.

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Principle

To view a specimen in dark field, an opaque disc is placed underneath the condenser lens, so that
only light that is scattered by objects on the slide can reach the eye. Instead of coming up
through the specimen, the light is reflected by particles on the slide. Everything is visible

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regardless of color, usually bright white against a dark background. Pigmented objects are often
seen in "false colors," that is, the reflected light is of a color different than the color of the object.
Better resolution can be obtained using dark field as opposed to bright field viewing.

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When to use dark field illumination

Dark field illumination is most readily set up at low magnifications (up to 100x), although it can
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be used with any dry objective lens. Any time you wish to view everything in a liquid sample,
debris and all, dark field is best. Even tiny dust particles are obvious. Dark field is especially
useful for finding cells in suspension. Dark field makes it easy to obtain the correct focal plane at
low magnification for small, low contrast specimens. Use dark field for
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 Initial examination of suspensions of cells such as yeast, bacteria, or cell and tissue
fractions including cheek epithelial cells, chloroplasts, mitochondria, even blood cells
(small diameter of pigmented cells makes it tricky to find them sometimes despite the
color).
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 Initial survey and observation at low powers of pond water samples, hay or soil infusions,
or metazoan cultures.
 Examination of lightly stained prepared slides. Initial location of any specimen of very
small size for later viewing at higher power.
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 Determination of motility in cultures

Working
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In a dark-field microscope, the object is brilliantly illuminated against a dark background. This is
accomplished by equipping a light microscope with a special kind of condenser.
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It is a condenser with a dark-field stop, which is an opaque disc obstructing the path of light from
the light source centrally, but allowing a peripheral ring of light.

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Thus, the condenser transmits a hollow cone of light from the light source. This cone of light
converges on the object and diverges from there again as an inverted hollow cone. Thus, no light
enters into the objective, as it remains in the dark cone and the field essentially appears dark in
absence of any object.
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However, if some objects such as microbial cells are present, some of the light rays are scattered
(diffracted) by them. These diffracted rays enter into the objective and reach the eye. Thus, the
object (microbial cells) appears bright in an otherwise dark microscopic field.
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Applications:

• Used to study marine organisms such as algae and plankton, diatoms, insects, fibers,
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hairs, yeast and protozoa as well as some minerals and crystals, thin polymers and some
ceramics.
• Used to view the blood cells.
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Phase Contrast Microscopy

Phase contrast microscopy, is a contrast-enhancing optical technique that can be utilized to

produce high-contrast images of transparent specimens, such as living cells (usually in culture),
microorganisms, thin tissue slices, lithographic patterns, fibers, latex dispersions, glass
fragments, and sub cellular particles (including nuclei and other organelles).

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In effect, the phase contrast technique employs an optical mechanism to translate minute
variations in phase into corresponding changes in amplitude, which can be visualized as
differences in image contrast. One of the major advantages of phase contrast microscopy is that
living cells can be examined in their natural state without previously being killed, fixed, and
stained. As a result, the dynamics of ongoing biological processes can be observed and recorded

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in high contrast with sharp clarity of minute specimen detail.

Working Principle

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Light passing from one object into another object of a slightly different refractive index or
thickness undergoes a change in phase.
pa
In a phase-contrast microscope, this difference in phase is translated into variation in brightness
of the image and hence is detectable by eye. With a phase-contrast microscope, the differences
among various cells with different refractive indices or thickness can be seen in unstained
condition.
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Microscopic objects can be seen in unstained condition, due to the difference in the refractive
index of the object and its surrounding medium. Unstained structures within cells, not discernible
by other microscopic methods can also be observed due to the slight differences in their
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refractive indices or thickness.

A phase-contrast microscope is a compound microscope fitted with a phase-contrast condenser

and a phase-contrast objective. An annular aperture in the diaphragm placed in the focal plane of
the sub-stage condenser controls the illumination of the object.
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The image of the aperture is formed at the rear focal plane of the objective. In this plane, there is
a phase-shifting element or phase- plate. The phase plate also has an annular ring of phase
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altering pattern, which can increase the wavelength of light passing through it.
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Light coming through the annular aperture of condenser passes through the object. Those rays,
which are not deviated by the object (solid lines in figure), pass through the phase-altering
pattern of the phase plate and acquire longer wavelength.
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Those rays, which are deviated by the object structures (broken lines in figure), due to different
refractive index, pass through the phase-plate not covered by the phase altering pattern. Thus,
their wavelength remains unchanged. The difference in phase (wavelength) gives the contrast for
clear visibility of the object.
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Applications:

• Small unstained specimens such as a living cell can be seen.

• It makes Highly Transparent objects more visible.

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• Examining Intracellular components of living cells at relatively high resolution. eg: The
dynamic motility of Mitochondria, mitotic chromosomes & vacuoles.
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• It made it possible for Biologists to study living cells and how they proliferate through
cell division.
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Fluorescent Microscope
A fluorescence microscope is much the same as a conventional light microscope with added
features to enhance its capabilities.

 The conventional microscope uses visible light (400-700 nanometers) to illuminate and

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produce a magnified image of a sample.
 A fluorescence microscope, on the other hand, uses a much higher intensity light source
which excites a fluorescent species in a sample of interest. This fluorescent species in turn
emits a lower energy light of a longer wavelength that produces the magnified image
instead of the original light source.

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Fluorescent microscopy is often used to image specific features of small specimens such as
microbes. It is also used to visually enhance 3-D features at small scales. This can be
accomplished by attaching fluorescent tags to anti-bodies that in turn attach to targeted features,

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or by staining in a less specific manner. When the reflected light and background fluorescence is
filtered in this type of microscopy the targeted parts of a given sample can be imaged. This gives
an investigator the ability to visualize desired organelles or unique surface features of a sample
of interest. Confocal fluorescent microscopy is most often used to accentuate the 3-D nature of
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samples. This is achieved by using powerful light sources, such as lasers, that can be focused to a
pinpoint. This focusing is done repeatedly throughout one level of a specimen after another.
Most often an image reconstruction program pieces the multi level image data together into a
3-D reconstruction of the targeted sample.
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• A microscope fitted with a source of ultraviolet radiation to aid in the detection and exam
ination of fluorescent specimens.

• A microscope equipped to examine material that fluoresces under ultraviolet light.

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• Fluorescence microscopy is based on the principle that fluorescent materials emit visible
light when they are irradiated with ultraviolet rays or with violet-blue visible rays.

Fluorescence is the emission of light by a substance that has absorbed light or

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other electromagnetic radiation. It is a form of luminescence. In most cases, the emitted light has
a longer wavelength, and therefore lower energy, than the absorbed radiation. The most striking
example of fluorescence occurs when the absorbed radiation is in the ultraviolet region of
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the spectrum, and thus invisible to the human eye, while the emitted light is in the visible region,
which gives the fluorescent substance a distinct color that can only be seen when exposed to UV
light.
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Working:

• 2 filters are used: an excitation filter, an emission filter.

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• Each fluorophore has a specific absorption or excitation wavelength band, the excitation
filter will transmit only that specific range of wavelengths.

• The fluorophore, once excited, will emit a different range of wavelengths.

• The emission filter transmits only the emission wavelengths.

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• A dichroic mirror that is specifically designed to reflect the emission wavelengths and
transmit the excitation wavelengths is used to separate the excitation and emission
channels.

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In most cases the sample of interest is labeled with a fluorescent substance known as a
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fluorophore and then illuminated through the lens with the higher energy source. The
illumination light is absorbed by the fluorophores (now attached to the sample) and causes them
to emit a longer lower energy wavelength light. This fluorescent light can be separated from the
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surrounding radiation with filters designed for that specific wavelength allowing the viewer to
see only that which is fluorescing.

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The basic task of the fluorescence microscope is to let excitation light radiate the specimen and
then sort out the much weaker emitted light from the image. First, the microscope has a filter that
only lets through radiation with the specific wavelength that matches your fluorescing material.
The radiation collides with the atoms in your specimen and electrons are excited to a higher
energy level. When they relax to a lower level, they emit light. To become detectable (visible to
the human eye) the fluorescence emitted from the sample is separated from the much brighter

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excitation light in a second filter. This works because the emitted light is of lower energy and has
a longer wavelength than the light that is used for illumination.

Most of the fluorescence microscopes used in biology today are epi-fluorescence microscopes,
meaning that both the excitation and the observation of the fluorescence occur above the sample.
Most use a Xenon or Mercury arc-discharge lamp for the more intense light source.

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Applications:
 Imaging structural components of small specimens, such as cells

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 Conducting viability studies on cell populations (are they alive or dead?)
 Imaging the genetic material within a cell (DNA and RNA)
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 Viewing specific cells within a larger population with techniques such as FISH

Electron Microscope
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A microscope with high magnification and resolution, employing electron beams in place of light
and using electron lenses.
 An electron microscope is a microscope that uses a beam of accelerated electrons as a
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source of illumination. As the wavelength of an electron can be up to 100,000 times

shorter than that of visible light photons, electron microscopes have a higher resolving
power than light microscopes and can reveal the structure of smaller objects.
Electron microscopes are used to investigate the ultrastructure of a wide range of biological
microorganisms, cells, large molecules, biopsy samples, metals, and crystals. Industrially,
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electron microscopes are often used for quality control and failure analysis. Modern electron
microscopes produce electron micrographs using specialized digital cameras and frame
grabbers to capture the image.
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 An electron-optical instrument that utilizes a beam of electrons, rather than light, to

focus on cell surfaces of a very thin specimen to produce an enlarged image on a
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fluorescent screen or photographic plate.

• Electron microscopes use a beam of electrons rather than visible light to illuminate the
sample.

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• They focus the electron beam using electromagnetic coils instead of glass lenses
because electrons can’t pass through glass.
• It can’t be used to look directly at living things because of the special preparation that
samples must undergo before they are visualised.
 2 Types.

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1. Transmission Electron Microscope(TEM)
2. Scanning Electron Microscope(SEM)

TEM
• It is a microscopy technique in which a beam of electrons is transmitted through a

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specimen to form an image.
• It is capable of imaging at a significantly higher resolution.

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Three essential systems:
o An electron gun, which produces the electron beam, and the condenser system, which
focuses the beam onto the object
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o The image-producing system, consisting of the objective lens, movable specimen
stage, and intermediate and projector lenses, which focus the electrons passing through
the specimen to form a real, highly magnified image.
o The image-recording system, which converts the electron image into some form
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perceptible to the human eye.
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Uses a high voltage electron beam to illuminate the specimen and create an image. The
electron beam is produced by an electron gun, commonly fitted with
a tungsten filament cathode as the electron source. The electron beam is accelerated by
an anode typically at +100 keV (40 to 400 keV) with respect to the cathode, focused
by electrostatic and electromagnetic lenses, and transmitted through the specimen that is in
part transparent to electrons and in part scatters them out of the beam. When it emerges from

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the specimen, the electron beam carries information about the structure of the specimen that is
magnified by the objective lenssystem of the microscope. The spatial variation in this
information (the "image") may be viewed by projecting the magnified electron image onto a
fluorescent viewing screen coated with a phosphor or scintillator material such as zinc sulfide.
Alternatively, the image can be photographically recorded by exposing a photographic
film or plate directly to the electron beam, or a high-resolution phosphor may be coupled by

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means of a lens optical system or a fibre optic light-guide to the sensor of a digital camera.
The image detected by the digital camera may be displayed on a monitor or computer.

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One major disadvantage of the transmission electron microscope is the need for extremely
thin sections of the specimens, typically about 100 nanometers.

SEM pa
• It produces images of a sample by scanning the surface with a focused beam
of electrons.
• An electron microscope that produces a high magnification image of the surface
of a metal-coated specimen by scanning an electron beam
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and building an image from the electrons reflected at each point.
• A beam of electrons is produced at the top of the microscope by an electron gun. The
electron beam follows a vertical path through the microscope, which is held within a
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vacuum.
• The beam travels through electromagnetic fields and lenses, which focus the beam
down toward the sample.
• Once the beam hits the sample, electrons and X-rays are ejected from the sample.
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• Detectors collect these X-rays, backscattered electrons, and secondary electrons and
convert them into a signal that is sent to a screen similar to a television screen. This
produces the final image.
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• SEM produces images by detecting secondary electrons that are emitted from the
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surface due to excitation from a primary electron beam. In more detail, SEM works by
rapidly scanning your sample with a focused electron beam. This causes electrons to
be knocked off the surface of your sample. These secondary electrons provide signals
carrying information about the properties of the specimen surface, such as its
topography and composition. And it is these secondary or backscattered electrons that
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are picked up by a detector and are used to produce your image.

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Bacteria Cell

Bacterial cells may be gram positive or gram negative. Both types of bacterial cells have cell
walls, but these have different compositions. Bacteria are prokaryotic, unicellular
microorganisms, which lack chlorophyll pigments. The average diameter of spherical bacteria is

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0.5-2.0µ. For rod-shaped or filamentous bacteria, length is 1-10µ and diameter is 0.25-1 .0µ .
Gram positive bacterial cells contain thick peptidoglycan walls that contain high concentrations
of peptides. Gram negative bacterial walls contain thin peptidoglycan walls and exhibit enzymes
that help them with mobility. Gram negative bacteria also contain lipid A, which is an endotoxin
that can protect the bacterial cell.

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Shape and Arrangement of Bacteria:
1. Spherical Bacteria:
Bacteria, which are spherical or ovoid in shape, are called ‘coccus’ (plural: cocci). Based on the

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arrangement of the cells they are of the following types.

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(a) Coccus:
The spherical bacteria cells, called cocci, are present as single individuals.
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(b) Diplococcus:

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The cocci are arranged in pairs.

(c) Streptococcus:

The cocci are arranged in chains, as the cells divide in one plane. (d) Tetrads:

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The cocci are arranged in packets of four cells, as the cells divide in two plains.

(e) Staphylococcus:
The cocci are arranged in grape-like clusters formed by irregular cell divisions in three plains.

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(f) Sarcinae (Octet):
The cocci are arranged in a cuboidal manner, as the cells are formed by regular cell divisions in
three planes.

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2. Rod-shaped Bacteria:
The cylindrical or rod-shaped bacteria are called ‘bacillus’ (plural: bacilli).

They are of three shapes as follows:

(a) Bacillus:
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They are rod-shaped bacteria. Based on arrangement they are of the following types.
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(i) Bacillus:
The rod-shaped bacteria cells, called bacilli, are present as single individuals.

(ii) Diplobacillus:
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The bacilli are arranged in pairs.

(iii) Streptobacillus:
The bacilli are arranged in chains, as the cells divide in one plane.
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(iv) Trichomes:
The bacilli are arranged in chains with larger area of end-to-end contact between the cells.
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(v) Palisades:
The bacilli bend at the points of division following the cell divisions, resulting in a palisade
arrangement resembling a picket fence and angular patterns that look like Chinese letters.
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(b) Coccobacillus:
These are so short and stumpy that they appear ovoid. They look like coccus and bacillus.

(c) Vibrios:

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They are comma-shaped bacteria with less than one complete turn or twist in the cell.

3. Spiral Bacteria:
Unlike the vibrios, which have less than one complete turn or twist in the cell, the spiral bacteria
are rod-shaped bacteria, which have more than one twist in the cell. They usually occur singly.

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They are of two types as follows:
(a) Spirillum:
They have rigid spiral structure. Spirillum with many turns can superficially resemble
spirochetes. They do not have outer sheath and endoflagella, but have typical bacterial flagella.

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(b) Spirochetes:
They are flexible and can twist and contort their shape. They have outer sheath and endoflagella,
but lack typical bacterial flagella.

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4. Filamentous Bacteria:
They are very long thin filament-shaped bacteria. Some of them form branching filaments
resulting in a network of filaments called ‘mycelium’.
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5. Box-shaped or Square-shaped Bacteria (Arcula):
They are flat, box-shaped bacteria with perfectly straight edges and sharp 90° angles at the
corners. Smaller cells are usually perfectly squares (2X2µ), while larger cells are rectangular;
about twice as long as they are wide (4X2µ).
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Each bacterium is a thin flexible sheet with smooth surface. After cell divisions, the cells remain
attached to each other, producing large sheets of squares. It was first discovered in 1980 in
natural salt ponds.
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6. Appendaged Bacteria:
They possess extension of their cells, as long tubes in the form of stalk or hypha, or as buds.

7. Pleomorphic Bacteria:
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These bacteria do not have any characteristic shape unlike all others described above. They can
change their shape. In pure cultures, they can be observed to have different shapes.
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Nutrition in Bacteria

Bacteria exhibit different modes of nutrition. On this basis, broadly two types of bacteria can be
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recognised autotrophic bacteria and heterotrophic bacteria.

Autotrophs are of two types:

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1. Photoautotrophs: The bacteria that use light energy, CO 2 as their carbon source and an
inorganic electron source (Examples: H2, H2S) are called Photoautotrophs
2. Chemoautotrophs: The bacteria that obtain energy by oxidizing inorganic compounds
and use CO2 as their sole source of carbon are called Chemoautotrophs. These include
nitrifying bacteria, iron bacteria and sulphur bacteria.

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Heterotrophs are of two types:

1. Photo heterotrophs: Bacteria that use light energy and organic electron donors as well
as simple organic molecules rather than CO 2 as the source of carbon are called as Photo
heterotrophs. These include Purple and Green Bacteria

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2. Chemo heterotrophs: Bacteria which use organic compounds as source of energy,
electrons, hydrogen and carbon for biosynthesis are called Chemo heterotrophs.

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They are as unrelated to human beings as living things can be, but bacteria are essential to human
life and life on planet Earth. Although they are notorious for their role in causing human
diseases, from tooth decay to the Black Plague, there are beneficial species that are essential to
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good health. For example, one species that lives symbiotically in the large intestine manufactures
vitamin K, an essential blood clotting factor. Other species are beneficial indirectly. Bacteria
give yogurt its tangy flavor and sour dough breads its sour taste. They make it possible for
ruminant animals (cows, sheep, and goats) to digest plant cellulose and for some plants,
(soybean, peas, alfalfa) to convert nitrogen to a more usable form.
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Bacteria are prokaryotes, lacking well-defined nuclei and membrane-bound organelles, and with
chromosomes composed of a single closed DNA circle. They come in many shapes and sizes,
from minute spheres, cylinders and spiral threads, to flagellated rods, and filamentous chains. In
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the late 1600s, Anton van Leeuwenhoek became the first to study bacteria under the microscope.
During the nineteenth century, the French scientist Louis Pasteur and the German physician
Robert Koch demonstrated the role of bacteria as pathogens (causing disease).

There are two different ways of grouping bacteria. They can be divided into three types based on
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their response to gaseous oxygen. Aerobic bacteria require oxygen for their health and existence
and will die without it. Anaerobic bacteria can't tolerate gaseous oxygen at all and die when
exposed to it.
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 Capsule - Some species of bacteria have a protective covering, a capsule made up of
polysaccharides (complex carbohydrates). Capsules play a number of roles, but the most
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important are to keep the bacterium from drying out and to protect it from phagocytosis
(engulfing) by larger microorganisms. The capsule is a major virulence factor in the
major disease-causing bacteria, such as Escherichia coli and Streptococcus pneumoniae.
Nonencapsulated mutants of these organisms are avirulent, i.e. they don't cause disease.
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 Cell Wall - Each bacterium is enclosed by a rigid cell wall composed of peptidoglycan, a
protein-sugar (polysaccharide) molecule. The wall gives the cell its shape and surrounds
the cytoplasmic membrane, protecting it from the environment. It also helps to anchor
appendages like the pili and flagella, which originate in the cytoplasm membrane and
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protrude through the wall to the outside. The strength of the wall is responsible for
keeping the cell from bursting when there are large differences in osmotic pressure
between the cytoplasm and the environment.
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 Mesosomes - are folded invaginations in the plasma membrane of bacteria that are
produced by the chemical fixation techniques used to prepare samples for electron
microscopy. It increases the surface area of the plasma membrane. This drastic increase
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in the surface area of the membrane mainly helps the cell to carry out cellular respiration
more efficiently.
 Cytoplasm - The cytoplasm, or protoplasm, of bacterial cells is where the functions for
cell growth, metabolism, and replication are carried out. It is a gel-like matrix composed
of water, enzymes, nutrients, wastes, and gases and contains cell structures such as
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ribosomes, a chromosome, and plasmids. The cell envelope encases the cytoplasm and all
its components. Unlike the eukaryotic (true) cells, bacteria do not have a membrane
enclosed nucleus. The chromosome, a single, continuous strand of DNA, is localized, but
not contained, in a region of the cell called the nucleoid. All the other cellular
components are scattered throughout the cytoplasm.

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One of those components, plasmids, are small, extrachromosomal genetic structures
carried by many strains of bacteria. Like the chromosome, plasmids are made of a
circular piece of DNA. Unlike the chromosome, they are not involved in reproduction.
Only the chromosome has the genetic instructions for initiating and carrying out cell
division, or binary fission, the primary means of reproduction in bacteria. Plasmids

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replicate independently of the chromosome and, while not essential for survival, appear
to give bacteria a selective advantage.

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Plasmids are passed on to other bacteria through two means. For most plasmid types,
copies in the cytoplasm are passed on to daughter cells during binary fission. Other types
of plasmids, however, form a tubelike structure at the surface called a pilus that passes
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copies of the plasmid to other bacteria during conjugation, a process by which bacteria
exchange genetic information. Plasmids have been shown to be instrumental in the
transmission of special properties, such as antibiotic drug resistance, resistance to heavy
metals, and virulence factors necessary for infection of animal or plant hosts. The ability
to insert specific genes into plasmids have made them extremely useful tools in the fields
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of molecular biology and genetics, specifically in the area of genetic engineering.

 Cytoplasmic Membrane - A layer of phospholipids and proteins, called the cytoplasmic

membrane, encloses the interior of the bacterium, regulating the flow of materials in and
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out of the cell. This is a structural trait bacteria share with all other living cells; a barrier
that allows them to selectively interact with their environment. Membranes are highly
organized and asymmetric having two sides, each side with a different surface and
different functions. Membranes are also dynamic, constantly adapting to different
conditions.
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 Nucleoid - The nucleoid is a region of cytoplasm where the chromosomal DNA is

located. It is not a membrane bound nucleus, but simply an area of the cytoplasm where
the strands of DNA are found. Most bacteria have a single, circular chromosome that is
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responsible for replication, although a few species do have two or more. Smaller circular
auxiliary DNA strands, called plasmids, are also found in the cytoplasm.
 Ribosomes - Ribosomes are microscopic "factories" found in all cells, including bacteria.
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They translate the genetic code from the molecular language of nucleic acid to that of
amino acids—the building blocks of proteins. Proteins are the molecules that perform all
the functions of cells and living organisms. Bacterial ribosomes are similar to those of
eukaryotes, but are smaller and have a slightly different composition and molecular

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structure. Bacterial ribosomes are never bound to other organelles as they sometimes are
(bound to the endoplasmic reticulum) in eukaryotes, but are free-standing structures
distributed throughout the cytoplasm. There are sufficient differences between bacterial
ribosomes and eukaryotic ribosomes that some antibiotics will inhibit the functioning of
bacterial ribosomes, but not a eukaryote's, thus killing bacteria but not the eukaryotic

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organisms they are infecting.
 Fimbriae: Most Gram-negative bacteria have fimbriae; hair-like projections, external to
the cell wall, that allow bacteria to stick to the cells they infect. Long, thin flagella are
used by some bacteria to move about. Sex pili allow bacteria to share genes. Shorter
extensions, called fimbriae (singular fimbria), enable bacteria to adhere to surfaces and
potentially infect the cells of their host.

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VIRAL CELL:

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A virus is a small parasite that cannot reproduce by itself. Once it infects a susceptible cell,
however, a virus can direct the cell machinery to produce more viruses. Most viruses have
either RNA or DNA as their genetic material. The entire infectious virus particle, called a virion,
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consists of the nucleic acid and an outer shell of protein. The simplest viruses contain only
enough RNA or DNA to encode four proteins. The most complex can encode 100 – 200 proteins.

Viruses are the smallest infectious organisms, and they are so tiny that millions of them could fit
inside a single human cell. Viruses are only capable of reproduction inside a living cell, called a
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host cell that they invade. A virus consists of little more than a single or double strand of genetic
material surrounded by a protein shell. However, some types of virus also have a protective outer
envelope.

How viruses reproduce

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To survive, viruses must reproduce inside living cells. The genetic material from an infecting
virus takes over the functions of the host cell to make millions of new virus particles. The new
viruses leave the host cell by bursting out of the cell or by budding out from the cell surface.
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Proteins on the virus attach to specific receptors on the surface of a host cell. The virus may enter
the cell by being engulfed by the cell membrane or by fusing into the cell membrane.
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When inside the cell, the virus sheds its protein shell. The genetic material of the virus

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reproduces, using substances from inside the cell.

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Each copy of the genetic material programmes the formation of a new protein shell. Once the
shells have formed, the new viruses are complete.
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The viruses leave the cell either by suddenly rupturing the cell membrane, which destroys the
host cell, or by slowly budding out from the surface of the cell membrane.
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Budding viruses
When certain viruses bud out from their host cell, they envelop themselves in host cell surface
membrane.
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Structure:

Without a host cell, viruses cannot carry out their life-sustaining functions or reproduce. They
cannot synthesize proteins, because they lack ribosomes and must use the ribosomes of their host
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cells to translate viral messenger RNA into viral proteins. Viruses cannot generate or store
energy in the form of adenosine triphosphate (ATP), but have to derive their energy, and all other
metabolic functions, from the host cell. They also parasitize the cell for basic building materials,
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such as amino acids, nucleotides, and lipids (fats). Although viruses have been speculated as
being a form of protolife, their inability to survive without living organisms makes it highly
unlikely that they preceded cellular life during the Earth's early evolution. Some scientists
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speculate that viruses started as rogue segments of genetic code that adapted to a parasitic
existence.

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All viruses contain nucleic acid, either DNA or RNA (but not both), and a protein coat, which
encases the nucleic acid. Some viruses are also enclosed by an envelope of fat and protein
molecules. In its infective form, outside the cell, a virus particle is called a virion. Each virion
contains at least one unique protein synthesized by specific genes in its nucleic acid. Viroids
(meaning "viruslike") are disease-causing organisms that contain only nucleic acid and have no

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structural proteins. Other viruslike particles called prions are composed primarily of a protein
tightly integrated with a small nucleic acid molecule.

Viruses are generally classified by the organisms they infect, animals, plants, or bacteria. Since
viruses cannot penetrate plant cell walls, virtually all plant viruses are transmitted by insects or
other organisms that feed on plants. Certain bacterial viruses, such as the T4 bacteriophage, have

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evolved an elaborate process of infection. The virus has a "tail" which it attaches to the
bacterium surface by means of proteinaceous "pins." The tail contracts and the tail plug
penetrates the cell wall and underlying membrane, injecting the viral nucleic acids into the cell.

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Viruses are further classified into families and genera based on three structural considerations:
1) the type and size of their nucleic acid, 2) the size and shape of the capsid, and 3) whether they
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have a lipid envelope surrounding the nucleocapsid (the capsid enclosed nucleic acid).

There are predominantly two kinds of shapes found amongst viruses: rods, or filaments, and
spheres. The rod shape is due to the linear array of the nucleic acid and the protein subunits
making up the capsid. The sphere shape is actually a 20-sided polygon (icosahedron).
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The nature of viruses wasn't understood until the twentieth century, but their effects had been
observed for centuries. British physician Edward Jenner even discovered the principle of
inoculation in the late eighteenth century, after he observed that people who contracted the mild
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cowpox disease were generally immune to the deadlier smallpox disease. By the late nineteenth
century, scientists knew that some agent was causing a disease of tobacco plants, but would not
grow on an artificial medium (like bacteria) and was too small to be seen through a light
microscope. Advances in live cell culture and microscopy in the twentieth century eventually
allowed scientists to identify viruses. Advances in genetics dramatically improved the
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identification process.
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 Capsid - The capsid is the protein shell that encloses the nucleic acid; with its enclosed
nucleic acid, it is called the nucleocapsid. This shell is composed of protein organized in
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subunits known as capsomers. They are closely associated with the nucleic acid and
reflect its configuration, either a rod-shaped helix or a polygon-shaped sphere. The capsid
has three functions: 1) it protects the nucleic acid from digestion by enzymes, 2) contains
special sites on its surface that allow the virion to attach to a host cell, and 3) provides
proteins that enable the virion to penetrate the host cell membrane and, in some cases, to
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inject the infectious nucleic acid into the cell's cytoplasm. Under the right conditions,
viral RNA in a liquid suspension of protein molecules will self-assemble a capsid to
become a functional and infectious virus.
 Envelope - Many types of virus have a glycoprotein envelope surrounding the
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nucleocapsid. The envelope is composed of two lipid layers interspersed with protein
molecules (lipoprotein bilayer) and may contain material from the membrane of a host
cell as well as that of viral origin. The virus obtains the lipid molecules from the cell
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membrane during the viral budding process. However, the virus replaces the proteins in
the cell membrane with its own proteins, creating a hybrid structure of cell-derived lipids
and virus-derived proteins. Many viruses also develop spikes made of glycoprotein on
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their envelopes that help them to attach to specific cell surfaces.

 Nucleic Acid - Just as in cells, the nucleic acid of each virus encodes the genetic
information for the synthesis of all proteins. While the double-stranded DNA is
responsible for this in prokaryotic and eukaryotic cells, only a few groups of viruses use

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DNA. Most viruses maintain all their genetic information with the single-stranded RNA.
There are two types of RNA-based viruses. In most, the genomic RNA is termed a plus
strand because it acts as messenger RNA for direct synthesis (translation) of viral protein.
A few, however, have negative strands of RNA. In these cases, the virion has an enzyme,
called RNA-dependent RNA polymerase (transcriptase), which must first catalyze the

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production of complementary messenger RNA from the virion genomic RNA before viral
protein synthesis can occur.

The Influenza (Flu) Virus - Next to the common cold, influenza or "the flu" is perhaps the most
familiar respiratory infection in the world. In the United States alone, approximately 25 to 50
million people contract influenza each year. The symptoms of the flu are similar to those of the

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common cold, but tend to be more severe. Fever, headache, fatigue, muscle weakness and pain,
sore throat, dry cough, and a runny or stuffy nose are common and may develop rapidly.
Gastrointestinal symptoms associated with influenza are sometimes experienced by children, but

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for most adults, illnesses that manifest in diarrhea, nausea, and vomiting are not caused by the
influenza virus though they are often inaccurately referred to as the "stomach flu." A number of
complications, such as the onset of bronchitis and pneumonia, can also occur in association with
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influenza and are especially common among the elderly, young children, and anyone with a
suppressed immune system.

The Human Immunodeficiency Virus (HIV) - The virus responsible for HIV was first isolated
in 1983 by Robert Gallo of the United States and French scientist Luc Montagnier. Since that
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time, a tremendous amount of research focusing upon the causative agent of AIDS has been
carried out and much has been learned about the structure of the virus and its typical course of
action. HIV is one of a group of atypical viruses called retroviruses that maintain their genetic
information in the form of ribonucleic acid (RNA). Through the use of an enzyme known as
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reverse transcriptase, HIV and other retroviruses are capable of producing deoxyribonucleic acid
(DNA) from RNA, whereas most cells carry out the opposite process, transcribing the genetic
material of DNA into RNA. The activity of the enzyme enables the genetic information of HIV
to become integrated permanently into the genome (chromosomes) of a host cell.
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BACTERIAL IDENTIFICATION TESTS

Identification of Bacteria: 7 Steps
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The following points highlight the seven steps for identification of bacteria isolated from a
specimen. The steps are: 1. Morphology and Staining 2. Cultural Characteristics 3.
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Biochemical Reactions 4. Antigenic Characters 5. Typing of Bacteria: Bacteriophage

Sensitivity 6. Animal Pathogen City 7. Antibiotic Sensitivity.

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Identification of Bacteria:
Step # 1. Morphology and Staining:
Serve as preliminary criteria. The Gram stained smear shows the Gram reaction, size, shape,
groupings of the bacteria and intracellular position of the endospore. Special staining reaction
can reveal the presence of capsule.

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Hanging drop wet preparation can be used to study the motility of bacteria. An unstained wet
film is examined under dark ground illumination microscope to observe the exact morphology of
delicate spirochaete. A smear is stained by Ziehl Neelsen method to demonstrate the acid fast
staining reaction.

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Identification of Bacteria:
Step # 2. Cultural Characteristics:
The growth requirement and the appearance of colonies on media to the naked eye are further

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criteria to assist the identification of bacteria. A culture is a growth of bacterium on artificial
nutrient medium or culture medium prepared in the laboratory.
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Attempts are made to grow (to cultivate or culture) the bacteria on media of different
compositions (glucose, inorganic salts mixture, meat extract or meat infusion with blood)
incubated under a variety of conditions (different temperatures, pH) in the presence of
atmospheric oxygen (aerobically).
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The ability or inability to grow on medium containing a selective inhibitor (e.g. bile salt,
optochin, tellurite, bacitracin, malachite green, low pH, high pH) may also be useful to identify
the bacteria.
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The growth of bacteria in liquid culture medium (e.g. nutrient broth) may show:
(1) A uniform turbidity;

(2) Little deposit at the bottom;

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(3) Surface growth (pellicle formation).

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The appearance of the discrete masses of growth or colonies that can be grown from isolated

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bacteria on the surface of the solid medium (nutrient agar) can be used to study the size of the
colonies (diameter in mm), their outline (whether circular, entire, indented or wavy or rhizoid),
their elevation (low convex, high convex, flat, plateau-like, umbonate, or nodular)—Fig. 7.1b),
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their transparency (clear and transparent) or opaque, whether they are colourless (white or
pigmented) or whether they produce any change in the medium (e.g. haemolysis in the blood
agar medium).
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Identification of Bacteria:
Step # 3. Biochemical Reactions:
E.g. fermentation of various sugars (carbohydrates). Morphology and cultural characters may not
be able to distinguish some species of bacteria; but these same species may exhibit distinct

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differences in their biochemical reactions e.g. typhoid and paratyphoid bacilli (glucose and
mannitol are fermented without gas production by typhoid bacilli, whereas paratyphoid bacilli
produce acid and gas).

Certain serotypes of the salmonella group may resemble one another in fermentation properties.

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The growth of the bacteria in liquid medium will ferment particular sugars (glucose, lactose,
mannitol) with the production of acid, which is detected by the changes of colour of Andrade’s
indicator dye incorporated in the medium; the gas production is detected by the collection of air

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bubble in a small inverted tube (Durham’s tube) immersed in the medium.

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Other tests are used to find out the ability of a bacterium to produce particular end products e.g
Indole, hydrogen sulphide, nitrite and certain enzymes (oxidase, catalase, urease, gelatinase,
collagenase, lecithinase, lipase) in culture media.
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Identification of Bacteria:
Step # 4. Antigenic Characters:
Species or types of bacteria can be easily and distinctly identified by “specific” antibody
reactions observed in serological tests performed on a glass slide. This specific antibody
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(antiserum) is obtained from the animal (rabbit) immunized against a particular type of
microorganisms which agglutinates with the same antiserum.
An unknown bacterium may thus be identified by demonstrating its reaction with one out of a
number of standard known antisera.
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Similarly, the serum of a person suffering from a bacterial infection may contain specific
antibody. The nature of the infection may thus be diagnosed by demonstrating that the patient’s
serum agglutinates one out of a number of known antigens of laboratory cultures, e.g. Widal test
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in Typhoid fever.

Identification of Bacteria:
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Step # 5. Typing of Bacteria:

Bacteriophage Sensitivity:
A single bacterial pathogenic species may include different types of strains which are
distinguishable in minor characters. Recognition of the type of a strain isolated from a patient

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may be of great importance in epidemiological studies related to the source and the spread of the
infection in the community.

The typing of stains may be done by special biochemical or serological tests. Another important
method of typing is by testing the susceptibility of the culture to lysis by each of a set of type

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specific, lytic bacteriophages.

Identification of Bacteria:
Step # 6. Animal Pathogen City:
Final identification of a toxigenic strain of tetanus bacillus may be done by injecting the toxin
liberated by tetanus bacillus into the base of the tail of two mice, one of them has already been

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protected by prior injection of specific antiserum to tetanus toxin (a soluble poisonous protein
secreted by the tetanus bacilli).

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The unprotected mouse shows the symptoms of tetanus, whereas the protected one without any
tetanus symptoms identifies the culture, as an organism producing toxin, as the injected
antiserum neutralized toxin liberated by tetanus bacilli. Similarly, diphtheria bacillus is also
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identified by inflammation and necrosis of the skin of guinea pig brought by diphtheria exotoxin.

Identification of Bacteria:
Step # 7. Antibiotic Sensitivity:
The organism is tested for its ability to grow on artificial nutrient media containing different
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antibiotics and chemotherapeutic agents in different concentrations. In disk diffusion test, the
culture to be examined is inoculated confluently with swabs over the surface of an agar plate and
six to ten paper disks containing different antibiotics are placed in different areas of the plate.
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Antibiotic diffuses outwards from each disk into surrounding agar. On incubation, the bacteria
grow on areas of the plate except those around the antibiotic disks to which they are sensitive.
The width of each growth-free “zone of inhibition” is a measure of the degree of the sensitivity
of the drug.
Information about the sensitivity patterns of strains (anti-bio-grams) isolated from the patient is
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required as a guide to the drug of choice for therapy and may also be used as an epidemiological
marker in tracking hospital cross-infection.
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