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PATHOLOGY
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BY DR. (PROF.). S.k. SAMANTA

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DEFINATION

Pathology is a scientific study of disease. It is divided into general and systemic pathology

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for pedagogical reasons. General pathology covers the basic mechanisms of diseases,

whereas systemic pathology covers diseases as they occur in each organ system. This unit

covers only general pathology. If one knows general pathology well, one can apply this

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knowledge to diseases in the various organ systems. Hence, your general pathology

knowledge will facilitate your understanding of systemic diseases.

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GENERAL PATHOLOGY

Pathology is the study of disease by scientific methods. The word of Pathology came from the Latin

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Words “PATHO” and “LOGY”. “Patho” means disease and” logy” means “Study”, therefore

Pathology is the scientific study of disease. Diseases may, in turn, be defined as an abnormal

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variation in structure or function of any part of the body.

As a field of general enquiry and research, Pathology addresses four components of disease :-

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cause/etiology, mechanisms of development (Pathogenesis), structural alteration of cells

(Morphologic Changes) and the consequences of changes (clinical manifestation).

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Pathology gives explanations of a disease by studying the following four aspects of the disease.

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1. Etiology

2. Pathogenesis

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3. Morphologic Changes

4. Functional Derangements & Clinical Significance.

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Etiology

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Etiology of a disease means the cause of disease. If the cause of a disease is known, it is called

Primary Etiology. If the cause of the disease is unknown it is called Idiopathic. Knowledge or

discovery of the primary cause remains the backbone or which a diagonises can be made, a disease

understood, and a

Treatment developed. There are two major classes of etiologic factors:- Genetic & Acquired (

infectious, nutritional, chemical, physical etc.). The etiology is followed by Pathogenesis.

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Pathogenesis :- Pathogenesis means the mechanism through which the cause operates to

produce the pathological & clinical manifestations. The path genetic mechanisms could take place in

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the latent or incubation period. Pathogenesis lead to morphologic changes.

Morphologic Changes:-

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The morphologic changes refer to the structural alteration in the cells or tissue that occur following

the path genetic mechanisms. The structural changes in the organ can be seen under the

microscope. Those changes that can be seen with the naked eye are called Gross Morphologic

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changes may only be seen in that disease, they may be specific to that disease. Therefore, such

morphologic change can be used by the pathologist to identify(to diagnose) the disease. In addition,

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the morphologic changes will lead to functional alteration & to the clinical signs & symptoms of the

disease.

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Functional Derangements & Clinical Significance :-

The morphologic changes in the organ influence the normal function of the organ. By doing so, they

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determine the clinical features course, and prognosis of the disease.

Cell In Health & Disease :-

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Cell injury underlies all diseases. So, to understand disease one, has to start by knowing what cell

injury is. When a cell is exposed to an injurious agent, the possible outcome are 1. The cell may

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adapt to the situation or 2. The cell may acquire a reversible injury & may die. The cell may die via

one of two ways- either Necrosis or Apoptosis.

Inflammation :-
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Inflammation is the body’s attempt at self- protection, the aim being to remove harmful stimuli,

including damaged cells, irritants or pathogens & being the healing process.

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When something harmful or irritating affects a part of our body, there is a biological response to

try to remove it, the signs & symptoms of inflammation, specifically acute inflammation, show that

the body is trying to heal itself.

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Inflammation does not mean infection, even when an infection causes inflammation. Infection is

caused by a bacterium, virus or fungus, while inflammation is the body’s response to it.

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Body Fluids & Electrolytes :-

Sodium retention & subsequently water retention occurs in the various clinical condition such as

congestive heart failure & renal failure. In these conditions, the retained sodium & water result in

increased capillary hydrostatic pressure which lead to the edema seen in these diseases.

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Distribution Of Fluids Among The Two Body Fluid Compartment.

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1)
The distribution of fluids among the body fluid spaces is determined by osmic pressures. The

homeostatic mechanisms of the body which maintain this function are designed to

maintain the osmotic pressure of the extracellular fluid. If E.C.F. osmotic pressure can be

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maintend,

it will also severe to maintain intracellular osmolarity. If the osmotic pressure of the E.C.F. is

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increased, water is removed from cells, producing cellular dehydration & anew state of

equilibrium between E.C.F. & I.C.F. at a new & different osmolarity.

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2)
The plasma proteins are confined to the vascular space & in that location, exert at osmotic

force that holds fluid in the vessels in opposition to the tendency of the blood pressure to

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force fluid out of the vessels.

3)
The tonicity (osmic pressure) of the I.C.F. & E.C.F. is determined by the total number of

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practical (electrolyte ions) dissolve in each of these fluids.

4)
Normal hydration of the body depends, therefore, not only on optimum water in the body,

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but on optimum protein , sodium, & potassium in the appropriate fluid compartments to

hold the Proper amount Of Water In Each Compartment.

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Shocks

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Shock is a state in which there is failure of the circulatory system to maintain adequate cellular

perfusion resulting in widespread reduction in delivery of oxygen & other nutrients to tissue . In

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shock, the mean arterial pressure is less than 60mmHg or the systolic blood pressure is less than

90mmHg.

Regardless of the underlying pathology, shock constitute systemic hypo perfusion due to reduction

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either in cardiac output or in the effective circulating blood volume. The end results are hypotension

followed by impaired tissue perfusion & cellular hypoxia.

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Adequate organ perfusion depends on arterial blood pressure (B.P.) which, in turn depends on :-

1) Cardiac Output (C.O.).

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2) Peripheral Vascular Resistance (PVR).

Classification Of Shock :-
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Shock can be divided into :-

*Hypovolemic Shock :- This is shock caused by reduced blood volume. Reduction in circulating

blood volume results in the reduction of the preload which leads to inadequate left ventricular

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filling, reflected as decreased left right ventricular and diastolic volume and pressure. The

reduced preload culminates in decreased cardiac output which leads to wide spread tissue

perfusion(shock).

A> HAEMORRHAGE B> DIARRHOEA AND VOMITING C> BURNS D> TRAUMA, ETC

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The effect of hemorrhage depends on the rate and amount of blood loss. Hypovolemic shock is

the mast common type of shock in clinical medicine. A normal healthy adult can loss 550ml(10%of

blood volume) without significant symptoms.

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But loss of 25% or more of blood volume(N=1250ml) result in significant hypovolemia.

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
CARDIOGENIC SHOCK :- This is shock that results from severe depression of

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cardiac performance. It primarily result from pump failure (myocardial failure) causes

of cardiogenic shock can be divided into:- myopathy:- includes acute myocardial

depression in septic shock etc.

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MECHANICAL :- 1> Intra cardiac :- left ventricle outflow obstruction, reduction in

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forward cardiac output, Arrhythmia. 2> EXTRACARDIAC :- Also called obstructive

shock, it can be caused by :- a> Gross fluid accumulation in the pericardial space

which result in a decreased ventricular diastolic filling . b> Gas accumulation in a

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plural space which decreases the venous returns by creating a positive pressure.

C> Acute massive pulmonary embolism occupying 50-60% of pulmonary vascular

bed. D> Severe pulmonary

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hypertension .

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
Distributive shock :- Distributive shock refers to a group of shock subtype

caused by profound peripheral vasodilation despite normal or high cardiac output.

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 Cause :- (i) Septic shock :- Distributive shock refers to a group of shock subtypes

caused by profound peripheral vasodilation despite normal or high cardiac output.

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(ii) Neurogenic shock :- usually occurs in the setting of on aesthetic procedure or spinal

cord injury owing to loss of vascular tone and peripheral pooling of blood.

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(iii) Anaphylactic shock :- Initiated by generalized IgE – Mediated hypersensitivity

response , associated with system vasodilatation and increased vascular pameability.

(iv) Endocrine shock :- This is a type of shock that typically occurs in adrenal

insufficiency.


Ischemia : Ischemia is a restriction in blood supply to tissues , causing a

shortage of oxygen and glucose needed for cellular metabolism . Ischaemia

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generally caused by problems with blood vessels , with resultant damage

to or dysfunction of tissue . if also means local anemia in a given part of a body

sometimes resulting from congestion .


Infection :- The anatomic pathologist performs an important role in the

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diagnosis or exclusion of infections diseases . the morphologic interpretation

of biopsies and cryptologic preparations allows for the definitive establishment

or exclusion of a wide variety of diseases . once the pathologist has

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
determined that a disease is likely to be due to an infection and has

characterized the inflammatory response , associated microorganisms or viral-

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associated psychopathic effects should be recorded . all though some

microorganisms or those psychopathic effects may be clearly visible on routine

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hemaloxylin and eosin stained sections, additional histochemical stains are

often needed for their complete characterization . highly specific molecular

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techniques , such as immunohistochemistry , in situ hybridization , and nucleic

acid amplification may be needed in certain instances to establish the diagnosis

of infection . through appropriate morphologic diagnoses and interlaboratory

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communication and collaboration , the anatomic pathologist contributes greatly

to the diagnosis and treatment of infectious diseases .

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
Neoplastic :- Literally, neoplastic means new growth and technically , it is

defined as abnormal mass of tissue , the growth of which exceeds and persists

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in the same excessive manner after cessation of the stimulus evoking the

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transformation .

Neoplasms are named based upon two factors :-

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(i) On the histologic types :- Mesenchyme and epithelial.

(ii) On behavioral patterns :- Benign and malignant neoplasms .

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ETIOLOGY/CAUSES :- The origin for many neoplasms is obscure . how ever, there are

several theories of origin. 1> Environmental causes 2> Hereditary cause 3> Obscure

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defects 4>Age 5> Altered DNA

1>
Environmental causes :- Chemicals including those that are man – made (such

as aniline dyes and bladder cancer ), drugs (cigarette smoke and lung cancer), and

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natural compounds( aflatoxins and liver cancer) which are carcinogenic .

2>
Hereditary causes :- Chromosome which have defective or no anti-oncogenes at

all that control growth of tumor . for example , retinoblastoma results from

defective chromosome 13.

3> Obscure defects :- Racial predilection.

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4>
Age :- Older person have a greater propensity to develop neoplasms from lack

of effective control mechanisms.

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Altered DNA :- All of the above are probably mediated by the cause, what ever

it is producing a mutation in , or damage to, cell , DNA. There can be mutation

involving tumor suppressor genes, which then fail to exert a controlling influence

upon probably arise via this mechanism.

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In some cases these mutation are probably mediated by proto – oncogenes that

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translocation & by gene amplification.

About 15 to 20% of human cancers have been linked to oncogenic activity. The ------------------------

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oncogene is the transforming gene found most frequently in human cancers.

Oncogenic viruses may bring oncogenes with them, so-called viral oncogenes.

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Pathogenesis :- Pathogenesis means the mechanism through which the cause

operable to produce the pathological & clinical manifestation. The pathogen etic

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mechanisms could take place in the latent or incubation period. Pathogenesis lead

to morphologic changes.

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Divisions Of Pathology :- Anatomical Pathology :- Emphasises on morphology of

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diseases including that, that of organic level & cellular level.

Patho –Physiology :- Emphasizes on homeostatic function & mechanisms of

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diseases including that of organic & cellular level.

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Sub Divisions Of Clinical Pathology :- Pathology is a vast subject with

many manifestation. In practice, however, it has major subdivisions :-

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*Histopathology :- Histopathology the investigation & diagnosis of disease from the

examination of tissue.

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* Cytopathology :- Cytopathology the investigation & diagnosis of disease from the

examination of isolated cells.

* Hematology :- Hematology the study of disorders of the cellular & coagulable

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components of blood.

*Microbiology :- Microbiology the study of infections diseases & the organisms

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responsible for them.

*Immunology :- Immunology the study of the specific defence mechanisms of the

body.

* Chemical Pathology :- Chemical Pathology the study &diagnosis of disease from

the chemical change in tissue & fluids.

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*Genetics :- Genetic the study of abnormal chromosomes & genes.

* Toxicology :- Toxicology the study of the effects of known or suspected poisons.

*Forensic Pathology :- Forensic Pathology the application of pathology to legal

purposes.

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Technique For Studying Pathology

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The conventional optical microscope has been the primary tool in assisting pathological

examination. The modern digital pathology combines the power of microscopy, electronic

detection, and computerized analysis. It enables cellular, molecular & genetic – imaging at high

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efficiency & accuracy to facilitate clinical screening & diagnosis. First reviews the fundamental

concept of microscopic imaging & introduces the technical features & associated clinical

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applications of optical microscopes, electron microscopes, scanning microscopes, & fluorescence

microscopes.

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*Microscope :- A microscope is an optical instrument that used a lens or a combination of lenses to

produce magnified images of small object which are too small to be seen by naked eye. The

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function of any microscope is to enhance resolution. The microscope is used to create an enlarge

view of any object such that can observe details not otherwise possible with the human eye.

Because of the enlargement, resolution is the often confused with magnification, which refers to the

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size of the image. In general, the greater the magnification, the greater the resolution, but this is

not always true. These are several practical limitations of lens design which can result in increased

magnification without increased resolution. Cells are smeared thinly into glass microscope slides to

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allow individual cells types to be recognized & counted. A number of stains are available that help in

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the identification of particular cells. Giemsa for example, in used in blood & bone – marrow

preparation & stains red blood cells pink, platelets pale pink, lymphocytes , cytoplasm blue &

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leukocytes are chromatin magenta in colour. Giema can also be used to detect blood parasites such

as malaria & other spirochete & protozoan microorganisms. Key of clinical microscopy techniques

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include bright – field microscopy, dark- field, fluorescence microscopy, immunocytochemistry,

immunohistochemistry, digital slide & tele-pathology.

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Modern microscope, for use in the haematology laboratory , is equipped with an illuminator system

,a sub- stage condenser system, an objective system, a projector (eyepiece or ocular system), an iris

diaphragm,nicle prisms, a lubular barrel (mononuclear or binocular bodies) & a mechanical stage.

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A compound microscope uses a combination of lenses, the objective lens (lens closer to the

object) & ocular lens (lens closer to the eye) to project the image to the retina of the eye. The

objective lens acts much like a small, projection lens which projects an enlarged primary image near

the top of the tubular barrel. This image formed in air, is known as an Arial Image. This object is

viewed through the projector or eyepiece that acts like a magnifier except that it magnifies an

aerial object instead of an actual object. The final Virtual Image because the light rays appear to

come from the image. The rays are actually created by an increase in magnification by the lens

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system.

IMPORTANT PART OF A MICROSCOPE

Eye – Piece : The lens the viewer looks through to see the specimen. The eyepiece usually

contains a 5X, 10X, & 15X power lens.

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Diopter Adjuustmjent: Useful a means to change focus on one eyepiece so as to

correct for any difference in vision between your two eyes.

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Body Tube: (HEAD) : The body tube connects the eyepiece to the objective lenses.

 Arm :- The arm connects the body tube to the base microscope .

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 Coarse adjustment :- Brings the specimen into general focus.

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 Fine adjustment :- Fine tunes the focus and increases the detail of the specimen.

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
Nosepiece :- A rotating turret that house of the objective lenses. The viewer spins the

nosepiece to select different objective lenses.


Objective lenses :- One of the most important parts of a compound microscope , as they

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are the lenses closest to the specimen.A standaed microscope has three , four or five

objective lenses that range in power from 4x to 100x. when focusing the microscope , be

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careful that the objective lens does not touch the slide, as it could break the slide and

destroy the specimen.

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
Slide :- The specimen is the object being examined . most specimens are mounted on

slides,flat rectangles of thin glass.the specimen is placed on the glass and cover slip is

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placed over the specimen. This allows the slide to be easily removed from the microscope. It

also allows the specien to be labeled, transported, and stored with out damage.

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 Stage :- the flat platform where the slide is placed.

 Stage clips :- metal clips that hold the slide in place.

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 Stage control:- these knobs move the stage left and right or up and down .

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
Aperture :- the hole in the middle of the stage that allows light from the illuminator to

reach the specimen.

 On loff switch :- this switch on the base of the microscope turns the illuminator off and on.


Illuminator :- the light source or a microscope. Older microscope used mirrors to reflect

from an external source up through the bottom of the stage , however, most of the

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microscopes now use a low – voltage bulb.

 Iris diaphragm :- adjusts the amount of light that reaches the specimen.


Condenser:- gathers and focuses light from the illuminator on to the specimen being viewed

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
Base :- the base supports the microscope and it’s where illuminator is located. All of the

parts of a microscope work together. The light from the illuminator passes through the

aperture, through the slide , and through the objective lenses where the image of the

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specimen is magnified.


Centrifuge :- In aclinical laboratory centrifugation is one of the initial steps in testing a

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patient’s blood . the blood is collected in tubes or bag , depending on the prescribed test

from the doctor . tubes can very in size from 1.2ml – 15ml, while bags are usally around

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300ml -500ml.

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Role of centrifuge:- the role of centrifuge in testing blood is to separate hole blood in to its

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various components . each components of blood has a specific use in the body and there

fore a diierent test may be require FOR EACH component. Blood can be centrifuged in a

tube bottle of or bag. Blood collected from blood donors are collected in blood collected at

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a hospital or testing lab is collected tubes. Blood collections in a bottle is for microbiological

testing and contains transport media to preserve the micro organism. Centrifuges apply

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centrifugal force to separate suspended particles from liquid of different densities. These

liquids can include body fluids (blood, serum, urine) commercial reagents of the two with

other additives. by creating forces many times greater than gravity, centrifuge can

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greatly accelerate separations that occur naturally as a result of density differsences. The

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microhemato-crit centrifuge, a special purpose verrion of a fixed- head unit, quickly attains

speeds of 11000rpm and rcfs(relative centrifugal forces)of up to 15000g to spin

microcapillary tube smples. These tube require only small blood smples. These tube

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require only small blood smples. These tube require only small blood smples taken from

puncture site or from an anticoagulated venous blood specimen.

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
Table top models:- these units are mounted on rubber feet that absorb vibration. The

speed is controlled by means of a rheoslat on the front panel. Top speeds of centrifuges

will vary and the top speed of a rheoslat on the front panel. Top speed of particular

instrument should be known in oder to use the speed control device. Those centrifuge have

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adapters to hold 6tubes and adapters for 12 tubes.


Floor mounted models :- the heavier floor mounted models accommodate a large number

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of tubes at one time . the top speed of these instrument is higher than that of table

models.

The centrifuge is an instrument used in nearly every research laboratory across the globe.

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Centrifugation is the process by which a centrifuge is used to separate components of a

complex mixture. By spinning laboratory samples at very high speeds,the components of a

given mixture are subjected to centrifugal force, which more dense particles to migrate

away from the axis of rolation and lighter ones to move to ward it. These particles can

sediment at the bottom of the tube into what’s known as a pellet, and this isolated

specimen or the remaining solution, the supernatant, can be further processed or analyzed.

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
A centrifuge is used to separate particles or macromolecules:- cells, sub-cellular

components, proteios,nucleic acid.

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 Basis of separation:-

 Size

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 Shape

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 Density

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Methodology:- utilizes density difference between the particles macromolecules and the

medium in which these are dispersed.

 Dispersed systems are subjected to artificially included gravitational fields.

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
Incubator:- an incubator is a device used to grow and maintain microbial cell cultures. The

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incubator maintains an oplimal temperature, humidity and other conditions such as the

carbon dioxide (co2)and oxygen(o2) content of the atmosphere inside.


The simplest incubators are insulated boxes with an adjustable heater, typically going up to

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60 to 650c(140 to 150 f) though some can go slightly higher(generally to no more than

100oc).

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The most commonly used temperature both for bacteria as well as for mammalian cells is

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approximately 37oc, as these organisms grow well under such conditions. For other organisms grow

well under such conditions. For other organisms used in biological experiments, such as the

building yeast saccharomyces cerevisiae, a growth temperature of 30oc is optimal.

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More elaborate incubators can also include the ability to lower the temperature or the ability

control humidity or Co2 levels . This is important in the cultivations of mammalian cells, where the

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relative humidity is typically > 80% to prevent evaporation & a Slightly acidic PH is achived by

maintaining a Co2 level of 5%.

HOT AIR OVEN : The oven uses dry heat to sterilize articles. Generally,

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they can be operated from 50 to 300 degree centigrade. There is a thearmostat controlling the

tgemparature. These are digitally controlled to maintain the temperature. These are digitally

controlled to maintain the temperature. Their double walled insulation keeps the heat in and

converse energy, the inner layer is being the poor conductor and other layer being metallic. There

are also an air filled space in between to aid insulation. An air circulating for helps in uniform

distribution. An air calculating for helps in uniform distribution of the heat. These are fitted with the

adjustable wire mesh plated trays or aluminium trays and may have an on/ off rocker switch, as

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well as indicators and controls for temperature and holding time. The capacities of these ovens vary.

Power supply needs vary from country to country, depending on the voltage and frequency used.

They are advantages to use from frequency used. They are advantageous to use as they do not

require water and there is not much pressure build up within the oven, unlike an auto clove,

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making them safer to work with. This also makes them more suitable to be used in a laboratory

environment. They are much smaller than auto cloves but can still be as effective. They can be more

rapid than be as effective. They can be more rapid than an auto claves and higher temperatures can

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be reached compared to other means. However, as they use dry heat instead of moist heat, some

organism like prions, may not be killed by them every time, based on the principal of thermal

inactivation by oxidation.

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These are highly used to sterilize articles that can withstand high temperatures and not get burnt,

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like glassware and powders.

Water Bath:- A water bath is a device that maintains water at a constant temperature. It is used in

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the microbiological laboratory for incubation.

At the beginning of the lab period, you should check the water bath to see if it is turned on, set at

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the right temperature and filled with water. If you are using the water bath for an experiment you

should check the temperature frequently to make sure that the water bath is maintaining the

proper temperature.

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Water Bath Control:- Temperature Control- All water baths have a control to set temperature. This

control can be digital. Often there is an indicator light associated with this control. When the light is

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on the water bath is heating. When the water bath reaches the set temperature, it will cycle on and

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off to maintain constant temperature.

Safety Control:- Most water baths have a second control called the safety. This control is set at the

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maximum temperature the water bath should attain. It is usually set just above the temperature

control. Often an indicator light is associated with the safety control. If the water bath reaches the

temperature that the safety control is set at, the light will go on.

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Anaerobic Jar:- An Anaerobic jar is an instrument used in the production of an anaerobic

environment. This method of anaerobic as other is used to culture bacteria which die or fail to grow

in presence of oxygen.

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Autoclave:- An autoclave is a device used to sterilize equipment and supplies by subjecting them to

high pressure saturated steam at 121O c for around 15-20 minutes depending on the size of the

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load and the contents.

The name comes from Greek ‘auto’- ultimately meaning self, and lalin ‘clavis’ meaning key- a self-

locking device. Autoclaves are widely used in microbiology, medicine mycology, dentistry and

prosthetics fabrication. They vary in size and function depending on the media to be sterilize.

Autoclaves is mainly used for sterilization of various microbiological media this helps in avoiding

crass contamination are used to get error free result as they can be used for specific purpose as

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required. Therefore autoclave is very important in any microbiology laboratory.

Colony Counter:- Biological procedure often rely on an accurate count of bacterial colonies and

cells. Colony counters are used to estimate a liquid culture’s density of microorganism by counting

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individual colonies on an agar plate, slide, mini gel, or petri dish. Typical applications include ames

testing, bacterial mutation assays, and E. Coli bacterial colonies.

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Instruments accommodate various size and formats, including units that can scan plates up to 300 x

300 mm, and are optimized for use with UV illumination, white light, fluorescent, and/ or green

fluorescent protein colonies. The counting can be accomplished manually, often with touch pressure

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and a digital counters improvements centre on increasing precision and accuracy, such as the ability

to detect smaller colonies in low- contrast media, illumination systems that increase visibility, direct

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image scanning and analysis, and IQ/OQ documentation.

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Introduction Chamber:- “Introduction” has a specific meaning for procedures done in vitro. These

includes the transfer of micro organisms into and from laboratory apparatus such as test tubes and

petri dishes in research and diagnostic laboratories, and also in commercial application such a

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brewing, baking and the production of antibiotics. In almost all cases the material inoculated is

called the inoculums, or less commonly the inoculants, although the term culture is also used for

work done in vitro.

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The hood or the chamber for inoculation has a bacteria fungae – free atmosphere in it with ultra

violet germicidal light and it is used in biological culture studies. It is made of ms sheet attractively

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painted. Two holes for two hands are provided.

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It has viewing glass. It is a tabletop model. Two door on two door on two sides to keep glassware.

Almost air light chamber.

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Glassware:- Handling and Care:- In microbiology, clean glassware is crucial to ensure valid result.

Previous used or new glassware must be thoroughly cleaned. Laboratory ware and equipment that

are not chemically clean are responsible for considerable losses in personnel time and supplies in

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many laboratories. These losses may occur as down time when experiments clearly have been

adversely affected and as invalid data that are often attributed to experimental error. Chemical

contaminants that adversely affect experimental results are not always easily detected. This practice

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describe the procedures for producing chemically clean glassware.

General Glassware used in a laboratory:-

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1. Beaker:- A flat bottomed, cylindrical, container, often displaying volume measurements on

the side used for heating mixing and stirring liquids.

2. Burettle:- A long tube, with volumetric markings on the wall of the cylinder and a top at the

base, used for dispensing to secure samples for spectroscopic experiments, it is closed at one end of

the tube.

3. Curette:- A small tube of circular or square cross section, designed o secure samples for

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spectroscopic experiments, it is closed at one end of the tube.

4. Flasks:- There are many different types, shapes and size of flask but in general a flask is a

large bodied container taping off to a thin bottle neck, used to hold, heat mix and measures

volumes of liquid.

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5. Gas Syringe:- An inner glass chamber within a glass holding barrel, the syringe is used to

measure resultant gaseous products from a chemical reaction.

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6. Glass Tubes:- Gain tubes, like flask comes in all shape and sizes for a variety of different of

different uses, such as storage or transportation of liquid.

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7. Graduated Cylinder:- Large thin measuring chamber, which allows more accurate

measurement than generally achieved with a beaker.

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8. Petri Dish:- A shallow circular glass container with a lid, used to culture cells.

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9. Pipette:- This is a measuring instruments which comes in various size, for a variety of uses.

Similar in concept to a burette a pipette is able to measure and dispense liquids to precision

accuracy.

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10. Soxhlet Extractor:- Initially designed to extract lipid molecules, this made scientists looking

glass ware instrument can be used to extract solvents.

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GENERAL PATHOLOGY

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Abscess:- It is defined as localised collection of bus in the living solid tissues and this is caused by

acute inflammation as a result of pyogenic organism or chemical irritants.

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Pyogenic Organism:- 1) Some Cocci like streptococcus haemolyticus, staphylococcus aureus,

Pneumococcus, gonococcus, anaerobic coccus etc. 2) Some bacilt like Escherichia coli, proteus

r5
group, pseudomonas group etc.

Atrophy:- Atrophy is an acquired condition and is defined as diminution in size of the organ due to

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decreased in number of cells, size of the cells without any degeneration change and little amount of

replacement by fibrosis.

There are two types of atrophy.

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1. General Atrophy:-

on

a) Senile Atrophy- This is a physiological atrophy. There will be atrophy of different organs and

tissues like endocrine glands. Sex organs, bones and tissues like endocrine glands. Sex organs,

bones, muscles, skin during senility. Atrophy of the breast and uterus in females takes place at the

age of menopause. In long continued wasting diseases and all old age, the breast becomes a smaller

in size, myocardium in slightly thinned out and nutmeg- brown in appearance. Microscopically, the

myocardial fibres show accumulation of small fat globes and lip chrome granules which are called

“wear and tear” pigment. Such type of atrophy is called brown atrophy of the breast.

PD

b) Starvation Atrophy:- This type of atrophy is found in famine, carcinoma of oesophagus,

anorexia nervosa attended with loss of appetite etc. In these condition, less nutrition is the main

cause and there will be utilisation of stored of energy metabolism such as carbohydrate, fat and

protein, cachexia or generalised wasting is seen in tuberculosis or carcinoma due to malnutrition.

12

ob

2. Pathological Atrophy:-

a) Ischaemic atrophy:- There will be atrophy of the affected part due to diminution of blood

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supply.

b) Pressure atrophy:- There will be atrophy of individual cells due to persistent pressure of

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some substances like intercellular infiltration of amyloid, glycogen or due to the pressure of tumor.

c) Discuss atrophy:- This type of atrophy is sen due to the restriction of the use of a part

is
followed by diminished metabolic activity. This is particularly observed in the muscle fibres where

there is immobility of the limbs as in ankylasis of a joint or hemiplegia.

eg
d) Neropathic atrophy:- This is due to destruction of neuromuscular activity where the

nutrition of tissues is not properly maintained as a result there will be presence of feature of

nr
atrophy as found in poliomyelitis, tabes dorsalis syringgomyelia facial hemiatrophy, lower motor

nurone paralysis.

U
e) Endocrine atrophy:- If pituitary gland is destroyed , the dependent endocrine glands like

adrenal gland, thyroid gland and gonads may undergo atrophy as this trophic harmonic stimulation

diminishes.

3
f) Atrophy due to over work:- Proper nutrition and over work may lead to the hypertrophied

.5
condition of muscle but less nutrition and over work will produce atrophy of the muscle.

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Degeneration:- Degeneration indicates unhealthy condition of cells or tissues. When the cells of a

tissue are subjected to sub-lethal or non- fatal injury or abnormal influence, certain retrograde

changes occur in the cells. It is characterised by the accumulation of abnormal substances, either in

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the cell protoplasm or in the tissue structure. The abnormal substances are not visible in those cells

normally. The degeneration may be defined according to the nature of the accumulated material in

the cells.

ve

Factors responsible for degeneration:-

on

1. Deficiency of oxygen supply to the cells-

a) Low oxygen tension in air.

b) Anaemia such as low oxygen capacity.

c) Varcular inefficiency

d) Inability of the cell to utilize oxygen

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e) Passive venous congestion

13

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2. Effects of poisons or chemicals:-

a) Effects chemical poison- Arsenic, lead mercury, gold, etc.

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b) Internal toxic substances-

(i) Toxaemia in pregnancy

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(ii) Toxin in chronic rheumatoid arthritis.

is
(iii) Chronic gout.

c) Bacterial toxins- In case of diphtheria, tuberculosis.

eg
d) Metabolic products- Acetone I diabetes mellitus.

nr
3. Effect of physical agents- such as trauma, electricity, X-ray, heat and cold etc.

U
a) Deficiency of nutrition- Particularly to protein and vitamin deficiency. Deficiency of fat and

carbohydrate are less important. Extra supply of fat to a primarily damaged cells enhance the

3
process.

Types of degeneration:-

1. Cloudy degeneration

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2. Hydrophic degeneration

3. Vascular degeneration

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4. Fatty degeneration

5. Hyaline degeneration

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6. Amyloid degeneration

on

7. Fibrinoid degeneration

8. Glycogen degeneration

9. Mucoid degeneration

Phases of degeneration:-
F

1. Cloudy Swelling

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2. Fatty Changes

3. Necrosis

14

4. Other degeneration changes.

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Termination of degeneration:-

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1. Recovery of cell when there is removal of the irritants, the cells come to normal both in size

and appearance.

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2. When the irritants is persistently present, the cells undergo progressive degeneration.

Ultimately there is lysis of the nucleus and the cell will die.

is
3. Cloudy swelling is the earliest type that is seen in a cell. The affected organs looks dull,

opaque like parboiled rice.

eg
Embolism:- Literal meaning of embolus is plug or a foreign body. An embolus is insoluble material

in the blood vessels and embolism is impact of an embolus carried by the blood stream from its

nr
point origin to different parts where it sets impacted resulting vascular obstruction.

Types of Emboli:-

U
1. Solid embolus:-

3
a) Detached thrombus- detached of an aseptic thrombus.

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b) Solid foreign body- Fragments of body specules, lead salt etc.

r5
2. Liquid embolus:-

a) Oily embolus- Liquid paraffin or oily injection.

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b) Fat containing cells (fat embolism)- Embolism is caused by fat, after the fracture of a long

bone or trauma to a fatty tissue.

ve

c) Amniotic will be secured in the uterine venous sinus. These droplets become covered up

with fibrin shell and develop emboli.

on

3. Gas embolus:-

a) Air embolus:- Such as embolism will be caused by the injury of veins of neck, ratified

pneumothorax, tubal insufflations, careless intravenous injection of air bubbles etc.

b) Nitrogen gas embolus- such as caison’s disease or diver’s sickness.

F
PD

4. Cellular embolus:-

i) Malignant cells embolus

ii) Red cell embolus in polycythaemia and white cell embolus in leukaemia.

15

ob

5. Parasitic embolus:- Such as malarial parasitic, lava of hook worm and ascaris lumbricoids

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etc.

te
Gangrene:- Death of soft tissues in mass with superadded putrefaction is called Gangrene. The

putrefaction is carried out by proteolytic enymes liberated by the dead bacteria or by the

is
saprophytic putrefying bacteria. There will be the formation of some gases like carbon dioxide,

hydrogen, ammonia, hydrogen sulphide, indole and skatole due to the proteolytic activity.

eg
Gangrene may be classified as follows:-

nr
1. Primary Gangrene:- This type of gangrene is produced by the invasion of some organism

U
and these organism are responsible for both death and putrefaction of the tissue. Best example is

gas gangrene where the causative organisms are claostrdium welchii, clostridium septicum etc.

3
2. Secondary Gangrene:- This type of gangrene is produced due to ischaemic condition of the

tissue. This ischaemic condition is due to the vascular obstruction . The secondary gangrene is

divided into:-

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a) Dry Gangrene- This type of gangrene is produced due to he obstruction of asterial path only

and presence of small number of putrefying bacteria. Senile gangrene is the best example.

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b) Maist or wet gangrene:-This type of gangrene is produced due to the obstruction of both

the arterial and venous path. Strangulated hernia is the best example. The aetiological classes of

gangrene may be as follows:-

ve

1) Gangrene due to acterial occlusion :- Peripheral artery may be occluded. It may be sudden

or gradual.

on

Sudden Occlusion:- due to the embolism or thrombosis in a great vessel like popliteal artery or

prolonged application of a tourniquet or complete tear of a major peripheral vessel will lead to

traumatic gangrene.

Hyperaemia:- Hyperanemia is due to opening of new capillary and collection of blood in capillaries

of tissue of any part of the body where as congestion is accumulation of blood in small vessels live

venules or arterioles. Hyperaemia may be divided into active and passive types:-
PD

1) Active Hyperaemia:- Due to the dilation of both arterioles and capallaries, there will be an

increased blood flow in a particular area. Hyperaemia in inflammation is the best example. The

features of active hyperaemia is, the part will be warm, bluish red in colour, swollen, the capillaries

are dilated and engorged.

e
ob

16

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2) Passive Hyperaemia:- This is also called passive venous congestion. It is due to the

stagnation of blood. Here the blood flow will be slow, the part will be purple in colour and cold due

to the less incoming of arterial blood. Passive hyperaemia may be:-

te
a) Local- It may be due to the obstruction in the venous passage and it may be:-

is
1) Acute obstruction:- A thrombus or an embolus may occlude the venous passage. Volvulus

and twisting of pedicle of an ovarian tumor may cause acute obstruction.

eg
2) Chronic local venous congestion:- Due to the portal obstruction, there will congestion in

liver.

nr
b) General:- It is due to the difficulty in venous return to the central area like cardiac and

pulmonary area. It may be due to:-

U
1) Acute passive venous congestion:- It is found in pulmonary oedema due to left ventricular

failure.

3
2) Chronic generalized passive hyperaemia :- It is found in congestive cardiac failure.

.5
r5
Aplasia:- Aplasia denotes that there will be complete or almost complete failure of development of

an organ. Due to aplasia, the organ may totally absent or may be replaced by a mass of fibrofatty

tissue. It is generally found in kidney, gonads and adrenal glands.

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Hypoplasia:-Hypoplasia is a congenital condition of failure of development of an organ, in which

ve

organ will fail to reach in adult form. Hypoplasia in kidney is an example where the kidney will not

grow sufficiently.

on

Hyperplasia:- It is defined as increased in size of the orgin due to increased in number of cells but

the size of the cell may or may not be altered.

Hyperplasia may be caused by:-

F

1. Chronic irritation by infection or foreign body.

PD

2. Chronic stimulation by hormone or other chemical agents.

Involved the tissue:-

e

1. Endocrinal glands such as pituitary, thyroid, parathyroid, adrenal glands, etc.

ob

2. Target organs such as breast, endometrium and prostate.

17

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3. Lining epithelial cells of the surface- such as skin mucous membrane of respiratory tract and

gastro intestinal tract.

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Types of Hyperplasia:-

is
Hyperplasia differs from neoplasm by the fact that the changes recedes when the stimulus is

eg
completely taken off. The types are:-

1. Physiological hyperplasia:- Hyperplasia f different endocrine and their target organs will take

place at puberty such as puberty goiter.

nr
2. Pathological hyperplasia:- Pathological hyperplasia will be found in the following endocrines

U
and clinical manifestation will be present.

i) In pituitary- Gigantism Acromegaly.

3
ii) In Thyroid- Grave’s disease.

iii) In Parathyroid- Osteoporosis

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iv) In adrenal Cortex- Cushing’s syndrome.

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Hypertrophy:- Hypertrophy is defined as increased size of an organ due to increased in size of

individual cells but number of cells remain normal. Hypertrophy is frequently observed in relation to

muscle tissue and this may be found in:-

ve

a) Heart:- Due to mitral sterosis, aortic stenosis, aortic incompetence, hypertension, etc,

hypertrophy of heart may be found.

on

b) Hypertrophy of stomach of gastro:- intestinal tract hypertrophy of stomach may be due to

pyloric stenosis. In case of intestinal obstruction, proximal part of obstruction will show

hypertrophy.

c) Uniary tract:- Due to enlarged prostage or stricture urethra, hypertrophy of bladder will be

found.

d) Hypertrophy of skeletal muscle:- Hypertrophy of biceps, triceps, deltoids etc. may be found

PD

in athlets or in manual worker due to over work.

Inflammation:- Literal meaning of inflammation is flaming or burning but pathologically it means a

dynamic process by which series of changes that take place in living tissues when it is insufficient to

cause immediate of the tissue.

e
ob

18

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Immunity:- Immunity means resistance or non susceptibility to contagion. The resistance offered by

the host to an infecting organism constitutes a complex subject to which the term immunity is

applied.

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is
Nature of immunity:- Immune is a protective mechanism in our body. Utllising this system, the body

kills micro organism, neutralizes toxin and eliminates anything which is foreign to the body. Recently

eg
it has been recognized that we have immunity against neoplasm and incidence of neoplastic growth

is high among those who have attered immune mechanism. But this system is not always without

harm to the body. It is described as a two edged sword. Immune reaction sometimes becomes so

nr
violent that it produces necrotizing inflammation. There may be some sort of haemato vascular

responses which simulate to those found in hypersensitive phenomenon. In fact hypersensitivity is

U
an altered mmune response, which s harmful to the body.

A person may be immuned by natural body defence by acquired means. Immunity may be acquired

3
from:-

i) Inheritance (since Birth)

.5
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ii) After an attack of small diseases like small pox.

iii) Antificial inoculation.

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Infarction:- Infarction is defined as, it is a process of coagulative necrosis as a result of ischaemia

ve

resulting from sudden arrest of blood supply due to the occlusion o physiological or anatomical

endarteries. The effect of vascular obstruction may be judged by the texture of the tissue, spread of

onset of obstruction, nature of the vessels involve and state of collaterals.

on

Both the artery and vein may be involved and they artery will be involved. The sites of infarction are

common in lungs, bowels, etc.

Inflammation:- Literal meaning of inflammation is flaming or burning but pathologically it means

dynamic process by which series of changes that take place in living tissues when it is injured by a

PD

various irritants provided the injury is insufficient to cause immediate death of the tissue.

Causes of inflammation:-
e

1. Living (Animate): (a) Bacteria like- streptococcus, staphylococcus, My. tuberculosis (c)

ob

diptheriae, etc and bacterial toxins.

19

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b) Protozoa- like E. Histolitice.

c) Helminths- like Ancylostoma duodenale, bancrofit.

te
d) Fungi- like mycoses.

is
e) Allergy- due to some organism.

2. Non-living (inanimate) (a) Physical agents- such as heat and cold, electric burns, x-ray,

eg
radium etc.

(b)Chemical agents-like corrosive acids, alkali or poisons.

nr
(c) Foreign body- ligature like silk worm, gut or any metallic agents.

U
(d) Dead tissue:- like sequestrum formed in astomyelitis

(e) Any type of trauma.

3
Jaundice:-
.5
Jaundice or Ictesus is a condition characterized by yellowish colouration of skin, selera

and mucous membrane due to hyperbilirubinaemia only when the bilirubin level in blood is above 2

r5
mgms per 100 cc of blood with the exception of saliva, tear, milk and secretion of common bile due

and C.S.F. due to bilirubin does not pass the blood brain barrier except in the immediate neonatal

period. Internal tissues are also stained.

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Clinical jaundice:- When blood, bilirubin level is above 2 mgms per 100 CC of blood, it clinically

ve

manifested such as yellowish colouration of skin, sclera and mueous membrane. This is called

clinical jaundice.

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Latent jaundice: When serum biliorubin level is less than 2 mgms per 100cc. of blood but above

normal range, its presence can be often detected by serum analysis. This is called Latent jaundice.

Shunt hyperbili:- In certain haematalogic diseases, notably thalassaemia, pernicious anaemia or

megaloblastic anaemia, refractory normoblastic anaemia, and enythropoietic porphyria there may

develop of haemoglobin due to defective metabolism of haem part of haemoglobin along with

PD

ineffective erythropoiesis in the bonemarrow. The excessive intermedullary destruction of

haemoglobin, which never appears in the circulating bloods, is known as ineffective erythropoietin.

Classified and types Juandice:-

e
ob

1. Mc Nee’s classification (based on actiology)-

20

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a) Haemolytic type or overproduction type.

b) Obstructive or cholestatic type.

te
c) Toxic or Hepatocellular type.

is
2. Rich’s classification (based on mechanism or pathophysiology)

a) Retention jaundice

eg
b) Regurgitation jaundice

nr
3. Modern Classification (Based on category of bilirubin in hyperbilinubinaemia.

A)Unconjugated category-

U
(1)Prehepatic type- Excessive production of bilirubin haemolytic or overproduction state.

(2) Hepatic type- (a) Defective transport of bilirubin from sinusoidal blood into hepatocyte- Gilbert

3
syndrome, toxicity.

.5
(b) Inability to conjugate- crigler- najjar syndrome, neonatal jaundice, drug toxicity.

r5
B) Conjugated category:-

(a)Hepatic type- (1) Injury to the hepatocyte- virehapatitis, cirrhosis. It is called Hepatocellur.

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(b) Cholestatic – (1) Intrahepatic cholestasis which is due to the defective bilirubin transport to the

canaliculus found in viral hepatitis, drug toxicity

(2) Pesthepatic type:-

ve

(a) Mechanical obstruction in the biliary tree found in carcinoma in pancreas or carcinoma in

common bile duct, etc.

on

Oedema:- Oedema means abnormal accumulation of fluid in the tissue space and serous sacs. The

fluid may be abnormally accumulated in loose sub- cutaneous tissue like lower eyelid, tissues in

external genitakia, serous part of the body like leg etc.

F
PD

Type of Oedema:-

1. Fluid Oedema:- In this type fluid collects in intercellar space. This type of oeema is pitting

type, there will be pit on pressure. Here the tissue will be pale, swollen, soft and inelastic.

2. Solid oedema:- In this typ, there will be no pit on pressure, so non- pitting type. The tissue

will be pale, swollen, tough and inelastic. This is found in filariasis due to lymphatic obstruction.

ob

21

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3. Tumour:- Literal measuring of tumor is swelling and pathologically it means new growth or a

neoplasm. A neoplasm is an automous and endless conco-ordinated new growth that means it

follows the low of natural growh can continue even after the slappage of the stimules which has

te
eveked he change.

is
Clinical Pathology

eg
Examination of urine

a) Collection of urine for screening purposes – for chemical and microscopic examination, a

nr
clear voided early morning specimen and also two hours after the principal meal are suitable.

U
b) Collection of urine for quantities analysis – a twenty four houses specimen is collected for

many assays such as for the detection of urobilinegen hormones, proteins, electrolytes and

quantitative cell count. Errors in the result of quantitative urine tests are more after related to

3
collection problems.

c)

.5
Outline of procedure for routine urine analysis

1.Physical Examination

r5
i) Quantity or amount or volume

ii) Colour or appearance

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iii) Odour

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iv) Transparency and Deposits

v) Reaction (pH) of urine

on

vi) Specific gravity

2.Chemical examination

i) Test for sugar

ii) Test for protein (albumin)

PD

iii) Test for kitones (Acetones)

iv) Test for Bile

v) Test for bite salt

vi) Test for bile pigment

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vii) Test for ehrlich’s reacting substance

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viii) Test for urobilinogen

ix) Test for porphysis

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x) Test for uric acid

is
xi) Test for creatinine

xii) Test for 5 Hydroxy Indole Acetic Acid (5 HIAA)

eg
xiii) Test for Melanin

nr
xiv) Test for Blood

U
3. Microscopical examination -

i) Cellular examination like R.B.C bucocyte, pus cells etc

3
.5
ii) Casts

iii) Cryslals and phosphates

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iv) Parasites

v) Spermatozoa

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vi) Bacteria

vii) Other elements

ve

After proper collection and identification of urine sample, urine should be examined under the

following headings and for the routine examination of urine, about 6 to 8 oz urine should be

on

collected in a clean container.

Physical Examination

A) Quantity or volume of urine

Volume of the urine in 24 hours collection has a great value. Average excretion of urine in 24 hours

PD

is about 1,500 c.c. to 2,500 c.c (The factor regulate urinary volume amount of solute in the body to

be eliminated, fluid intact and power of kidney to concentrate urine.

B) Parnchumal disease of kidney Olgaria :- These will be formation of urine less than 500ml in

24 hours, the condition termed as oliguria.

e
ob


Causes :- In several infection, these may be urinary output 2 to 3 liters in 24 hours but it is

insufficient to excrete solute present in the body.

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i) In hypotension

ii) In cardiac failure

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iii) In acute glomerulonephritis

is
iv) Toxic or inchaemic nephrosis

v) Bilateral hydronephrosis

eg
vi) In those disease given rise to water and salt depletion such as in

nr
vii) Pyclonephritis

viii) Addison’s disease

U
ix) Hypopituilarisum

x) The diurectic phase of acute renal failure

3
.5
xi) Acute or choronic diarehoea

xii) Intestinal fistulae

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
Dehydration - In prolonged vomiting, diarrhoea, or excessive seating in febric state, severe

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dehydration and haemocencentration will occur.

ve

 Anuria : Anusia is the absence of excretion of urine for 12 hours

 Causes : Acute renal failure

on


Circulatory insufficiency - Excessive blood loss, severe shock, diarrhoea, vomiting, burns,

transfusion reaction and crush injury.

 Nephrotoxic agent like heavy metals, sulphonamides, etc


Other disorders – Acut glomerulonephsitis, malignant hypertension, septicaemia,

bilateral renal cordical necrasis and eclampsia, etc.

PD

2. Obstructive Unopathy-

i) Acute pyclonephritis

ii) Stone in the kidney

ob

iii) Carcinoma in the pelvic organs

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iv) Polyuria - Persistent increase in urinary output is polyuria and it may be more than 2000ml.

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 Causes : Diabetes mellitus

is
i) Diabetes insipidus

ii) Chromic parenchymal renal disease with uromia

eg
iii) Hyperparathyroidism

nr
iv) Excessive drinking of water

v) Nervousness or emotional disturbance

U
vi) Hydronephrosis

vii) Recovery stage of congestive cardiac failure.

3
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viii) Recovery state of nephritic syndrome

ix) Polassium depletion

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x) Addison’s disease

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
Diabetes insipidus : It is a condition caused by deficient or absence of Anti- diurrtic

hormone (A.D.H) characterised by excretion of huge amount of dilute, pale but otherwise

normal urine, excessive thirst and polydipsia.

ve

 Investigation will be done in case of Diabetes insipidus

on

 Urine examination :

i) Total daily urinary output- increased

ii) Specific gravity- 1005 to 1010

iii) Colour – pale

PD

iv) Sugar, albumin, casts - Nil

v) Blood sugar level – Normal

Colour or Appearance

ob

The usual colour of urine is pale – yellow. Sometime the colour of urine is changes due to the

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presence of urobilins urochromes, urocrythrix and prophyrin. Yellow brown clour of mine is mainly

due to the pigment. Acid urine is usually darker than alkaline urine. Diluted urine is pale colour and

concentrated urine is highly coloured.

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 The changes occur of urine in different condition.

is
1. Cloudy urine due to presence of

eg
a. Phosphates, carbonates, urates, uric acid

b. Leucocytes, red cells

nr
c. Spermatozoa, prostatic fluid

d. Mucin, nucous threads, pus

U
e. Faecal contamination

3
2. Milky white due to presence of

a.

b.
Many neutrophils (Pyuria)

Fat

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c. Lipiduria

d. Chyluria, milky

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e. Vaginal cream

3. Yellow – Due to presence of acriflavine

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4. Deep yellow - Due to presence of bile pigment in case of obstructer jaundice

on

5. yellow – orange - Due to presence of

a. urobilin in excess

b. bilirubin

c. concentrated urine

6. Yellow – green - Due to presence of bilirubin and biliverdin in case of obstructive jaundice

PD

and hepatocellular jaundice

7. Yellow – brown – Due to presence of bilirubin and bilivendin

8. Red - Due to presence of

a. Haemoglobin

ob

b. Red blood cells

c. Myoglobin

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d. Porphyrin

e. Aniline dye

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f. Menstrual contamination

is
9. Red – purple - Due to presence of prophyrins

10. Red – brown - Due to presence or

eg
a. Red blood cells

nr
b. Myoglobin

c. Haemoglobin on standing

U
 Odour -

3
1. The normal odour of urine Aromatic odour is the characteristic odour of normal urine and it is

due to the presence of vilatilo acids and ‘urinod’ like substance

2. The abnormal odous of urine

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a. Ammoniacal odour - Decomposition of proetisn

b. Fauity odour- In diabetes ketosis where acetone liberates

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c. Putrid odour- Decomposition of pus and formation of Hydrogen sulphide

d. Faccal odour - contamination of faced matter in urine

ve

e) Transparency and Deposits - Normal urine is clear and leaves no deposit

f) The different types of deposits present in abnormal urine

on

1. Unorganised deposits

a. In acid urine

i. uric acid

ii. amorphous acid, potassium, sodium, urates

PD

iii. choesteroal, sulphonamide, leucine

iv. calcium oxalates

b. In alkaline urine –

i. Triple phosphate

ob

ii. Amor.phosphate

iii. Ammonium biurate

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2. Organised deposits

a. R.B.C. in microscopic haematuria, lencocytes or pus cells

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b. Epithelial cells

is
c. Tube casts - all the casts

d. Spermatozoa

eg
e. Parasites like micro-filaria bancrofti in chylous urine

nr
f. Malignant of urine

U
 Reaction of urine - Urine is acidic reaction. Urine pH is 6.

3

Detect the reaction - with the help of litmus paper – Blue or Red Indicators are methyl red

and bromthymol blue

i) Blue litmes paper terms Red – Acidic

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ii) No change with Blue litmus paper – Neutial or Alkaline

iii) Red litmus paper turns blue- Alkaline

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iv) No change with Red litmus paper- Neutral, such as no change with both blue and red litmus

paper- Amphoteric.

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 Causes of urine Acidic and alkaline –

on

i) The urine in acidic due to the presence of Monosodium salts of phosphoric acid and a little

amount of free organic acid. The ratio is Na H2 PO4 : Na2 HPO4 = 4 :1

ii) In urinary tract infection, except in Esch, cali and tuberclebacilli (T.B) infection where urine is

acidic,

iii) After vomiting

iv) After a meal (alkaline tide)

PD

v) When urine decomposes, urea which is present in urine, decomphoses to from ammonia.

vi) After intake of alkali

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ob

 Specific Gravity

Specific gravity of urine is measured with the help of urinometer. This is a hydrometer adopted to

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measure the specific gravity of urine at room temperature. The specific gravity is a measure of the

weight of particles in solution. If indicates the degree of the urinary total solute concentration.

Inability to conceeatrate or dilute urine is an indication or renal disease or hormonal deficiency

te
(ADH).

is
 Normal Specific gravity of urine

1. In case of Newborn (first few days) - 1012

eg
2. in case of Infants – 1002 to 1006

3. In case of Adult - 1005 to 1033

nr
4. In case of Adult (normal fluid intake) – 1016 to 1022

U

Specific gravity of distilled water is 1 and it is converted into 1000, so specific gravity of

urine in adult is aclually 1.016 to 1.022.

3
 On an average specific gravity of urine in adult is 1010 (1.010)

.5
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 The identification points

1. Bulb at the bottom filled with necessary

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2. constriction above the bulb

3. Graduation –Starting from 1000 above and increasing downwards upto 1060.

ve

 Chemical Examination

on

 Test for sugar – I. Fehlings test.

C
F

 Composition of Fehling’s solution

PD

Fehling’s No. – 1

Crystalline copper sulphate 34.64 gm

Distilled Water 500.00 ml

Fehling’s No. 2

ob

Potassium sodium tartrate 175.00 gm

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Potassium hydroxide 100.00 gm

Distilled water

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Experiment –

is
Equal volume of Fehling’s No. 1 and Fehling’s No 2 are taken in a test tube and it is boiled. Then

equal volume of Fehling’s No. 1 and Fehling’s No 2, urine will be added to it and then the whole

eg
mixture is boiled.

 Observation

nr
Heavy red or yellow perceptible will appear

Inference –

U
 Presence of sugar or dextrose

3
2. Benedi ) :-

Composition of Benedicts Solution (Qualitative)

a. Copper Sulphate
.5
17.30 gm (cryslallised)

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b. sodium citrate 173.00 gm

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sodium carbonate 200.00 gm

Distilled water to make upto 1000.00 cc with the aid of heat (a) is dissolved in 100CC water and (b)

in another beaker in 600 C.C water. It is mixed thoroughly and then it is made upto 1000 C.C

ve

 Experiment –

on

5 C.C of Benedict’s solution is taken in a test tube and then it is heated and boiled. Then 8 to 10

drops of urine are added. It is vigorously heated for 1 to 2 minutes and then it is cooled under tap

water.

The entire body of the solution will be filled with a collcidal precipitate which may be green, yellow

or red depending on the presence of increased sugar.

PD

Inference –

Presence of sugar or dextrose. Depending on the colour of the solution, percentage of sugar present

in urine can be calculated.

1. Green opalescent fluid blue in standing - 0.1%

ob

2. green opalescent fluid with slight yellow precipitate - 0.2%

3. Green colour with slight orange precipitate -0.3%

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4. Definite orange precipitate, the supernatant fluid still tinged with blue – 0.5%

5. Hexy orange brown precipitate, the supernatant fluid still tinged with blue - 1.0%

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6. Heavy bright red precipitate -2.0% or more

is
* Different type of sugar often found in urine –

1. Glucose - Presence of glucose in urine is called glycosuria

eg
2. Lactose - Lactosuria may be detected

nr
i) In normal pregnancy or during laclation

ii) In intestinal lactose deficiency

U
iii) In intestinal disease where lactose activity may be depressed

iv) In lactose intolerance in infant

3
.5
3. Fructose - Fructosuria which is autosomal recessive, found in –

i) During parenteral feeding with fructose and in association with inherited enzyme

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deficiencies.

ii) Fructose intolerance associated with severe vomiting and kidney and liver disease.

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4. Galactose- Galactosuria is due to inherited enzyme deficiency

5. Pentose - Pentosuria may be occur due to

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i) Ingestion of large amount of fruits, causing the excretion of L-xylose and –arbinose in amounts

upto 0.1gm/day.

on

ii) L-xylulose is excreted in bring essential poentosuria in amount of 1 to 4 gm / day.

iii) Familial disorder

6. Surcrose = Sucrosuria may be found in :-

i. After ingestion of very large amounts of sucrose

ii. Sucrose deficiency which associated with intestinal diseases such as sprue.

PD

iii. Sucrose intolerance which is inherited disorder

* Carbohydrates : The carbohydrates are complex non – nitrogenous compounds containing

carbon. Hydrogen and oxygen, the test two components remaining in the same proportion as in

ob

water. They are abdehyde or ketone derivative of polyhydric alcohols. They can be broadly

represented by the formula (C6 H10 05).

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te
1. Menosaccharides - [one unit of simple sugar, [C6H12O6]

is
i) Monose, Diose, triose, tetrose etc (ii) Glucose

ii) Fructose (iv) Galaclase (v) Monnose etc.

eg
nr
2. Disaaccharides - [Two units of simple sugar (C6H10O5)]

i. Lactose - One molecule of glucose and one molecule of galactose

U
ii. Maltose - Two molecules of glucose

3
iv. sucrose - one molecules of glucose and one molecule of fructose.

.5
3. Polysaccharides - [More than two units of simple sugar (C6H100¬5)]. (i) starch (ii) Dextrin

(iii) Glycogen (iv) Inuline (v) cellulose (vi) Agar agar

r5

Hyperglycaemia – Hyperglycaemia is a condition when glucose level in blood is above

120mgs per 100 cc. of blood by glucose oxidase method or is above 140mgs per 100 c.c of

blood by method including non-sugar reducing substances.

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 Causes of Hyperglycaemia

1. Primary –

ve

a. Insulin- dependent diabetes mellitus

b. Non-insulin – dependent diabetes mellitus

on

2. Secondary –

A. Hyperglycaemia resulting from disease of pancreas

1. Inflammation

a. Actue pancreatitis

PD

b. Chronic pancreatitis

c. Pancreatitis due to mumps

2. Pancreatic infiltration by tuonours

3. Trauma to pancreas

ob
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B. Hyperglycaemia resulting from endocrinal diseases

1. Acromegaly

te
2. cushing’s syndrome

is
3. hyperthyroidism

4. hyperthyroidism

eg
nr
C. Hyperglycaemia caused by drugs

1. steroids

U
2. oral countraceptives

3
.5
D. Hyperglycaemia related to the major diseases

1. Chronic ranal failure

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2. chronic liver diseases, cirrhosis of liver

3. infection

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E. Miscellaneous hyperglycaemia

ve

1. During pregnancy

2. myocardial infarction

on

3. head injury

4. Subarachnoid haemorrhage

5. increased intracramial tension

F
PD


Hypoglycaemia - Hypoglycaemia is defined as a syndrome characterised by low plasma

glucose and associated group of symptoms which are relived by ingestion of food or

carbohydrate. In the hypoglycaemic condition, glucose level in the blood is below 50mgs per

100 cc of blood by glucose oxidase method or is below 60 mgs per 100 cc of blood including

non-sugar reducing substance.

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 Common causes of hypoglycaemia

1. No anatomic lesion present

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A Fasting plasma glucose level normal

a. Relative hypoglycaemia

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i. Functional hypoglycaemia

is
ii. Mimentary hypoglycaemia

iii. Diabetic and impaised glucose tolerance

eg
B. Fasting plasma glucose level low

nr
1. Ethanol – induced hypoglycaemia

2. Drug induced hypoglycaemia such as in insulin, thanol, sulphonylurea.

U
11. Anatomic lesson present

1. Insulinoma

3
.5
2. Extrapancreatic neoplasm

3. Adrnocortical insufficiency

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4. Hypopituitarism

5. Hypothyroidism

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6. Glycogen storage disease

7. Massive live disease

ve


Glycasuria - Glycosuria is a condition in which glucose in present in urine and this amount of

glucose will reduce Fehling’s solution on its will reduce Benedict’s solution.

on


Diabetes mellitus - It is the disorder of carbohydrate metabolism caused by relative or

absolute deficiency of insulin leading to the alteration secondarily in the metabolism of fat,

protein, water, electrolytis and acid base balance and conically may be mainfestoted by

polyuria, polyphagia, polydipsia, hyperglycaemia and glycasuria affect the eye, the kidney

and the nervous system.

PD

 Physiological changes produced in diabetes mellitus

1. Alteration in the carbohydrate metabolism –

a. Hyperglycaemai - It is due to the diminished utilisation of glucose by tissue cells and

ob

increased synthesis of glucose in the liver. If may be characterised by glycosuria and polyuria.

b. Increased synthesis of mucopolysaccharide

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2. Alteration in the fat metabolism –

Excessive breakdown of depot fat, ketone body formation hypercholesterolemia are the result in

te
diabetes mellitus.

3. Alteration in the protein metabolism –

is
Due to breakdown large amount of protein, there will be gross weakness of the patient.

eg
4. Alteration in water, electrolyge and acid and base balance Dehydration, hyponatraemia,

hyper polassaemia and acidesis are the results in Diabetes mellitus

nr
 Pathological changes in different organs due to diabetes mellitus.

1. Pancreatic lesion –

U
a. The number of islets greatly diminised

3
b. Hyalinisation of the islets

c.

.5
Hydropic degeneration or vacuolation of the cytoplasm of the islets, or glycogen in filtration

in islets.

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d. Neutrophil infiltration in islets.

2. Renal lesions -
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a. Glycogen in the renal tubules

b. Arteriosclerosis following hypertension

ve

c. Diabetic nephrosis

d. Pyelitis and pyelonephritis

on

e. Papillitis necroticans which occurs in association with acute pyelonephritis. Here the renal

papillae may be increased passably due to ireteral obstruction

3. Vascular Lesion

a. All varieties of arteriasclerosis leading to gangrene coronary artery disease, renal disease,

retinal disease, cerebral haemorrhage etc.

PD

b. Arteriosclerosis followed by hypertension

c. Diabetic microangiopathy

4. Retinal lesion

a. Diabetic retinopathy

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i. Punctate haemorrhage and exudate

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ii. Globular microaneurysms of capillaries in the inner molecular layer of retina

iii. Proliferative retinopathy with fibrosis

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5. Nervous Systems

is
a. Peripheral neuropathy

b. Diabetes amyotrophy

eg
c. Automomic neuropathy leading to sexual impotence and disturbance in bowl movement

and bladder function

nr
6. Skin Lesion –

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i. Lacalised infaction

ii. Yellow colour and yellow modules in the skin due to lipemia result of abnormal fat

3
metabolism

7. Metabolic disorder

.5
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a. Kitoacidesis

b. Diabetic coma

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c. Hypoglycaemic come due to excessive administration of insulin during the treatment of

diabetes

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
Insulin - It is an anti-diabetic hormone secreted by B-cells of islets of Langerhans. It is

albumin in nature. It contains almost all the essential amino-acids except tryptophan. It

on

does not contain carbohydrate. The molecular weight of it is 35,000 dalton. pH of it is 5.4. It

contains sulphur as cystine. It is soluble in delute alcohol. It is soluble in acid but no alkali. It

is destroyed by proteolytic enzymes. It is isolated in pure crystalline from as salts of zinc,

cobalt, cadmium or Nickle.

 Function of Insulin –

a) it stimulates glycolysis

PD

b) it stimulates glycogenesis

c) it prevents neo glucogenesis

d) It presents the formation of ketone bodies.

e) It stimulates lipogenesis

ob

 Complication of Diabetes mellitus –

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1. Diabetes ketasis or Diabetic coma

2. Genito – urinary system

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a. K.W. syndrome

is
b. Pyclitis

c. Cystitis

eg
d. Renal arteriosclerosis

nr
e. Chronic renal failure

f. Vaginitir in case of female

U
g. Fangal infaction in genital organ

h. Impotence and sterility

3
.5
3. Nervous system –

a. Peripheral neuropathy (b) cerebro – vascular accident which will lead to paralysis (c)

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Diabetes anyotrophy

4. Cardio – vascular sytem

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a. Myocardial inchaemia,

b. Hypertension

ve

c. Peripheral arteriosclerosis (senile gangrene)

d. Diabetic microangiopathy

on

5. Respiratory system

a. Pulmonary tubuculosis

b. Pneumonia

c. Broncho pneumonia

PD

d. Lung abscess

e. Gangrene

6. Skin

a. Carbuncle, abscess, boils, fungal infection

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b. Itching

c. Gangrene

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7. Eye –

a. Diabetic retinopathy, (b) Diabetic cataract

te
is
 Parts of Pancreas

Pancreas is a racemose gland. This has two parts.

eg
1. Exocrine part - The pancreatic juices will be secreted by this part.

nr
2. Endocrine part- There are three types of cells such as a, B, y cells islets of Langerham. αcells

which are oxyphil cells and will secrete Glucagon or hyperglycaemic factor. βcells which are

basophilic cells will secrete insulin or hypoglycaemic factor, and y cells are the mother cells of α and

U
β cells. It has been said that the hormone gastrin is secreted by y – cells.

3
 Test for alubumin

1. Caogulation Reaction
.5
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a.Heat and Acetic Acid Test –

Experiment :-
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i)Three fourth portion of a test tube is filled up with clear and fresh urine. Then upper part of the

urine is heated.

ve

Observation :

i)A haziness or or turbidity appears

on

Inference :-

Albumin or earthy phosphates may be present

Experiment : -

A few drops of 10%Acetic Acid is added to the hazy part

Observation :-
PD

Maziness increases, (i) Albumin is present, Haziness disappears (ii) Earthy phosphate is present

b) Heller’s cold Nitric Acid test :-

e

Experiment :-
ob

Some concentrated Nitric Acid is taken in a test tube. Some urine is poured on it through a pipetle

Observation :-

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Sharp white ring of precipitate at the junction which does not disappear on heating due to

formation of acid metaprotein of albumin is present

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Inference

Albumin is present

is
eg
2. Precipitating Reaction

Experiment

nr
Some part of urine is treated with Esboch’s solution

U
Observation

Turbidity or precipitate appears

3
Inference :-

Albumin is present

.5
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Normally urine’s protein is present a scanty amount, upto about 10mg/dl or 150 mg/24 hours. This

amount can not be detected in qualitative test of albumin.

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Different state of protenuria

Heavy proteninusia - heavy protein loss, more than 3 to 4 gr per 24 hours, is characteristically seen

ve

with the nephritic syndrome

a) Primary renal diseases, including idiopathic disease

on

i)Minimal change nephritic syndrome

ii)Membranous glomesulonephritis

iii)Systemic diseases causing renal involvement

F
PD

Causes –

i)Diabetic nephrosis

ii)Lupus nephritis

iii)Malignant hypertension

ob

iv)Toxaemia in pregency

v)Multiple myeloma

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vi)Congestive cardiac failure

vii)Renal vein thrombasis

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viii)Drugs like penicillamine

is
2. Moderate proteinuria – Moderate proteinuria, when protein los sis in between 1.0 to 3 or 4gr per

eg
days is found in –

a) Glomerulae diseases

nr
b) Pyctonephritis

U
c) Multiple myoloma

d) Toxic nephropathy

3
e) Radiation nephritis

.5
3. Mineral proteinuria – Minimal proteirusia, less than 1.0gr per day may be noted in –

r5
a) Chromic pyelonephritis

b) Chronic intestinal nephritis

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c) Renal tubular disease

d) Congenital disease

ve

4. Functional proteinuria - This is usually less than 0.5g. per day. It is found in

a) Heavy exercise

on

b) Cold exposure

c) Fever

d) Dehydration

e) Congestive heart failure

PD

5. Bence Jones Proteinuria - It is found in

a) Multiple mycloma

b) Macroglobulinaemia

c) Malignant lymphomar

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Measurement of Bence Jones protein

a) Heat test- On heating the urine at the temperature between 400c and 600c the protein

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precipitates and redissolves near 1000c due to heat solubility property of this protein. On cooling

the precipitate will appear again.

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b) Protein electrophoresis test - For electrophoretic analysis the urine must be concentrated.

When some part of urine is run over electrophoreic field, a homogeneous band in the globulin

is
region may be seen and this is followed by immunoelectrophoresis to identify the light chain.

eg
• Albumin- Albumin is a simple protein, which is amphoteric in reaction, soluble in distilled

water and salt solution, coagulated by heat acid and alkalies.

nr
• Protein- Proteins are complex nitrogenous substance containing carbon 54%, Hydrogen 7%

Nitrogen 16% oxygen 22% and sometimes phosphorus 0.6% and sulphur 1%. They are essential

U
constituents of protoplasm. The protein molecule is built up by the union of Amino acids molecule.

• Albuminuria – Albuminuria means presence of albumin in urine and it may be due to

3
accidental or renal cause.

.5
Esbach’s Albuminometer : It is used for the quantitative estimaton of Albumin in urine, that

is the total amount of albumin percentage. Albumin in any fluid can be estimated with the help of

r5
this instrument

• Identification points - 1. Graduation - ½ to 12 (from below upward)

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2. ‘U’ Marking

3. ‘2’ Marking

ve

Precautions for the experiment

1. Urine sample must be clear. Filtration should be done if it is not clear, such as turbid or presence

on

of deposit. If it is turbid or there is presence of deposit, the precipitate along with deposit may give

false result.

2. Urine must be acidic and if not, a few drops of 4% Acetic acid should be added to make it acidic. If

the urine is alkaline, the acidity of Esbach’s reagent will be neutralised and the yellow precipitate of

albumin picrate will be diminished.

3.Specific gravity of urine should be in normal limit or 1010. If the specific gravity is high (more than

1025), the precipitate of albumin picrate may float instead of settling at the bottom.

PD

• Composition of Embach’s reagent

Picnic Acid 10 gms

Citric Acid 20 gms

ob

Distilled Water 1000 C.C.

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Yellow precipitate of albumin picrate caused by picric acid.

Citric acid helps in the reaction since the reaction takes place best in medium.

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is
• Estimate the Albumin

Apparatus - Esbach’s Albuminometer

eg
Chemical - Esbach’s Reagent

nr
• Procedure :-

U
i. The given urine to be taken in the instrument upto the mark ‘U’

ii. Then Exbach’s Reagent to added upto mark ‘R’.

3
.5
iii. Then the tube is closed with rubbercork and shaken well.

iv. Then the tube is then set aside on the stand in a vertical position for 24 hours.

r5
v. Yellow precipitation of albumin picrate will be found and this will be measured.

vi. The graduation on the tube represents dry albumin in grams per litre of urine

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• Reading :-

It is 1 (suppose). Then 100 cc urine will contain 1gm of albumin, So, 100 c.c. urine will contain 0.1

ve

gm% of Albumin. If the urine is diluted with water due to high specific gravity, the result will be the

present reading multiplied by the degree of dilution.

on

Normally, urine’s Albumin is not present in urine - But low molecular weight of albumin and in

some physiological conditions, albumin may be present in urine, as in

1. Excessive muscular exercise

2. excessive ingestion of protein

3. prolonged cold bath

PD

4. later stage of pregnancy

5. prolonged standing

Filtering pores of kidney allow only those substance, the molecular weight of which is less than

70,000. The molecular weight of albumin is 70,000. So, it cannot pass pathological conditions when

the membrane is damaged the size of the filtering pore in enlarged and albumin may come in urine

ob

• Pathological conditions

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organic changes in kidney :-

a) acute glomerulorephritis

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b) Sabacute glomerulomophritis

is
c) Tuberculosis in kidney

d) Pyclorephritis

eg
2. Circulatory changes in the kidney :-

nr
a) Congestive cardiac failure

b) Febrile albuminuria

U
c) Torsion of renal view

3. Any cause of systemic Haematuria (blood in urine) like leukaemia

3
.5
4. Tumours of kidney

a) Hypernephroma

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b) Wilm

c) S Tusmour

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5. Endocrional cuases

d) Hyperthrcidism

ve

e) Pituitary disease like Acromegaly

f) Tumours of suprarenal glands

on

6. Irrigation of kidney

g) Bacterial infections

h) Mercury poisoning

• Acute glomerulonephritis : - Acute glomarulonephritis is a non-suppurative inflammation of

PD

kidney in which the burnt falts on the glomeruli. It is caused by an immune process triggered by

streptococcus ‘B’ haemolyticus type.

• Sub acute glomurilonephritis : The aetiology of sub-acute glomerulonephritis is unknown.

An autommune process may be responsible to damage the kidney. The patient may be infected as

there may be lowering of gamma globulin and water logged condition of the tissue.

ob

• Test For Acetone

1. Rothera’s Test :

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Experiment : 5ml of urine is saturated with Ammonium Sulphate (or Ammonium chloride). Then a

few drops of freshly prepared sedium Nitropruside solution is added. Then strong Ammonia (Liquor

Ammonium forte) is added with the help of a piptte

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Observation : A violet or permanganate ring at the junction of saturated urine and Ammonia

is
solution.

Inference : Acetone is present

eg
2. Iodoform Test :-

Experiement: 5ml, of urine will be taken in a test tube. Equal volume of Iodine solution will be

nr
added to it and the colour of the mixture will be iodine colour . then sodium hydroxide (NaoH)

solution is added drop by drop.

U
Observation : Colour of the mixture will be discharge with a yellow white precipitate of idoform

3
Inference : Acetone is present

.5
3. Legal’s Test :-

Experiment : 5ml of urine will be taken in a test tube. English sodium or potassium hydroxide is

r5
added to make it alkaline. Then a few drops of freshly prepared sodium Nitroprusside solution is

added. Then a few drops of glacial acetic acid is added to it.

Observation : A purple or violet red colour appears

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Inference : Acetone is present.

ve

• Acetone and it’s produced in our body

Acetone is a ‘Ketone’ produced by oxidation of n-propyle alcohol (propanel).

on

CO3

CH3CH2CH2OH Co (acetone)

CO5

In our body, increased metabolism of fat result in the accumulation of ‘B’ hydroxybatyrie acid (not a

PD

ketone), Acetocetic acid (ketone) and acetonic in blood. Acetone is also produced partly due to

decomposition of Acetoacetic acid outside the body in urine.

• Sources of ketone bodies

Liver is the main size for formation of ketone bodies.

ob

• Ketosis :- Ketosis is due to overproduction of ketone bodies due to oxidation of fatty acid

with carbohydrates burning and it is not due to their non utilization.

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• Causes of Ketonuria-

1. Diabetic keto-acidosis

te
2. Hypoglycaemia due to anti-diabetic drug application.

is
3. Cerebral haemorrhage

4. Uraemia

eg
5. Hyper asmotic non-ketolic hyperglycaemic coma.

nr
6. Toxaemia in pregnancy

7. Glycogen strong disease

U
8. Starvation

9. In case of severe vomiting and diarrhoea.

3
• Causes of Acetone present in urine
.5
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1. Diabetic mellitus

2. Starvation

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3. In alkalosis

4. Due to ammoniacal decomposition resulting from long standing urine.

ve

• Test for Bile

a) Test for bile salt-

on

1. Hay’s surface tension test:-

C

Experiment:-To some part of urine in a test tube, a little flour of sulphur is sprinkled.

Observation:- The flour rapidly sinks to the bottom of the urine.

Inference:- i) If flour of sulphur sinks at once, bile salts are presents, in a concentration of 0.01% or

PD

more.

ii) If it sinks after gentle shaking, bile salts are present in concentration of 0.0025% or more.

iii)If it remains floating after gentle shaking, bile salts are absent.

b) Test for Bile Pigment-

ob

1. Fouchet’s Test:-

Experiment:- 5 ml of urine is taken in a test tube and 3ml of 10% Barium chloride solution is added

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to it. Then it is mixed and filtered completely. The filter paper is opened out and it is spread on

another filter paper. Then one drop of Fouchet’s regent is added on the centre of the filter paper.

te
Observation:- A greenish blue colour is obtained.

Inference:- Bile pigment is present.

is
Composition of Fouchet’s Regent-

eg
i. Trichlor Acetic Acid:- 25gm in 100c.c. distilled water

ii. 10c.c. of 10% Ferric chloride solution.

nr
2. Gmelin’s test:-

U
Experiment:- A little in pure nitric acid is term in a test tube and urine is added slowly over it down

the side of the tube.

3
Observation:- A play of colours green, blue, purple and yellow.

Inference:- Bile pigment is present

[ This test can be done on a filter paper]

.5
r5
3. Smith’s Test:-

Experiment:- About 1 C.C. urine will be taken in a test tube. A few drops of one percent alcoholic

rte

iodine is added to it.

Observation:- An emerald green forms at the junction.

ve

Inference:- Bile pigment is present.

4. Hunter’s Test:-
on

Experiment:- i) About 5 c.c. of urine is taken in a test tube. Equal volume of 10% Barium Chloride

solution is added to it.

Observation:- i) A precipitate of barium sulphate will appear spun down and washed with water

several times. The precipitate will be taken on a filter paper and few drops of fuming nitric acid is

added to it.

PD

Observation:- A display of colour will be noticed such as green, red, yellow, etc.

Inference:- Bile is a complex fluid contain various substance and it is both a product of secretion and

excretion of the liver. Formation of bile by liver is continuous process. Human bile is yellowish green

in colour, bitter in test, viscid mucoid liquid in consistency. Liver bile is alkaline (ph 8 to 8.6)and gall

bladder bile is neutral or slightly alkaline (ph 7 to 7.6) of slightly acidic (ph 6.8). it is composed

mainly if bile salts, bile pigments, cholesterol, lecithin and inorganic salts like chloride, carbonates

ob

and phosphates of sodium, potassium and calcium. There are two type of bile salts and two type of

chief bile pigments are present in urine. Two types of Bile salts:-

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1) Sodium taurocholate

2)Sodium glycocholale.

te
1)Sodium taurochalate is composed of:-

is
i) Sodium: – it will come from blood.

ii) Tomocholic acid:-is formed by the union of taurine and cholic acid. Taurine is derived from

eg
sulphur containing amino acid cystine.

iii) Cholesterol

nr
2) Sodium glycocholate is composed of

U
i) Sodium - it will come from blood

ii) Glycocholic acid- It is found by the combination of Glycine and cholic acid. Glycine is an amino-

3
acid

iii) Cholesterol

•
.5
Sile of formation of bile salts- liver is the main site for formation of bile salts.

r5
The two chief pigments are:-

1. Bilirubin (golden yellow)

rte

2. Biliverdin (green)

Other pigments are:-

ve

Biliprassin, Bilicyanin, Bilifuscin, Choletelin

on

• Other tests for urine examination

1. Test for urobilinogen:-

C

Experiment:- About 5 c.c. urine will be taken in a test tube. About I.C.C. Ehrlich’s aldehyde reagent is

added.

Observation:- Red colour will appear

PD

Inference:- Urobilinogen is present

Composition of Ehrlich’s reagent:-

i) Para-dimethyl amino-benzaldehyde- 0.7gm

ii)Concentrated Hcl- 150 c.c.

ob

iii) Deionised water- 100 c.c

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Represents of urinary urobilinogen:-

Urobilinogen in urine represents more than one closely related tetrapyrrole derived from bilirubin.

te
It is normally present in urine. It is colourless and lowbile and reacts with Ehrlich’s aldehyde reagent

forming red- purple colour but urobilin which is the oxidation product of urobilinogen is 0.5 to 2.5

is
mg. units per day.

The causes of abnormal variation of urobiliongen in urine:-

eg
1. Urobilinogen in urine will increase in alkaline urine, such as with alkaline tide after meal where

tubular reabsorption is decreaded.

nr
2. it is also decreased in acid urine

U
3. it is increases in liver damage such as in-

a) viral hepatitis

3
b) with drugs or toxic substances

c) some cases in portal cirrhosis

.5
r5
d) fever

e) Dehydration

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4. Absence or diminished urinary urobilinogen found in obstruction jaundice.

5. variable amount of urobilinogen is found in hepotocellular jaundice.

ve

• Tests for Urobilin

on

1. Schlesinger’s test:-

Reagents:-
C

i) Compound solution of iodine (Lugal’s iodine solution).

ii) A saturated alcoholic zinc acetate or zinc. Chloride solution.

iii) A 10% solution of calcium chloride.

PD

Experiment:-

i) 5 ml of urine will be taken in a test tube.

ii) 3 drops of Lugal’s are added to it.

ob

iii) Then 5 ml of 10% alcoholic zinc acetate is added.

iv) It is mixed well and filtered.

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v) If bile pigment is present is present, it should be removed previously by adding 1/5th volume of

10% solution of Calcium chloride.

te
Observation:- A greenish fluorescene develops.

Inference:- Urobilin is present.

is
Test for Porphyrins or Porphobilinogen:-

eg
Reagent:-

Composition:-

nr
(i) 20gm Para dimethyl amino benzaldehyde

U
(ii)1000ml of Hcl

Experiment:-

3
(i) About 2ml of the reagent is taken in a test tube.

.5
(ii) Two drops of fresh urine are added to the reagent.

r5
Observation:- Cherry red colour appears predominantly on the lap of the solution.

Inference:- Porphobilinogen is present

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Porphyrias:- If there is any defective metabolism in porphyrin, the metabolic products will be

accumulated in the system and then these will come out through urine. These substance are called

‘Porphyrias’.

ve

Test for 5 Hydroxy, Indoleacetic acid (5-HIAA)

Reagent:-
on

1. 1gm of 1 Nitroso, 2 naphthol in 100ml of 95% ethanol.

2. Freshly prepared Nitrous acid (0.2ml of 2.5ml sodium nitric solution added to 5 ml of

2NH2SO4

3. Ethylene dichloride.

F
PD

Experiment:-
e

1. In a test tube 0.2ml, urine, 0.8ml distilled water, 0.5ml, 1-nitroso, 2-naphthal solution are taken

and mixed well.

ob

2. Then 0.5ml fresh nitrous acid is added and mixed.

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3. it is allowed to stand at room temperature for ten minutes.

te
Observation:-

is
A purple colour in the upper aqueous layer is observed.

eg
Inference:- 5-HIAA is present in urine.

nr
Interpretation:- Normal excretion of 5-HIAA in 24hours is 1 to 5 mgs. To avoid the hazard, the

U
patient should not take any drugs for 75 hours before the test.

Patients with malignant cascinoid tumors may excrete 5-HIAA upto 350 mgs per day and the test

3
will show a black colour.

Test for Test-

.5
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Reagent- 10% Fecl3 in 10% HCL

rte

Experiment:- 5 ml of urine is taken in a test tube to which 1ml of the reagent is added.

ve
on

Observation:- A gray or black precipitation will form.

Reference:- Melanin is present.

Interpretation: Melanogens are oxidized to melamine. The Hcl prevents phosphate precipitation.

PD

Homogentic acid will also cause a dask colour in urine give a transient blue- green colour with ferric

choride.

Melanin:- Melanin is a pigment derived from tyrosine which is normally present in herirs, skin, and

eye.

ob
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2. Nitroferricyanide Test:-

te
Reagents:-

is
(i) Freshly prepared solution of nitroferricyanide.

[(a) A few crystals of nitropresside, about 3 gm

eg
(b) 10ml of distilled water].

nr
(ii)10% NoaH

(iii) Glacid acetic acid.

U
Experiment:-

3
.5
(i) 2ml of urine is to be taken in a test tube to which three or four drops of freshly prepared sodium

nitro prusside solution are added.

r5
(ii) Then two drops of 10% NaoH are added to it to make the solution alkaline and it is shaken.

rte

Observation:- A red colour will develop if acetonic, creatinine or melamine is present.

(iii) Then two drops of glacial acetic acid are added to it to make it acidify.

ve

Observation and inference:-

on

(i) Green colour- presence of small amount of melanogen.

(ii)Blue turns into black- pressure of large amount of melanogen.

(iii) Purple colour- Pressure of acetone.

(iv) Amber colour- Pressure of creatinine.

PD

Uric acid:- Uric acid is the major product of purine catabolism in man and is formal from xanthine by

the action of xanthine oxidase. Uric acid is derived from three sources (i) catabolism of ingested

nucleoproteins (ii) catabolism of endogenous nucleoprotein and (iii) direct transformation of

endogenous purine nucleotides.

ob

The average adult excerts appromimately 0.4 to 0.8gm of uric acid in urine every 24 hrs. normal uric

acid in blood is 1.6 to 5.5 mgs per 100 c.c

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Test for uric Acid:-

te
1. Murexide test:-

is
Experiment:-

(i) Some portion of urine will be concentrated by boiling and then it will be filtered. The filtered will

eg
be taken in a porcelain basin. Then 2 drops of Nitric acid will be added to it. Then it will be heated.

nr
Observation:- (i) Precipitate will form.

U
Experiment:- (ii) One portion of the precipitate will be added with ammonium hydroxide solution.

3
Observation:- (ii) Purple red colour appears.

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Experiment:- (iii) other portion of the precipitate will be added with sodium hydroxide or Potassium

hydroxide solution

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Observation:- Purplish violet colour appears

ve
on

Experiment:- (iii) other portion of the precipitate will be added with sodium hydroxide or Potassium

hydroxide solution

Observation:- Purplish violet colour appears.

F
PD

Inference:- Uric acid is present.

Test for Creatinine:-

e

Experiment:- some portion of urine will be taken in a test tube and concentrated by boiling. Then a

ob

few drops of sofium nitroprusside solution is added. Then sodium hydroxide solution is added to it.

Observation:- A ruby colour appears which will change to yellow colour.

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Inference:- Creatinine is present.

Creatinine:- Creatinine is anhydride of creatine and is formed by a spontaneous and irreversible

te
reaction. Two percent of creatine is transformed into creatinine. Both creatine and creatinine are

filtered into creatine and creatinine are filtered out through glomeruli but most of cratine will be

is
absorbed from proximal tubules and creatinine will not be absorbed and passes through urine.

Excretion of creatininie through urine will not be absorbed and passes through urine.

eg
Excretion of creatinine is about 0.6 to 1.2 mg/dl

nr
Indicates the pressure of creatinine in urine

U
1. A useful index of renal function

2. Intake of protein in diet

3
3. Degree of hydration

4. Protein metabolism
.5
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5. Reliable screening test or index of renal function test than the blood urea nitrogen (BUN)

test.

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Creatine:- creatine is important in metabolism in that it provides storage of higher energy

phosphate through synthesis of phophocreatine. This synthesized from glycocyamine which take

plae in the kidney, small intestinal mucosa, pancreas and probably in the liver. Normal value of

ve

creatine in blood is 0.2 to 0.6 mgm/dl. Normal excretion of creatine through urine per day in 0 to 40

mgms.

Causes:-1. Skeletal muscle necrosis or atrophy found in

on

a) Trama

b) Poliomyelitis

c) Amyotrophic lateral sclerosis

d)dermatomyosistis

PD

e) starvation

2. Methyltestosterone stimulates increased creatine synthesis by the liver

3. Hyperthyroidism

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4. Diabetic acidosis

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Tests for Blood in Urine

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1. Benzidine test-

is
Experiment:-

Some portion of urine will be boiledand then cooled. Then saturatedsolution of benzidine in

eg
glacialacetic acid is added to it. The mixture will be shaken well. Then hydrogen- peroxide is added

to it.

nr
Observation:-Blue colour will appear.

Inference:- Blood is present

U
3
2. Orthotolidine test:-

Experiment:-

1.
.5
A pinch of orthotolidine is dissolved in 2ml of glacial acetic acid.

r5
2. To this, 10 to 15 drops of urine are added and shaken well.

3. Then 4 ml of 30% hydrogen-peroxide is added.

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Observation:-

ve

Greenish blue colour appears

on

Inference:-
C

Blood is present

3. Guaiac test:-
PD

Reagents:-

i. 1.60 solution of gum guaiac in 95% ethyle alcohol.

ii. Glacial acetic acid.

iii. 3% hydrogen peroxide

ob
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Experiment:-

i. 10 to 15 drops of urine will be in a test tube, the 0.5ml of glacial acetic acid added to it and

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shaken.

ii. About 2ml of gum guaiac is added and mixed well.

is
iii. About 2ml of hydrogen peroxide is added and mixed well.

eg
Observation:- Blue colour appears

nr
Inference:- Blood is present.

U
[Orthotolidine and benzidine test are more sensitive for blood than guaiac test. Benzidine test is not

routinely recommended due to its carcinogenic effect.]

3
Microscopical Examination:-

.5
The microscopically examination will be done after centrifuging the urine. The supernatant fluid will

r5
be poured off and a small amount of sediment is to be taken on a glass slide and covered with cover

glass. Then the urine should be examined under high power objective of microscope.

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The following things can be identified by microscopical examination.

a) R.B.C. in microscopic haematuria

ve

b) Pus cells

c) Casts

on

d) Parasites

e) Spermatozoa

f) Crystals and Phosphates

g) Bacteria

F
PD

Blood in Urine:-

When red cells present in urine, it is called haematuria.

e
ob

Causes:-

Renal Causes:- Tumors

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Calculus

Tuberculosis

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Bladder:- Papilloma

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Carcinoma

Prostate:- Senile hyperplasia

eg
Carcinoma

nr
Other Causes:- Same as R.B.C. in urine.

U
Pus cells in urine:-A few pus cells may be found in the normal urine . but the excess number

indicates that there may be inflammation in any part of the urinary tract such as in bladder, urethra

3
or renal pelvis. Pus cells from vagina may come in urine. A dead neutrophil is a pus cell. Here

neutrophils appear as granular spheres about 12u in diameter. Nuclear segments appear as small

.5
round discrete nuclei. When cellular degeneration occurs, the nuclear detail may be lost.

r5
Epithelial cells in urine:- A few epithelial cells may be present in normal urine. Increased number of

epithelial cells indicates the pathological condition of urinary tract.

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There are three types cells are found in acute tract.

1. Small round:- These cells are found in acute nephritis and they come from deeper layer of urinary

ve

tract.

on

2. Transitional Cells:- these cells are spindle shaped or round shaped or pear- shaped or caudate

from or tadpole shape. Traditional epithelium contains these types of cells. This type of epithelium

is found in the pelvis of the kidney in the ureter, in the urinary bladder and the upper part of the

urethra.

3. Squamous epithelial cells:- These cells are also found in the alveoli of the lungs, the serous

PD

membrane like peritoneum, pleura etc.

Urinary casts in urine:-

e

Casts:- Casts are cylindrical structures present in urine due top coagulation of protein in the renal

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tubules.

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Different types of Casts:-

1. Cellular casts:-

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a) Renal tubular epithelial cell casts:- It is formed due to random disturbance of the tubular

cells and found in

is
(i) Pure renal tubular necrosis.

eg
(ii) Acut and subacute glamerulonephritis with paraenchymal degeneration.

(iii) Viral disease, such as cytomegalovirus disease,

nr
(iv) Heavy metal poising

U
(v) Pyelonephritis.

3
2. Lencocyte casts or Pus casts- It is found in

(i) Acute pyelonephritis

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(ii) Acute glamerulonephritis

(iii) Nephrotic syndrome.

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3. Red Blood cell casts:- It is found in

ve

(i) Acute glomerulonephritis

(ii) IgA nephropathy

on

(iii) Lupus nephritis

(iv) Subacute bacterial endocarditis

(v) Renal infraction

F
PD

4. Mixed cellular casts:- When tubular casts and while blood cells are iidentified in a casts, the

resulting hybrid is called mixed cast, and it is found in tubule interstitial disease.

2. Cast Matrix

1. Hyaline casts:- Hyaline cast are composed of protein and fine granules are present in the cast.

ob

They are found in

(i) Renal disease like chronic glomerulonephritis

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(ii)Congestive heart failure

(iii) Diretic therapy

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(iv) Normal urine particularly in old age.

is
2. Waxy cast:- They can be easily visualized because of their high refractive index. With bright field

eg
microscopy, waxy casts are homogeneously smooth in appearance. These are found in- Tabular

inflammation and degeneration.

nr
3.Inclusive casts:- These are the granular bodies small or large, formed by the aggregation of

U
plasma protein and also cellular elements from leucocyts, red cells remnants and damaged renal

tubular cells. Protein aggregates include fibrinogen, immure complexes and globulins and they pass

3
through damaged glomeruli.

.5
4. Granular casts:- these casts will form due to the aggregation of coarsely granular degenerating

r5
leucocytes or epithelial cells and found in

(i) Chronic glomerulonephritis

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(ii) Chronic pyelonephritis

(iii) Viral infection

ve

(iv) Renal papillary necrosis

on

5. Fatty casts:- fatty material incorporated into the cast matrix from lipid laden renal tubular cells.

These contain refractive fat droplets or fatty degenerated cells. These are found in nephritic

syndrome and heavy proteinuria.

C
F

6. Haemosiderin casts:- Haemosiderin granules are derived from pigment renal tubular cells and

may present in cast.

PD

7. Crystal Casts:- Casts containing urates, calcium or oxalate are occasionally seen and indicate the

diposition of crystals in tubules or collecting ducts and may produce obstruction and haematuria.

e
ob
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Pigments Casts:-

te
1. Hemoglobin Casts:- It appears to red and found in glomerular disease.

is
2. Myoglobin casts:- these casts are red- brown in colour and found in acute renal failure.

3. Bilirubin Casts:- In case of obstructive jaundice, bilirubin casts may be seen.

eg
nr
Crystals in Urine:-

1. Crystal found in normal acid urine:-

U
(a) Amorphous urates (calcium, magnesium, sodium and potassium urate) – In urine the precipitate

may appear pink- orange to reddish brown and is sometimes called “brick dust”.

3
.5
(b) Crystalline Urates:- (Sodium, potassium, and ammonium)- These are small, brown, or colourless

needles like crystals found in slightly acid urine.

r5

(c)Crystalline uric acid:- These are typical four sided, flat and yellow or reddish brown coloured.

rte

Other shapes are- rhombic plates or prisms, oval forms, with poinled ends, wedges, irregular plates.

ve

(d) Calcium Oxalate:- they are typically small, colourless octahedrous which resemble envelops.

Rarely dumbbell or avoid forms are found.

on

2. Crystals found in normal alkaline urine:-

C

(a) Amorphous Phosphate (calcium and magnesium) colourless amorphous granules are seen in

urine in clumps or masses.

F
PD

2. Crystalline Phosphate:- Triple phosphate (ammonium magnesium phosphate)- these are

colourless, three to six sided prism with oblique ends referred to as coffin lids, occasionally fern leaf

like stellar phosphate crystals.

3.Calcium Carbonates:- these are small granules or colourless crystals. These are not small granules

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or colourless spherical crystals. These are not commonly found.

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4. Ammonium Biurate:- These crystals look yellow brown spheres referred to as thorn apples.

te
3. Crystals found in abnormal urine:-

is
(a) Crystals:- It is colourless, refractive, hexagonal plate, sometimes twinned. It is soluble in water. It

is confirmed cyanide- nitroprusside reactions. Presence of cystine in urine is called “Cystinuria” and

eg
indicates metabolic disorder due to congenital renal tubular defect.

(b) Leucine:-these are yellow, oily appearing spheres with radial and concentric striation. These are

found in sever lever damage.

nr
(c) Tyrosine:- These are needless which may be arranged in sheaves or clumps. These may be

U
colourless or yellow. It is found in liver damage.

3
Supermotozoa in Urine:-

.5
A considerable supermotozao in may be present in the normal urine of man.

r5
Parasites:- Parasites like micro- filaria bancrofti in chylos urine.

rte

Bacteria in urine:- Normally bacteria is present in urine. But increase number indicates infection in

urine tract. These bacteria such as Esch. Coli can be.

ve
on

Examination of stool:-

Stool should be collected in clear , clean and dried vessel and should not be mixed with urine. It

should be examined within 2 hours after passage of stool. Purgative or laxative enema may be

induced for the proper collection of stool.

The stool should be examined under the following headings:-

F

1. Physical examination

PD

2. Chemical examination

3. Microscopical examination

Physical Examination:-
ob

1. Amount

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2. Colour

3. Consistency

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4. Odour

is
5. Blood

6. Mucus and Pus

eg
7. Helminths

nr
1. Amount:- Normally 100 to 200gm of stool is passed per day.

U
2. colour:- Normally the colour of the stool is light or dark brown due to pressure of sterecobilirubin.

3
.5
Different colour of stool:-

(i) Yellow colour:-due to pressure of unchanged bilirubin and milk diet of infants.

r5
(ii) Golden yellow colour:- Due to unchanged bilirubin

(iii)Green stool:- Found in diarrhea of infants and may be found intake of green vegetables or may

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be result from the presence of bilirubin.

(iv) Clay colour:- Found in obstructive jaundice

ve

(v) Tarry black colour:- due to the presence of blood and the point of haemorrhage is high up in the

intestine as in gastric ulcer or duodenal ulcer. Oesophageal varix gastric carcinoma, enteric fever

etc.

on

(vi) Dark brown to red stool:- Due to the presence of blood and the point of haemorrhage is near or

in the rectum.

3. consistency: The normal consisting of stool is firmed stool or semisolid.

F
PD

4. Odour:- The colour of stool is offensive and it is due to pressure of indole and skatole.

5. Blood:- Frank blood, present or not, should be observed presence of frank blood in stool is mostly

due to anal fissure, rectal polyp, haemorrhoids rectals, carcinoma, ulcerative colitis and lower gastro-
ob

carcinoma, ulcerative colitis and lower gastro- intestinal tract lesion.

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6. Mucus and Pus:- Presence of translucent gelatinous mucus clinging to the surface of the formed

stool indicates spastic constipation or mucous colitis. It may be found in emotionally disturbed. It is

also found in dysentery, bacillary or amoebic dysentery.

te
is
7. Helminths:- Thread worm, round worm or segment of tapeworm may be found in stool.

eg
2. Chemical Examination:-

nr
(a) Reaction

(b) Occult blood

U
(c) Test for reducing substance

3
.5
(a) Reaction:- Normally stool is acidic or alkaline in reaction acidic is due to carbohydrate in diet and

alkaline is due to protein in diet

r5
With the help of litmus paper or pH paper will detect the reaction of stool. pH of stool is 6.

rte

There are two type of litmus paper such as blue, and red.

(i) Blue litmus paper turn red- Acidic

ve

(ii)No change with blue litmus paper- Alkaline

(iii) Red litmus paper turn blue- Alkaline

on

(b) Occult blood test:- Normally negative

C
F

For the occult blood test of stool the following tests are done:-
PD

(i) Orthotoliline test

(ii) Benzidine test

(iii)Guaiac test

Out of these tests orthotolidine test is more sensitive than the other. Due to carcinogenic effect of

ob

benzidine, use of this test is not recommended now a days.

False positive test may often be seen due to presence hemoglobin or myoglobin derived from

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dietary meat or fish.

So, prior to the test, the patient should be advised not to take meat or fish.

te
is
1. Orthotolidine test:-

Procedure:-

eg
(a) A suspensionof faeces (stool) (1gm in 10ml of distilled water) is made and boiled for a few

minutes (to avoid pseudopositive reaction).

nr
b) After cooling , 2ml of orthololidine, 1% solution in glacial acetic acid is added and mixed well.

U
c) then 2ml of hydrogen peroxide is added.

d) A deep blue or green colour will appear within a minutes indicates presence of blood.

3
2. Benzidine test:-
.5
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Procedure:-

a) A suspension of faeces (stool) is made and boiled for a few minutes

rte

b) after cooling, saturated solution of Benzidine in glacial acelic acid is added and mixed well.

c)A deep blue colour will appear within a minutes indicates presence of blood.

ve

3. Guaiac test:-
on

Reagents:-

i) 1.60 solution of gm guaiac in 95% ethy alcohol.

ii)Glacial acetic acid

iii)3% hydrogen peroxide

PD

Procedure:-

a) 0.5gm of stool will be taken in a test tube and 2ml of distilled water is added to it and mixed well.

b) then 0.5ml of acetic acid is added to it and mixed it well.

ob

c)after that 2ml of gum guaiac solution is added and mix it well.

d)within a few minutes blue colour will develop.

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Strongly positive reaction will rapidly

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Significance of the presence of occult blood in stool:-

is
The appearance of black colour or tarry consistency in stool is great importance of diagnosing

different gastrointentinal diseases. Loss of more than 50 to 75ml of blood from the upper G.I. tract

eg
generally appears a dark red or black or tarry consistency in stool. Presence of blood in tarry balck

stool is to be done by occult blood test and the following diseas may be indentified

i) oesophaged varix or malignancy

nr
ii)paptic ulcer where melaena or black stool is present.

U
iii) carcinoma syomach

iv)Duodenal ulcer

3
v)Malignant disease of G.I tract

vi)Typhoid ulcer in small intenstine

.5
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vii)Parasitic infection like Ancylostomiasis

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3. Test for reducing substance:- Normally, in stool0.25gm/dl or less reducing substance are present.

More than this level indicates intestinal carbohydrate malabsorption syndrome. It is also associated

with intestinal disaccharidase deficiency. Lactose intolerance and glucose- galactose intolerance

ve

may be found in malaabsorption syndtome and may be detected by oral lactose tolerance test and

oral glucose- galaclose test respectively.

on

For the reducing substance test of stool, the following tests are done-

(i) Clinitest

(ii) Benedict’s test

(i) Clinitest for reducing substance in stool:-

PD

Procedure:-

a) one volume of stool is added with two volume of distilled water and mixed thoroughly

b) 15 drops of this suspension is transferred to a clean test tube and a cinitest tablet is added to it.

c) positive result means, the colour of suspension will be orange colour and change into original

ob

dark shade of greenish brown colour.

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2) Benedict’s test for reducing substance in stool:-

te
Procedure:-

is
a) 2 to 3 gms of stool will be taken in a test tube and 5ml of distilled water is added to it and mixed

eg
well, then filtered completely

b) then the supernatant fluid about 0.5ml taken in another test tube, 5ml of benedicts solution

nr
added it and then it is heated and boiled. It is vigorously heated for 1 to 2 minutes and then it is

colled under tap water.

U
c) the entire body of the solution will be filled with a colloidal orecipitate which may be green,

yellow ir red depending on the presence of reducing substances in stool.

3
Microscopical examination:-
.5
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Microscopical examination is dome by making a thin coverslip preparation of stool with normal

saline and lugol’s iodine preparation and should be examined by low and high power objective,

condenser will be down and concave mirror should be fitted.

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Preparation of stool:-
ve

1. Collection of stool:-The following important points are to be observed for the collection of stool-

a)the specimen of stool should be always fresh and examined within two hours.

on

b) The container should be cleaned and no antiseptics should be used to wash it.

c)The specimen should not be contained with urine.

d) No oil, oily emulsion should administered the patient before hand.

F
PD

2. Covership preparation:-

a) Unstained preparation:- this preparation of stool is very useful for the demonstration of the

actively motile forms of entamseba histolytica.

a) A clean glass slide will be be taken and a drop of freshly prepared normal saline will taken on it.

b) A portion of stool along with mucus or blood if present is to be picked up with a broom stick and

ob

mixed with the normal saline present on the slide. The mixture should be thin or not be too thick.

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c) A clean covership is to be placed over it.

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B. Satined Preparation:- this preparation of stool is important for the identification of nuclear

character and glycogen mass of cyst of amoeba and also flagellated protozoa.

is
eg
Iodine preparation:- this type of stool is important for the identification of nuclear character and

glycogen mass of cyst of amoeba and also flagellated protozoa.

nr
[Composition of Lugal’s Iodine solution

U
a) Iodine crystals powder- 5 gms

3
b)Potassium Iodide -10gm

Distilled water -100 ml]

.5
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Microsocipical Finding:-

1. Epithelial cells:- Normally it is present is stool. number will be increased in catarrnal condition of

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G.I. tract

2. Pus cell:- Any ulcerative condition in G.I. tract may show pus cells in stool

ve

3. Red blood cells:- In hookworm infection, bacillary dysentery, amoebic dysentery, R.B.C may be

found in stool.

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4. Crystals:- Charcot- leyden crystals may be found in amoebic dysentery

5. Bacteria:- some bacteria may be found in normal stool. Lactobacillus, acidophilus group of bacillus

may be found in nursing infants

6. Fat:- It may appear as neutral fats, fatty acid etc.

7. Muscle fibres:- It may be present

PD

8. Vegetable cells:- These may be present specially in amoebiasis

9. Starch granules:- may be found

10. Protozoa

a) Entamoeba histolytica

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b) Enlamoeba coli

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c) Endolimax nana

d) Intestinal flagellates

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i) Giardia intestinlis

is
ii)Trichomonous intestinlis

iii)Chilomastix mesnili

eg
11. Ova

nr
a) Ova of Asearis lumbricoides

b) Ova of Trichuris trichiura

U
c) Ova of hook worm

d) Ova of Enterrobius vermicularis

3
.5
e) Ova of Hymenolepis nana.

f) Ova of Taenia solium, taenia saginata, echinococcus bgranulosus (in dog)

r5
12. Larva- such as larva of strongyloids stercoralis.

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Methods for the examination of Ova of Helminths:-

1. Direct methods:- A smear of stool is done with the help of broome stick, a drop normal saline

ve

may be used for the preparation of smear. Microscopical examination will be done.

2. Concentration Method:-
on

Simple floatation technique

Material required

1) Small “pill box” or glass “container” of 15 to 20ml capacity the diameter of which will be 1.5

inches

PD

ii)Glass slide

iii)Saturated salt solution of specific gravity 1200.

Here common salt or table is used

e
ob

Technique-

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1) About gm of stool will be taken in the container and a few drops of saturated salt solution will be

added to it. With the help of stick, an emulsion will be made.

te
ii)then the container will be filled upto the brim by adding the saturated slat solution with a pipette

drops by drop till aconvex meniscus of the fluid will form

is
iii) then aglass will be carefully laid on the lop of the container.

eg
iv) the preparation is allowed to stand for 8 to 10 minutes

v) Then the glass slide be placed over the fluid on the glass slide

nr
vi) it will be examined with low power objective.

U
The egg of the helminthes float on saturated salt solution

3
i. Ancylostoma americanus

ii.

iii.
Necator americxanus

Hymenele[is nana

.5
r5
iv. Fertilized ove of Ascaris lumbricoides

v. Trichuris trichiura

rte

vi. Hymenalepis diminuta

ve

The eggs of Helminths do not float on saturated salt solution

on

i) unfertilized ova of Asearis lumbricoids

ii)Taenia solium

iii)Enterobius Vermicularis

iv) Taenia saginate

F
PD

Parasite:- Any living organism derived from animal species and drawing nutrient by living another

organism is called parasite.

Classes of parasite:-
ob

iii) Iodamoeba butschlii and (iv) Dientamoeba fragilis

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Classification of Protozoa (according to the degree of locomotive)

1. Class Rizopada (Photozoa with pseudopodia)-

te
(Rhizos means blunt and podos means foot)

is
a) Pathogenic

i. Entamoeba histolytica

eg
b) Non-Pathogenic

nr
i)Entamoeba coli

ii)Entamoeba gingivalis

U
iii)Endolimax nana

iv)Iodamoea butschlii

3
.5
v)Dientamoeba fragilis

r5
2. Class Mastigophora (Protozoa with flagella)

(mastrix means whip and pherein means to beer)

rte

A.. In antentine a) pathogenic

i. Giardia intestinals (Giardia lamblia)

ve

ii. Trichomonas vaginalis

b) Non-pathogenic

on

i)Trichomous hominis

ii)Trichonous Tenax

iii)Chilomastic mesnili

iv)Enteromous homimis

PD

B. In blood and tissue:-

1) Pathogenic

i)Trypanosome gambiens

ob

ii)Trypanosome cruzi

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iii)Leishmania donovani

iv)Leishmania tropica

te
v)Leighmania brasiliensis

is
2) Non- pathogenic

eg
1)Trypansoma rangeli

nr
3. class sporozoa (Protozoa without any oragns of locomotion)

1. In intestine

U
i)Isospora hominis

ii)Eimeria gubleri

3
2. In blood and tissue:- .5
r5
i) Plasmoium vivax

ii)Plasmodium falciparum

rte

iii)Plasmodium falciparum

iv)Plasmadium ovale

ve

3. In liver

on

i) Toxoplasma gondii

In small intestine:-
F

Pathogenic

PD

1. Giardia instestinalis:- Gairdia instestinalis is under class mastigophora. It generally present in

duosenum and the upper part of ileum of man. It produces diarrhea, dynestery, duodenitis etc.

2. isopora hominis;- It is under class sporozoa or coccidian,. It is present in the lower portion of

ileum. It produce intestinal “symptons” like colic and diarrhea.

3. Eimeria gubleri:- It is under class sporozoa or coccidian. It is present in the ,lower part of ileum

ob

and responsible for the occurrence of hepatic coccidiosis in man.

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In large intestine:-

A. pathogenic-

te
1. Entamoeba histolytic- it is under class, Rhizopada. Trophozoite of entamoeba histolytica lives in

the mucous and sub- mucous layers of the large intestine intestine in man. It cause diarrhea,

is
dysentery, hepatitis and liver abscess in man.

eg
2. balantidium coli:- It is under class ciliate. It produce dysentery.

nr
B. Non-Pathogenic:-

1. Entamoeba coli:- it is under class Rhizopoda. It exist as a harmless commensal to man.

U
2. Enlamoeba hartmanni:- it is under class Rhizopodo. It is harmless.

3
3. Endolimax nana:- it is under class Rhizopoda. It is commonly encountred in fiarrhoeic and

dysenteric stool otherwise it is harmless.

.5
4. Enteromonas hominis- It is under Mastigophora. It is harmless

r5
5.Embadomonas intestinalis:- It is under class Mastigophora. It is harmless.

rte

In Caecum and ileocaecal region:-

1. Trichomonas hominis:- It is under class mastigophora. It lives in ilecaecal region. It is commensal

ve

of the intentine and often appears in stool of diarrhoa.

on

2. chilomastix mesnili:- It is under class mastigophora . it is non- pathogenic flagellate and lives in

caecum and is found in cases of diarrhoea due to some other causes.

Non- parasitic object:-

1. Polymorphonuclear leucocytes- It look like E,histolytica cyst, found in dysentery and other

inflammatory bowel diseases.

PD

2. Mocrophages:- it looks like amoebic trophozoite, espically E.histalytica, found in dysentery and

other inflammatory bowel diseases.

3. Sqyamas epithelial cells- It appears from anal nucosa and look like amoebic trophozoite

4. Yeasts- It is normally present in stool. It looks like protozoan cyst.

5. Starch granules- It is normally is normally present in stool and looks protozoan cyst.

ob
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(c) Ziehl-Neelsen Method for Acid fast bacilli –

te
Material required – (1) Carbal fuqohsin solution.

is
Composition (a) Basic fuchsin – 1gr.

eg
(b) Absolute alcohol – 10 ml.

(c) Solution of phenol (5% in water) – 100

nr
ml.

U
3
(II) 20% Sulphuric acid (H2SO4)

(III) Methylene blue solution.

.5
r5
Procedure – (1) A smear will be prepared and fixed by passing it

rte

three times over the flame.

(II) It will be stained with steaming carbal-fuchsin for 10 minutes

ve

and washed with water.

on

(III) Next it will be decolourised with 20% Sulphuric Acid and

decolourisatoin will be continued until after washing the smear is

a faint pink.

(IV) After washing, with water, it will be counter-stained with

PD

methylene-blue for 30 seconds to 1 minute.

Use – It is used for the identification of mycrobacterial group of

bacteria and after staining, the bacteria will take red stain and

other bacteria and the tissue-cells will take the methylene-blue

ob

counter stain.

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(d) Leishman’s Stain –

te
Composition of leishman’s stain

is
(I) Leishman Powder (0.15 to 0.2 gr.) composed with

eg
(a) Methylene blue for basic stain.

(b) Eosin for acid stain.

nr
(II) Acetone free methyl alcohol – 100 c.c.

U
Material requirement for Leishman’s staining –

3
(I) Leishman’s Stain

(II)
.5
Distilled water (must be neither acidic nor alkaline

r5
pH is 7.0) or Buffer solution.

rte

Composition of Buffer Solution –

ve

(a) Disodium hydrogen phosphate – 5.447 gr.

on

(b) Potassium dihydrogen phosphate – 4.752 gr.

(c) After mixing (a) and (b) in a mortar, 1 gr. of the mixture is

added to 2000 ml of distilled water. This gives a pH of 7.0.

F
PD

Procedure –

(I) A blood film will be drawn on a glass slide and the film is

ob

covered with sufficient quality of Leishman’s Stain and is

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kept for 2 minutes.

te
(II) About double quantity of distilled water or buffer solution

will be added gently or with the help of a capillary

is
pipette, drawing in and blowing out the solution. A

eg
golden scum will form. It is kept for 10 minutes.

nr
(III) Then the slide is washed under running tap water and

dried in air.

U
3
Use Blood film, bone marrow may be stained with

.5
–

Leishman’s Stain. In the blood film, different blood cells can be

r5
identified and also abnormal blood cells parasites like malaria,

Leishmania denovani.

rte

(E) Giemsa’s Stain –

ve

Composition –

on

(I) Azure II easin – 0.3 gr.

(II) Azure II – 0.08 gr.

(III) Acetone free methyl alcohol (methanol) – 25.0 ml.

(IV) Anhydrous pure glyerine – 25.0 ml.

PD

Azure II easin and Azure II will be dried in adesiccater. Then the

dry powder will be dissolved in 25 ml. of anhydrons pure

glycerine at 600C for about two hours and 25 ml of Acetone free

ob

methyl alcohol will be added and kept it at 370C or room

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temperature for about a week. It is then filtered.

te
Material required –

is
(I) Giemsa’s Stain

eg
(II) Acetone free methyl alcohol (methanol).

(III) Distilled water (neutral) or phosphate buffer (pH 6.8 to 7).

nr
Procedure –

U
(I) The smear (blood film) is fixed treatment with acetone free

3
absolute methyl alcohol for 3 to 4 minutes.

(II) .5
Diluted stain (I part stain to I part buffer solution or

r5
water) is allowed to act for ½ hour.

rte

(III) The film is then washed with distilled water and dried in

air.

ve

Use – It is used mainly for staining spirochaetes, malarial

parasites.

on

3. Special stains and also used as Differential stains –

(a) vital stain for microfilaria.

(b) McGuire stain or Ponder’s stain.

PD

(c) M. R. Stain for the study of parasitic protozoa and fungas.

(d) Neisser’s method for Diphtheria bacilli.

e
ob

(e) Albert’s stain for Diptheria bacilli.

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(f) Staining of Spore.

te
(i) Moeller’s Method.

is
(ii) Fleming’s Method.

eg
(g) Craigie’s method for staining of flagella.

(h) Staining of capsule –

nr
(i) Hiss’s Method.

U
(ii) Muir’s Method.

3
(i) Staining of spirochaetes –

(i) Foutana’s Method.\

.5
r5

(ii) Levaditi’s Method of staining of spirochates in tissues.

rte

(j) Staining of tissue –

ve

(i) Weigherts Iron haematoxylin stain.

(ii) Van Geison’s stain.

on

(k) Paschen’s Stain for virus.

(l) Macchiavello’s stain for Rickettsia.

(m) Papanicolaou Stain for Malignant cells.

PD

(n) Supravial Staining by Osgood’s method for enumeration of

reticulocyte.

(a) Vital Staining

ob

Material Method –

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(I) Methylene blue Solution (1 in 5000 in physiological saline)

te
is
Procedure –

eg
The fresh blood or dehaemoglobinised blood will be mixed with

methylene blue solution.

nr
Use – The living microfilariae of L. loa, 0.volvulus, A. perstans

U
will be stained within 10 minutes and microfilariae of w.

banerofite and w. malayi will take more time to stain.

3
(B) McGuire Stain or Ponder’s Stain

.5
r5
McGuire Stain is Ponder’s Stain, four times concentrated.

Composition of Ponder’s Stain –

rte

(I) Taluidin blue – 0.2 gm.

ve

(II) Acetic Acid – 10 ml.

(III) Ethanol – 20 ml.

on

(IV) Distilled Water – 1000 ml.

C
F

Procedure –

PD

(I) A drop of McGuire Stain will be added on the thick blood

film and keep it for 3 to 5 minutes.

(II) Then it will be blotted for drying.

ob

(III) A coverslip will be put on it.

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Use – It is used for the details of microfilaria.

te
is
(C) M. R. Stain (Mukherjee and Roy) –

eg
Composition –

(i) Solution I – composed with

nr
(a) Saturated Solution of Mercuric Chloride – 64 ml.

U
(b) Absolute alcohol – 32 ml.

3
(c) Glacial acetic acid 5.2 ml.

.5
(d) Formalin (40% formaldehyde) 4.2 ml.

r5

(e) Thionine – 0.04 gm.

rte

(f) Azure – 0.02 gm.

ve

(ii)Solution 11 composed with

(a) 70% alcohol – 93 ml.

on

(b) Glacial acetic acid – 7 ml.

(c) Thionine – 0.06 gm.

(d) Azure 1 – 0.06 gm.

PD

(e) Methyl green (chloroform extracted) – 0.04 gm.

(f) Eosin – 0.24 gm.

e
ob

Solution I and II are mixed in the proportion of 1 : 1 and filtered

after 24 hours. This is a fixative and also staining solution.

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Procedure –

te
(I) A faecal smear will be done and that should be called from

culture of protozoa and fungi.

is
(II) The slide will be dipped in a jar containing the solution

eg
for 10 minutes.\

nr
(III) Next, it will be rinsed with tap water.

U
(IV) The smear is then dehydrated by passing successively

through two changes of tertiary butyl alcohol for 1

3
minute each.

(V)
.5
Then it will be cleared in toluol and xylol for 1 minute.

r5
(VI) Next it will be mounted in Canada balsam.

rte
ve

Use – It is used for the study of parasitic protozoa and fungi.

(d) Neiner’s Method for Diphtheria bacillus

on

Composition of stain –

(I) Solution A – composed with

(a) Methylene blue – 1 gm.

PD

(b) Absolute alcohol – 50 ml.

(c) Glacial acetic acid – 50 ml.

e
ob

(d) Distilled water 1,000 ml.

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(II) Solution B – composed with

te
(a) Crystal violet – 1 gm.

is
(b) Absolute alcohol – 10 ml.

eg
(c) Distilled water 300 ml.

Procedure –

nr
(I) Two part of solution A and one part of solution B will be

U
mixed and smear will be stained for 30 to 60 seconds.

3
(II) Next it will be washed in water.

(III) .5
It is then stained with Bismarck brown (0 to 2%) for 30 to

r5
60 seconds.

rte

(IV) Next it will be washed in water and dried in air.

ve

Use – The granules of Diphtheria bacilli will tack bluish-black

on

colour and the protoplasm light brown colour.

(E) Albert’s Stain for Diphtheria bacilli

Composition –

(I) Solution I (Albert’s Stain) – composed with

PD

(a) Teluidin blue – 0.15 gm.

(b) Malachite green – 0.2 gm.

e
ob

(c) Glacial acetic acid – 1 c.c.

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(d) Alcohol – 2 c.c.

te
(e) Distilled water – 100 c.c.

is
The dye will be dissolved in alcohol and water will be added.

Then glacial acetic acid will be added and it will be allowed to

eg
stand for a day and filtered.

nr
(II) Solution 2 (Albert’s Iodine) – composed with

U
(a) Iodine – 2 gm.

3
(b) Potassium iodine – 3 gm.

.5
(c) Distilled water – 300 c.c.

r5
Procedure –

rte

(I) A smear will be prepared and it will be dired in air.

(II) It will be fixed by flaming.

ve

(III) It is the stained in solution 1 for about 5 minutes.

on

(IV) Then the excess stain will be poured off and the smear

will be blotted to dry.

(V) Next the smear will be stained with solution; for about 2

minutes.

PD

(VI) The excess stain will be pured off and the smear will be

blotted to dry.

Use – It is used for the details of the diphtheria bacilli. The

ob

metachromatic granules stain blue-black, protoplasm green and

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other organisms light green.

te
(f) Moeller’s Method for spore staining

is
Material required

eg
(I) Carbal-fuchsin

(II) Absolute alcohol

nr
(III) Methylene-blue. (Loeffler’s).

U
Procedure –

3
(I) The smear will be made from the spore culture and dried

.5
and fixed by passing over the flame.

r5
(II) Steaming carbol-fuchsin will be poured on it and allowed

rte

to stand for 5 minutes.

(III) Next it will be decolourised with alcohol till to the smear

ve

is just pink in colour.

on

(IV) It will be washed well.

(V) It is then counterstained with Loeffler’s methylene – blue

for 2 minutes.

(VI) Then the smear is washed in water and dried in air.

PD

Use – The spore are stained bright red and the bacterial

protoplasm will take blue stain.

(g) Craigie’s Method for Flagella staining

ob

Material required –

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(I) 2% Formal-Saline

te
(II) Mordant Solution composed with

is
(a) Tannic acid (light and pure) – 10 gm.

eg
(b) Tartar emetic (aqueous 5% solution) – 30 ml.

(c) Distilled water – 200 ml.

nr
(d) Add a crystal of theymal.

U
(III) Silver solution composed with

3
(a) Silver sulphate 1 gm.

.5
(b) Distilled water 250 ml.

r5

(IV) Gold chloride solution composed with

rte

(a) Gold chloride (1%) – 20 days

ve

(b) Distilled water – 20 ml.

Procedure –

on

(I) Material should be collected from condensation water of a

very your culture and kept into 2% formal-saline and

allowed to fix overnight.

F
PD

(II) Then this will be diluted with distilled water and a thin

film will be made on glass slide.

(III) The film will be dried and fixed by heat at 1000C for 5

e
ob

minutes.

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(IV) Then the mordant solution will be added and kept for 5

to 10 minutes at 1000C.

te
(V) The film will be washed in tap water and then distilled

is
water.

eg
(VI) Next it will be treated with silver solution. Before adding

nr
the silver solution, 20 ml. of this solution will be added

with 33% mono-ethylamine until the precipitate

U
produced just clear. The slide will be flooded with this

and heated gently until the preparation becomes brown

3
.5
black.

r5
(VII) Then it will be washed in distilled water and dried in air.

rte

Use – The flagella of bacteria can be identified.

ve

(h) Muir’s Method of capsule staining

on

Material required –

(I) Carbol-fuchsin.

(II) Absolute alcohol.

(III) Mordant – composed with

PD

(a) Saturated solution of corrosive sublimate – 2

Parts.

(b) 20% Solution of tannic acid – 2 parts.

ob
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(c) Saturated solution of potassium alum – 5 parts.

te
(IV) Counter-stain – Methylene blue.

is
Procedure –

eg
(I) The smear will be stained with carbol-fuchsin for 1 minute

and heated gently.

nr
(II) Then it will be washed with absolute alcohol and then in

U
water.

3
(III) It will be then treated with absolute alcohol for about 20

to 30 seconds.

.5
r5
(IV) Then it is washed well in water.

(V) Counter-stain methylene blue is added for 30 to 60

rte

seonds.

ve

(VI) It will washed in water and dried in air.

Use – Capsules of bacteria are stained blue and the body of the

on

bacteria red.

(j) Staining for Spirochaetes -

F

(a) Fontana’s Method –

PD

Material required –

(I) Fixative – composed with

(a) Acetic acid – 1ml.

ob

(b) Formation – 20 ml.

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(c) Absolute alcohol.

te
(II) Absolute alcohol

is
(III) Mordant – composed with

eg
(a) Carbolic Acid – 1 gm.

(b) Tannic Acid – 5 gm.

nr
(c) Distilled water – 100 ml.

U
(IV) Silver Solution – composed with

3
(a) 10% Ammonia Solution

.5
(b) 0.25% Solution of silver nitrate.

r5

Ammonia Solution will be added to silver nitrate solution drop by

rte

drop till the precipitate which from, does not dissolve.

Procedure –

ve

(I) The film will be treated three times, 30 seconds each time

on

with the fixation.

(II) Next it will be washed with absolute alcohol for 3 minutes

and heated until the film in dry.\

(III) Then the mordant will be added and this will be heated

PD

gently until steam rises and allowed to act for 30

seconds.

(IV) Next if will be washed well in water and again dried.

ob

(V) After this, it will be treated with silver nitrate solution,

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heating till steam rises, for 30 seconds, when the film

becomes brown.

te
(VI) Then it will be washed well in water and drive air.

is
Use – The spirochaetes are stained brownish-black on yellowish

eg
background.

nr
(b) Levaditi’s Method of staining spirochaetes in Tissue.

U
Material required –

3
(I) 10% formation.

(II) 96% to 98% alcohol.

.5
r5
(III) 1.5% silver nitrate solution

rte

(IV) Reducing Solution composed with

(a) Pyrogallic acid – 4 gm.

ve

(b) Formation – 5 ml.

on

(c) Water – 100 ml.

Procedure –

F
PD

(I) A small piece of tissue will be fixed in 10% formation for 24

hours.

(II) It is then washed in water for one hour and placed in 96

e
ob

to 98% alcohol for 24 hours.

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(III) Then it is placed in 1.5% silver nitrate solution in a dark

bottle and is kept in incubator for 3 days

te
(IV) Next, it will be washed for 30 minutes.

is
(V) It is then placed in the reducing solution and is kept in a

eg
dark bottle at room temperature for 2 days.

nr
(VI) Next it will be washed and dehydrated with increasing

strength of alcohol and embedded in paraffin.

U
3
.5
Use – The spirochaetes are stained black and the tissue

brown.

r5
(j) Staining of Tissue

rte

For the staining of tissue, the following steps should be

adopted.

ve

(a) Fixing and hardening of tissue.

on

(b) Dehydration, clearing and impregnation.

(c) Embedding

(d) Section of the tissue –

PD

(I) Paraffin Section

(II) Frozen Section

(e) Staining of tissue

ob

(f) Fixing and hardening of tissue

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Small pieces of fresh tissue should be kept in the any one of

the following fixation before section and staining.

te
(I) Alcohol – The tissue may be fixed in 95 to 100%

is
alcohol for 24 hours and then it should be kept in

eg
water overnight.

nr
(2) Formal Solution – 10% formal-solution is used.

Composition :-

U
(I) Formalin (Commercial 40%) – 10 volumes.

3
(II)
.5
Water of normal saline – 90 volumes.

r5
It is not an ideal fixation but can be used for routine work.

It fixes tissues in a few hours to 24 hours depending on the

rte

thickness of the tissue.

(3) Formal Sublimate –

ve

Composition :_

on

(I) Formalin (Commercial 40%) – 1 Part.

It is a better fixation. It fixes the tissue within 8 to 18

hours and then the tissue should be washed for 2

hours. It may be used for routine work.

PD

(4) Zenker’s fluid

Composition :-
e
ob

(I) Potassium bichromate – 2.50 gm.

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(II) Hydrarg perchloride – 5.00 gm.

te
(III) Water – 100 C.C.

is
It is a good fixation and gives good results. Before fixation

with this fluid, 5 c.c. of glacial acetic acid will be added

eg
whoch makes the solution into

nr
‘Zenker’s formal’

U
Then the tissues will be fixed in the fluid for 3 to 12

hours and washing is to be done in running water over

3
night to remove bichromate and then to alcohol

.5
containing sufficient iodine is to be added to keep the

r5
solution brown.

(5) Formal nitric acid

rte

Competition :-
ve

(I) Formalin (Commercial 40%) – 5 c.c.

on

(II) Nitric acid – 5 to 10 c.c.

(III) Water – 80 to 85 c.c.

It is used for the bony and calcified tissue. The bone will be

sawed into small discs, 3 to 5 mm. in thickness, then it will be

PD

fixed in 5% formal saline, then it will be decalcified in the formal

nitric acid solution. It requires 4 days or more for decalcification.

After decalcification, it will be punctured by a needle and washed

e
ob

in running water and hardened in 70% alcohol.

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(B) Dehydration, clearing and impregnation

te
The fixed tissue are cut into size of about 1 cm. square with

thickness of about 5 mm. Then it will be washed overnight under

is
running tap water.

eg
(a) Then the tissue will be dehydrated by passing through

nr
ascending grades of alcohol for 12 to 24 hours in each (70%

and 90% alcohol). Two changes will be done in absolute

U
alcohol, 2 hours each.

3
(b) Then clearing will be done by two changes, first change

.5
after hour and second change after 2 hours in xylol or

r5
chloroform.

(c) Next, it will be kept in saturated solution of paraffin

rte

(melting point 54 to 56%) in xylol or chloroform.

(C) Embedding

ve

(a) After dehydration and clearing, the tissue should be

on

embedded in paraffin (melting point 54 to 560C) in constant

temperature paraffin bath at 560C. Thorough and uniform

impregnation is necessary.

(b) Then a block is to be prepared.

PD

(D) Section

(a) Paraffin section – After embedding, thin section of 4 to 6 in

thickness, are cut with the help of rotary microtome and

ob

spread over glass slide which will be smeared previously with

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egg albumin. For this, the white portion of an egg will be

collected and filtered and eqaual part of pure glycerine will be

te
added to it. Then the slides with the sections are dried in the

is
incubator overnight.

eg
Use – This method is routinely used for histopathological

examination.

nr
(b) Frozen Section (Cryocut method)

U
(I) First the small pieces of tissue will be fixed in formal saline

3
for 12 to 24 hours.

.5
(II) Then they will be transferred in gum solution and kept for

r5
half an hour or more in warm oven.

(III) Next, the tissue will be freezed in gum solution on the

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microtome platform by passing CO2 or liquid nitrogen over it.

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(IV) After some times, the sections of 6 to 8 µ thickness are

prepared quickly when the snow is just thawing.

on

(V) Then the section are stained and then mounted on glass

slides with glycerine.

Use – Frozen Section technique may provide rapid diagnostic

help. This method is helpful for histochemical on

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immunohisogical study. Here detail histologic architecture of

the tissue may not be obtained.

(E) Staining of histological Sections –

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For the haematoxylin – eosin stain of tissue, one the fellow

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Haimatoxylin solution may be used.

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(a) Delafield’s Haematoxylin –

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Composition :-

eg
(I) Haematoxylin – 4 gm.

(II) Absolute Alcohol – 25 c.c.

nr
(III) Saturated solution of ammonium alum – 400 c.c.

U
For complete oxidation, it should be kept for one

3
month in a stoppered battle when the exposure to light

.5
and air will be sufficient. Then it will be filtered. Then,

r5
100 c.c. each of glycerine and methyl alcohol are to be

added to the filtrate.

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The solution will be ripened till it is dark purple

colour. Then, for use,k this will be diluted 1 in 4 of

ve

water.

on

(b) Ehrlich Haematoxylin (acid).

Composition –

(I) Haematoxylin – 2 gm.

F
PD

(II) Absolute alcohol – 100 c.c.

(III) Glycerine – 100 c.c.

(IV) Distilled water – 100 c.c.

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(V) Glacial acetic acid – 10 c.c.

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With this composition, potassium alum will be added in

excess to saturate and make the solution blue. Then it

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should be ripened as for Delafield’s

is
(1) Staining of paraffin sections with haematoxylin and

eg
easing

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Preliminary treatment –

(I) The slide will be warmed just to melt the paraffinand

U
should be dipped in xylol (Two changes in xylol 5

3
minutes each).

(II) Then the xylol .5

will be removed by putting in

r5
absolute alcohohl (Two changes, 5 minutes each).

(III) Then it should be passed through descending

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gardes of alcohol (90%, 70%, 50%) 5 minutes each

and then to water.

ve

(IV) The section which are fixed with Zenker’s fluid or

on

formal sublimate, should be treated with 70%

alcohol containing iodine for 10 minutes and then

passed through alcohol to water.

(V) After washing the water for 5 minutes, 0.5% water

PD

solution of sodium hyposulphate is to be added and

kept for 10 minutes.

(VI) Finally it will be washed under running tap water

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for 10 minutes.

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Staining with haematoxylin and eosin –

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(I) First section will be stained with haematoxylin for 3 to

5 minutes.

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(II) Then it will be washed in running tap water, till a

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blue colour develops.

nr
(III) Then the section should be examined under low

power microscope and differentiated with acid

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alcohol (1 c.c. of HCL in 99 c.c. of 70% alcohol) for a

3
few seconds. It should be continued till the

.5
maximum nuclear details are seen.

r5
(IV) Then it should be kept running tap water in the

section is blue.

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(V) Next it will be counter stained with 1% aqueous

solution of eosin for a minute.

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(VI) Then it should be passed through graded alcohol

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and absolute alcohol.

(VII) Then it will be dried in air and cleared in xylol and

mounted in neutral canda balsam.

Finding after staining –

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(I) Nuclei will take blue colour.

(II) Acidophilic nuclei will take red colour.

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(III) Basophilic nuclei will take blue colour.

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(IV) Collagen and fibrils will take pink colour.

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(V) R. B. C. will take pink colour.

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(VI) Eosinophilic granules will take red colour.

eg
(VII) Basophilic granules will take blue colour.

(2) Weigert’s Iron Haematoxylin Stain-

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Reagents –

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(I) Solution A –

3
Haematoxylin – 1 gm.

.5
Absolute alcohol – 100 c.c.

r5

(II) Solution B –

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Liquor Ferriperchlor – 30% (aqueous) – 4 c.c.

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Distilled water – 100 c.c.

Procedure –

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(I) Before staining, equal volume of solution A and solution B

will be taken in a test tube and the slide will be stained

with the misture and it is kept for about 3 minutes.

F
PD

(II) Then, it will be washed under running tap water till blue.

If the section stained deep, it should be differentiated

with that acid alcohol solution.

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(III) Next, it will be counterstained with 1% aqueous solution

of eosin for 1 minute.

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(IV) Then, it should be dehydrated in absolute alcohol and

then in dry alcohol.

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(V) Next, it will be cleared in xylol and mounted in Canada

is
balsam.

eg
(3) Van Gieson’s Stain –

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Composition –

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(I) Solution A

3
1% of acid fuchsin – 5 to 10 c.c.

(II) Solution B

.5
r5
Saturated aqueous solution of picric acid – 95 c.c. Before,

use, these solution will be mixed.

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Procedure :-
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(I) The section should be taken down to water.

on

(II) Next it will be stained with weigert’s Iron haematoxylin

for 5 minutes.

(III) Then it will be washed in water till blue and blotted out.

(IV) Next it will be stained with Van Gieson’s stain for 3 to 5

PD

minutes.

(V) The degree of staining should be observed under

microscope.

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(VI) Then it will be dehydrated, cleared and mounted in

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Canada balsam.

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(4) Special stain used for specialized structures –

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(I) For the staining of Fat.

eg
Here frozen section should be used and stained with

nr
saturated solution of Suden III – shows pink.

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Saturated solution of Suden – IV shows pink.

3
Scarlet red stain – shows orange red.

.5
Osmic acid – shows black.

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(II) For the staining of Pigment (Haemssiderin)

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Potassium ferrocyanide, HCL and eosin stain –

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Haemosiderin will take blue stain and the cell and

appear red.

on

(III) For the staining of connection tissue –

Van Gieson stain – Collagen fibres take red colour and

other will will take yellow colour.

F
PD

(IV) For the staining of Mucin and glycogen –

Periodic Acid Schiff (PAS) Stain – carbohydrate

components will take Pink colour.

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(V) For the staining of Reticular fibres –

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Foot stain or Gomori stain (By silver impregnation

technique) – Reticulin fibres will take black colour.

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(VI) For the staining of Basement membrance –

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PAS Stain – It appears pinkish colours.

eg
(VII) For the staining of RNA –

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Methyl Green Pyrenin (MGP) Stain – RNA componenst

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shows pinkish colour.

3
(VIII) For the staining of DNA –

.5
Fuelgen stain – DNA components will shows pinkish

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colour.

(IX) For the staining of Amylaid material –

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Methyl violet stain will be shows pink colour. Iodine

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stain will show mehogny brown colour. PAS stain will

be pink colour. Van Gieson stain will s how khaki

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colour congo red stain will show greenish birefringence

on polarized light.

Thioflavin – T stain will show fluorescence on

fluroscent microscope.

PD

SEMINAL FLUID

Sinological examination is routinely done for investigation of a

sterile couple to find out the defect in the male partner before

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performing an extensive and expensive investigation of the

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female. Defect in the male partner is considered to be responsible

for infertility in near about 40% cases.

te
Method of collection :-

is
Strict abstinence for 6 to 7 days before collection of the semen is

eg
always preferred. The material in collected in a properly cleansed

nr
test tube or Petridis after manual ejaculation by masturbation. If

the method fails the semen may be collected by coitus

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interruptions. Care should be taken to collect whole sample at a

time and not in part to avoid erroneous result. First part of the

3
.5
ejaculation is poor in sperm content, second part is rich and

third part is again poor in sperm population.

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Examination of the specimen should be done as early as possible

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without freezing or keeping in an incubator. Incubation of the

samle at 370C does more harm than any benefit.

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Macroscopic Examination :-
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(a) Amount :- The volume is measured with the help of a small

graduated cylinder. Amount per ejaculation usually varies

between 3 to 5 ml. Volume below 1.5 ml is considered

abnormal though infertility can not be related unless

deficiencies are present.

PD

(b) Appearance :- Freshly collected material appears thick,

mucoid and viscid fluid. It undergoes coagulation shortly

and selfique faction takes place within 30 minutes.

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Improper liquefaction of the sample is related with

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inhibition of movement of spermatozoa in vaginal canal and

thus interfares fertilization.

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(c) Reaction :- The reaction of seminal fluid in always towards

is
alkaline side and pH ranges between 7.5 to 8.5.

eg
Abnormalities in relation to pH of semen is rare.

nr
Microscopic Examination :-

(a) Motility of spermatozoa – A drop of semen is taken over a

U
clean glass slide, covered by a coverslip and examined

3
under low power and high power objective respectively. A

.5
few abnormalities can be detected and described as –

r5
Aspermia – Complete absence of spermatozoa in all the

microscopic fields.

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Oligaspermia – Only a few motile spermatozoa are found in

different fileds.

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Necrospermia – Presence of plenty of red blood cells and

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pus cells usually associated with chronic gonococcus or

tuberculous infection of prostate or seminal vesicles. In

normal condition, 80 to 90 per cent sperms are found

motile and motility with less than 60 per cent is considered

PD

abnormal. Examination of sperm motility different hour

intervals is of no practical importance.

(b) Total sperm count – Total sperm count is done with the help

of haemocytometer and WBC pipette using semen diluting

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fluid.

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Composition –

te
Sodium bicarbonate – 5 gms.

is
Formalin – 1 ml.

eg
Distilled water – 100 ml.

Semen is collected upto the mark 0.5 in WBC pipette and

nr
the filuent is sucked upto the mark 11. The mixture in the

U
pipette is well stirred for 5 minutes and first few drops are

discarded. The counting chamber is then charged and the

3
sperms are counted at 2 big squares at two corners as used

.5
for WBC count. To obtain total sperms per ml of the sample,

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the figure is multiplied by 50.

In normal condition, total sperm count varies between 80 to

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130 million per ml. Count below 20 million per ml. is

considered abnormal and is more likely to be related with

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infertility.

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Sperm morphology – For evaluation of sperm morphology,

smear are prepared with a drop of semen on a glass slide in

a manner identical to blood films and stained by

Papnicolaou technique. Other stains often used are

PD

haemotoxylin, Giemsa and basic fuchsin.

Basic fuchsin method –

(I) Fix the smear in 95 per cent alcohol and dry in air.

ob

(II) Stain the smear for 3 minutes with dilute Zichl-

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Neelsen carbal fuchsin with equal amount of 95 per

cent alcohol.

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(III) Was in running tap water for 2 minutes, dry in air and

is
examine under oil immersion objection. Terminal part

eg
of sperm head appears light blue, basal parta a dark

blue and tail appears light red or pink. At least 200

nr
sperms are counted to note morphologic.

U
Configuration with their abnormal forms. Different

abnormalities in the form of very small head, giant

3
head, head with irregular countour, double head, bifia

.5
tail and acute tapering are often identified.

r5
Normal semen might show presence of 20 to 25 per

cent abnormal forms of spermatozoa. The stained

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smear also helps in identification of red blood cells,

lencocytes or germinal epithelial cells if present.

ve

Postoital (Sims-Huhnex) Test :- The test is often done to note the

on

degree of sperm – penetration in cervical mucus during

investigation of a case of infertility. It is routinely undertaken in

midcycle ovulatory phase when the amount of cervical mucus is

maximum and its viscosity is significantly diminished.

PD

The female partner is examined 2 – 3 hours after coital but the

result of the test remains constant even for 8 hours. The external

cervical OS is wiped clearly with a cotton swab and the mucus is

aspirated from endocervix with the help of a thick glass cannula

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and a rubber teat or a syringe.

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Volume, colour, viscosity and tenacity of the mucus is noted. It

should be clear, watery and tenacious in normal condition. A

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drop of the mucus is taken on a glass slide, covered by a

is
coverslip and exmined under microscope for presence of sperms

eg
and their motility. Total number of spermatozoa per high power

field with the percentage of motile sperms should be reported. It

nr
also helps in demonstrate of trichomonas vaginalis if present.

U
Tests for the presence of semen )medicolegal_ :_

Detection of semen in vaginal aspirate, skin surface or clothing is

3
.5
of medicolegal importance in cases of alleged rape or sexual

assault and the pathologist is very often requested to take part in

r5
the investigation.

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Collection of material :- Vaginal secretion or the saline wash from

vagina is aspirated with the help of a glass pipette. Material from

ve

skin surface is also collected by saline wash. Stained piece of

cloth immersed in physiologic saline solution for at least one

on

hour and then the extract is tested.

(a) Florence Test :- It is routinely performed with the stained

clothing or hair. The material is extracted with distilled

water by gentle heating. A few drops of the extract is taken

PD

over a glass slide and mixed with equal amount of iodine

solution (iodine – 2.5 gms.; potassium iodine – 1.6 gm.

Distilled water – 30 ml.) High concentratin of choline in

semen develops rhombic or needle shaped crystals of

ob

choline preiodide and are well demonstrated within a few

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minutes.

te
(b) Acid phosphatase estimation – Semen is rich in acid

phosphatase content and average level is 2500 K. A. units

is
per ml. All other body fluids contain less than 5 K.A. units

eg
of acid phosphatase per ml. Demonstration of high acid

phosphatase content in the washing is considered as a

nr
reliable indication for the presence of semen.

U
(c) Demonstration of Sperm – Direct smear from the vaginal

secretion or the smear prepared from the centrifused

3
.5
deposit of vaginal wash help in demonstration of

spermatozoa after staining as usual.

r5
(d) Precipitin test – This is an immunological test done with

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saline wash either from vagina, skin surface, or stained

clothing and high titre antiserum raised in rabbit by

ve

immunizing with human seminal fluid. It is performed

either by capillary tube method or by gel-diffusion

on

technique.

(e) Determination of A, B, or H blood group substain the

collected sample helps in identification of the male

individual as the source.

PD

MALARIAL PARASITES

Malaria – The word of Malaria comes from Mal means bad and

Aria means air. In 1753 the name malaria was introduced by an

ob

Italian writer.

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Plasmodium – Plasmodium means a mass of naked protop

formed by fusion or aggregation of amoeboid bodies without

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fusion of their nuclei. It may be called as haemamoeba, was

is
introduced amoeba in blood.

eg
Different types of malarial parasites –

nr
There are flour types of malarial parasites –

I. Plasmodium Viax – The specific name falciparum derived

U
from L. Vivere to live and indicates the active movement.

3
This will produce benign tertin malaria.

.5
II. Plasmodium falciparum – The specific name falciparum

r5
derived from Latin word falx or a sickle. The gametocy of

this group is sickle-shaped. This will produce malignant

rte

tertian malaria.

III. Plasmodium malaria – This will produce quartan malaria

ve

and the name was given by Loveran and the same name

on

is still retained.

IV. Plasmodium Ovale – The specific name of this parasite is

derived from its oval shape and the blood cells also

become oval with the shape of the parasite.

PD

Malignant Malaria – Plasmodium falciparum produced malignant

malaria. It has the property of agglutination and embolic effects

and it thus proves fatal to human life within a short period of a

few hours to few days. It can also produce pernicious malaria

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and black water fever.

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Trophozoite – A trophozoite is defined as the vegetative form of an

unicellular parasite. In this phase, the chromative mass in cell is

te
solitary but the cytoplasmic outline is irregulate due to

is
pseudopodia. Examples are Malarial parasites, Entamoeba

eg
histolytica etc.

Schizont – Schizont is the next stage of trophozoite where a

nr
sexual division takes place in liver and red cells. In this stage

U
chromatin mass starts to divide into fragments and may form

into immature and mature schizont.

3
.5
Haemozoin pigments – These are metabolic products resulting

from amoeboid activity of parasite. A malarial parasite utilizes

r5
haemoglobin as food (Protein part) and yellowish brown pigment

granules appear in the cytoplasm of parasite after a period of

rte

about 10 hours growth of trophozoite.

ve

Schuffner’s dots – These are fine pinkish, delicate, innumerable

granules in the infected red blood cells. These can be identified

on

under microscope after Leishman’s staining. It is present in

plasmodium vivax.

Maurer’s dot or clefts – The brick-red dots in the red blood cells

infected by plasmodium falciparum under microscope after

PD

Leishman’s staining are called Maurer’s dots or clefts. In

Plasmodium falciparum Schuffner’s dots are not seen. The colour

of the corpuscle is reddish violet and the corpuscle shows

crenated appearance at the periphery.

ob

Life cycle of malarial parasite

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The life cycle of malarial parasites in two different hosts is as

follows –

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(1)In man – Man represent the intermediate host of malarial

is
parasite. The parasite resides inside red cell and liver cell

eg
and reproduces by asexual methozoites by the bite of

infected female anopheles mosquito. Sporozoites are found

nr
in the salivary gland of a mosquito and it may vary from

U
950 to 2,00,000 or more. But a mosquito can infect 3000

sporozoites at each bite. The sporozoites are thread like

3
curved organs measuring about 12µ in length and an

.5
elongated nucleus is present centrally. There is no pigment.

r5
The sporozoites will come in to the blood after the bite of the

mosquito and will enter the liver after 30 to 60 minutes.

rte

(2) In Female Anopheles Mosquito – Mosquito represents to

definitive host of the malarial parasite as there is sexual

ve

method of reproduction. The stages inside the mosquito are

on

as follows –

(I) A female Anopheles will draw blood of an infected

person and take both sexual and asexual form of

parasite. But asexual form will die immediateoy in the

PD

stomach of the mosquito.

(II) Inside the stomach of the mosquito, the pigment of

microgametocyte (male gametocyte) will show violent

movement and exflagellation occurs and it is

ob

chancterised by 4 to 8 thread-like filamentous

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strucers. They become detached and form

microgametes. From one microgametocyte, 4 to 8

te
microgametes will form the other hand, there is no

is
exflagellation of macrogamatocyte (female gametocyte).

eg
So, from one macrogametocyte, one macrogamete will

form.

nr
(III) By the process of chemotaxis, microgametes are

U
attracted towards the macrogametes and unite

together and form zygote.

3
.5
(IV) Then the zygote which is an actively moving body,

develops into coxinete or travelling vermicule.

r5
(V) The oocyst will mature and it will increase about 6 to

rte

60 µ in diameter. By the successive nuclear division

there develops a large number of sickle-shaped bodies

ve

called sporozoites.

(VI) The sprozoites will come in the body cavity of mosquito

on

except ovary. The sporozoites will be distributed to

various organs and tissues of mosquito through the

circulating fluid. Then they will come to salivary

glands and ducts. These sporozoites infect another

PD

person during the blood-meal of mosquito.

(VII) Development of sexual cycle in the mosquito in

different species –

ob

(a) P. vivax – 8 to 10 days.

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(b) P. falciparum – 10 to 12 days.

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(c) P. Malariae – 18 to 21 days.

is
(d) P. Ovale – 14 to 18 days.

eg
nr
---------------------------------------------------------------------------------------------------------

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S .K . Sam an ta

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