Histopathology

Histology is the microscopic study of normal tissue and histopathology is

the study of abnormal or diseased tissue.
This the most important tool of the anatomical pathologist in routi8ne
clinical diagnosis of cancer and their disease. Histopathology deal with
diagnosis of diseased tissue.
There are various techniques and methods designed and employed in
histopathological investigation as it impossible to study all normal and
abnormal tissue components in a single preparation.

FLOW OF SAMPLES PROCESSING IN HISTOPATHOGY.

Received samples from OT in container containing proper fixative
Giving identification numbering to sample.
Grossing of received sample by pathologist.
Placing gross section in cassettes.
All cassettes are placed in automati8c tissue processor.
 On the second day, cassettes are remove from automatic tissue
processor.
Processed section then removed from cassettes and ate embedded
in mould using paraffin wax. Cool by placing it on cold plate
Trimmed the paraffin block using microtome.
Take thin section on clean glass slide using microtome and then
place on hot plate for fixation.
Staining with H and E stain and some special stain
Mounted with DPX reagent.

COLLECTION OF TISSUES
Histopathological examination of tissues starts with surgery, biopsy or
autopsy. The tissue is removed from the body or plant, and then placed
in a fixative which stabilizes the tissues to prevent decay. The most
common fixative is formalin (10% formaldehyde in water)

GROSSING ROOM
Following fixation, the specimen of tissue blocks for sectioning is done by
the pathologists who briefly decries the gross specimen and also tag a
number with pencil on it. The tag is placed in the cassettes with cover
along with the tissue.

FIXATION
Once the specimen arrives in the lab it is first put into a fixative to
prevent autolysis and putrefaction. The usual fixative for paraffin
embedded tissues is neutral buffered formalin.
DECALIFICATION
Immerse tissue cassette in 11% formic acid with stir bar overnight in a
fume hood. Rinse in running water for 30-60minutes (the smell should be
gone) once the tissue has been fixed, it must be processed into a form in
which it can be made into thin microscopic section.

TISSUE PROCESSOR
A tissue processor is programmed for fixation, dehydration and clearing
of electron microscopy size particles and utilizes receptacle for each
particle or group of particles of tissue and a processing chamber adapted
to contain a plurality of the receptacles. The chamber is connected to a
plurality of containers, some of which contain the various processing
solution. The solution are individually piped to, and from beneath, the
chamber through a remotely controlled valve and remotely controlled
valve and manifold arrangement which also connects with a metering
pump. These arrangements minimizes fluid contamination and allow
each solution to be precisely metered, brought to, retained in and
drained from the chamber thus allowing the particle to be bathed is the
solutions according to an automatic programmed time sequences. The
program may be varied as to number of cycles per solution and terminal
solution in the program.

TISSUE PROCESSING
Dehydration
Wet fixed tissues (in aqueous solutions) cannot be directly infiltrated
with paraffin, Frist the water from the tissues must be removed by
dehydration. This is usually done with a series of alcohol, say 70%to
95%to100%. Sometimes the first step is a mixture of formalin and
alcohol. Other dehydrates can be used, but have major disadvantages.
Acetone is very fast, but a fine hazard, so is sage only for small, hand-
processed sets of tissues. Dioxane can be used without clearing, but has
toxic fumes. That’s why used alcohol it is easily available.

CLEARING
The next step is called “clearing” and consist of removal of the
dehydration with substances that will that will be miscible with the
embedding medium the commonest clearing agent is xylene. Chloroform
use to be used, but this a heal hazard, and is slow.

INFILTRATION
Finally, the tissue is infiltrated with the embedding agent, almost always
paraffin.
Paraffin can be purchased the differ in melting point 600-650cfor various
harnesses, depending upon the way the histotechnologist likes them and
upon the climate. A product called parables contains added plasticizers
that make the paraffin blocks easier for the some technicians to cut. A
vacuum can be applied inside the tissue p0rocessor to assist penetration
of the embedding
AUTOMATED TISSUE PROCESSOR
This equipment is an excellent device. It is used to carry out the
procedure like:

i. Dehydration
ii. Cleaning
iii. Infiltration

This equipment decrease human error and saves time.

COMPONENTS:
1. A time clock
2. Basket carrier in a circular superstructure
3. Receptable Basket and receptable.

WORKING:
1. Small blocks of tissue are enclosed in the perforated capsules and
the capsules are placed in the basket.
2. The basket is attacked to one of the 12 yokes in the super structure.
3. The entire superstructure decent and ascent at scheduled intervals
controlled by the time clock.
4. The basket oscillated and moves up and down to keep the tissue and
reagent in a state of controlled agitation.
 5. The time required for routine processing is 16hrs. Hence after
setting the machine in the afternoon the processed material has
been taken out for embedding on the following morning.

PROCEDURE:
1. Place the solution and paraffin in their respective beakers of the
equipment
2. The timing level is set as zero and machine was started 4.30 p.m.
3. The basket with the cassettes automatically changes position and
takes a bath in

 Dehydration
 Clearing
 Infiltration

PROTOCOL:

SR.NO TIMING SOLUTION PERIOD

1. 4.30pm 80% alcohol 2hrs
2. 6.30pm 95% alcohol 1hrs
3. 7.30pm 95% alcohol 1hrs
4. 8.30pm 100% alcohol 1hrs
5. 9.30pm 100% alcohol 1hrs
6. 10.30pm 100% alcohol 1hrs
7. 11.30pm Xylol 1hrs
8. 12.30pm Xylol 1hrs
9. 2.30pm Paraffin 2hrs
10. 4.30pm Paraffin 2hrs
11. 6.30pm Paraffin Stop

EMBEDDING

Tissues processed into paraffin are melted by placing the entire cassette
in 650c. Paraffin bath for 15 minutes. Turn the heat block on to melt the
paraffin on hour before adding the tissue block molds on the hot plate.
The paraffi zed specimens may be dissected with a razor blade and
placed with the cut surfaces down towards the bottom of the mold and
from the paraffin pot. Use heated forceps to orient the tissues in the
mold. When the tissue is in the desired orientation add the labeled tissue
cassette on top of the mold as a backing. Be sure there is enough paraffin
to cover the face of the plastic cassette. Slide the mold off the hot plate
onto a cold aluminum heat sink. When the wax is completely cooled and
hardened (20min) the paraffin block can be popped out of mold. If the
wax cracks or the tissues are not aligned well, simple melt them again
and start over. Tissue blocks can be stored at room temperature for
years.

SECTION CUTTING.

Block to be sectioned are placed face down on an ice block for 10 mins.
Place a fresh blade on the microtome. Insert the block into the
microtome chuck so the wax block faces the blade and is aligned in the
vertical plane. Set the dial to cut 4-10µm sections. The blade should be
angle 4-60. Face the block by cutting it down to the desired tissue plane
and discard the paraffin ribbon. If the block is ribboning well then cut
another four section and pick them the forceps or a fine paint brush and
folt them on the surface of the 370 water bath. Float the sections onto
the surface of clean glass slides. If the block is not ribboning well then
place it back on the water bath then it may be too hot.

Place the slides with paraffin sections in a 650c oven for 20 minutes ( so
the wax just starts to melt ) to bond the tissue to the glass slides can be
stored overnight at room temperature.

REHDRATION

Commonly referred to as taking the section to water. The goal is to

replace the water insoluble paraffin wax with water prior to staining with
water soluble stains.
TISSUE FLOATING BATH

The sections were transfers to the water bath of temperature 560 so the
section flat on the water. This is done to remove the flat & wrinkles
produced in the section during cutting

PICKING UP THE SECTIONS.

The sections of ribbon was picked by emerging the slide tightly smeared
with adhesives, vertically in water bath to 3 quarter of its length and
manuaring the section into the contact with the slides. The section were
correctly positioned on the slide and bottled lightly with moistened
blotting paper to remove excess ant to increase contact between section
and slide.

DRYING OF SECTION CUTTING

Drying of the staining of the section enables one to study the physical
Characteristics and relationship for the tissue and of their constituent’s
cell as the slides were dried in the oven in the oven, the slide are then
transferred to the staining to stain with different stain.

The different types of staining procedures carried out in the histology

departments are as follows.
 H & E staining
Masson trichromes sating
Retic staining
Prussian blue staining
Orcien staining
Periodic acid Schiff’s etc.

Mounting of slides

Mounting of slides with different mounting medium is done is done to

prevent stained slide from any kind of distortion or disaffect caused by
environment or any other thing routinely used mounting media is DPX
 STAINING
HAEMATOXYLIN ANE EOSIN STAINING
 H & E stain, HE stain or haematoxylin and Eosin stain, is popular
staining method in histology.
 It is the most widely used stain in medical diagnosis, for example
when a pathologist looks at a biopsy of a suspected cancer; the
histological section is likely to be staining with H&E.
 The staining method involves application of the basic dye
haematoxylin, which colures basophilic structures with blue- purple
hue , and alcohol – cased acidic eosin which colures eosinophil
structures bright pink.
STAINING PROCEDURE
1) Immerse section in the filtered Harris haematoxylin for 5 minute.
2) Rinse with tap water.
3) Dip the slide in acid alcohol immediately.
4) Rinse with tap water.
5) 5. Immerse section in Eosin stain for 1min.
6) Exchange tap water until the water is clear.
7) Dehydrate in ascending alcohol solution 70%, 80%, 90%,1oo%×2).
8) Clear with xylene (2×).
9) Mount coverslip onto a labeled glass slide with permanent.

RESULTS

Nuclei and other basophilic structure are blue.

Cytoplasm and acidophilic structures are light to dark red or pink colour.

SPECIAL STAIN

MASSON TRICOM
Weigert’s iron haematoxylin stains the nuclei in black, Biebrich scarlet –
acid fuchsine stains cytoplasm and muscle fiber in red and after
treatment with phosphotungstic and phosphomolybdic acid, collagen in
stained in blue with aniline blue.
Procedure

 Deparaffinise slide & hydrate to water.

 Keep in haematoxylin for 10min.
 Wash in running water for 2-3 min till section becomes blue colour.
 Add poncean acid fuchsine solution for 15 min.
 5. Rinse in water.
  Add 3% phosphotangstic acid for 5min.
 Rinse in water.
 Add light green solution 35 min.
 Rinse wash with alcohol.
 Dehyate, clear in xylene & mount in DPX

Results

Nucleus – Blue
Muscles-Red
Rbcs-Orange

Retic

In pathology the retic stain is a popular staining method in histology. It is

used to visualize reticular fiber and used extensively in liver
histopathology.
PROCEDURE

 1% potassium permanent for 3 min.

 Rinse water 2 min. Oxalic acid for 2 min.
 Wash with 5 min.
 Iron alum for 20 min.
 Wash with water.
 Retic complex- 2cc 10% silver nitrate (AgNo3) +0.2cc 10% NaoH then
liquid ammonia put in drop by drop filled the dissolved +equal
amount of D/W.
 Then filtered & put on the slide &wait 10 min.
 Then wash under running water.
 Put on 10% for malin on slide.
 Then filled the formalin when tissue coloured in brown then do not
wash the slide.
 Then put on gold chloride 10 min.

RESULTS

Reticulie –Black
Nuclear –Grey
 PRUSSIAN BLUE

 The reaction occurs with the treatment of section in acid solution of

Ferro cyanides. Any ferric ion (+3) in the tissue combines with the
Ferro cyanides and result in the formation of a bright called
“Prussian blue” or ferric Ferro cyanide.

PROCEDURE
A. Mix on 2% equal quality of potassium ferricynide 1ml + 2% Hcl
1ml acid for 20 min& wait.
B. Wash under tap water put on rack.
C. Then slide put on eosin for q1 min.
D. The dip 10-15 time on alcohol.
E. The clean slides help on cotton and put on rack.
F. Then mounting.
 G. Put on coverslip DPX then put coverslip on the slide and
observed under microscope.

RESULT

 FIBRIN –BLUE
 NUCLI- RED.

ORCEIN
 Orcien is a natural dye obtained from lichens which are found to
stain copper associated protein, elastic fibers and hepatitis B surface
antigen, all of when stain is used for diagnosis of hepatitis B
infection as well as copper accumulation in Wilson’s disease or in
chronic biliary diseases.

PROCEDURE

1. 1 % potassium permangent 10 min.

2. After 10 min wash under the tap water 5 min.
3. Then put on slide oxalic acid for 2 min.
4. Then was under tap water 5 min.
5. The put on slide orcine stain for 2 hrs.
6. After slide dip on acid alcohol and wash under tap water 5 min.
7. Mounting.

PREPARATION OF ORCIN SOLUTION.

(35 ml Absolute alcohol + 0.5g orcein powder + 15 mi D/W).

(Cover jar & keep in over for 20 min & then cool the solution & add 0.5 ml
conc.HCI)
 PERIODIC ACID SCHIFF’S
(PAS)

 This all around useful stain for many things. It stains glycogen,
mucin, mucoprotein, glycoprotein, as well as fungi. A predigestion
step with amylase will remove staining for glycogen.
 PAS is useful for outlining tissue structures-basement membranes,
capsules, blood vessels, etc. It does stain a lot of things and
therefore can have a high background. It is very sencitive, but
specificity depends upon interpretation.
PROCEDURE
a) Deparaffinise slide & hydrate to water.
b) Oixidise with 0.5% periodic acid solution for 5 min.
c) C) Wash in running water for 5 min.
 d) D) Cover slide with Schiff”s reagent for 10 mins till tissue becomes
pink colour.
e) E) Wash in running water for 10 min.
f) Do acid alcohol (just dip) and then wash in running water for 5 min.
g) Counter stain with haematoxylin for 1 min.
h) Wash in water for 1 min
i) Dehyrate in alcohol.
j) Clear in xylene.
k) Mount in DPX
Result

Glycogen fungus – magenta.

Nucleus – blue.
 ACID FAST BACILLI
(AFB)
Principle

The organisms such as mycobacterium tuberculosis and

mycobacterium leprae are extremely difficult to stain by ordinary method
because of the lipid containing cell wall. The bind carbol-fuchsin tightly
and resist destining with strong decolorizing agent such as alcohol and
strong acids.
PROCEDURE

a) Flood slide with carbon fuchsine solution put on the slide & heat the
slide filled the steam.
b) Then wash the slide.
c) After wash acid alcohol in proper cleaning & wash with running
water for 10 min.
d) Then put on slide methylene blue for 2-3 min.
e) Air dry and mount.

Result

 ACID FAST BACILLI :- BRIGHT RED

 BACKGROUND :- BLUE
 GOMORI METHANMINE SILVER NITRATE
(GMS)

In pathology the Gomori methenamine silver stain abbreviated GMS is a

popular staining method in histology
It is used widely as a screen for fungal organisms particularly useful in
staining carbohydrates. It can be used to identify the yeast like fungus
pneumocystis jiroveci which causes a form of pneumonia called
pneumocystis pneumonia (PCP) or pneumocystis.
The cell walls of these organisms are outlined by the brown to black stain.

PROCEDURE
 Add chromic acid 1 hr. and wait.
 Wash under tap water.
 Then put on 1% sodium Bisulphate for 1 min.
 After 2 min wash under tap water.
 Then put on slide GMS complex.
 Then 33 min for 60c and wash.
 Then 33 min add gold chloride for 10min.
 Wash y=under tap water 2-3 min.
 Then sodium thiosulphide tap water.
 After 3 min wash under tap water.
 Then put on light green for 3 min and wash under tap water.
 Mounting

RESULT

 FUNGI – BLACK
 BACKGROUND – GREEN.
 LEICA ST4040

 The leica ST 4o4o linear staining system is designed to meet the

current and future needs of today’s high throughput staining
laboratories.
 Routine applications such as H&E can be run at a throughput
capacity of up to 2000 slides-per hour.
 The modular design of the system allow the customer utmost
flexibility in instrument configurations to meet the needs in both
staining volume and workflow of histopathology and cytopathology
laboratories.
User convenience

 A special storage container keeps slide rack carriers conveniently

located next to the slide rack loading zone. Continuous agitation of
the slide racks during processing enhances reagent penetration and
ensures the highest quality staining result. The leica ST4040 slide
rack design significantly reduces reagent carryover.
 THERMO SCIENTIFIC

The thermo scientific brand provides anatomical pathology labs with the
broadest portfolio of instrument and consumable solutions, from
specimen collection and grossing to advanced staining and cover slipping.
Labeling And Tracking, Grossing, Tissue Processing And Embedding,
Primary Staining, Microtome And Cryotomy, Immunohistochemistry, And
Cover Slipping And Associated Consumables Are The Key Product
Categories Offered The Anatomical Histopathology Laboratory.

CRYOSTAT
CRYOSTAT – (-15 TO -25)
IT’S USED ONLY EMERGENCY CASES.
 Take the tissue in popper
 Take tissue holder put one drop of water at the center kept tissue
sample and hold into holder (temperate -20oc
)
 After tissue fixing remove tissue holder and take proper section (-
25oc)
 Terming and then sectioning set as the slide and staining is done by
H&E staining and Toluidine blue stain mounted with DPX
Staining for frozen section only –
Toluidine blue-
COMPOSITION - Toluidine blue – 0.1gm
Distilled water -100ml

PROCEDURE
 Stain in 1 % toluidine blue for 1 min.
 Rinse in distilled water (D/W).
 Mount in glycerin.
CYTOLOGY
CYTOLOGY
Cytology is the examination of individual cell and small clusters of
cells, and may be used for the diagnosis and screening of diseases,
including cancers. Cytology also refers to the study of diseases at the
cellular level. For diagnosing disease such as cancer, cytology may be
referred to as cytopathology.

FNAC (fine needle aspiration cytology)

Fine – needle aspiration biopsy (FIAB, FNA OR NAB), or fine –

needle aspiration cytology (FNAC).
Is a diagnostic procedure used to investigate superficial (just under
the skin) lumps or masses.
In this technique, a thin, hollow needle is inserted into the mass for
sapling of cells that, after being stained, will be examined under a
microscope. There could be cytology exam of aspirate (cell specimen
evolution, FNAC) or histological (biopsy- tissue specimen evaluation,
FNAB. Fine- needle aspiration biopsies are very safe, minor surgical
procedures. Often, a major surgical (excisional or open) biopsy can be
avoided by performing a needle aspiration biopsy instead, today; this
procedure is widely used in the diagnosis of cancer.

APPLICATION OF FNAC:-
 FNAC is very valuable for preliminary diagnosis of carcinomas as
well as inflammatory conditions.
 FNAC is particularly useful in the dealing with suspected masses
or lesion on the skin, lymph node, breast, thyroid, liver, kidney,
lung and bone.

PROCEDURE:-

a) To perform the aspiration, first of all, the skin is cleaned with a

suitable antiseptic.
b)The masses are then immobilized with one hand. Without this
piston of the syringe being retracted, the needle is inserted into
the mass.
c) The cells are than aspirated into the needle.
d) The needle can be inserted from different angles if the
material from one site is too scanty.
e) Release the piston before withdrawing the needle.
f) This is to equilibrate the pressure in order to prevent the
material being drawn into the barrel of the syringe.
 g) The collected specimen is expressed on to a clean pre-labeled
slide and spread evenly.
h) Fixation is done immediately and on stained with
papanicoloau, H and E stain and geimsa stain.
i) Air dry and mount.
Two types of staining are use for fnac

1. PAPANICOLOAU STAINING
2. GIEMSA STAINING.

PAPANICOLOAU STAINING
Papanicolaou stain (also papanicolaou’s and pap stain) is a
multichromatic staining histological technique developed by George
papanicolaou, the fater of cytopathology.

Pap staining is used to differentiated cell sin smear preparations of

various bodily secretion; the specimen can be gynecological smears
(pap smear), sputum, cerebrospinal fluid, urine, washings, abdominal
fluid, synovial fluid, seminal fluid, fine needle aspiration material,
tumors touch samples, or other materials. Pap staining is a very
reliable technique. As such it tis used for cervical cancer screening in
gynecology.
Papanicolaou staining is a polychrome counter-stain method, based
on dye completion in the cytoplasm combined with a nuclear
hematoxylin and two cytoplasmic stains OG-6 AND EA-36.

PROCEDURE

A. Fixation of slides in absolute alcohol _

20min.
B. Running Tap water ` _ 5min.
C. Dip in Gill’s Haematoxylene _ 10min.
D. Running Tap water _ 5min.
E. Dip the slide in Orange-6 stain _ 1½
min.
F. Ist dips Absolutes Alcohol _ 8-10min.
G. IInd dips Absolutes Alcohol _ 8-10min.
H. Keep the slide in Eosin Azure-36 (E-A 36) _
2 min.
I. Ist dips Absolutes Alcohol _ 8-10min.
J. IInd dips Absolutes Alcohol _ 8-10min.
K. After dips keep in running Tap water _
1-2 min.
L. Dry & mount in D.P.X.

RESULT
Nuclei: - Blue
Cytoplasm: - Varying shades of pink, blue, yellow, green, grey.

GIEMSA STAINING
There are a variety of “romanowsky-type” stains with
mixtures of methelene blue, azure, and eosin compound. Amo9ng these
are the Giemsa stain and the Wright’s stain (or Wright-Giemsa stain). The
latter is utilized to stain peripheral blood smear. The giemsa stain can be
helpful for identifying components in a variety of tissues.

One property of methylene blue and toluidine blue dyes is

metachromasia. This means that a tissue component stain a different
color than the dye itself. For example, mast cell granules, cartilage, mucin
will stain purples and not blue, which is helpful in identifying these
components.

PROCEDURE
I. Fixation of slides in methanol _ 20min.
II. Flood the slide with working _ 1:9 dilution with Tap water for 30
min. Giemsa solution
III. Wash slides with Tap water.
IV. Dry & mount in D.P.X.
CARDIO VASCULASR THORACIC CENTER
(CVTC)
IN THIS DEPARTMENT THE FOLLOWING TEST ARE PERFORMED -
1) Prothrombin Tine Test
2) Lipid Profile Test.
3) Serum Analyses Test
4) LDH Test
5) Cpk Test

 PROTHROMBIN TIME DETERMINATION.

This test is done by lyoplastin it is semi-automated prothrombin time
determine.
PRINCIPLE
Tissue thromboplastic in the presence of calcium activates the
extrinsic pathway of human blood coagulation mechanism.
When lyopastin reagent is added to normal citrated plasma, the
clotting mechanism is initiated forming a solid gel clot within in
specified period of time. The time required for clot formation would
be prolonged it there is aquried or congenital deficiency of factor
activity in the extrinsic pathway of the coagulation mechanism or
reduction in the activity of vitk depend clotting factor.
REAGENT
Lyophilized calcified thromboplastin reagent.
PROCEDURE
 1) Aspirate from the reagent vial enough
reagent for immediate testing requirement in thoroughly clean
and dry test tubes.
2) Bring the reagent to room temperature
before prewar Ming at 300c for testing purpose
3) Recap the reagent vial and replace
immediately to 2-80c
4) To a 12×75mm tube add 0.1ml of plasma
and place the tube in a waterbath for 3-5m at 370c
5) To the tube forcibly and 0.2ml of lyoplastin
reagent and simultaneously start a stopwatch. Shake tube gently
to mix contents.
6) Gently till the tube back and forth and stop
the stopwatch as soon as the first fibrin strand. Recode
time. this is prothrombin time
NORMAL VALUE
11-15 second

Calculation
R =Mean of the patient plasma PT in sec
MNPT for reagent
 SERUM AMYLASE TEST

Principle
An alpha- analysis hydrolyzes p- nitro phenyl-alpha-D-maltoheptoside
to p-nitrophenylmaltotriose and maltotetrose Glucoanaylse
hydrolyzes PNPG3 to p- nitrophenlyglycoside and glucose and p- nitro
phenol, which produce yellow color.
REAGENT
Amylase reagent.
SPECIMEN
Unhemolyzed serum
NORMAL VALUE
10-93IU
PROCEDURE
1) Dispense 1.0ml of reconstituted analyses
reagent in a test tube
2) Precincubation at 370c for 5 min.
3) Add 0.02ml of serum and mix well.
4) Read absorbance every 30 sec for two min.
5) Determine mean absorbance
(∆𝐴)dufference per minute
6) Multiply ∆ A/min by 7/33 to obtain serum
anaylase
CALCULATION
Serum analyses increase in traumatic lesion of pancreases and
carcinoma of pancreas etc.
Serum amylase, IU =∆Abs/min×total×1000
Mill molar absorptivity
sample volume LP
CLINICAL SIGNIFICANCE

DETERMINATION OF SERUM LACTATE DEHYDROGENASE (LDH)

METHOD – KING’S METHOD.

PRINICPLE
Addition of dinitrophenyl hydrazine (DNPH)
dinitrophenyl hydrazine is formed. The formation of D.N hydrazone
gives measure of LDH concentration and can be measured after
alkaline dilution at 440nm
SAMPLE SPECIMEN
Serum
REAGENT
Glycine reagent
Buffered substrate
NAD solution
DNPH reagent
0.4N sodium hydroxide.
NORMAL VALUE
70-240IU.

PROCEDURE
SR.NO TEST BLANK
1 Substrate 1.0ml 1.0ml
Incubate at 370c
for 5 min
2 NAD+ solution 0.2ml 0.2ml
3 Serum 0.2ml
Incubate at 370c
for 5 min
4 D.NPH 1.0ml 1.0ml
5 0.02ml
Mix, keep at
room tem
(250c) for 15
min
6 0.4N sodium 10ml 10ml
hydroxide
 Mix and measured OD (optical density) at
440nm.

CLINICAL SIGNIFICANCE
High serum LDH observed in neoplastic state, hemolytic anemia,
megaloblastic anemia etc.
In disease of liver, renal infarct, muscular dystrophy etc.

CPK (CREATINE PHOSPHOKINASE)

PRINCIPLE
Creatine phosphate + ADP CPK creatine + ATP
The creatine formed in the reaction, reacts with diacetyl and α-
naphthol in alkaline medium to give coloured complex. The intensity
of color is proportional to CPK activity and it measured at 520 nm.

SAMPLE
Serum (free from hemolysis)
METHOD
Huge method
NORMAL VALUE
Men – 20-50 IU.
Women -10-37IU.
PROCEDURE

SR.NO. TEST STANDARD BLANK

1 Substrate 0.4ml 0.4ml 0.4ml
2 D/W - 0.2ml 0.2ml
3 Serum 0.05ml - 0.05ml
4 Reagent 2 0.2ml - -
Mix well incubate at 370c
for 30 min.
5 Working standard - 0.05ml -
6 Reagent 5 0.5ml 0.5ml 0.5ml
7 Reagent6 3.75ml 3.75ml 3.75ml
8 Reagent9 1.0ml 1.0ml 1.0ml
9 Reagent10 0.5ml 0.5ml 0.5ml

Mix and keep dark RT for 30 min measure O.D to test standard
and blank

CALCULATION
Serum CPK IU =
O.D Test – O.D Blank
×66.7
O.D std –
O.D Blank
CLINICAL SIGNIFICATION

Increase levels may also be found in polymyositis

Motor – neuron disorders and in acute cerebrovascular

DETERMINATION OF ESR
ESR is determined by with the help of method.
CBC (complete blood count).
CBC is done by ERMA machine.

URINE ANALYSIS

Determination of glucose by Benedict’s test

PRINCIPLE
When benedict’s qualitative reagent is heated with
8drop of urine, glucose present in urine reduces cupric ion
present in the reagent to cuprous ion. Alkaline medium is
provided to the reaction by sodium carbonate present the color
to green yellow, orange or red so conformed glucose present in
the urine
 PROCEDURE
 Take 5ml of benedict’s reagent .
 Add 0.5 ml of urine sample.
 Mix well and keep it warm water for 8 min
 Observed the changing color.
NEUROPATHOLOGY
Neurology is a Brach of medicine dealing with disorder of the
neverous system. Neurology deals with the diagnosis and treatment of
all categories of condition and dissolve involving the central and
peripheral nervous system ( and its subdivision , the autonomic
nervous system and the somatic nervous system ) Neurological
Patrice relies heavily on the field of neurosciences, which is the
scientific study of the nervous system. Neuropath it is the type of
histopathology.
In neuropathology department work done by fully manually method.

SAMPLE COLLECTION
It is collected by a surgeon in Operation Theater the routine
histopathology lab receives these specimen in the form of small pieces
of tissue (Biopsy or whole section).

FLOW OF SAMPLE PROCESSING IN NEUROPATHOLOGY

 Grossing
 Tissue preservation.
 Dehydration and clearing.
 Infiltration and Embedding.
 Terming & sectioning
 Floating
 Staining (special sating in NP sample like , GMS
 Mounting & PB etc.
(All this process written by histopathology department)
TISSUE PROCESSING
In the KEM hospital post mortem samples are processed manually in
this department.
In this process the tissue sample is changed from one container of
reagent to another by hand.

AUTOMATED TISSUE PROCESSING

(Same processing as histopathology department).

TEST FOR CSF & BODY FLUID

In neuropathology lab of kem hospital sugar and protein tests are
carried out of CSF another body fluid like peritoneal fluid, synovial
fluid ascetic fluid etc.

 PROTEIN TEST FOR CSF –

Reagent
 3% trichloron acetic acid
 Sample

PROCEDURE
1. 3% trichloron acetic acid 3ml
2. 5µl of CSF sample wait for 20 min.
3. Take reading against 470 nm with blank
  SUGAR TEST FOR CSF

PROCEDURE
1. 5µl GOD +50 µl sample of CSF wait for half hours.
2. Take the reading at 540 nm (GOD as blank )

PROTEIN TEST FOR FLUIDS.

PROCEDURE
1. 12.5 % TCA for 1ml + 4 ml normal saline + 40 µL fluid sample.
Wait for 15-20 min.
2. Take the reading with blank.
BLOOD BANK
A blood is a center where blood gathered as result of blood donation,
stored and preserved for later use in blood transfusion.
The term ‘blood bank’ typically refers to adivison of hospital where
the storage of blood product occurs and where proper testing is
performed (to reduce the risk of transfusion related adheres event.) It
sometime refers to a collection center, and indeed some hospital also
performed collection.

IN KEM HOSPITAL BLOOD BANK DIVIDED, IN TO FOUR

PART

 Donor room
 Red cell room
 Component room
 Transmitted transfusion disease
 WHY TO DONATE BLOOD?????
There are several health benefits of donating blood …….
 Reduces the chances of heart diseases. Regular blood donation
helps, especially in males in loosing iron on regular basis. It
reduces the chances of heat attack to one third.
 Enhances the production of new RBC’S
 Helps in fighting hemochromatosis (Iron overload disorder )
 Burns calories: one print of blood (450ml) when donated, it

burans 650 calories in donor’s body.

Except all these health benefits, the reason to donate blood is that.
As we know blood cannot be harvested, it can only be donated; this
means only we can save a life that needs blood the time taken by a
patient’s body to replace it could cost his /her life. Sometimes the
body might not be in a condition to replace it all.
Saving a life does not require heroic deeds.
 BLOOD DONATION

CRITERIA FOR SELECTION OF DONOR

Following are some of the criteria accepted for selection of the
donor :
 Age: above 18 years (18-60 year).
 Hemoglobin: should not be less the 12.5g/dl.
 Interval: between donations should be minimum 90 days.
 Pulse rate: should be regular and between 80-120/min
 Accepted :blood pressure,
Systolic:-between 90-150mm/hg
Diastolic: between 50-100mm/hg
 Temperature: of the donor should be normal and should be
absence of any chronic disease.

Table: deferment of blood donation

 Conditions Period of deferment
1) Abortions 6 Months

2) History Of Blood Transfusion 6 Months

3) Surgery 12 Months

4) Typhoid 12months After Recovery

5) Malaria 3 Months
6) Tattoo 6 Months

7) Breast Feeding 12months After Delivery

8) Immunization (Cholera), Plague Gammalobin
Tphoid,Diptheria,Tetanus

9) Rabies Vaccination 1 Year After Vaccination

10)History Of Hepatitis In Family 12 Months

Of Close Contact
11 ) Hepatitis A 1 Year

12) Tuberculosis 5 Year

13)Alcohol Consumption 24 Hours

14) Tobacco Consumption 6hours

 COLLECTION OF BLOOD
Basic Requirements:
Before the collection of blood it is necessary to fill a form, which
includes the following data of the donor.

Types of containers:
Mainly two types of disposable bags are there according to the
presence of anticoagulants:
a) CPDA Bags
b) SAGM Bag
According to the volume, there are four types of Bags:
a) Quadra Bags
b) Penta Bags
c) Triple Bags
d) Double Bags

BLOOD COLLECTION PROCEDURE:

I. Venipuncture site: The donor’s forearms are preferably
the cubital region. If the donor is right handed, it is
preferable to collect the blood from left cubital region.
II. Venipuncture is performed.
III. Blood is collected in respective bags as per requirement.
IV. After blood collection, mixed it adequately with
anticoagulant.
 V. Donor is asked to lie down for 10-15 minutes; a sterile
cotton ball can be placed over the site of venipuncture.
VI. After the donation of blood, refreshments are given to the
donor.
The most important tests performed after the collection of blood are:
 Blood group and Rh – typing
 Hepatitis B
 Hepatitis c virus
 Malaria parasites
 Syphilis

STORAGE OF BLOOD
After all the testing of blood, bags are labeled by using appropriate
labels as per international regulation as:
ABO Type Colour of label
O Blue
A Yellow
B Pink
AB white

The label should contain following particulars:

 Date of expiry.
 The ABO and Rho (D) type.
 Date and time of collection.
 STORAGE TEMPERATURE OF BLOOD COMPONENT:
 Pack cell volume : 2 to 6 degree celcious
 Plasma: -30 to -80 degree celcious
 Platelets :20to 24 6 degree celcious with agitation
 Cryo: -30 to -80 degree celcious
 Fresh frozen plasma:- 30 to -80 degree celcious

EXPIRY DATES:
 Platelets : 5 days
 Fresh frozen plasma : 1 year
 SAGM bag: 42 days
 CPDA bag: 35 day
 RED BLOOD CELL

 ABO GROUPING AND RH-BLOOD TYPE GROUPING

The ABO blood grouping system is the most important blood type in
human transfusion.
Human being can be divided into 4 major blood groups depending
upon reaction which are obtained by mixing the blood of a person
with2 different antisera’s known as Anti- A and Anti- B. and group are
A,B,AB and O.
Naturally occurring antibodies in their plasma that are directed
against the missing antigens. For this reason, ABO grouping should
include both forward (cell) and reverse (serum) procedures

FORWARD GROUPING (CELL GROUPING):

In forward groping the suspension of red cell reacts with
reagent antisera (anti A and anti-B). Agglutination indicates
the presence of corresponding antigen on red cells.
REQUIREMENTS-
 Glass slides
 Test tubes
 Pasteur pipette
 Antisera’s
SAMPLE
Clotted whole blood.

PROCEDURE
1. Prepare 5% cell suspension of red cells in isotonic saline
2. Take four rows of test tube.
3. Add 1 drop of anti-A sera to the first tube.
4. Add 1 drop of anti –B sera to the second tube.
5. Add 1 drop –D sera to the fourth tune.
6. Add 1 drop of anti-AB sera to the third tube.
7. With the help of Pasteur pipetted add1 drop of 5% cell
suspension in each tube.
8. Mixed well with gentle shaking, incubate it or centrifuge for
observing agglutination.

REVERSE GROUPING (SERUM GROUPING)

In reverse grouping mixing of serum+ known A cells and known B
cells. Agglutination indicates the presence of corresponding
antibodies in the serum.

REQUIREMENT-
 Glass slide
 Test tube
 Pasteur pippte
 Antisera’s

SAMPLE
Clotted whole blood
PROCEDURE
Prepare a saline suspension of pooled A cell, B cell, O cells.
1) Add 1 drop of patient’s serum in 3 test tubes.
2) Add 1 drop off pooled A cell to first tube.
3) Add 1 drop off pooled B cell to second tube.
4) Add 1 drop off pooled O cells to third tube.
5) Mix well gentle shaking, incubate it or centrifuged for observing
agglutination.

OBSERVATION
Forward grouping Reverse grouping ABO grouping
unknown cell test unknown serum
against tested against
Anti-A cell Anti-B A-Cell B-Cell

O O + + O
+ O + + A
O + O O B
+ + O O AB

 CROSS MATCH
Cross matching is a procedure performed prior to a blood transfusion
to determine whether donor blood is compatible with recipient blood.
Compatibility is determined through matching of which are the ABO
and Rh system and by directly testing for presence of antibodies
against a sample of donor tissues or blood.

PRINCIPLE
Cross- matching will detects incompatibles between the donor and
recipient that will not be evident on blood typing. The are two types of
cross- matching
The Major cross match involves testing the patient’s serum with
donor cell to determine whether the patient has an antibody which
may cause a hemolytic transfusion reaction or decreased cell
survivals of donor cell. This is most important cross- match.

PROCEDURE

1) Prepare donor and recipient blood sample.

2) Donor’s red cell and recipient serum or plasma.
3) Prepare 3-5% cell suspension of red cells.
4) Label a test tube. Add two drops of the patient serum and
one drop off the appropriate donor cell suspension

PRINCIPLE
Cross- matching will detects incompatibles between the donor and
recipient that will not be evident on blood typing. The are two types of
cross- matching
The Minor cross match involves testing the patient ’cell with donor
plasma to determine whether there is an antibody in the donor’s
plasma directed against an antigen on the patient’s cells.

PROCEDURE
 1) Prepare donor and recipient blood sample.
2) Recipient red cell and donor’s serum.
3) Prepare 3-5% cell suspension of red cells.
4) Label a test donor serum and one drop of the patient cell
suspension.
5) Mix the tube and incubate at 370c for about 45 min.
6) Add two drops of AHG (antihuman globulin) and mix well.
7) Centrifuge for 1 m at 1500 rpm
8) Rea macroscopically and microscopically and record the result.

COOMBS TEST
Coombs test is also known as ant globulin test.
The coombs test for antibodies that may stick to the blood cell and
cause red blood cell to die too early. It was discovered by coombs,
mordant and race in 1945. Coombs reagent is antihuman globulin. It is
made by injecting human antibodies specific for human
immunoglobulins and human complement system factors.
Principle
Red cell coated with complement or IgG antibodies do not agglutinate
directly when centrifuged. These cells are said to be sensitized with
IgG or complement. In order for agglutination to occur additional
antibody, which react with Fc portion of the IgG antibodies. With the
c3b or c3d component to complement, must be added to the system.
 TYPE OF INDIRECT TEST
1. Direct test
2. Indirect test
 DIRECT COOMBS TEST
The direct coombs test is used to direct antibodies (IgG or C3 ) that are
stuck to the surface of red blood cell. Many disease and drugs can
cause this. This antibody something destroys red blood cells and
cause anemia.
This test that is done on the newborn’s blood sample. Usually in the
setting of a newborns with antibody mediated hemolysis in newborn
are Rh incompatibility and ABO incompatibility.

PROCEDURE
I. Prepare a 5 % suspension in isotonic saline of the red blood
cells to be tested.
II. With clean pipette add one drop of the prepared cell
suspension to small tube.
III. Wash three times with normal saline to remove all the traces
of serum.
IV. Decant completely after the last washing.
V. Add two drop of antihuman serum.
VI. Mix well and centrifuge for one min at 500 rpm
VII. Resuspend the cell by gently agitation and examine
macroscopically and microscopically or

INDIRECT COOMBS TEST.

Indirect coombs test looks for free-flowing antibodies against certain
red blood cells. It is most after done to determine.
This is the test that is done on the mothers blood sample the test
identifies a long list of minor antigens that could either cause
problems in the newborns or cause problems in the mother if
transfusion is necessary.
PROCEDURE
I. Label there test tubes as test serum (T) positive control and
negative control
II. In the tube labeled as T. add two drop of anti –D serum.
III. In the tube Pc add one drop of saline.
IV. Add one drop of 5% saline suspension of the pooled ‘O’ Rho
positive cell in each tube.
V. Incubate all the three tube for one hrs. at 370c
VI. Wash the cells three times in normal saline to remove excess
serum with no free antibodies.
VII. Add two drops of coombs serum to each time
VIII. Keep for 5 min and then centrifuge at 1500rpm for one min
IX. Resuspend the cell and examine macroscopically well as
microscopically

INTERPRETATION

NEGATIVE RESULT
No clumping of cell this means no antibodies to red blood cell.
POSITIVE RESULT
Clumping of the blood cell during a direct coombs test. Antibodies on the
red blood cells and that cause the destruction of red blood cells.
This may be due to hemolytic anemia. Chronic lymphocytic leukemia,
syphilis.

PLATELETPHERESIS
Plateletpheresis is the process of collecting thrombocytes, more
commonly called platelets, a component of blood involved in blood
clotting. The term specifically refers to the method of collecting the
platelets, which is performed by a device used in blood donation that
separates the platelets and returns other portion of the blood to the
donor. Platelet transfusion can be a life –saving procedure in preventing
or treating serious compilations form bleeding and hemorrhage in
patients who have disorders manifesting as thrombocytopenia (platelet
count) or platelet dysfunction. This process may also be used
therapeutically to treat disorders resulting in extraordinarily high platelet
counts such as essential thrombocytosis.

PLATELET TRANSFUSION
Platelet transfusions are traditionally given to those undergoing
chemotherapy for leukemia, multiple myeloma, those with aplastic
anemia, AIDS, bone marrow transplant, radiation treatment, organ
transplant or surgeries such as cardiopulmonary bypass. Platelet
transfusions can worsen neurologic symptoms and acute renal failure,
presumably due to creation of new thrombi as the platelets are
consumed. It should be avoided in those with heparin- induced
thrombocytopenia (HIT) or disseminated intravascular coagulation (DIC).

ADVANTAGE
There are several advantages of separating the platelets at the time of
collection. The first advantage is that the whole –blood platelets,
sometimes called “random” platelets, from a single donation are not
numerous enough for a doses to give to an and patient. They must be
pooled form several donor to create a single transfusion, and this
complicates processing and increases the risk of diseases that can be
spread in transfused blood, such as human immunodefiency virus.
Collecting the platelets from single donor also simplifies human leukocyte
antigen (HLA) matching, which improves the chance of a successful
transfusion. Plateletpheresis products are also easier to test for bacterial
contamination, a leading cause of transfusion-
Associated deaths. Pooling of whole – blood platelets is often done in an
‘’open” system where the platelet containers are connected in a way that
could expose the platelets to air, and pooled platelets must be transfused
promptly so that any contamination does not have time to grow.
The basic principles of automatic platelet apheresis are the same as in
the manual procedure, but the whole procedure is performed by a
computer-controlled machine. Since the donor’s blood processed in a
sterile single – use centrifuge, the unwanted components can be
returned to the donor safely. This allows the apheresis machine to repeat
the draw-centrifuge-return cycle to obtain more platelets. The bulk of the
machine and the length of the donation process mean most platelet
Donations are done in blood centers instead of mobile blood drives.
Most newer apheresis machines can separate a maximum donation of
platelets in about 60 to 120 minutes depending on the donor’s health
condition.

PLATELET DONATION
After a short physical examination, the donor is taken into the donation
room and sits in a chair next to the machine. The technician cleans one or
both arms with iodine, or other disinfectant, and inserts the catheter into
a vein in the arm. With some procedures both arms are used, one to Drau
blood and the other to return it. The process takes about one to two
hours while blood I pulled into the machine, mixed with with an
anticoagulant such as sodium citrate, spun around, and returned to the
donor “double needle” procedures using both arms tend to be shorter
since the blood is drawn and returned through different catheters; with
“single needle” procedures a set volume is drawn and processed in the
first part of the cycle and turned in the second part. The donor’s blood
undergoes repeated cycles of draw and return.
 TRANSMITTED TRANSFUSION DISEASES

1) HIV
INTENDED USE –
HIV 1+2 ELISA kit is an in vitro quantitative test to diagnose HIV infection
using human serum or plasma. It is intended for screening of blood
donors and HIV suspected individuals.

PRINCIPLE
HIV antigens are immobilized on a porous immunofiltration membrane
and are absorbed into the underlying absorbent .As p0atients sample
pass through the membrane, HIV antibodies .if present binds to the FC
portion of the HIV antibodies to give distinct blue or yellow color.

KIT CONTAINS.
 Sample diluent
 Conjugate
 Washing control
 Negative control
 Positive control
 Micro well strip
PROCEDURE
 Add 200µl of sample diluent to required number of well except
blank well
 Add 10µl of negative control and positive control and sample to
the respective wells mix thoroughly.
 Incubate for 30 min at R.T
 Decant and wash the well 5 time with diluted wash buffer.
 Add 50µl of conjugate to each well except blank.
 Incubate for 30 min at R.T
 Repeat the washing steps number 4
 Add 100µl of color reagent to each well.
 Incubate for 15 min at R.T.
 Add 100 µl of stopping solution to each well.
 Read the absorbance at 450-630 nm within 30 min after
blanking with blank well.
 2) HBsAg
ERBA LISA PICO HBsAg is an immunoassay for the detection of pictogram
to Nano gram quantity of HBsAg in human serum & plasma quantity of
ERBA LISA Pico HBsAg use polylonal antibodies to HBsAg as coating
material and monoclonal antibodies to HBsAg as conjugate material.

PRINCIPLE:-
The ERBA LISA Pico HBsAg test kit is solid phase immunoassay for
the qualitative detection of addition HBsAg human serum and Plasma
Addition of positive control or HBsAg containing human serum or plasma
will form a stable complex with the bound antibody present in the well
and with anti HBsAg HRPO. A washing step with remove the unbound
conjugate molecule. Addition of color reagent will developed blue colour
only I positive control well and well containing HBsAg in test specimen
upon addition of stopping solution blue colour change to yellow.

CONTENTS OF THE KIT:-

i) Anti HBsAg coated plate
ii) Conjugate
iii)HBsAg negative control
iv)HBsAg negative control
v) Color Reagent
vi)Sample diluent
vii) Stopping solution
viii) Washing solution
 ix) Black cover
x) Adhesive strip

PROCEDURE:-
 Bring all the reagents and specimens at RT befo9re use
 Add 25 ml sample diluent to each well in each run, there will be one
blank (100ml sample diluent plus 50ml conjugate) thre3e negative
control and one positive control. Add 75ml of control and test
specimens to the respe3ctiv3e well add 50ml of conjugate to each
well cover the plate with black cover and incubate3 60min at
37degree Celsius.
 Wash the plate as per micro plate washing procedure
 Add 50ml of color reagent cover the3 plate with black cover and
incubate 60min in dark at 20-30degree Celsius
 Add 100ml of stopping buffer to each well
 Read absorbency at 450nm

INTERPERATION OF THE RESULT

Non-Reactive
If the absorbency of the test serum is less then cut off value then the
sample is consider a non-reactive
Reactive:-
If the absorbency of the test serum is equal or greater than the cut
off value then it is consider as reactive
 3) SD HCV ELISA
Intended used:-
SD HCV ELISA test kit is a sensitive and qualitative in vitro
diagnostics test for the detection of antibodies to Hepatitis virus in
human serum or plasma.

PRINCIPLE:-
HCV antigens are immobilized on porous immunofiltration
membrane. Sample and the reagents pass through the membrane
and are absorbed into the absorbent pad. As the patients sample
pass through the membrane HCV antibodies if present is
serum/plasma bind to the immobilized antigens in the subsequent
washing step unbound serum/plasma proteins are removed the
protein a conjugate is added which binds to the FC portion of the
HCV antibodies to give a distinct yellow or blue color.

KIT CONTENTS:-
 Coated micro plate
 Enzyme conjugate
 Sample Diluent
 Positive & negative control Tetra methyl benzidine substrate
A
 TMB substrate B
 Washing solution
  Stopping solution
PROCEDURE:-
 Prepare the strip well for negative control 3 well positive
control 2 well and sample.
 Pipette 100ml of Sample diluent each well
 Add 10ml of negative control 3 well positive control 2well
and sample each well
 Cover the micro plate with adhesive plate sealer and mix
well on vibrating mixer mixing is very imp to get the
reproducible results.
 Incubate the well at 37degree Celsius for 30min
 Wash the well 5 time with 350 diluent washing
 Pipette 100ml enzyme conjugate each well
 Cover the mi8croplate with adhesive plate sealer
 Incubate the wells for 30min at 37degree Celsius
 Wash the wells 5 time withy 350ml of diluent washing
 Gently mix the TMB substrate A & B at the ratio of 1:1 and
pipette 100ml of mixed substrate solution to each
 Incubate the well for 10min at R.T.
 Pipette 100ml of stopping solution each well
 Read the absorbance at 450min
 MALARIA PT/PV ANTIGEN TEST

CLINICAL SIGNIFICANCE:-
Malaria is a serious parasite disease characterized by fever
chills and anemia and is caused by parasite that transmitted from
one human to another by the bite of infected Anopheles
mosquito. There are four kinds of malaria that can infected
human. Plasma dim falciparum. P vivax, P ovale, P Malaria

PRINCIPLE:-
The malaria antigen Test contains a membrane strip, which
is pre coated with two monoclonal antibodies as two separate
lines across a test strip. One monoclonal antibody is specific to
the P falciparum hi8stidine rich protein (Pf HRP ll) and another
monoclonal antibody is pan specific to lactate Dehydrogenase
(Pan LDH) of Plasmodium species (P. Falciparum, Vivax, Malaria,
ovale).

KIT COMPONENT:-
Test Device, Assay Buffer, Sample dropper & product
insert.
PROCEDURE:-
 Add 5ml of whole blood into sample well
 Add 3 drops of assay buffer into developer well
 Read the test Result at 20min
MICROBIOLOGY
INTRODUCTION:-
The science of microbiology is the study of microorganism
and their activities it is concerned with their 1) form 2) structure
3) Physiology 4) Metabolism 5) Identification and 6) Reproduction
The microorganism are3 classified as Protista the microorganism
included in the kingdom Protista are 1) Bacteria 2) Algae 3) Fungi
and 4) protozoa

BACTERIOLOGY:-
Bacteriology is the scientific study of bacteria most bacteria
are not pathogenic. In pathological lab, successful classification of
pathogenic organisms may provide the direct route to their
elimination. Bacteria that cause disease is known as bacteriology.

Samples received in the microbiology department:-

Blood samples
Ear swab
Nose swab
Eye swab
Urine sample
Wound swab
Fluids

Primary section (Day 1)

Samples are received from OPD & from wards they are labeled
appropriately. These samples are stricking on different plate
media & inoculated in Broth tubes.

Different Agar plates are used in microbiology:-

Urine3 samples are stick on nutrient agar and Mac conk eyes
Agar plates

1) NUTRIENT5S AGAR PLATE:-

Nutrient agar is used as a general purpose medium for the growth
of a wide variety of non-fastidious mo9croorganims. It consists of
peptone, beef extract and agar. Nutri8ent Agar/broth is used for
the cultivation and maintenance of non-fastidious organisms.
COMPOSITION OF NUTRIENT AGAR:-
Beef extract - 3.0g
Peptone - 5.0g
Agar - 15.0g
D/W - 1000ml

Composition of Nutrient Broth Nutrient broth contains same

ingredients except agar