DRV/Pub-801/2018-19/88

LABORATORY MANUAL ON
FIELD DIAGNOSIS OF ANIMAL
AND AVIAN DISEASES

First Edition

Published by

CORE LABORATORY-I
ADVANCED ANIMAL DISEASE DIAGNOSIS AND
MANAGEMENT CONSORTIUM (ADMaC)
Directorate of Research (Vety)
Assam Agricultural University
Khanapara, Guwahati-781 022 
  
 

First Published : 2018

Published by

Core Lab I,
Advanced Animal Disease Diagnosis
And Management Consortium (ADMaC)
Directorate of Research (Vety)
Assam Agricultural University
Khanapara, Guwahati-781 022

Printed at
Digicraft, Ghy.
  
 

LABORATORY MANUAL ON FIELD

DIAGNOSIS OF ANIMAL AND AVIAN DEPARTMENT OF
BIOTECHNOLOGY

DISEASES

Edited by

Dr N. N. Barman
Professor
Department of Microbiology
College of Veterinary Science
Assam Agricultural University
Khanapara, Guwahati
Assam, INDIA
  
 

FOREWORD
 
I have immense pleasure in writing the foreword note for the
laboratory manual of disease diagnosis. The DBT Centre in NE on
Advanced Animal Disease Diagnosis and Management Consortium
(ADMaC) is a tripartite concept and has already completed four years of its
establishment. The Project was established to strengthen Animal Disease
Monitoring and Surveillance system in North Eastern region of the country.
In regards to the animal disease scenario of the country, the North Eastern
Region is very important because of its strategic geographical location. The
North Eastern Region shares approximately 4500 km of porous
international boundaries with Myanmar, Bangladesh, Bhutan, Nepal and
China. Uncontrolled migration of animals from the neighbouring countries
can be a great threat for incursion of emerging and transboundary diseases
to this region. Rapid globalization and increasing trade has resulted free
movement of humans and animals and seems to be another precipitating
factor for increasing threat of infectious diseases crossing the borders.
Coupled with global warming, massive deforestation resulting in increased
vector population, the region becomes more vulnerable for occurrence of
many exotic viral, bacterial and parasitic diseases. The region has already
been experienced with the outbreak of virulent form of H5N1 in poultry,
PRRS, circo virus infection in pigs, pox and PPR in goats. There are reports
of occurrence of Nipah virus infection in our Country as well as in
neighboring country. Therefore, we need to build up our capabilities and to
strengthen ourselves in the direction of developing latest diagnostics and to
undertake rigorous surveillance for the highly contagious and ravaging
(selected) diseases. We must have a complete vigil on the disease situation
of emerging and exotic infections and build a formidable defense to guard
our territories and thereby saving the livestock wealth and livelihood of
millions of human beings involved with it.

To achieve this target, one of the prime objectives of ADMaC is to

strengthen the human resources of the region involved in Animal Disease
Monitoring and Surveillance by training of State Veterinary personnel’s on
the use of advanced and molecular diagnostic techniques. Since at the base
of the pyramid of animal disease monitoring and surveillance are the
paravets of the region, therefore, it was felt that the paravets should be
  
 
trained first with the modern techniques of laboratory diagnosis of animal
diseases. Accordingly the ADMaC organized several training programmes
for the paravets and veterinary officers of all eight North Eastern states.
During the course of different trainings, many trainees expressed the need
of a comprehensive laboratory manual for use in the field labs of North
East. I am happy to learn that the scientists and research workers of the
Core Lab I of the Advanced Animal Disease Diagnosis and Management
Consortium (ADMaC) have taken initiatives with active support from all
other core labs and national labs in preparing a comprehensive manual
entitled “Laboratory manual on field diagnosis of animal and avian
diseases”. The manual contains much important information like laboratory
safety rules, collection, preservation and dispatch of samples, preparation of
laboratory items, laboratory reagents etc. and some routine diagnostic tests.
The use of different illustrations/ colored photographs, flow chart has made
the manual more attractive.

Since limited copies will be printed and distributed to few ADMaC

attached field labs, I hope that the different State AH & Veterinary
Departments may replicate some more copies or may download from the
website www.neradslab.res.in with due acknowledgements to DBT and Core
Lab I of the ADMaC project.

I congratulate the scientists and scientific staffs and hope that with
their guidance Good Laboratory Practice (GLP) will be routinely practiced
in all district level laboratories of all North Eastern Region and timely and
accurate diagnosis can be given to the needy farmers.

(A.Chakraborty)

 
 
  
 

PREFACE
Herd health is a key and highly sensitive determinant of productivity
within any livestock husbandry system. Deviations from the maintenance of
optimal herd health can cause significant consequences for farmers and
livestock as a whole. Emerging and endemic infectious diseases in animals
and birds are occurring in our country and causing serious economic loss to
this unique industry. Diagnosis of disease is pre-requisite to contain as well
as control such outbreaks. However, trained manpower, laboratory facilities
at field level are still in infancy in this North Eastern part of India. DBT-
NERBPMC provided financial support for development of skilled
manpower, creation of infrastructure facilities through a tripartite concept
involving three NE Institutes, four National Labs and eight Directorates of
Animal Husbandry and Veterinary in NER.

Diagnosis of diseases at field level is an important job as it can be a

practical guidance for the farmers to take early action to contain the
outbreaks. Authentic disease data generated at field level helps in depicting
real-time disease map in the region. To train up field veterinarians and
create diagnostic facilities in peripheral labs are challenging tasks in this
region. With limited facilities our field veterinarians can make use of
certain simple techniques for quickly diagnose of diseases. To overcome the
difficulties commonly faced in field laboratories, this laboratory manual is
compiled providing comprehensive knowledge and information on selection
of suitable samples, appropriate collection procedure and proper dispatch
method, diagnosis of microbial and parasitic agents, clinical pathology and
histopathology from a single source. Standard disease diagnosis and
reporting formats, contact details of diagnostic laboratories and companies
are also included. Procedures are described briefly in simple language with
the help of sketch, colour illustration so that it can be utilized by different
range of laboratory workers including veterinary officers, students and
technicians.

It is hoped that, this laboratory manual on diagnosis of animals and

avian diseases would immensely benefit its users in the field of disease
diagnosis.

N. N. Barman
  
 

ACKNOWLEDGEMENTS
On behalf of the Core lab-I of ADMaC project, Assam Agricultural
University conveys our deep sense of gratitude to the Department of
Biotechnology, NER-BPMC, Govt. of India for providing necessary funds
under “DBT-NER Centre for Advanced Animal Disease Diagnosis and
Management Consortium”.
We also express our sincere gratitude to Dr K. M. Bujarbaruah,
Vice-Chancellor, Assam Agricultural University who happens to be the
architect of this project. His encouragement and support inspires the scientists
and staff to achieve the desired goal.
Dr Madhan Mohan, advisor, DBT also duly acknowledged for
involvement and interest of the project ADMaC since its incubation period to
the present state.
Our special thanks to Prof Dr C Balachandran, Vice-Chancellor, and
TANUVAS for contributing a valuable chapter in this manual.
Dr A. Chakraborty, Director of Research (Vety), Assam Agricultural
University is the guiding force behind publication of this hand book and the
help and co-operation received from him is duly acknowledged.
Dr R. N. Goswami, Dean, FVSc, Khanapara is also always there to
encourage the activities of ADMaC project and his good gesture is also
acknowledged.
The help and support extended by the Directors of co-operating
ICAR institutes like ICAR-NIVEDI, ICAR-NRCE, ICAR-NIHSAD, ICAR-
NRC pig is duly acknowledged. The contributions from PIs, Co-PIs of the
project from the above ICAR Institutes including ICAR-VTCC is also duly
acknowledged.
The Core Lab II of ICAR-NEH, Barapani and the Core Lab-III of
CAU, Aizwal deserve similar acknowledgement for their active co-operation
and help. We also offer our due acknowledgement to all the Directors of
Animal Husbandry and Veterinary from all eight North Eastern States, their
disease investigating officers and state level PIs and Co-PIs.
Special thanks are due to Dr Arun Verma, former ADG, ICAR, Sr.
Consultant, Animal Biotechnology, NER-BPMC, DBT, Govt. of India for his
keen interest in materializing the project proposal.
Finally, we express our thanks to Dr S. K. Das, Professor & Head,
Department of Microbiology, the scientists, research scholars, project staffs
of the Core Lab I and other members for their helps and co-operation in
bringing out this practical hand book.

N.N. Barman

Editor
  
 

LIST OF CONTRIBUTORS

Prof Dr C. Balachandran, PhD

Vice-Chancellor, TANUVAS

Dr A.Chakraborty, PhD
Director (Vety), AAU, Khanapara

Dr. S.K.Das, PhD

Professor, CVSc, AAU, Khanapara

Dr. N.N.Barman, PhD

Professor, CVSc, AAU, Khanapara

Dr.(Ms) Rajeswari Shome, PhD

Principal Scientist, ICAR-NIVEDI, Bengaluru

Dr. Arnab Sen, PhD

Principal Scientist, ICAR-NEH, Barapani

Dr. Tapan Kumar Dutta

Professor, CVSc & AH, CAU, Selesih, Aizawl

Dr. R. K. Veid, PhD

Principal Scientist, ICAR-VTCC, Hisar

Dr. Sharmita Doley, MVSc

Jr.Scientist, ADMaC

Dr. Pallabi Pathak, PhD

JRF, ADMaC

Dr. Parikshit Kakati, MVSc

JRF, ADMaC

Dr. Arpita Bharali, MVSc

JRF, ADMaC
  
 
CONTENTS
Chapters Page No.

Foreword
Preface
Acknowledgements
1 Laboratory safety rules 1
2 Guidelines for collection of biological samples 2
2.1 List of items in field sample collection kit 3-5
2.2 Handling and collection of samples from pigs 6-8
3 Collection, preservation & dispatch of samples 9-12
3.1 Type of materials to be collected in various disease 13-16
conditions
3.2 Check list for collection of samples 17
3.3 In-situ distribution of appropriate tissue samples 18-19
3.4 Collection of blood from animals 20
3.5 Collection of blood from birds 21
3.6 Packing and transportation of samples 22
4 Preparation of laboratory items/materials 23-24
4.1 Preparation of inoculating loop 25
4.2 Sterilization 26-27
5 Preparation of laboratory reagents 28-31
5.1 Preparation of bacteriological culture media 32-33
5.2 Inoculation of sample for bacterial/fungal isolation 34
6 Preparation of smear from liquid bacterial culture 35

6.1 Preparation of smear from solid bacterial culture 36

7 Staining of bacterial smear by simple stain 37
7.1 Staining of bacterial smear with differential stain 38
7.2 Staining of acid fast bacteria with Ziehl Neelsen stain 39
7.3 Staining of fungus with lactophenol cotton blue stain 40
  
 
8 Diagnosis of bovine mastitis 41

8.1 California mastitis test 42-43

8.2 Somatic cell count 44-46
8.3 Detection of mastitis using mastitis detection card 47
9 Antibiotic sensitivity test 48
10 Diagnosis of brucellosis 49
10.1 Rose Bengal Plate Test 49-51
10.2 Milk ring test 52-53
11 Diagnosis of bovine tuberculosis 54
12 Agar gel precipitation test (AGPT) for detection of 55
IBD in poultry

13 Viral haemagglutination test (HA) 56-57

13.1 Viral Haemagglutination inhibition test (HI) 58
14 In-house indirect ELISA for detection of CSFV 59-61
antibody in serum & plasma from pigs
15 Microscopical examination of helminth ova 62-70
(qualitative methods)
15.1 Microscopical examination of protozoa in faecal 71-74
sample
15.2 Microscopical examination of blood smears for 75-76
diagnosis of haemoprotozoan parasites

15.3 Examination of skin scrapping for ectoparasites 76-77

16 Cytopathological diagnosis of diseases in livestock 78-91

and poultry in clinical and necropsy samples
17 Processing of tissue for histopathological 92-101
examination
18 Standard formats for disease survey and disease 102-112
reporting
19 Contact details of veterinary disease diagnosis 113-116
laboratories/ institutes
20 Contact details of companies/suppliers of 117
laboratory items and reagents
 
 
 1 
 
1. LABORATORY SAFETY RULES

 
¾ Treat all microorganisms as potential pathogens.

¾ Sterilize equipment and materials.

¾ Disinfect work areas before and after use with 10% bleach or 70%
ethanol.

¾ Wash your hands with soap.

¾ Use gloves and mask as personal protective equipment for

performing routine laboratory works.

¾ Never pipette by mouth and use pipette bulbs or pipetting devices.

¾ Do not eat or drink in the laboratories, nor store food in areas

where microorganisms are stored.

¾ Label cultures, chemicals, and media clearly and securely with

names and dates.

¾ Arrange chemicals according to their nature.

¾ Autoclave or disinfect all waste material before discarding.

¾ Incinerate or dump it in hollow pit.

¾ Clean up spills with care. Cover any spills or broken culture

tubes use with10% bleach /70% ethanol.
 2 
 
2. GUIDELINES FOR COLLECTION OF BIOLOGICAL
SAMPLES
Accurate diagnosis is based on:

• The understanding of the host systems involved in the course of the

disease.

• Localization/ site of predilection of infection in the body.

• Proper and timely collection of sample following strict aseptic

measures.

• Correct packaging, proper storage and transportation of sample.

Common errors:

• Improper selection of site

• Poor accessibility of sites

• Contamination of samples by normal micro flora of animal body

system

Sample collection is based on:

• Clinical signs observed

• Significant and clinical history

• Pathological changes

• Disease outbreak of a disease or

• Disease surveillance
 3 
 
2.1 List of items in field sample collection kit
Sl Name of the Item Quantity
No.
1 Ice box with ice pack / vaccine carrier 1
2 Needle and syringe (2ml, 5 ml, 10 ml) 10 each
3 Needle ( 18, 21, 24 gauge) 10 each
4 Vacutainer (serum in yellow cap and whole blood in red cap ) 20 each
5 Alcohol swab in plastic vial 2
6 Transport swab in broth / virus transport medium 10-15
7 Cotton swabs 10-15
8 Glass slide with box 10-15
pieces
9 Sterile normal saline in plastic bottle 500 ML
10 Plastic beaker 25 ml/ 50ml 1
11 Phosphate buffer glycerine in vials 10-15
12 Tissue collection vial for microbial isolation 10-15
13 Zip lock bag 20-30
14 10% formaline in sample vials 10
15 Serum storage vials (1.8, 2 ml) 20-30
16 Pasteur pipettes ( disposable) 20-30
17 Rubber teats 2
18 Spatula for shearing /BP Blade 1
19 Spirit lamp & match box 2
20 Post mortem set [Knives-small, medium, large; sharpening 1
stone; forceps; scissors-small, medium, large ; scalpel; B P
blade with handle; axe; hack-saw with frame; plastic sheet;
measuring tape; camera; thread ball; gloves; syringe; sterile
vials; sample collection bottle; cotton; glass slide box, cover
slip; marker]
21 Restraining rope 1
22 Adhesive tape & thread 2-3
23 Marker 2
24 Data entry sheet
25 Hand gloves 10 pairs
26 Mask 10
27 Towel & soap 2 sets
28 First aid box 1
29 Medicine
30 Pen & note book 2
31 Camera 1
32 Apron 1
33 Disposable bag 5
 4 
 

 
 5 
 

 
 6 
 
2.2 Handling and collection of samples from pigs

In comparison to other large animals, adequate restrain of pig is

necessary for any injection to be given or any sample to be collected.
Reduction of stress and providing comfort to pigs during repeated handling in
the experiment minimize error in generation of scientific data.

During collection of oral or nasal swabs proper restraint is necessary

to prevent excessive head movement and sneezing. Pigs of 40 kg weight and
larger can be restrained by use of hog snare. Animals less than 10 kg or 10-20
kg can be restrained manually. The animal should be lifted with fore legs
with back towards the handler and place into the bucket, caudal end first,
ensuring that the hind legs pointing up. The front legs are placed down into
the bucket. With this position handler could sit on top of the bucket with
pig’s head and nose protruding out between the legs. Appropriate sample
could be collected with minimum stress.

For collection of blood, the small pig can be picked up and secured on
a lap. For large animal certain tools like hog snare, locally prepared snare
string/ rope can be used. Pig should be calmed down by touching their
belly/teats or scratching base of the ear. Then loop of the hog snare/pig
catcher string/rope is placed around the animal’s nose and the upper jaw and
tighten properly. The pig reacts to this restraint device by pulling back
against the snare, reaching a stalemate with the operator. Squeeze the pig
against wall/post/tree and place leg of the handler behind fore legs of the
animal. Alternatively, pig of 10-20 kg can be restrained in dorsal
decumbency. The common sites for collection of blood are the ear vein,
cephalic, saphenous veins or the cranial vena cava. The lateral, intermediate
and medial ear veins are superficial and visually accessible. Marginal/lateral
 7 
 
ear vein is stable and it can be engorged by placing rubber band/ tourniquet
around base of the ear. One must grasp the ear securely to prevent movement
during collection of blood. After proper swabbing the engorged vein, a 20-22
gauge needle/ butterfly needle attached with rubber tubing can be inserted
into the vein and collect 5-8 ml blood. For large volume of blood samples,
venipuncture of the cranial vena cava is the method of choice. This may be
done either on a standing pig or one in dorsal recumbency. To make thoracic
inlet accessible on standing position, the head should be held high and on
dorsal recumbency, forelegs should be drawn back along the body. The
needle (20-22 gauge) of 3.8 cm is inserted on the right side in the depression
between the point of the shoulder and the manubrium sterni, in order to avoid
pricking vagus nerve. The needle is aimed at opposite shoulder and passed
into the cranial vena cava.
 8 
 

       RESSTRAINING TO
OOLS          REESTRAINING O
OF PIGS   

Restraining of you
ung pigs 

 
 
 
Resstraining of Ad
dult Pig 
 

 
 9 
 
3. COLLECTION, PRESERVATION & DISPATCH
 
OF SAMPLES
General Considerations for collection of specimens
™ The diseases most commonly encountered in animals are of bacterial,
viral, parasitic, fungal and metabolic origin. Diagnosis based on
symptoms and laboratory examination of the relevant materials is
essential for initiating treatment at the proper time. In general the
following points should be duly considered while collecting materials for
laboratory diagnosis.

™ All materials collected should be accompanied by detail history of

disease, type of species affected, duration of disease, clinical signs,
morbidity and mortality rates, disease suspected etc in standard format.

™ The collected biological specimens should be transported on ice to the

nearest laboratory as early as possible for diagnosis.

™ Materials collected for bacteriological examination should be kept at

refrigeration temperature (4o C) and if a viral etiology is suspected the
material can be stored at –20 to -80o C in case of delay of transportation.
™ For sero-diagnosis collect paired serum samples (about 2 ml sera). One
serum sample should be collected at the onset of disease and second sera
after recovery (2-4 weeks) from disease, preferably on 14th /21st day.

™ If death is reported, the post-mortem examination should be conducted at

the earliest as putrefied materials are unfit for laboratory examination.

™ Detailed post-mortem report should be attached along with the samples

collected during postmortem.

™ Different virological transport media like 50% sterile phosphate buffered

glycerine saline solution and phosphate buffer saline (pH 7.2-7.4) may
be used. Collect samples in sterile containers if transport media is not
available or transport on ice.
™ For histopathology studies, tissues should be preserved in 10% formalin.
The volume of formalin used should be approximately 10 times the
 10 
 
volume of material. Specimen bottles with wide mouth should be used
for collecting tissues.

™ The specimen bottles should be sealed well so as to avoid leakage and

mark clearly indicating the fixative/transport media used.

™ All the impression smears, should be fixed in methanol for 1-5 minutes
unless otherwise specified.

™ In case of outbreaks, collect materials from ailing animals (5-6 or more)

and in-contact animals at the height of body temperature or flaring up of
clinical signs and symptoms.

™ Refer check list to arrange vials, buffer and other tools (Page No. 3).

   
 
 
   
 11 
 
 12 
 
 13 
 
3.1 Type of materials to be collected in various
 
disease conditions

BACTERIAL DISEASES
Haemorrhagic Blood smear, smears of fluid from swelling blood in sterile
Septicaemia (HS) container, impression smear from heart, lungs, liver,
submaxillary swellings, smears from heart blood, lymph
nodes and spleen on ice
Anthrax Blood smears from ear vein, smear of the discharges from
natural orifices, smear from swelling, ear tip or a piece of
muzzle in saline/ charcoal
Black Quarter Impression smears from the affected muscle, exudate from
(BQ) the swelling on ice, pieces of affected muscle on ice,LN,
muscle in 10% formaline
Enterotoxaemia Blood, smears from contents of small intestine, contents of
intestine
Brucellosis Milk, blood, serum sample (paired serum sample), vaginal
mucus, uterine fluid, stomach contents of foetus, aborted
foetus

Johne’s Disease Rectal pinch swab or smear, faecal sample, terminal

(JD) portion of ileum with ileo-caecal valve, mesenteric lymph
gland in 10% formal saline
Glander Nasal discharge on ice, pus from skin lesions on ice,
affected tissue in 10% formalin
Tuberculosis (TB) Sputum swabs in sterile container on ice, milk in sterile
vials on ice, faeces, heat fixed impression smears from
lymph glands, affected tissue for histopathology in 10%
formalin. lymph glands or lung lesions in sterile container
in 50% glycerol phosphate buffer

Leptospirosis Blood, blood smear, serum, urine, tissue from kidney, liver
and spleen in 10% formalin, milk or urine in vials by
adding 1 drop of formalin per 20 ml
Salmonellosis Blood, faeces, intestinal content, heart blood and bile in
separate sterile vials, tissues like mesenteric lymph nodes,
kidney and gall bladder in 10% formalin.
Actinomycosis Pus smear, pus in sterile container on ice, tissue in 10%
formalin
Actinobacillosis Pus smear, Pus in sterile container on ice, affected tissue
in 10% formalin
 14 
 
Listeriosis Blood, cerebrospinal fluid, brain, aborted foetus or
placenta, all internal organs in 10% formalin or on ice

Mycoplamosis/ Swabs from sinus/trachea, nasal and vaginal swabs in PBS

CCPP/CBPP/ on ice, piece of lung preserved in 10% formalin and on ice
Coryza separately, serum samples (paired serum)

Chlamydia/ Nasal swab, lung pieces in sterile vials on ice, impression

Psittacosis smears from lungs, liver, conjunctiva and foetus, serum
samples (paired serum samples), internal organs in 10%
formalin
Campylo- Vaginal mucus, stomach content of aborted foetus
bacteriosis
VIRAL DISEASES
Blue tongue (BT) Blood at the height of body temperature in heparin or
EDTA for isolation. Paired sera in sterile vials on ice for
serology.
Spleen, lung and lymph nodes on ice for isolation. spleen,
lymph nodes, intestine in 10% formol saline for
histopathology
Bovine Viral Nasal discharge for isolation, blood in EDTA for isolation,
Diarrhoea disease paired serum samples for serology, semen for isolation.
(BVD) Lymph nodes and spleen in sterile vials on ice for isolation,
intestinal swabs for isolation
Canine Distemper Blood smears / impression smears of conjunctiva or
(CD) vaginal epithelium for viral inclusion body.
Impression smears from liver for viral inclusion body,
pieces of liver & spleen in sterile vials on ice for isolation,
pieces of lung, urinary bladder, liver, trachea, stomach wall
and brain in 10% formol saline
Foot and Mouth Vesicular epithelium in 50% phosphate buffered glycerine,
Disease oeso-pharyngeal fluid in 50% phosphate buffered glycerine
Infectious Bovine Paired sera for serology, Swabs from vaginal and nasal
Rhinotracheitis lesions for isolation.
(IBR) Pieces of trachea, lungs in PBS on ice for isolation, pieces
of trachea, liver, turbinate bone, lungs in 10% formol saline
Infectious Canine Spleen and liver in sterile containers on ice for isolation.
Hepatitis Liver, gall bladder and kidney in 10% formol saline for
histopathology. Impression smears from liver fixed in
methanol
Parvo Viral Rectal swab and faeces in PBS, paired serum samples.
Infection of Pieces of intestine, heart on ice for isolation. Liver, heart,
Canines intestine in 10% formol saline
 15 
 
Peste des Petits Eye, mouth and rectal swabs in PBS on ice for isolation.
Ruminants (PPR) Citrated Blood for isolation. Paired sera for serology.
Spleen, lymph nodes, pieces of intestine on ice for
isolation. Lungs, liver, spleen, tonsil in 10% formalin

Rabies Head / Whole carcass on ice for demonstration of viral

antigen, viral inclusions and isolation of virus. Brain on ice
for demonstration of viral antigen, viral inclusions and
isolation of virus
Note: It is not advisable to open the skull by persons not
protected by vaccination
Swine Fever Heparinised blood at the height of temperature for
(CSF) isolation. Pieces of spleen, mesenteric lymph glands,
intestine especially ileo-caecal region in 50% glycerol
saline for isolation. Pieces of brain, lung, intestines, ileo-
caecal region and kidney in 10% formol saline
Infectious Bursal Ailing bird, paired serum sample.
Disease (IBD) Bursa of Fabricious in 50% buffered glycerine saline.
Bursa of Fabricious, kidney, spleen in 10% formol saline
Marek’s disease Ailing bird. Paired serum sample.
(MD) Trachea, ovary, liver, kidney, spleen in 10% formol saline
Ranikhet disease Serum sample from ailing bird. From dead animals –fresh
(RD) carcass on ice. Liver, spleen, trachea, bronchi, lungs in
50% buffered glycerine saline. Proventriculus in 10%
formol saline
BLOOD PROTOZOA, FUNGAL INFECTION
Theileriosis Blood smears, biopsy smears from swollen lymph nodes
from early stage of disease fixed with methanol
Babesiosis Thin blood smears fixed in methanol
Anaplasmosis Thin blood smears fixed in methanol
Filariasis Direct blood smear (wet film), blood smear
Trypanosomiasis Wet film examination of blood by hanging drop, fixed
blood smears, blood in anticoagulant on ice
Schistosomiasis Dung sample (avoid contact of water which may aid in
hatching of the egg). Nasal schistosomiasis –nasal
discharge in normal saline, nasal granuloma in normal
saline
Trichomoniasis Vaginal or uterine discharges, prepuce scraping/ washing
Gastro-Intestinal Faecal sample in clean vial, Affected internal organs in
Parasitic Diseases 10% formalin. Intact nematode parasite in 70% warm
alcohol. Trematode, cystode gently press between clean
glass slides, tie with rubber band and put in 5% formol
saline
 16 
 
Ectoparasitic Deep skin scrapings in sterile vials, affected hair, skin,
Infestations nails in dry container
(Ringworm,
Mange, Mites)

Rhinosporidiosis Nasal discharge in sterile vial, nasal swab, granulation

tissue in 10% formol saline
External Fungal Skin scrapings in sterile vials
Infections
Aflatoxicosis 100 gm of suspected feed (specially groundnut cake), liver
and spleen on ice, liver and spleen in 10% formol saline
OTHER DISEASE CONDITIONS
Poisoning Cases For chemical analysis fresh tissues(a loop of intestine,
stomach ) and fluids should be sent as soon as possible on
ice. Avoid addition of preservatives to the samples.
Stomach/ intestinal content (10 gm), liver, kidney,
intestine, urinary bladder in saturated salt solution (45%
sodium chloride) for forensic analysis. Duplicate tissue
samples in 10% formol saline

Plant Poisonings Sample of suspected grass/fodder/plants, liver on ice,

stomach contents on ice
Mastitis Milk samples (mid-stream) before onset of treatment in
sterile vials on ice
Abortion Whole foetus/ all internal organs of foetus on ice, Vaginal
swab in PBS, pieces of placenta in sterile vials on ice,
pieces of placenta in 10% formalin, paired serum samples
Infertility and Semen in sterile vials, prepuce swab on ice, paired serum
Sterility sample on ice

Diseases of Feed/ fodder, blood smears, urine sample, faecal sample,

Unknown blood samples collected in EDTA on ice from live animals,
Etiology serum samples from live animals, stomach contents,
spleen, lung, lymph node, liver, kidney, intestine in sterile
vials from dead animals on ice, stomach contents, spleen,
lung, lymph node, liver, kidney, intestine in 10% formol
saline
 
 
 17 
 
3.2 Checklist for collection of samples

Type of Sample Clinical samples

Sample on ice for Haematology Parasitology
ELISA PCR Microbial In
Isolation EDTA/Heparin
Whole blood
CSF
Serum
Nasal Swab
CLINICAL

Ocular swab
Oral swab
Genital swab
Rectal swab
Faecal sample
Milk sample
Skin biopsy
Wound swab
Post-Mortem Examination
Sample on Ice for Insitu Antigen Histopathology
Type of Sample ELISA PCR Microbial detection, Tissue in 10%
Isolation Tissue snap Formalin
frozen in LN2
Heart blood
Tonsil
Lymph node
Mesenteric LN
Spleen
POSTMORTEM SAMPLES

Liver
Lung
Kidney
Small intestine
Large  
intestine/Colon
Urinary  
bladder
Rib bone  
Tarsal gland  
Nictitating  
membrane
Skin  
Thymus  
Brain  
 18 
 
3.3  In-Situ distribu
ution of apprropriate tisssue sampless

A. Location of organ
ns showing paathological changes in largee animal

 
 
 
 
 
 
 
 
 
 
 
 
 
 19 
 
B.  Locattion of organss showing typ
pical lesions in
n birds 

 
   
 
 
 
 
 
 
 
Bursa of 
    Fabricius 

 
 
 
 
 
 20 
 

 3.4 Collection of blood from animals

The common sites for collection of blood in large animals are the ear
vein, cephalic, saphenous veins, abdominal vein or the cranial vena cava. The
lateral, intermediate and medial ear veins are superficial and visually
accessible. Marginal/lateral ear vein is stable and it can be engorged by
placing rubber bang/ tourniquet around base of the ear. One must grasp the
ear securely to prevent movement during collection of blood. After proper
swabbing the engorged vein, a 20-22 gauge needle/ butterfly needle attached
with rubber tubing can be inserted into the vein and 5-8 ml blood can be
collected. For large blood samples, venipuncture of the cranial vena cava is
the method of choice. In case of pigs this may be done either on a standing or
one in dorsal recumbency. To make thoracic inlet accessible on standing
position, the head should be held high and on dorsal recumbency, forelegs
should be drawn back along the body. The needle (20-22 gauge) of 3.8 cm is
inserted on the right side in the depression between the point of the shoulder
and the manubrium sterni, order to avoid vegus nerve. The needle is aimed at
opposite shoulder and passed into the cranial vena cava

Location of blood vessels

 21 
 
3.5 Collection of blood from birds

In poultry wing vein and additionally in duck in leg vein should be

attempted to collect blood.

 Wing Vein 

 
   
 
 22 
 
3.6 P
Packaging a transporrtation of sa
and amples
Precauttion to be takeen during tran
nsportation

• To prevent furtther contaminaation.

T
• T maintain viiability of cellss & there
To
n
number ratio as
a it is.
• T avoid sampple drying.
To
D 
Care to be taken in Packaging
P

• Submit samplees individuallyy in separate leeak proof contaainers.

S
• S
Second containner around firsst in case of leaak chances, fillled with
a
absorbent mateerial.
• L
LABEL-type o animal, typee of sample and date of colleection, phone
of
n
numbers (writee using permannent marker orr typed it).
• S
Secure/tighten the lids propeerly.
• I delayed, trannsport at 4°C on
If o ice by ice packs.
• A
Avoid sendingg on weekends.
• Syringe can bee used for fluidd sample by reemoving needdle and puttingg
c expel air.
cap,
• D not use celllo tape the lidss of the tubes.
Do
• P
Primary contaainer: containning the specim men (A), should be clearly
labelled, waterrtight and leak proof. Wrapp ped in sufficiennt absorbent
m
material to prevent breakagee and placed in n a secondary container (B).
• O
Outer shippin ng package-shhould be a rigid d box (C): place the
s
secondary receeptacle in an ouuter shipping package,
p with suitable
c
cushioning thaat protects it annd its contents from physicall damage and
w
water etc.
• O
Outer packing (D) write receeiver’s addresss and sender’s address.
W
Write in bold letter
l caution words
w - GLASS HANDLE WITH W CARE,
B
BIOLOGICAL L MATERIAL LS, NO COMM MERCIAL VA ALUE

C
B
 23 
4.
  PREPARATION OF LABORATORY ITEMS/
MATERIALS
For routine microbiological diagnostic works various glass wares,
cotton swabs, inoculating loop are necessary. Glass wares should be
thoroughly washed with detergent followed by plain tap water and finally
air dried. Mouth of test tubes, bottles, flasks is to be plugged with non-
absorbent cotton, wrapped with paper and tied securely with thread. Cotton
swabs are prepared by wrapping absorbent cotton on one end of a bamboo stick 
and put in a test tube plugging with cotton. 

   
 24 
 
Two different qualities of cotton
T c are useed in microbioological work--
absorbeent and non-aabsorbent cottton. Non-abso orbent cotton will not soakk
water annd source is seeeds of tall sim
mli tree. It is used
u to plug opening
o of testt
tubes, boottle, flask etcc. Absorbent cotton
c is obtainned from cottoon plant and itt
soaks waater. Absorbennt cotton is useed for preparin ng swabs.

Non
n- absorben
nt cotton useed for plugg
ging of tubees/flask

NON ABSOORBENT
  COTTON USED
FOR PLUG
GGING
 
  
 
  Absorben
nt cotton useed for prepa
aring swabss

 
A
Absorbent Cottton Abso
orbent Cotton Balls
 
 25 
 
4.1 Preparation of inoculating loop
Inoculating loop is a small but essential tool used in any
bacteriological laboratory for isolating bacteria from clinical as well as post-
mortem samples. A 3mm diametre loop is prepared at one end of a platinum
wire or suitable metal wire and fixed in a handle. Various steps are illustrated
below to prepare an inoculating loop.
 26 
 
4.2 Sterilization

Sterilizzation by hoot air oven : any type of microbiologica

m al works, glasss
wares, plastic
p wares, media and otther reagents should
s be freee from micro--
organism ms and the proocess is calledd sterilization. Commonly glass wares aree
sterilizedd by dry heatt i.e by Hot air a oven. Liqu uid media, reaagents, plasticc
wares arre sterilized byy moist heat i.ee. Autoclave or pressure coooker.
 27 
 
Sterilizzation of meedia & reaggents by auttoclave :

An autoclavve is used to
sterilized utelssils, containers,
media,soil, used infectious
materials andd other waste

Never autooclave toxic,

flammable or radiological
ageents
 28 
 

5. PREPARATION OF LABORATORY REAGENTS

A. Hanks’ balanced salt solution
Stock solution A:
1. NaCl … 160g
KCl … 8g
MgSO4.7H2O … 2g
MgCl2.6H2O … 2g
Distilled water … 800ml

2. CaCl2 … 2.8g
Distilled water … 100ml

Mix these two solutions slowly and make up volume to 1000ml with distilled water.
Add 2ml Chloroform, store at 4oC.

Stock solution B:
Na2HPO4 … 3.04g
KH2PO4 … 1.2g
Glucose … 20g
Distilled water … 800ml

When chemicals have dissolved add 100ml of 0.4% phenol red in NaoH. Make up
volume to 1000ml with distilled water. Add 2ml chloroform and store at 4oC.

For use: Stock solution A … 100ml

Stock solution B … 100ml
Distilled water … 800ml

Sterilize by autoclaving at 15lb for 20 minutes & store at 4oC. Add antibiotic in
sterile solution

Penicillin … 250IU/ml of solution

Streptomycin … 100 μg/ml
Nystatin … 25 units/ml

C. 50% Glycerine-PBS solution used for preserving tissue samples :

Glycerine pure … 200ml
Phosphate buffered saline solution … 200ml

The solution should be sterilized at 10lb pressure for 30 minutes and store at 4oC
 29 
 
D. Phosphate buffer saline solution (pH 7.4):

Solution A:

NaCl … 8.00g
KCl … 0.20g
Na2HPO4 … 1.15g
KH2PO4 … 0.20g
Distilled water … 800ml

Solution B:

CaCl2 … 0.10g
MgCl2 … 0.10g
Distilled water … 200ml

Dissolve the chemicals in water in the order listed.

Sterilize the solutions A & B separately by autoclaving .Store at 4oC till use

 
E. EDTA solution for blood collection: A 10% sodium salt of
EDTA is dissolved in distilled water. Use one drop for
each 5ml blood.

F. Dextran solution (5%) for lymphocyte :

separation

Dextran sulphate … 5g
1.5% EDTA solution … at 100ml

Dissolve at 45oC and autoclave before use

G. Alsever’s solution collection of RBCs:

Glucose … 2.05g
Sodium Chloride … 0.42g
Sodium citrate … 0.80g
Citric acid detergent fiber … 100ml

Sterilize by autoclaving 10lb pressure for 15 minutes and store

at 4oC
 30 
 

H. Staining for gel slides:

Comassie brilliant blue staining solution (A)

Comassie brilliant blue … 0.1g

Acetic acid … 10ml
Ethanol … 45ml
Distilled water … 45ml

Destaining solution (B)

Acetic acid … 10ml

Ethanol … 25ml
Distilled water … 65ml
 
 
I. Phenol saline for Brucella agglutination test :

Sodium chloride … 0.85g

Phenol … 0.5g
Distilled water … 100ml
 
J. Phosphate buffered saline (pH-7.4):

Sodium chloride … 8.0g

Potassium di-hydrogen phosphate … 0.2g
Disodium hydrogen orthophosphate … 1.16g
Potassium chloride … 0.2g
Distilled water … 1000ml

Adjust pH to 7.4 with 4% NaOH or 8% HCL and autoclave at 10 lb

pressure for 30 minutes.
 31 
 
K. Lugol’s Iodine Stain- is used with wet mount preparations and
concentration techniques for the detection of intestinal protozoa and
helminth ova and larvae.
Composition :
Iodine crystal – 5.0 gm
Potassium iodide – 10.0 gm
Dist water - 100 ml

Working dilution – 1:5 in dist water. Use for 3 weeks.

L. Lactophenol cotton blue stain (45 ml) -is used to stain fungus
Composition :
Phenol – 10 gm
Cotton blue, water soluble- 0.04 gm
Lactic acid – 10 ml
Glycerol – 20 ml
Dist water – 10 ml

M. Nutrient Agar –is used to grow bacteria

Composition:
Peptone – 0.5 gm
Beef extract/yeast extract - 0.3 gm
Agar agar- 2.5 gm
NaCl- 0.5 gm
Distilled water – 100 ml
pH is adjusted to neutral (7.4)

N. Nutrient Broth –is used to grow bacteria

  ( To prepare use same composition of Nutrient agar without adding agar 
agar) 
O. Sabouraud Dextrose Agar – is used to grow fungus
Composition :
Dextrose (glucose) - 4.0 gm
Peptone - 1.0 gm
Agar - 2.5 gm
Dist water – 100 ml
Final pH 5.6
 32 
 

 5.1 Preparation of bacteriological culture media

The survival and growth of microorganisms depend on available and

a favourable growth environment. Culture media are nutrient solutions used
in laboratories to grow microorganisms. For the successful cultivation of a
given microorganism, it is necessary to understand its nutritional
requirements and then supply the essential nutrients in the proper form and
proportion in a culture medium.

Preparation of agar plates:

Materials and equipments:
1) Distilled water 7) 1N HCl solution
2) Measuring cylinder 8) pH meter
3) Flask 9) Gloves
4) Bacteriological 10) Sterile, empty Petri
powder media (Page dishes
No-31) 11) Bunsen burner
5) Spatula 12) Autoclave
6) 1N NaOH solution 13)Incubator

Procedure:
™ Measure the components of the medium into a flask. Use a clean spatula
for every measurement. Dissolve the solid components and fill with the
remaining solvent up to final volume. If the medium contains heat
sensitive components (like sugars), they must be separately sterilised in
solution (e.g. by filter sterilisation), and then mixed with the already
sterilised and cooled agar medium. Add agar between 2.5% depending
upon the climatic conditions.
™ Check the pH of the medium with an indicator paper or with a pH meter
and adjust to the proper value with NaOH or HCl solution.
™ Close the flask with cotton plug and cover with aluminium foil, label the
flask and put into the autoclave and start a sterilisation under 15 psi
pressure, at 121°C for 15minutes.
™ Cool the sterilized medium.
™ In a laminar air flow or inoculation hood, remove the cotton plug and
flame the mouth of the flask over a bunsen burner and dispense the
medium into sterile Petri dishes (15-20 ml into each Petri dish) or test
tube (for preparation of slant).
™ Keep on the floor of the work bench until complete solidification.
 33 
 
™ Turn the Petri dishes upside-down and stack them. The slants can be
stacked separately.
™ Perform a sterility test: incubate the medium at 37°C for 24 hours to
check for sterility.
™ In case of longer storage, Petri plates must be placed into plastic bags or
boxes to avoid drying out.
  Preparation of media plate

 
 34 
 

 5.2 Inoculation of sample for bacterial/fungal isolation

Isolation of Bacteria -a loopful of sample is smeared on the sterile solid

media at one corner and sterilized the loop by burning . Streak out three
parallel lines from pooled smear after cooling down the loop, burn the loop
again and draw another three parallel lines from first streak as shown in the
diagram. In broth, loopful of sample shake inside tube containing sterile
broth.  

Isolation of fungus
Sabouraud’s dextrose agar media (Page No- 31) is used to isolate fungus.
Suspected sample is burrowed into the media and incubate at room
temperature for one to two weeks.
 35 
 
6. PREPARATION OF SMEAR FROM LIQUID
BACTERIAL CULTURE
Materials required:

• Microscope and micro slides

• Bunsen burner/ Spirit lamp
• Inoculation loop
• Glass marking pencil
• Broth culture (Page No-31 )

Procedure:

™ Take a clean micro slide and pass it over the flame for 3-4 times.
™ Make the surface grease free. Handle the clean slide by its edges.
™ Write the identification number/ initial of organisms on the side of the
slide with glass marking pencil.
™ Make a circle in the centre of the slide, where the organisms are to be
placed (target circle). Flame the inoculating loop until red-hot and let it
cool.
™ Shake the culture vigorously and transfer a loop full of organisms at the
target circle. Spread the organisms over the area of the target circle. Be
sure to flame the inoculating loop before placing it aside.
™ Allow the slide to air dry. Do not apply heat.
™ Pass the slide over the flame 3-4 times exposing the lower surface to the
flame (i.e. the surface in which there is no smear) to heat kill and fix the
organisms to the slide. Now the smear is ready for staining. 

 
 
 36 
 
6.1 Preparation of smear from solid bacterial culture

Materials required:

• Microscope and micro slides

• Bunsen burner/spirit lamp
• Inoculating loop
• Glass marking pencil
• Culture plate with organisms
Procedure:
™ Take a clean micro slide and pass it over the flame for 3-4 times.
™ Make the surface grease free. Handle the clean slide by its edges.
™ Write the identification number/ initial of organisms on the side of the
slide with glass marking pencil.
™ Mark a target circle in the centre of the slide with glass marking pencil.
™ Flame the inoculating loop until it is red hot, let it cool and transfer a
loopfull of sterilized NSS to the slide over the target circle.
™ Flame the inoculating loop, let it cool and pick up a fraction of isolated
colony of the target organism from the culture plate and mix it with saline
on the slide. Disperse the mixture over the area of the target circle. Be
certain that the organisms have been well emulsified in the liquid. Be sure
to flame the inoculating loop before placing it aside.
™ Allow the slide to air dry. Do not apply heat.
™ Pass the slide over the flame 3-4 times exposing the lower surface to the
flame (i.e. the surface in which there is no smear) to heat kill and fix the
organisms to the slide. Now the smear is ready for staining.

 
 37 
 
7. STAIINING OF
F BACTER
RIAL
  SME
EAR BY SIMPLE
S
STAIIN

The use of a single staain to colour a bacterial orgaanism is comm

monly referredd
mple stain.
to as sim

Materiaals required:

• Bacterial cultuure
B
• M
Methylene Bluue
• W
Wash bottle
• B
Blotting paper
• M
Micro slide, Microscope
M
• G
Glass marking pencil
• B
Bunsen burnerr
• I
Inoculating looop

ure:
Procedu
™ Prepare
P a bacteerial smear
™ Stain
S the smeaar with Methyleene blue for 1 minute
™ Wash
W the slidee with water
™ Allow
A the slidee to dry at room
m temperaturee
™ Examine
E underr oil immersioon and record the
t observationn

Interpreetation: Organnisms take bluue stain. Pasteu

urella organism
m takes
bipolar stain.
s
 38 
 
7.1 Staining of bacterial
b sm
mear with diffferential sttains
Gram sttaining methood

Materiaals required:

• Slides with
w prepared smears
• Gram staining
s kit
• Wash bottle
b
• Blottingg papers
• Microscope, cedar wood oil.
Grram +ve Bacteriaa stained as
violet coloour
Procedu
ure:

™ Preppare a smear from

fr bacterial culture
c as desccribed (Page-334)
™ Floood the smear with
w crystal vioolet solution an nd let it stand for 45 secs
™ Wassh with distilleed water and drain
d off excess water
™ Floood the smear with
w Gram’s Ioodine solution and allow to act a for 1 min
™ m’s iodine and wash with disstilled water
Pouur off the Gram
™ Floood with ethyl alcohol
a (70 %)) and gently roock the slide foor 15-20 secs
™ Wassh with water
™ Floood the smear with
w safranin (00.5%) or basicc fuchsin for 45 4 secs
™ Wassh gently with water, blot wiith blotting paaper and dry
™ Exaamine under oiil immersion and
a record the observation

Steps of Gram’s stain

n Gram –ve Bacteeria stained as
pink coolour
 39 
 
7.2 Sttaining of accid fast bactteria with Ziehl
Z Neelsen
n stain
Materiaals required:
• Slides with
w prepared smears
• Acid faast staining kit (HiMedia)
• Wash bottle
b
• Blottingg papers
• Microscope, cedar wood oil.

Procedu
ure:

1. Preppare a smear from

fr suspectedd bacterial culture.
2. Placce the numbereed slides on a staining rack with
w the smearred side
facinng up.
3. Floood the entire suurface of slidee with Ziehl-Neelsen 1% Carrbol fuchsin
soluution.
4. Heaat the slide sloowly until steam m arises. Main
ntain steamingg for 5 minutess
by using
u intermitttent heat.
5. Rinsse the slide unnder a gentle fllow of water until
u all free staain washes
awaay. Drain off exxcess water byy tilting the sliide.
6. Floood the slide with
w the decolorizing solution n (25% Sulphuuric acid) for 3
minnutes. (If the slide is under deecolorized afteer 3 minutes, further
f
decoolorize for 1 minute.)
m
7. Rinnse the slide thhoroughly withh water. Drain off excess waater from the
slidee. Wipe the baack of slide wiith cotton soakked in decoloriizer to clean
the dried
d stains.
8. Rinsse the slide aggain with waterr and drain offf excess water by tilting the
slidee.
9. Floood the slide wiith 0.1% Methhylene blue and d counter-stainn for 60
secoonds.
10. Rinnse the slide thhoroughly withh water and draain off excess stain by
placcing the slide under
u gentle sttream of runninng water.
11. Staand the rinsed slide
s on the sliide holding blo
ock and allow the smear to
air dry.
d
12. Exaamine under oiil immersion and a record the observation

Acid Fast Baacilli stained as

a
bright pink colour
 40 
 
7.3 Sttaining of fu
ungus with lactophenol
l l cotton bluee stain

S
Staining of funngal cells likee Dermatophyytes, Rhizopuss, Aspergillus,,
Penicilliium, Fusariumm, Candida, Mucor
M etc. Sttain fungal cells by usingg
Lactophenol cotton bluue method.

Requireement

1. Young fungal culture

Y c
2. G
Glass slide
3. C
Cover slip
4. N
Needle
5. L
Lactophenol cootton blue.

Procedu
ure

1. T
Take a clean grrease free slide.
2. Add
A a drop of mounting fluidd that is lactop phenol cotton blue
b solution
o a slide.
on
3. Sterilize
S the neeedle and cool it then transfeer a 40olostr mat
m on fluid
a press it genntly so that it easily
and e mix with the stain.
4. Take
T a clean coover slip and with
w the help of o a forceps plaace the cover
s on 40olosttr mat.
slip
5. Take
T a blottingg paper and wiipe the excess stain .
6. Observe
O underr low to high power
p objectivees of microscoope.

Observaation

After staaining fungus cell

c observe different
d parts of a cell like conidia,
c
hyphae asa well as 40ollostrums40es etc.
e
Result

On basiis of observatiion the type fu

ungal organissms identified
d.

Dermatoophyte Pen
nicillium
 41 
 
8. DIAGNOSIS OF BOVINE MASTITIS
The term mastitis refers to inflammation of the mammary gland
which is characterized by physical, chemical as well bacteriological changes
in the milk and pathological changes the udder tissue.

Detection of Mastitis

Physical examination of the udder: In clinical mastitis the udder may turn
hard, red, and hot to the touch. Palpation of the udder may be painful to the
cow. These symptoms arise from the changes in vascularity and blood flow
of the gland when inflamed.

Visualization of the milk in clinical mastitis: Gross changes in the milk

may be observed at the time of milking such as the presence of flakes, clots
or serous milk. This is the most common means of detection of clinical
mastitis. Stripping the first few squirts of milk from each quarter into a strip
cup at the beginning of milking is a preferred method of detecting flakes or
clots in the milk.

Visualization of the milk in sub-clinical mastitis: Sub-clinical mastitis is

the most economically devastating condition in diary industry. There is no
apparent gross change in the milk at the time of milking. There will be drop
in milk yield, formation of curd in boiling, bitter or salty taste.

Aseptic milk sample collection:

i. Wear disposable gloves, arrange marking pen, milk sample tubes, cotton
soaked in 70% alcohol, cooler with ice or freezer packs.
ii. Label the sample tube with the date, the cow ID and the quarter.
iii. Clean the udder and the teat and dry using cloth towel.
iv. Discard 3 to 4 streams of milk on the floor to minimize chances of
contaminating the sample with bacteria in the teat canal.
v. Scrub teat ends using a cotton ball or gauze pad soaked in 70% alcohol.
vi. Open the sample vial immediately before the sample is taken and collect
direct streams of milk into the vial without touching the teat end.
Immediately place the sample vial on ice or in the refrigerator and dispatch
to the lab.
 42 
 
8.1 California mastitis test (CMT)
Principle: The mastitis reagent reacts with genetic material of somatic cells
present in mastitis milk to form a gel/precipitate.

Requirement:
1. Mastitis reagent
2. Milk samples
3. Mastoid paddle/ Glass slide
4. Pasteur pipettes

Procedure:

A. Using mastoid paddle

¾ A plastic paddle having four shallow cups marked A, B, C and D for easy
identification of the individual quarter is to be used.
¾ Add approximately 1 teaspoon (5 ml) of milk in equal amount of the
reagent.
¾ Mix the contents thoroughly by a circular rotating motion. Formation of gel
will appear within ten seconds.
¾ Reading should be completed within 20-40 seconds.

Interpretation of result:

Gel formation in mastitic milk No gel formation in normal

milk
 43 
 
B. Using microscope slide

¾ Mark slide 1 as LF & RF, slide 2 as LH and RH.

¾ Add 3 drops of mastitis reagent to each labeled area.
¾ Add 3 drops of milk sample from each quarter to identified area.
¾ Mix thoroughly with clean individual stick for few seconds.
¾ Categorize milk sample based on degree of gel formation.

Interpretation of result:

LF (+ve)  RF (+ve) 

Gel formation in mastitic milk

  
 
 
 

          
 
 
 
 
 
  LH (‐ve)  RH (‐ve) 
    
  No gel formation in normal milk
  (LH & RH)
 
 
 
 
 44 
 
8.2 Somatic cell count (SSC
C) test
Due to infectiion in udder somatic cells,, mainly epithhelial cells off
D
mammarry gland and white blood cells
c m secretion..
are enterred into the milk
Higher the number of o somatic ceell count (SC
CC) higher thhe severity off
inflamm
mation of the uddder.
Requireements:
1. Milk
M samples.
2. Clean
C glass sliddes.
3. Staining
S solution.
4. Compound
C miccroscope.

Preparaation of smearr and stainingg:

| M
Mark a clean microscopic
m sllide as 1 cm x 1 cm
a
area
w a marker// diamond penccil/ glass mark
with ker.
| Transfer
T one looopful of propperly mixed milk
m to the marrked area withh
the help of wirre loop .
| Make
M a thin smmear by spreaading the milk to cover the 1 cm2 area onn
the glass slide and allow to air
a dry.
| Cover
C the smmeared area witth staining reaagent (Loeffleer’s Methylenee
B
Blue) and alloww to react for 2 – 3 minutes
| Wash
W the stainn and allow tot air dry. Examine under oil immersionn
m
microscope and count the nuumber of Somaatic cells in tottal 50 fields ass
indicated beloww.

Som
matic Cells sttained with
methylene blu ue stain
 45 
 
Calculation of SCC:
Total number of cell counts in 50 field = Y
Working factor= 10,000.
Total SCC per ml of milk = Y x 10,000

Interpretation:
| 1,00,000 cells/ml is often considered to be ‘normal’, reflecting a
healthy mammary gland
| SCC of >2,50,000 cells/ml is suggestive of bacterial infection
(Subclinical mastitis).
| Milk with an SCC of more than 4,00,000 is deemed unfit for human
consumption.

Summary of test results

Test Suggested Description of test Reaction picture in CMT Somatic cell

Score meaning reaction count with
interpretation

The reaction mixture 0-2,00,000

0 Negative remains liquid cells/ml

Normal Milk

A distinct slime
adhering towards
1 Weak periphery, but with >1,50,000-
positive no tendency towards 5,00,000
gel formation. The cells/ml
reactions tend to
disappear with Sub-clinical
continued movement Mastitis
of the fluid.
 46 
 

The reagent mixture

thickens immediately >5,00,000
with gel formation. cells/ml
2 Distinct The mixture spread
positive out again when the Clinical
motion is stopped Mastitis
and covers the
bottom of the cup.

This type of
The reaction mixture milk reflects
Alkaline is indicated by a depression of
+ milk, pH 7.0 contrasting deeper secretary
or over purple colour. activity and may
results either
from
inflammation or
in drying off of
the gland.
The bromocresol Acidic milk in
purple is distinctly the udder is
yellow at pH 5.2. rare. It indicates
Y Acid milk This type of fermentation of
indication should be lactose by
added to the score bacterial action
when the mixture is within the
yellow. gland.
 47 
 
8.3 Detection
D of mastitis usiing Mastitiss Detection Card
C
Potential users:
– Dairy farmers
f
– Milk coooperative soccieties
– Para-veeterinarians
– Veterinnarians
– Byre-siide test
 

Proced
dure:
1. Take out
o a piece of drry Mastitis Deteection Card Test paper.
2. Make sure
s that the staamped surface is on top.
3. Put onne drop of milkk from each quaarter of the uddder on the four marked
m cornerss
(already treated
t with inddicator) of card//paper.
4. Observve the colour chhange, if any.
5. After brief drying, innterpret the chaange of colour according to thhe colour guidee
provided with the kit.

Colour of paper Mastittic condition

Colourr remain as suchh Noo mastitis
(yellow)
Greeenish-yellow +

Green ++
Grreenish-Blue +++

Bluue-Deep Blue ++++

PRECAUT
TION

1. Avoid using kit with wet

w hands.
 48 
 
9. AN
NTIBIOTIIC SENSITIVITY TEST
T
Once the causaative organism
O m of a disease is isolated andd identified, itt
is essenttial for cliniciaans to know which
w antibiotiic will be mosst effective forr
treatmennt. Antibiotic sensitivity teesting by discc diffusion method (Kirby--
Bauer Method)
M can reaadily provide this
t informatio on.

Materiaals required:
1. Nutrieent agar plate
2. Nutrieent broth cultuure to be testedd in tube
3. Antibiotic disc
4. Sterilee swab
5. Forcepps
6. Bunseen burner
Procedu ure:
1. Select the organism thhat you are goinng to test
2. Label the
t plate with issolate number and a date
3. Inoculate the surface of the medium with swab afteer expressing exxcess fluid from m
the sw
wab by pressing and rotating thhe swab against inside wall of tube t above fluidd
level. Cover the surfface of the agaar evenly by sw wabbing in threee directions. A
final sweep
s should made
m on the agarr rim with the sw
wab.
4. Allow 3 to 5 minutes for the agar surrface to dry befo ore applying dissc
5. Dispennse discs as follow:
Steriliize forceps first by flaming before
b picking discs.
d Keep eacch disc at leastt
15mm m away from eddge of plate. Appply gentle pressure to each disc d on the agarr
with thhe tip of the steerile forceps.
6. Invert and incubate thhe plate for 16-118hrs at 37oC.
7. After incubation
i meaasure the diameeter of the zone of inhibition too the nearest inn
mm. Record
R the zonne of inhibition and consult th he code chart suupplied by discc
manuffacturing comppany and accoordingly determ mine resistant and sensitivityy
patternn of the test orgganism.
 49 
 
10. DIAGNOSIS OF BRUCELLOSIS

10.1 Rose Bengal Plate Test (RBPT)

Principle
RBPT is a simple spot agglutination test used in many species as a
screening test prescribed for international trade. The buffered acid Rose
Bengal antigen interacts with serum antibody to produce agglutination, which
is used for the detection of Brucella specific antibodies.

Materials required

a. Rose Bengal Antigen

b. Test serum
c. Positive Control serum
d. Negative Control serum
e. Slides, tips, micropipettes, magnifying lens, etc.
f. Wooden pricks

Procedure

1. Bring the serum samples and antigen to room temperature (22 ± 4°C);
only sufficient antigen for the day’s tests should be removed from the
refrigerator.
2. Place 25–30 µl of each serum sample on a microscopic slide, white tile,
enamel or plastic plate.
3. Shake the antigen bottle well and place an equal volume of antigen near
each serum spot.
4. Mix the serum and antigen thoroughly using a clean glass/ plastic rod/
micro tip for each test to produce a circular or oval zone approximately
2 cm in diameter.
5. The mixture is agitated gently for 1-3 minutes at ambient temperature on
a rocker or clock and anticlock wise direction 5-10 times.
6. Read for agglutination immediately within 3-minute period. Any visible
reaction is considered to be positive. A control positive and negative
serum that gives a minimum positive reaction should be tested
simultaneously each day to verify the sensitivity of test conditions.
 50 
 
Interprretation

9 Any degree of agglutination

n varying from
m mild (+),
moderate (++
+) and strong (+
+++) positive = recorded as
positive

9 No agglutinattion = recorded
d as negative.
9 Include posittive and negaative controls while testingg
each time.

The RBP
PT is very sennsitive. False-nnegative reactio
ons occur rarely, mostly duee

to pro-zoning and cann sometimes be

b detected by
y diluting the serum samplee

(1:2, 1:44, 1:8 dilutionn) or retesting after 3 week

ks time. Neverrtheless RBPT
T

appears to be adequatte as a screeniing test for deetecting infecteed herds or too

guaranteee the absence of infection inn brucellosis-ffree herds.

 
 51 
 

Colour antigen 
Serum  
 52 
 

10.2 Milk ring test (MRT)

Principle
Milk ring test is a screening test of great value for locating infected
cattle herds, especially in areas of low prevalence. An efficient means of
screening dairy herds is by testing milk from the bulk/ pooled tank. The
principle of milk ring test is immunoglobulins present in the milk attached to
fat globules via the Fc portion of the molecule. If antibody to Brucella sp. Is
present it move to the top along with the fat globules and combine with
antigen to form a ring in the milk and fat interface. If no antibody is present,
the fat layer will remain as a buff colour and the antigen will be distributed
throughout the milk. This test may be applied to individual animals or to
pooled milk samples using a larger volume of milk relative to the pool size.
Milk can be collected easily and more frequently than blood samples
and is often available centrally at dairies. When a positive test result is
obtained, all dairy cows contributing milk should be tested using specific test.

Materials required for test

a) Brucella Milk Ring Antigen (B. abortus S99)
b) Pooled milk samples 5ml each
c) Known Brucella positive milk sample
d) Known Brucella negative milk sample
e) Serological tubes, tips, micropipettes, test tube racks, incubator etc.

Procedure:
1. The test is performed on pooled milk sample preferably 1 ml of sample
from 100 litres of milk ( one pooled milk sample from 10 cows). If
necessary, samples could be pretreated with preservative (0.1% formalin)
for 2–3 days at 4°C prior to use.
2. If pooled milk samples from large herds exceeding 100litres up to 300
litres are to be examined, the volume of milk should be increased to 2 or 3
ml.
 53 
 
3. Bringg the milk sammples and antiggen to room teemperature (200 ± 3°C); only
sufficcient antigen for the day’s tests shoulld be taken out o from the
refriggerator. Gentlyy shake the anttigen bottle weell.

4. The milk sampless must not get g frozen, heeated, subjecteed to violentt
shaking or stored for
f more than 72 hours. Testt should be doone with fresh
milk (not pasteurizeed/ boiled/ currdled).
5. The test
t is perform
med in a serollogical test tub be where the height of the
milk column in thee tube must be b at least 25 mm. The vollume of MRT
antigeen should be 2 drops / 1 mll milk to be teested from poooled milk orr
50-755 μl of MRT antigen
a to a 2 ml volume of milk obtainned from more
than 100
1 litres and less than 300 litres
l of pooledd milk.
6.The milk/antigen
m m
mixtures are mixed
m gently and
a incubate at a 37°C for 1
hour, together withh positive annd negative working
w controols. However,
furtheer 2 hr incubation at 4°C increases the sensitivity off the test andd
allow
ws for easier reeading

Milk ring test: tube 1, positivve control, tube 2 & 3 positive samples, tuube 4,
negative sample, tube 5, negattive control

Interpreetation
Color of
o cream ringg Color of milk
m column MRT Reading
Definite red white ++++
Definite red slightly redd +++
Definite red definite redd ++
Same color
c as milk same color as Cream millk + or – ve
White or
o slightly red red milk - ve

False poositive reactionns may occurr in cattle vacccinated less than

t 4 monthss
prior to testing, in casse of colostrum
m and mastiticc milk (OIE Manual,
M 2008)..
Thereforre, it is not recommended too use this testt in very smalll farms wheree
these prooblems have a greater impacct on the test reesults.
 54 
 
11. DIAGNOSIS OF BOVINE TUBERCULOSIS
Single intra dermal test:
Requirements:
i. Tuberculin (Mammalian Tuberculin)/PPD
ii. Vernier calipers
iii. Intra dermal needle
Procedure:

™ An area on the side of the neck of the suspected animal is clearly

marked.

™ Shave the area aseptically.

™ Measure the thickness of the skin with the aid of a vernier caliper and
record the thickness.

™ Inject 0.1 ml of the tuberculin intra-dermally using a intra dermal needle.

™ Observe for any diffuse swelling or redness in the area after 48- 72 hours
and also record the thickness of the skin with a vernier caliper.

Observation:
Increase in the thickness of the skin by > 4 mm & diffuse swelling: Positive
result.
Increase by 3mm: Doubtful reaction.

Step -1 Step-2 Step-3(After 48-72 hrs)

 55 
 
12. AGAR GEL PRECIPITATION TEST (AGPT)
FOR DETECTION OF IBD IN POULTRY

Principle: Antigen and antibody placed in the wells of the agar or agarose
gel diffuse towards each other and form a visible opaque band of
precipitation in the optimal proportion.
Materials required:
1. Microslides
2. 1% agarose in PBS (pH-7.2-7.4) 0.02% sodium azide
3. Antigen (Bursa of Fabricius suspension)
4. Serum sample (Known positive IBDV sera)
5. Gel puncher
6. Template
7. Needle
8. Micropipette
Precoating of slides:
1. Dissolve the agarose by boiling and add sodium azide@0.02%.
2. Place the slide on a horizontal surface and slowly pour the agarose on
the slide.
3. Allow the agarose to solidify.
Procedure for AGPT:
1. Prepare the slide as described above.
2. Place the slide over a template and punch wells of 2 to 3 mm in
diameter and 4 to 5mm apart with gel punch.
3. Remove the cut gel from the wells by scooping with needle.
4. Fill the central well to brim with 20μl neat serum and peripheral
wells with 20 μl antigen. Alternatively fill one peripheral well NSS
or PBS as control.
5. Place the slides in humid chamber and incubate overnight at room
temperature.
6. Examine the slides for precipitin lines.
Interpretation: Precipitin lines appear between antigen and antibody wells
in positive reaction and interpreted as bird infected with IBDV.
 56 
 
13. VIRAL HAEMAGGLUTINATION TEST
Principle: The test is based on the binding of certain viruses to the
erythrocytes receptors of certain avian and mammalian species and thereby
causing agglutination of the erythrocytes. Unlike other serological test, viral
haemagglutination test does not involve any antibody. It is used for
identification and quantification of certain viruses like Newcastle disease
virus in infected tissues of poultry.

Materials required:

1) U/V bottomed microtitre plate

2) Pipette (20-200 µl)
3) Disposable syringe-2ml
4) Tips-20-200 µl
5) Centrifuge tube-15ml
6) Ice Box
7) Centrifuge machine

Chemicals required:

Sl HAEMAGGLUTINATION HAEMAGGLUTINATION
No TEST INHIBITION TEST
Reagents Storage Reagents Storage

1 Phosphate buffer saline 40C Chicken sera (Hyper immune -20 0C

(pH-7.5) sera)
2 1% Chicken red blood 40C Antigen 4HAU -20 0C
cells (RBCs) in Alsever’s
solution (Page No. 29)
3 Antigen -20 0C Chicken red blood cells 40C
(RBCs) in Alsever’s solution
 57 
 
Preparation of antigen:
A 20% suspension of spleen and lung tissue of dead bird is prepared in PBS.
Procedure:
o 25µl of PBS is dispensed into each well of a plastic V-bottomed
microtitre plate.
o 25µl of the viral antigen is placed in the first well of A1.
o 25µl of respective test samples are distributed in the first well of
B1,C1…..G1.
o Two fold serial dilutions of 25µl volume of the virus antigen is prepared
separately across the plate (from well-1 to well-12 of each row) using
individual tip and 25µl volume of virus suspension from the last well is
discarded.
o 25µl of 1% (v/v) chicken RBCs is dispensed to each well along with
control RBCs well in H1 & H2.
o The solution is mixed by tapping the plate gently. The RBCs are allowed
to settle for about 30 minutes at room temperature.

Observation and interpretation:

In positive cases, a layer of agglutinated RBCs in the form of a matt
on the bottom of the well is formed, whereas no agglutination of RBCs, it
appears as sharp button of RBCs. The highest virus causing complete
agglutination of RBC is termed as one haemagglutination unit-HAU. HA is
determined by tilting the plate and observing the presence or absence of tear
shaped streaming of the RBCs.
 58 
 
13.1 Viral haemagglutination inhibition test
Principle: When certain viruses like ND virus mixed with serum containing
specific antibodies, an Ag-Ab reaction occurs which subsequently inhibits the
haemagglutinating property of the virus. It is used for detection and
estimation of antibody in the serum of birds infected with ND virus.
Procedure:

o 25µl of PBS is dispensed into each well of a plastic V-bottomed

microtitre plate.
o 25µl of serum is placed into the first well of the plate (A1) and test
serum samples are in B1 to G1 of the plate.
o Two fold serial dilutions of 25µl volumes of the serum are made
across the plate and discard from the last well.
o 4 HAU virus/antigen in 25µl is added to each well and the plate is left
for a minimum of 30 minutes at room temperature.
o HAU control is kept at wells H9, dilute serially up to H12 yielding 4
HAU, 2 HAU, 1 HAU and ½ HAU, respectively.
o 25µl of 1% (v/v) chicken RBCs is added to each well including HAU
control (H9 to H12) and RBC control (H1 and H2). After gentle
mixing, the RBCs are allowed to settle for about 30 minutes at room
temperature.

Observation and interpretation:

The HI titre is the highest dilution of serum (i.e. prior to agglutination
well) causing complete inhibition of 4HAU of antigen. The agglutination is
assessed by tilting the plates where the RBCs stream at the same rate as the
HAU control wells.
 59 
 
14. In-House Indirect ELISA for detection of CSFV antibodies
in serum and plasma from pigs

Principle of the kit

The CSFV antigen used is a RK-13 adapted cell culture derived viral antigen
stabilized using the Lactalbumin hydrolysate (LAH) stabilizer. This assay is an
indirect ELISA which utilizes microplate coated with CSFV antigen. The antigen-
antibody (specific to CSFV) forms a complex and an anti-porcine horseradish
peroxidase conjugate is added which binds to the antibody indicating color

Preparation of reagents (one plate)

Antigen preparation
• Stock Antigen- dilute component 2 with 1ml of component 7 (Mix
properly)
• Working Antigen (10ml)- take 500μl of the stock antigen and dilute with
9.5 ml of component 3 (Mix properly)
Wash solution
• The wash concentrate (10X) (Component 4) must be diluted 1:10 in
distilled water. Example: for 500 ml wash solution, dilute 50 ml Wash
Concentrate (10X) with 450 ml distilled water and mix well to give 1X
Wash solution
 60 
 
Blocking solution
• 0.6 g of component 5 and 1.5 g of component 6 to be dissolved in 30ml of
the Wash solution (1X)
• Test sera (To be prepare in a deep well plate)
• Add 100 μl of component 7 to the entire well. Add 0.5 μl of component 8
to wells A1 & B1 and component 9 to wells C1 & D1. Add 1 μl of the test
sera into the remaining wells
Conjugate (Prepare before use)
• 1 μl of the conjugate (Component 10) added to 15 ml of 1X Wash Solution
and mixed well
Substrate (Prepare before use)
• Take 10 ml of distilled water and add 1 tablet (light sensitive) (Component
11). Dissolve the tablet; add 40μl of component 12. (To be prepared away
from direct light)

TEST PROCEDURE

¾ Add 100μl of the diluted Antigen (working Antigen) to all the wells
¾ Seal the plate and incubate at 37oC for 1 hour
¾ Wash each well with approximately 300μl of Wash solution 5 times.
Aspirate liquid contents of all wells after each wash. Following the
final aspiration, firmly tap residual wash fluid from each plate onto
absorbent material. Avoid plate drying between washes and prior to
next step
¾ Add 300μl of Blocking solution to all the wells
¾ Seal the plate and incubate at 37oC for 1 hour
¾ Repeat Step 3
¾ Add 100μl of the Control sera and the Test sera from the deep well
plate into the corresponding wells of the Test plate. (Contents should
be mix by pippetting before adding to the test plate). Use a separate
pipette tip for different Controls and Test sera
¾ Seal the plate and incubate at 37oC for 1 hour
¾ Repeat Step 3
¾ Add 100μl of the diluted Conjugate to all the wells
¾ Seal the plate and incubate at 37oC for 30mins
¾ Repeat Step 3
¾ Add 100μl Substrate (light sensitive) to all wells and incubate for
15mins in dark
¾ Stop the reaction by adding 100μl Stop Solution (Component 13). Add
the Stop Solution in the same order as the Substrate Solution was
added
¾ Measure the absorbance at 490 nm on a microplate reader.
 61 
 
N.B.: Alll reagents muust be allowedd to come to ro
oom temperatuure before usee
and mixxed by gentle swirling or vortexing.
v Usee separate tip for each testt
sample

CUT-OF FF CALCULA ATION

Positive cut-off= Negaative control x 2.5

INTERP
PRETATION
 62 
 
15. MICROSCOPIC EXAMINATION OF
HELMINTH OVA (QUALITATIVE METHODS)
A) Direct method:

Requirements:

• Faecal Sample
• Microscopic slide & cover slip
• Match stick or bamboo stick
• Forceps
• Compound microscope

Procedure:

Take clean microscopic slide

Place a drop of water at the centre of the slide

With a match stick or bamboo stick place a mass of the faecal

sample over the drop of water

Mix the sample with water

Remove coarse materials with a stick or forceps

Put a cover slip over the sample

First examine under low power and then under high power of a
compound microscope for confirmation
 63 
 
C. Salt floatation method of faecal examination:

Take 2-3 g of faecal sample in a clean mortar

Add a little amount of tap water and gently triturate the mixture to make it
uniform using a pestle

Add 10-15 ml of floatation fluid & mix thoroughly with the faecal sample

Strain the suspension through a sieve in a clean floatation tube and Allow to
stand undisturbed for 10-15 minutes

With the help of a clean wire loop, transfer a drop of suspension from the
surface on to a clean microscopic slide, put a cover slip over the drop and
examine under low power and then under high power using a compound
microscope
 64 
 
Alternative method:
 Make a suspension of the faecal sample with saline solution in a mortar
using a pestle
 Strain the solution to a centrifuge tube and centrifuge for 1500 rpm for 5
minutes
 Discard the supernatant and add floatation fluid up to the brim
 Put a cover slip over the tube and centrifuge again for 5 minutes at 1500
rpm
 Carefully remove the cover slip and place it over a microscopic slide and
examine under a compound microscope.

C) Sedimentation method:
Requirements:
ƒ Pestle & mortar
ƒ Water
ƒ Strainer/sieve
ƒ Microscopic slide
ƒ Centrifuge tube
ƒ Centrifuge machine
ƒ Compound microscope
Procedure:
¾ Place about 1 g of faeces in a mortar and add a little quantity of water,
triturate it using pestle
¾ Mix it thoroughly and strain to a centrifuge tube
¾ Fill the tube with water and centrifuge at 1500 rpm for 5- 10 minutes
¾ Discard the supernatant and repeat the above step by adding and
discarding the water until the supernatant becomes clear
¾ Using a pipette, take a drop of the sediment onto a clean microscopic
slide and add a drop of 1% iodine
¾ Place a cover slip and examine under the low and high power of a
compound microscope
Alternative method:
• Place about 1 g of faeces in a mortar and add a little quantity of water,
triturate it using pestle
• Mix it thoroughly and strain it to a petridish
• Allow to stand undisturbed for 10-15 minutes
• Discard the supernatant and examine the sediment directly under a
compound microscope

 
 65 
 

Courtesy: Veterinary Clinical Parasitology, 8th Edition by Anne M. Zajac and Gary A. Conboy
 66 
 

ne M. Zajac and Gary A. Conboy 

Courtesy: Veterinary Cliniccal Parasitology, 8th Edition by Ann

 
 67 
 

Courtesy: Veterinary Clinicaal Parasitology, 8th Edition

E by Anne M. Zajac and Gary A. Conboy 
 68 
 
 

Courtesy: Veterinary Clinical Parasitology, 8th Edition by Anne M. Zajac and Gary A. Conboy 
 69 
 

  

 
 70 
 

 
 71 
 
15.1 Microscopic examination of protozoa in faecal sample

A) Direct examination of fresh faecal smear:

Requirement:
• Fresh faecal sample
• Microscopic slide
• Cover slip
• Tooth pick/ wooden prick
• Saline solution
• 1:1000 aqueous solution of eosin
• Lugol’s iodine (Page no-31)

Procedure: (Without staining)

¾ Place a drop of saline solution on a clean glass slide
¾ Take a small amount of freshly collected faecal sample with a tooth pick
on a glass slide and mix thoroughly with the salt solution
¾ Remove the coarse particles aside using the tooth prick
¾ Place a cover slip over the drop of wet smear
¾ Examine over low and high power of a microscope

Procedure: (With stain):

 Place a drop of saline solution on a clean glass slide
 Take a small amount of freshly collected faecal sample with a tooth pick
on a glass slide and mix thoroughly with the salt solution
 Remove the coarse particles aside using the tooth prick
 Add a drop of 1:1000 eosin and mix thoroughly to stain the debris pink
leaving the trophozoites and cysts unstained
 Add a drop of iodine to stain the nucleus and the chromatoid bodies
 Place a cover slip and examine under low and high power of a compound
microscope

B) Floatation method:
Requirements:
• Faecal sample • Wire loop
• Pestle & mortar • Compound microscope
• Strainer/sieve • Floatation solution
• Narrow cylindrical • Centrifuge tube
container • Centrifuge machine
• Coverslip
• Micro slide
 72 
 
Procedure:
¾ Take 1 gm of faecal sample in a clean mortar
¾ Add a little quantity of tap water and homogenize the mixture using a
pestle
¾ Add 10-15 ml of floatation fluid and mix well
¾ Strain the suspension through a strainer to a narrow cylindrical tube
¾ Allow to stand in the tube undisturbed for 10-15 minutes with or without
putting a cover slip
Or
¾ Strain the suspension to a centrifuge tube and centrifuge at 1500 rpm for
5 minutes
¾ Transfer a loop of the suspension from the surface of the tube onto a
clean microscopic slide and place a cover slip
¾ Examine under low and high power of a compound microscope

B) Sedimentation method:

Requirements:

• Pestle & mortar • Microscopic slide

• Strainer/sieve • Pipette
• Petridish • Centrifuge tube
• Tap water • Centrifuge machine
• Microscope

Procedure:
9 Place 1 gm of faeces in a mortar and mix thoroughly with a little quantity
of water with a pestle
9 Strain in to a Petridish or a centrifuge tube
9 Allow the petridish to stand undisturbed for 10-15 minutes or centrifuge
the tube at 1500 rpm for 5 minutes
9 Discard the supernatant gently from the petridish or centrifuge tube
9 Examine the petridish directly under the microscope or place a drop of
the sediment from the centrifuge tube on a clean micro slide and examine
under the microscope.
 73 
 
C) Formol ether technique for detection of Giardia & Entamoeba cysts:

Requirements:
• Pestle & mortar • Pipette
• Strainer • Centrifuge tube
• Petridish • Centrifuge machine
• Tap water • Formol saline
• Microscope • Ether
• Microscopic slide • Iodine (1%)

Procedure:
¾ Take about 1 g of faeces in a mortar and emulsify with 5 ml of 10%
formol saline
¾ Strain through a strainer in to a clean centrifuge tube
¾ Add equal volume of ether and shake vigorously after inserting a rubber
stopper
¾ Remove the stopper and let it stand for 2 minutes
¾ Centrifuge at 2000 rpm for 2 minutes and a ring of faecal debris will
appear between the ether and formalin layer, leaving the sediment at the
bottom
¾ Loosen the debris ring with an applicator
¾ Pour off the supernatant and debris ring carefully without disturbing the
sediment
¾ Add a drop of saline solution to the sediment and mix well
¾ Take a drop of sediment with a pipette on a clean glass slide and add a
drop of iodine solution
¾ Place a cover slip over the drop and examine under the low and high
power of a compound microscope.
 74 
 

D) Ziehl Neelsen staining technique for detection of cryptosporidium

oocysts:

Requirements:
Faecal sample Immersion oil
Acid alcohol Microscopic slide
Carbol fuchsin Cover slip

Procedure:
 Prepare a thin faecal smear, air dry or pass over a flame
 Stain with carbol fuchsin for 2 minutes
 Rinse with tap water
 Rinse with acid alcohol for a 3-5 seconds
 Rinse again with tap water
 Counter stain with brilliant green or 1 % aqueous methylene blue for 1-
2 minutes
 Rinse again with tap water
 Air dry
 Apply cover slip and examine under 100 X using oil immersion.

Commonly used floatation fluids:

1. Sodium chloride: sp. Gravity-1.2

2. Zinc sulphate: Sp. Gravity-1.18 (386 gm in 1000 ml dist. Water)
3. Sheather’s sugar solution: Sp. Gravity-1.12-1.30 (Sucrose-500 gm,
Phenol -6.5 gm melted in hot water bath and dist. Water-320 ml)
 75 
 
15.2 Miicroscopic examination
e n of blood sm
mears for diiagnosis of
haaemoprotozzoan parasittes

A) Prep
paration of th
hin blood smeear :

Req
quirements:
• Anti coaguulated blood saample in EDTA/ HEPARIN
N
• Microscoppic slide
• Methanol

Procedure:

Airr dry the smeaar and dip in methanol

m in a co
oupling jar forr fixation

A dry the metthanol fixed sm

Air mear before sttoring for furthher use.
 76 
 
B) Giemsa staining of blood smear:
Requirement:
• Methanol fixed blood smear
• Sorensen Buffer (pH:7.2)
• Giemsa stain (Stock solution)
• Staining rack
Procedure:
¾ Prepare a fresh working solution of Giemsa stain by mixing with
Sorensen buffer @1:10
¾ Place the methanol fixed blood smear on a staining rack
¾ Pour the stain solution over the smear to cover the whole slide
¾ Allow it to stand for 30- 45 minutes
¾ Wash under running tap water directly without pouring off the stain
¾ Air dry the slide
¾ Observe under low, high and finally under oil immersion of a compound
microscope

Sorensen buffer: (working solution)

DiSodium hydrogen phosphate solution: 72 ml
Potasium dihydrogen phosphate solution: 28 ml
Distilled water: 900 ml.

15.3 Examination of skin scrapping for ectoparasites

A. Direct method:
Requirements:
• Scalpel
• Lubricating oil
• Clipping scissors
• Microscopic slide
• Cover slip
• Microscope
Procedure:
™ Clip off the hairs of the affected area
™ Smear the area with liquid paraffin or lubricating oil
™ Scrap the lesion with the help of an oily scalpel till blood oozes out
™ Transfer the scrapping material from the scalpel onto a clean
microscopic slide containing a drop of oil
™ Examine the preparation under low and high power of a compound
microscope
 77 
 
B) Indirect method:
Requirements:
Scalpel Test tube holder
Lubricating oil Spirit lamp
Clipping scissors Centrifuge tube
Micro slide Centrifuge machine
Cover Glass Sodium or
Test tube potassiumhydroxide (10%)

Procedure:
 The materials should be collected as described earlier in the direct
method
 Transfer the scrapping materials to a clean test tube/ centrifuge tube
 Add required quantity of Sodium/ potassium hydroxide and boil the
contents of the test tube over a spirit lamp till the hairs and tissue
debris are dissolved
 Discard the supernatant and take a small amount of sediment on a
clean micro slide
 Cover the material with a cover slip and examine under low and
high powers of a compound microscope

Fig: Common haemoparasites encountered in cattle

 78 
 
16. CYTOPATHOLOGICAL DIAGNOSIS OF
DISEASES IN LIVESTOCK AND POULTRY IN
CLINICAL AND NECROPSY SAMPLES
Diagnosis of diseases in animals is an interesting job that too under
field conditions. Field veterinarians can make use of certain simple
techniques which will aid in quick diagnosis of diseases. One among them is
the cytological technique which does not require elaborate procedure and
equipment. All you need are suitable syringes and needles, glass slides,
staining solutions and a good microscope. Usually lower and higher
magnification examinations are sufficient and in certain situations, oil
immersion objective is required e.g. identification of microorganisms like
bipolars and blood parasites.

Cytology is defined as the examination of cells without regard to the

tissue’s architectural details. This diagnostic process is suggested, when
clinical examination, radiographic evaluation and laboratory data could not
confirm the diagnosis. The advantages of cytological sampling technique are
quick, easy, inexpensive, less invasive than surgical biopsy, no need of
general anaesthesia, if required light sedation, short processing time, results
available in short time (5 to 30 in minutes) and a fewer complications. Be
cautious about haemorrhagic tendencies and gain expertise under suitable
guidance.

The limitations of cytological technique are no tissue architecture,

tissue of origin, biologic behavior, grading, degree of tissue infiltration,
presence and absence of necrosis and not a representative of the lesion. Small
mobile or very firm lesions don’t yield sufficient materials. In obesity,
perilesional fat may come instead of the tissue of interest. In large lymphoma,
lymph node may be necrotic and is non–diagnostic. While examining the
cytological preparations, note that the cellular cytoplasm and nucleus are
clearly visible. In poor smear preparation, do not attempt diagnosis. The bad
smears will show smudged cells, “bare” nuclei without cytoplasmic details
and nuclear and cytoplasmic vacuoles. Artifacts will be a problem with dirty
slides, water, starch granules from gloves, ultrasound gel and stain
precipitates.

Sample collection techniques: various approaches are adopted to collect

cytopathological samples.

i). Fine needle aspiration biopsy (FNAB): It is the most used method. In
short, it is known as FNAB and is employed to identify cells and differentiate
 79 
 
inflammation and neoplasia. The advantages are less time consuming,
multiple site sampling in single procedure, consecutive sampling at intervals
are possible. Diagnosis may eliminate surgery and specimens can also be
used for histopathology.
“Stop collecting sample if blood appears”“Metastasis is remote with FNAB
sampling; Hence, do not hesitate to get sample”

Procedure:
 Clean the area aseptically
 Anaesthetize the area
 Hold the mass firmly in hand
 Use 3 to 20 mL syringe with 21 to 25 gauge needle
 Insert needle into the mass, apply negative pressure
and move the needle in various directions while aspirating
 Release negative pressure, withdraw needle out
 Dislodge needle and air drawn into the barrel
 Push the contents of the needle on clean glass slides
 Prepare smear

This technique is referred to as “continuous suction” type.

 80 
 
Preparaation of slides
9 Fluid – Smearss
F
9 S
Small fragmennts – Squash
9 L
Large tissue fraagment – Imprrints/ histopath
hology
9 C
Culture – Swirrl the needle inn medium befofore fixation foor
h
histopathologyy /slide preparaation

Thhe next proceddure known ass “intermitten nt suction” is more suitablee

for smalller lesions, whhere it is not possible
p to adv
vance or redirrect the needlee
without exiting the mass.
m Use 23G G needle and 5 ml syringe. Withdraw
W andd
release the
t plunger sevveral times wiith the needle inserted
i in thee mass, releasee
the suctiion, remove thhe needle fromm the mass, dissconnect the neeedle from thee
syringe and
a obtain smears as describbed earlier.

Annother proceduure called as “non-suction”

“ ” type causes less
l damage too
fragile cells
c e.g. lymmphoid cells. This proced dure also minnimizes bloodd
contaminnation, useful for aspiratingg highly vascullar masses andd lymph nodess
and also obtaining ultrrasound guidedd samples of leesions in bodyy cavities.

ii). Imprression smearrs with microsscope slide

ƒ Ulcerated
U surfaace
ƒ Cut
C surface of excised tissuees or surfaces of o tissues in biiopsy or PM
s
specimens
ƒ Carefully
C blot the cut surfacee of the tissue with absorbennt
m
material/paper towel to remoove the blood and
a other bodyy fluid
ƒ Make
M impressiion smears by gently touchin ng flat cut surfface
ƒ Make
M several imprints
i on thee same slide
ƒ Air
A dry or wet fix
ƒ Remaining
R porrtions sliced innto thin section
ns and fixed foor
h
histopathologyy
 81 
 
Impression - Disadvantages

It collects relatively a few cells. Cells collected may not necessarily be

representative of the lesion under investigation e.g. Ulcerated lesions are
often secondarily inflammed or infected which may result in only
inflammatory cells of interest or may induce in the cells of interest dysplastic
changes that mimic those associated with neoplasia. It has limited use in
superficial neoplasia but, useful for intraoperative confirmation of neoplasia
in biopsy samples or excised tissue.

iii). Impression with cello tape– “No swelling but surface lesions are
present”

 Useful technique in diagnosis of malasseziasis in dogs

 Take a regular cellophane tape and stick on to the affected area
(Erythematous to hyperpigmented skin)
 Leave it for a minute
 Remove the tape, reverse and stick on to a glass slide with the
impression smear facing out
 Stain with Leishman-Giemsa stain as described below
 Examine under microscope

C. Impression smear taken from lesion paste B. Stain the impressed

on slide keeping impressed smear upside cello tape
 82 
 

iv). Scraapings

¾ T
Touch imprintss from solid tissues acellularr
¾ Harvesting
H cellls from lesioons unlikely to t yield largee no. cells onn
F
FNAB (Hard tiissues - Mesennchymal tissuee tumours – fibbroma)
¾ So
S scrape smeaars are prepareed
¾ Scrape
S cut surfface carefully with a scalpel blade
¾ Discard
D the firsst scraping conntaminated wiith the blood annd again
s
scrape carefullly
¾ Spread
S materiaals in a thin layyer/squash on glass slides
¾ Air
A dry or wet fix

v). Incissional Biopsy

Smmall portion off a lesion surgiically removed

d is utilized. Im
mpression or
scrapingg smear can bee prepared and used.

n of smears
Fixation

i. Dry fixation is generallyy adopted and is done by rappidly agitatingg

the slide in air
a (As in the case
c of blood smear
s preparaation).

ii. Wet fixatioon is advised iff staining is to be delayed foor a few days
to enhance cell
c preservatioon.
• Fix slide within
w 30-60 seconds / Spray y
• 95% ethannol/absolute isoopropanol /Meethyl alcohol/ for a few
minutes too 30 min
• RBC lysiss improves exaamination of clumps of cellss
 83 
 
Cytological stains

For routine staining, use Leishman-Giemsa (LG) staining. On

occasions, haematoxylin-eosin staining procedure can be adopted.

Leishman-Giemsa Staining

Preparation of LG stain
Leishman powder 120 mg
Giemsa powder 30 mg
Absolute methanol 100 mL

¾ Cover the smear with LG stain for a minute

¾ Dilute with tap water/Distilled Water /Phosphate Buffer Saline pH
6.8-7.2 in that order if there is a problem in staining character of cells
i.e. cytoplasm and nucleus
¾ Allow for about 20 min
¾ Wash in tap water/Distilled Water /Phosphate Buffer Saline
¾ Air dry
¾ Examine under microscope first under low power and then at high
power field (Place a cover slip over the smear while examination)
¾ If oil immersion objective is to be used, take out the coverslip

Haematoxylin and Eosin Staining

Preparation of Harris’ haematoxylin

Hematoxylin crystals 5.0 g

Alcohol, 100 % 50.0 mL
Ammonium or potassium alum 100.0 g
Distilled water 1000.0 mL
Mercuric oxide (red) 2.5 g

Dissolve the hematoxylin in the alcohol, the alum in the water by the
aid of heat. Remove from heat and mix the two solutions. Bring to a boil as
rapidly as possible (Limit this heat to less than 1 minute and stir often).
Remove from heat and add the mercuric oxide slowly. Reheat to a simmer
until it becomes dark purple, remove from heat immediately and plunge the
vessel into a basin of cold water until cool. The stain is ready for use as soon
as it cools. Addition of 2 – 4 mL of glacial acetic acid per 100 ml of solution
increases the precision of the nuclear stain. Filter before use.
 84 
 
™ Wet fixed smears kept in Harri’s haematoxylin for 20 min
™ Wash in running water
™ Dip in 1% acid alcohol, wash immediately
™ Keep in running water for 5-10 min for “bluing”
™ Stain with 1% aqueous solution of eosin for 1-2 min
™ Wash, dry and dip in xylol
™ Mount on DPX mountant with a clean cover slip

If Diffquik stains are available, they can be used much easily

following manufacturers instruction. May take only a few minutes.

Special stains like Gram (bacteria), acid fast (TB, JD), toluidine blue
(Mast cells) and Periodic Acid Schiff (PAS) staining and Grocott-Gomori
silver staining (Fungi) as it demands.

Staining characters

Romanowsky stains and combinations with Giemsa stain nucleus

purplish and cytoplasm bluish. Haematoxylin & eosin stain the nucleus
bluish and cytoplasm eosinophilic.

Mast Cells – Toluidine Blue Staining

Toluidine blue stock solution

Toluidine Blue O 1.0 g

70% alcohol 100.0 mL
Mix, solution is stable for 6 months

1% Sodium chloride: 0.5 g

Sodium chloride
Distilled water 50.0 mL

Make fresh

Working solution

Toluidine blue, Stock 5.0 mL

1% Sodium chloride 45.0 mL

Make fresh, discard after use

 85 
 
Procedure
¾ To the smear add working toluidine blue, 1 – 2 minutes
¾ Rinse in distilled water, 3 changes
¾ Dehydrate quickly through the 95% and absolute alcohols
¾ Clear in xylene and coverslip

Result
Mast cells will take violet stain of the cytoplasmic granules with a
background of shades of blue.

Diagnosis and interpretation of results

Any swelling and mass may be an
Abscess: Pus may come into syringe depending on the consistency
Haematoma: Blood tinged fluid will flow into the syringe
Cyst : Clear fluid will flow into syringe
Granuloma: TB, parasitic, pyogranuloma
Or A tumour :
Any surface swellings which are commonly observed and need proper
diagnosis. Hence, the condition has to be differentiated whether it is
inflammatory / infection or neoplastic.

Infections

Infectious agents like bacteria, virus, parasite and fungi may be

identified. In malasseziasis affected dogs (Fig. 1), basophilic, peanut or
double bottle or foot print shaped organisms are found. In abscess, bacteria
will be detected – cocci, bacilli; Gram +ve or Gram –ve (Fig. 2,3,4).

In pox virus infection, epithelial cells may show intracytoplasmic

inclusions. In granulomas, bacteria (TB / JD: Acid fast bacilli – Necropsy:
TB-Lungs, lymph nodes-{Caseous / calcified nodules}, intestine {JD –
Corrugated appearance}; Actinomycosis: Chinese letter appearance of bacilli
or fungi {Aspergillosis: Branching hyphae, Blastomycosis: Spherical bodies,
branching elements}). Necropsy: Suspected canine distemper cases, urinary
bladder / stomach / lung impressions, intranuclear / intracytoplasmic
inclusions see (Fig. 5); ICH – Intranuclear basophilic inclusions; PPR: Lung
lesions, you may find intracytoplasmic inclusions.
 86 
 
Inflammations

In acute inflammation, the neutrophils predominate in non-septic

condition. In septic condition neutrophil associates with bacteria. Neutrophils
may be intact or degenerate (swollen nucleus). Macrophages predominate in
chronic inflammation and accompanied by epithelioid cells and giant cells
in granulomatous inflammation. Subacute inflammation is characterized by
the presence of lymphocytes and plasma cells. In allergic conditions,
eosinophils predominate.

Diagnosis of lymphadenopathies – “Do not sample when lymph node is

not swollen”

Lymph node enlargement is commonly encountered condition in

animals. FNAB smears are handy in making the diagnosis. Five different
observations can be made

i. Specific diseases: Cattle: In theileriosis, the Koch’s blue bodies can

be detected in the cytoplasm of lymphocytes (Fig. 6). Bacterial
infections (Abscesses) and blastomycosis may be seen.

Fig. 1. Dog-Malassezisasis-Skin Fig.2. Dog-Skin-Chronic (active)

impression-Bluish peanut / double inflammation-Granuloma-
bottle / foot print shaped Multinucleate cell and neutrophils
organisms (arrow). Red (arrow)
keratinized cells
 87 
 

i. Reactive hyperplasia: Wherein plasma cells are found. These are round
to oval cells having eccentric nuclei and hyper-basophilic cytoplasm.

ii. Inflammations-lymph adenitis: As described earlier, it may be acute or

chronic conditions.

iii. Primary tumours: In lymphomas especially dogs, mixed lymphocytic

cell population is seen (Intermediate lymphoma). Sometimes, large
neoplastic lymphoid cells with multiple nuclei will be present (Poorly
differentiated lymphoma). At times, large undifferentiated cells may be
seen (Undifferentiated lymphoma).

iv. Secondary tumours: Tumours of skin may spread to local lymph nodes.
Look for characteristic of epithelial or mesenchymal cells and presence
of tumours nearby the lymph nodes.
 88 
 
Neoplastic conditions

Evaluation criteria

Nuclear criteria - These are anisokaryosis (Variation in size of

nucleus), multinucleate cells, giant cells (Large sized cells), mitotic figures,
abnormal shape, N : C ratio decreased due to enlargement of nucleus,
vacuolation.

Chromatin criteria- coarse, reticulate or sometimes fine.

Nucleolar criteria- size, shape and number variation.

More than three characters indicate malignancy.

Classification

Based on cytology, neoplastic conditions are classified into three

categories viz.
i. Discrete cell tumours ii. Epithelial cell tumours and iii Mesenchymal cell
tumours.

Discrete cell tumours (cells are discrete) -useful in dogs – easy to

diagnose

These tumours can be easily diagnosed and yield high cellularity. Cells
are discrete and mostly round in shape. Cells of mast cell tumours show
cytoplasmic granules (Leishman-Giemsa), seen in a pole or either pole or
distributed throughout the cytoplasm. Histiocytomas (Fig. 8.) will show
indented nucleus and multinucleate cells (Binucleate / trinucleate or more
number). TVT cells show cytoplasmic vacuoles (Fig 7). Cells of plasma cell
tumours are round to oval and cytoplasm is deeply basophilic and show pale
Golgi zone near nucleus. Melanoma cells contain brownish to black
cytoplasmic pigments (Fig. 9). Lipomas will show variable sized cells with
cytoplasmic vacuoles seen almost occupying the entire cytoplasm with
eccentrically placed nucleus (Fig.10).
 89 
 

Fig.8. Dog-Skin-Histiocytoma-Large cells,

Fig. 7. Transmissible veneral tumour (TVT)-Cells
abundtan cytoplasm and indented
showing punctate vacuoles in the cytoplasm;
nuclues(arrow) LG stain. Right-
Mitotic figure (arrow)
Multinuclate cells H&E stain

Fig 9: Dog-Skin-Malignant melanoma-Variable

Fig.10. Dog-Skin-Lipoma-Variable sized
sized cells showing blue-black granules in the cells with vaculated cytoplasm
cytoplasm-Anisokaryosis and nucleoli present.

Fig. 11. Dog-Skin-Squamous cell carcinoma-

Fig.12. Dog-Skin-Basal cell carcinoma-
Polyhedral cells and a few intermediate cells
Cluster of cylindrical cells
(arrow)
 90 
 
Epitheliial cell tumou
urs – moderateely easy to dia
agnose

Sqquamous cell carcinoma will w show clusster of squam mous cells andd
intermeddiate cells (Fiig.11.). Look for tadpole cells.
c Basal ceell carcinomass
will reveeal cluster of cylindrical cellls (Fig.12). In
n benign tumoours, cells andd
their willl show slight variation in size
s and carcinomas will shhow cluster off
cells (annd nuceli) varyying in size and
a shape. Perrianal adenom mas will reveall
hepatoidd cells (Fig. 133).

Mesench
hymal tumou
urs – needs exp
pertise gained
d over a periood of time

Thhese tumours yield

y low cellularity. Fibrom
mas will show w spindle cellss
and fibroosarcomas spiindle to plumpp cells (Fig.14
4.). However, osteosarcomas
o s
(Fig.15) can be diagnoosed better annd the cells will be discrete, round to ovall
and showw eccentric nuuclei (plasmacyytoid appearannce).

Fig.13. Dog-P
Perianal adenocarrcinoma-Variable sized
s Fig.14. Dog-Skin-Fib
broma-Note spindle
hep
patoid cells and nu
ucleoli (arrow) shapedd cells

Fig.15. Dog-B
Bone-Osteosarcom ma-Variable sized oval Fig
g. 16. Dog-Lymph node-Acute lymph
cells, eccentric nuclei, highly basophilic cytopllasm adennitis-Note many neeutrophils (arrow) are
(arrow) present. H&E
H stain
 91 
 
Poultry disease diagnosis

Impression smears from liver of suspected cases of fowl cholera

(Multifocal necrotic hepatitis) will reveal bipolars (LG stain). Similarly,
impression smears from nodular or diffuse (greyish-white) lesions will show
heterogeneous population of neoplastic lymphoid cells in Marek’s diseases
(MD) and homogeneous neoplastic lymphoblastic cells in lymphoid leucosis
(LL). One will find septate hyphae crushed preparations from lesion of lungs
and air sacs in aspergillosis. In granulomatous lesions, FNAB smears will
show macrophages and multinucleated giant cells (Look for any bacterial
cocci). In duck plague, attempts can be made to find out inclusions in
epithelial cells of intestinal impressions.

Conclusion

A judicious use of cytology will be beneficial to the practicing

veterinarians as diagnosis is made available within a short period of time and
can decide on proper treatment, prognosis and further investigative process
e.g. TVT can be successfully treated medically without surgical intervention.

Thanks are due to Dr. K.Krithiga, Dr.M.Thangapandiyan,

Dr.S.Vairamuthu, Dr.N.Jayanthi and Dr.B.Murali Manohar, TANUVAS for
their help.
 92 
 
17. PROCESSING OF TISSUE FOR
HISTOPATHOLOGICAL EXAMINATION
Collection:

Tissue for histopathological examination should be collected

immediately upon removal from the carcass as soon as after death as
possible. The portion representing the characteristics lesion should be
selected together with a healthy portion and that should not be more than
5-10 mm in thickness. After removal it should be kept in fixative, which
should be at least 10 times more than volume of the tissue. The tissue
specimen should be properly labelled and stored.

Fixation:

The foundation of all good histological preparation depends on complete

fixation. Fixation is required to-

1. Prevent post mortem changes such as putrefaction and autolysis.

2. Preserved various cell constituents in as life like manner as possible.
3. Protect by hardening the naturally soft tissue, thereby allowing easy
manipulation during subsequent processing.
4. Convert normal semi-fluid consistency of cells to an irreversible semi
solid consistency.
5. Aid visual differentiation of structure by application of biological
dyes and stains.

Choice of fixatives:

Selection of fixative depends on purpose for which the tissue is preserved.

Following are few commonly used fixatives:

1. 10% formalin: Commonest fixative widely used in the laboratories.

Concentrated formalin (37- … 100ml
40%)
Water … 900ml
2. Formalin saline solution:
Concentrated formalin (37- … 100ml
40%)
Sodium chloride … 9g
Water … 900ml
 93 
 
3. Buffered neutral formalin solution:

Concentrated formalin (37- … 100ml

40%)
Distilled Water … 900ml
Sodium phosphate … 4.0g
monobasic
Sodium phosphate dibasic … 6.5g

4. Ethyl alcohol 70-100%

It is used for preservation of glycogen. It is slow in penetration, hardens and

shrinks the tissue

5. Zenker’s solution:

Distilled Water … 1000ml

Mercuric chloride … 50.0g
Potassium dichromate … 25.0g
Sodium sulphate … 10.0g

Add 5 ml glacial acetic acid to 95ml of Zenker’s solution before use

(The solution does not keep well after the adition of acetic acid).

Tissue preserved by this method stain well with many techniques, but
it is suggested that they be post fixed in 2.5% aqueous solution of potassium
dichromate for 2 hours following Zenker’s fixation.

6. Bouin’s solution:

Picric acid, saturated … 750.0ml

aqueous solution
37-40% formalin … 250.0ml
Glacial acetic acid … 500.0ml

Fix blocks for 4-12 hours. Wash the tissue thoroughly in 50% alcohol for 4-
6hrs, agitating constantly for proper removal of the picric acid. Removal of
picric acid is essential for proper staining of the tissue section.
 94 
 
7. Carnoy’s solution:

  Absolute
  alcohol … 60.0ml
Chloroform … 30.0ml
Glacial acetic acid … 10.0ml

One of the best penetrating and quickly acting fixatives. Generally 3 hours is
adequate for normal size tissue. No washing is necessary and tissue may be
transferred to absolute alcohol + haemolyzes RBC.

8. Glutaraldehyde: 2.5 percent glutaraldehyde in 0.1 M Sodium

cacodylate buffer solution is used as fixatives for electron
microscopy.

Decalcification:

Calcium salt provides hardness and rigidity to the bone and other
tissue and that must be removed to ensure the specimen is soft for microtome
section.

Bone and other calcified tissue should be cut into small pieces before
fixation. After adequate fixation, place the tissue in a large quantity of
decalcifying solution. The tissue should be checked every day by pricking
with a pin and if decalcification is not proper the solution should be changed.
After decalcification is over the tissue should be washed in running water for
several hours to remove the traces of the decalcifying solution. Since the
decalcifying acids continue to act on tissue during subsequent processing.
One of the decalcifying solutions is

Perenyi’s fluid:

10% nitric acid, aqueous … 40.0ml

Absolute alcohol … 30.0ml
0.5% chromic acid, aqueous … 30.0ml

Processing of tissue:

A specimen brought to the laboratory is usually marked with

identifying number and name. Keep this identification with the specimen
thorough out the processing. All identifying marks should be made with a
soft lead pencil. For processing of tissue, tissue capsules are used. Tissue
 95 
 
from different animal can be processed together by keeping them separately
in different capsule with individual identification numbers.

a) Washing:
The first step of tissue processing is washing. The fixed tissue should be
washed thoroughly in running tape water at least for 6-8hrs preferably
overnight to remove the traces of fixatives. It is important, to obtain a desired
tissue section.

b) Dehydration:
It is the process of removal of all extractable water from the tissue. It
is usually accomplished by treating the tissue with ascending grades of
alcohol i.e.

50% Alcohol --- 1 hour

70% Alcohol … 1 hour
80% Alcohol … 1 hour
90% Alcohol … 1 hour
Absolute alcohol I … 1 hour
Absolute alcohol II … 1 hour
Absolute alcohol III … 1 hour

c) Clearing:
The clearing reagents miscible with dehydrant. As the dehydrant is
removed, the tissue clear, becoming translucent signifying the completion
of the process. Xylene is most wide used clearing reagent. Clearing with
xylene is done by

Absolute alcohol xylene mixture … 1 hour 

(50 parts each)
Xylene I … 1 hour
Xylene II … 1 hour
Xylene III … 30 minutes or until clear

If clearing time is not properly maintained the tissue become hard

and leading to difficulty in sectioning of it.
 96 
 
d) Impregnation:
It is complete removal of the clearing reagents by substitution with melted
paraffin. A paraffin bath at 60oC is used for this purpose with at least three
paraffin cups. Paraffin (58-60oC melting point) are allowed to melt in the
cups. The cleared tissues are immediately transferred to the paraffin cup and
impregnation in accomplished by three changes of melted paraffin of one to
two hours each.
e) Embedding:
It is the orientation of the tissue in melted paraffin, which after
solidification provides a firm medium for keeping intact all parts of the tissue
when section are cut. For this purpose, a pair of “L” mould is used. A
rectangle is made over a plain surface with the pair of “L” mould. Freshly
melted paraffin (60oC) is poured in the rectangle and the tissues are placed
immediately after removing from the paraffin bath. While placing the tissue
care must be taken so that the surface of the tissue from which sections are
required facing downward. Then it is allowed to cool down for solidification
of the paraffin. After solidification tissue blocks are ready for sectioning.

f) Sectioning:
Tissue blocks are sectioned at 4-5 micron thickness with the help of a
microtome. The blade of which should be very sharp with even edge.
The tissue blocks are fitted to the block holder at opposite side of the
surface of the tissue to be cut. Then the block holder is fixed in the
microtome and sections are made gently rotating the microtome. This tissue
sections are the put in to 50% alcohol for initial stretching. Thereafter they
are allowed to spread in tissue floatation bath, temperature of which is
maintained at 56-58oC. After proper spreading the tissue sections are
mounted in clean microslide and allowed to dry either at room temperature or
in hot air oven. Before mounting, the microslide should be smeared with a
small drop of Mayer’s egg albumin as an adhesive.
Composition of Mayer’s egg albumin
Egg white … 50ml
Glycerine … 50 ml

Mix well and filter through a coarse filter paper and add crystal of
thymol as preservative.
 97 
 
g) Staining:
A) Routine staining:
The tissue sections are stained routinely with Haematoxyline and
Eosine (H&E). Haematoxyline stains the acidic part of the cell i.e. Nucleus
(contains nucleic acid) while the eosine imparts colour to the basic part of the
cell i.e. cytoplasm. There are different types of haematoxyline e.g. Mayer’s,
Delafield’s, Harri’s, Ehrlich’s etc. Delafield’s haematoxylin is most
commonly used in the laboratory.

Preparation of Delafield’s haematoxylin:

Haematoxyline crystals … 8.0g

Alcohol 95% … 50 ml
Ammonium or potassium alum, saturated … 800ml
aqueous solution (approx. 15g/100ml)

Add the haematoxyline dissolved in alcohol to the alum solution and

expose to light and air in an unstopped bottle for 3-5 days. Filter and add

Glycerine … 200ml
Alcohol 95% … 200ml
Allow the solution to stand in the light approx. 3 days, filter and keep
in tightly stoppered bottle.

Preparation of Eosin Solution:

Stock Eosin Solution:

Eosin Y, water soluble … 1.0g

Distilled water … 20.0ml
Dissolve and add alcohol 95% … 80.0ml
Working Eosin Solution:

Eosin stock solution … 1part

Alcohol 80% … 3 parts
Just before use add 0.5ml of glacial acetic acid to each 100ml stain
 98 
 
Routine staining procedure:

The dried tissue sections are taken in a rectangular staining jar and
staining is proceeded as follows:

a) Deparaffinization: The first step is the removal of the paraffin from the
sections. This is done by immerging in xylene, 3 changes of 10 minutes
each.
b) Rehydration: This is the addition of water, done with descending grades
of alcohol, as follows
Absolute alcohol I … 2min
Absolute alcohol II … 2min
Alcohol 90% … 2min
Alcohol 80% … 2min
Alcohol 70% … 2min
Tape water … 6 minutes

c) Staining with haematoxyline: After hydration the sections are

immersed in Delafield’s haematoxyline for 15-20 minutes.

Rinse in tape water.

Differentiate in acid alcohol, 3-5 quick dips.

( Conc.HCL-1ml70% alcohol-99ml).

Wash in tape water.

Dip in ammonia water until sections are bright blue in colour(4-6 dips)

Wash in running tape water for 10-20 minutes. If washing is inadequate

eosin will not stain evenly.

d) Staining with eosin: Stain with eosin for 15 second to 2 minutes

depending on the age of the eosin and the intensity of the stain required.
e) Dehydration: It is done with 2 changes of 90% alcohol of 2 minutes
each and then 2-3 changes of absolute alcohol for 2 minutes each.
f) Clearing: Is done with 3 changes of xylene of 2 minutes each.(Slides
may remain in xylol until cover slipped.)
g) Mounting: The stained sections are then cleaned with a soft and clean
cloth and mount a cover slip with DPX.
 99 
 
h) Mounted slides are then allowed to dry and after drying it is ready for
examination.
Result: Nuclei: Purple
Cytoplasm: Pink
RBC: Red

B) Special Staining

In histopathological techniques, i.e. in tissue sections one can

demonstrate bacteria, fungus, chlamydia, rickettsia or viral inclusions by
using special staining procedure. Though it requires specific expertise it can
be done in diagnostic laboratories as a routine procedure.

i) Brown and Brenn stain for bacteria in tissue (Fig 1,2,3).

Result – gram positive - bacteria blue coloration
gram negative bacteria – red coloration
nuclei – red
other tissue element – yellows.

ii) MacCullum – Goodposture stain for bacteria in tissue.

Result – gram positive organism – blue
gram negative organism – red
other elements – various shade of red to purple.

iii) Ziehl-Nelsen stain for acid fast bacteria.

Result – acid fast bacilli – bright red
erythrocytes – yellowish orange
other elements – pale blue.

iv) Levaditis method for staining spirochetes in blocks

Result – spirochetes – intensely black
background – yellow to light brown

v) Silver method for spirochetes in sections

Result – spirochetes – black
background – yellow to brown

vi) Gridley’s stain for fungi.

Result – mycelia – deep blue
conidia – deep red to purple
background – yellow
 100 
 
vii) Grocott’s method for fungi (Fig. 5)
Result – Fungi – sharply delineated in black
mucin – deep gray
inner part of mycelia & hyphae – old rose.
background – pale green

viii) Phloxine Tolidine Blue stain for Malaria parasite.

Result – malaria parasite – pale blue cytoplasmic structures
within erythrocytes

ix) Giemsa stain for Rickettsia

Result – Rickettsia – violet
nuclei – blue

x) Schleifstein stain for Negri bodies

Result – Negri bodies – deep magenta
cytoplasm – bluish violet

xi) Haematoxylin – Shorr S3 stain for inclusion bodies (Fig.4)

Result – inclusion bodies – brilliant red.

The detail procedure for these special stains one can refer any book
on manual of histologic and special staining techniques.
 101 
 

 
Fig 1: Mixed infection of Candida & E. Fig 2: Streptococcal colonies. Brown &
coli. Brown & Brenn X400  Brenn X400 

 
Fig 3: Bacilli in Necrobacillosis. Liver Fig 4:Plant material and Blantadium coli in
.Brown & Brenn X400  bronchi H &E X400 

 
Fig 5: Mycelia of Aspergillus with Fig 6: Mycelia of Aspergillus sp. PAS x400 
conidiopore with in bronchi. Grocotts
X100.Coniodiospore in inset. Grocotts
X1000 
 102 
18.  STANDARD SURVEY FORMATS
Survey on Cattle herd & Health Management
Sl No. ___ Name of Village ________________________ ___________Tehsil____________________ 
Dist.______________State_____________________ GPS location :    Longitude _________________  
GPS location :    Longitude ______________________   and    Latitude     _______________________ 
Name of Veterinarian / AHW________________________ Doc Ph number_____________________ 
Date form completed:     /       /        
Number herd/ Farm/Unit (Frequency %)
Variables Semi-commercial Backyard Free grazing
Farms (n= ) Farms (n= ) Farms (n= )
Farm Attributes
Dry Plane
Wet land
Hills
Forest
Production system
Milk purpose
Draught purpose
Breeding Farm
Animal raising
Indoor
Partially Outdoor
Scavenging
Tethering
Pen type
Breeds of cattle
Local
Exotic
Cross-breed
Origin of stock
Own Farm bred
Live market
Breeder - other farm
Breeding
Natural service
Artificial insemination
Total Herd size / Categories
0—6 months
6 Month—2 Years
2 Years—6 Years
Above 6 Years
Bull
Feeds
Commercial
Vegetable /Grass/leaves
Market waste
 103 
 
Cattle Health & Farm Hygiene

Number herd/ Farm/Unit (Frequency %)

Variables
Semi-commercial Farms Backyard Farms (n= Free grazing Farms
(n= ) ) (n= )
According to you which are the common vaccines used
BQ
FMD
HS
Anthrax
Brucellosis
Regular deworming
According to you which are the top ten infectious diseases in your locality
CBPP
FMD
BQ
Pneumonia
Diarrhoea
Abortion/still birth
Nervine symptoms
Impaction
Mastitis
Hump sore/ skin disease
Babesiosis
Plant toxicity
Ectoparasite, vector prevalent
Any other diseases
According to you, which of these actions farmers in your area do when they notice a contagious disease
Quarantine of new cattle introduced
Isolation Sick animals
Treatment even healthy to sick
animals
Sale of animals without signs
Sale of diseased animals only
Slaughtering diseased animals
Slaughtering all animals
Share bullock in agriculture
Burying of carcass in the farm
Cleansing and disinfection of shed
Continue selling milk
 104 
 
Goat Health & Farm Hygiene

Number herd/ Farm/Unit (Frequency %)

Variables Semi-commercial Backyard Farms Free ghrazing
Farms (n= ) (n= ) Farms (n= ) 
Farm Attributes
Dry land/ Plain  
Wet land  
Hills  
Forest  
Production system  
Meat purpose  
Dual purpose    
Breeding Farm  
Animal raising  
Indoor  
Partially Outdoor
Scavenging
Tethering  
Pen type    
Breeds of Goats
Local  
Exotic
Cross-breed
Origin of stock
Own Farm bred  
Live market
Breeder - other farm  
Breeding
Natural service  
Artificial insemination
Total Herd size / Categories
0—3 months
3 Month—1 Year
1 Year—2 Years
Above 2 Years
Buck
Feeds
Commercial  
Vegetable /Grass/leaves  
Market waste  
 105 
 
Goat Health & Farm Hygiene

Number herd/ Farm/Unit (Frequency %)

Variables Semi-commercial Backyard Farms Free grazing
Farms (n= ) (n= ) Farms (n= )
According to you which are the common vaccines used
PPR
FMD
HS
Anthrax
Enterotoxaemia
Regular deworming
According to you which are the top ten infectious diseases in your locality
PPR
FMD
Enterotoxaemia
Pneumonia
Diarrhoea
Abortion/still birth
Nervine symptoms
Coccidiosis
Enteric Parasites
Mange
Anaemia
Ectoparasite, vector prevalent
According to you, which of these actions farmers in your area do when they notice a contagious
Quarantine of newly introduced goats
d Sick
Isolation d animals
Treat sick animals
Sale healthy animals without signs
Sale of diseased animals only
Slaughters diseased animals
Slaughters all animals
Consumption of dead/slaughtered
animals
Burying of carcass in the farm
Cleansing and disinfection of pen

Owner sales meat

 106 
 
Pig Herd & Management

Number herd/ Farm/Unit (Frequency %)

Variables Semi-commercial Backyard Farms Scavenging
Farms (n= ) (n= )  Farms (n= ) 
Farm Attributes
Dry/Plain  
Wet land  
Hills  
Forest  
Production system  
All – in- all out  
Continuous cycle    
Breeding Farm  
Animal raising  
Indoor  
Partially Outdoor
Scavenging
Tethering  
Pen type    
Breeds of Pigs
Local  
Exotic
Cross-breed
Origin of stock
Own Farm bred  
Live market
Breeder - other farm  
Breeding
Natural service  
Artificial insemination  
Total Herd size / Categories
0—3 months
3 Month—1 Year
1 Year—2 Years
Above 2 Years
Boar
Feeds
Commercial  
Industrial and agricultural by
products (e.g Rice Beer)  
Hotel/ house hold waste  
 107 
 
Pig Health & Farm Hygiene

Number herd/ Farm/Unit (Frequency %)

Variables
Semi-commercial Backyard Scavenging
Farms (n= ) Farms (n= ) Farms (n= )
According to you which are the common vaccines used
Swine fever
FMD
HS
Anthrax

Regular deworming

Others
According to you which are the top ten infectious diseases in your locality
Swine fever
FMD
Swine pox
Pneumonia
Diarrhoea
Abortion/still birth
Nervine symptoms
Coccidiosis
Enteric Parasites
Skin infection
Anaemia
Ectoparasite, vector prevalent
According to you, which of these actions farmers in your area do when they notice a contagious disease
Quarantine newly introduced pigs
d Sick
Isolation d pigs
Treat sick animals
Sale healthy animals without signs
Sale of diseased animals only
Slaughtes diseased animals

Slaughters all animals

Consumption of dead/slaughtered animals

Burying of carcass in the farm

Cleansing and disinfection of pig sty

Owner sales pig meat/pork

 108 
 
Poultry Flocck & Manageement

Number Farm/Unit (Frequ

uency %)
V
Variables Semi-coommercial Backyard Free graziing
Farmss (n= ) Unit (n= )  Unit (n= ) 
Farm
m Attributes
Dry Plain
D  
W land
Wet  
Hills  
Forest  
Produuction system  
Broiler  
Layer    
D type
Dual    
Breeeding Farm  
Raissing of birds  
I
Intensive  
Seemi-intensive
F ranging
Free
Cage type  
Breed
ds of Poultry
Local  
Exotic
Cross-breed
Origgin of stock
Ownn Farm bred  
Liive market
Breedeer - other farm  
B
Breeding
N
Natural service  
Artifiicial inseminationn
Total Flock
k size / Categoriies
Lesss than 1 week
1 wk-2wks
2 wks – 3 wks
3 wks- 4 wks
A
Above 4 wks
Spent bird
Feeds
Coommercial  
Hou
use hold waste  
 109 
 
Poultry Flocck & Farm Hygiene
H   

Number Farm/Unit (Frrequency %)

Variables Seemi-commercia
al Backyardd Free grazzing
Farms (n= ) Unit (n= ) Units (n=
= )
According to you which are the
t common vacccines used
RD
IBD
FP
Mareks
ILT
IB
According to you
y which are th
he top ten infectiious diseases in your
y locality
RD
AI
Coryza
DP
Pox
Coccidia
Nervine symptoms
Coccidiosis
Fungal infectiion
Mange
Deficiency
Ectoparasite, vector prevalent
According to you,
y which of theese actions farm
mers in your area
a do when they notice
n a contagioous
Quarantine off newly introduceed birds
Isolation Sick birds
Treatment of ailing
a birds
Sale of birds without
w signs
Sale of  in‐con
ntact  birds
Slaughtering o
of diseased birds and sale
Slaughtering o
of all birds on farrm
Consumption of dead/slaughteered animals
Burying of birdds /slaughtered iin farm
Cleansing and d disinfection of p
poultry shed
Owner sales birds
b in market
Visitors allowed
d on your farm
 110 
 

 
 111 
 

 
 112 
 

 
 113 
  19. Contact Details of Veterinary Disease Diagnosis
Laboratories/ Institutes
Designation Address Phone No/
Email Id
National Institutes/Laboratories
Director Indian Veterinary Research Institute +91-581-230096
(IVRI) Deemed University, dirivri@ivri.res.in
Izatnagar-243122(U.P)
Director  ICAR -National Institute 0755-2759204
of High Security Animal director.nihsad@icar.gov.in
Diseases, Anand Nagar, scd11@yahoo.in
Bhopal- 462 022 (MP), India
Director  ICAR - National Institute of 080- 2309 3100 / 2309 3110
Veterinary Epidemiology and director.nivedi@icar.gov.in 
Disease Informatics(NIVEDI)  
Ramagondanahalli, Post Box
No: 6450,Yelahanka,
Bengaluru 560064, Karnataka
State, India
Director  ICAR-National Research Centre on 01662-275787
Equines, Sirsa Road, nrcequine@nic.in 
Hisar-125 001 (Haryana) India.
Director ICAR-Veterinary Type Culture +91-1662-275787/ 278790
Collection, Sirsa Road, nrcequine@nic.in
Hisar-125 001 (Haryana) India
Joint Director Central Disease Diagnostic 0581-2302188 / 2310074
Laboratory, Centre for Animal
Disease Research and Development jdcadrad@rediffmail.com
(CADRAD) Indian Veterinary
Research Institute. Izatnagar-
243122(U.P)
North Eastern Institutes
Director  ICAR Research Complex for 0364-2570257 
NEH, Barapani, director.icar-
Umroi Road, Umiam, neh@icar.gov.in
Meghalaya. Pin –
793103,Meghalaya 
Director ICAR-National Research Centre on 0361-2847195, 2847221
Pig, Rani (Near Airport), nrconpig@rediffmail.com
Guwahati- 781 131

ICAR- National Research Centre on

Director Yak,Dirang-790101, +91-3780-242220,242387
West Kameng District, Arunachal yakdirector@gmail.com
Pradesh.
 114 
 
Director ICAR-National Research Centre on 03862 247340
Mithun, Jharnapani, Medziphema director.nrcmithun@icar.go
Dist.: Dimapur,Nagaland v.in
797106
Central Institutes

Dean College of Veterinary

  Sciences & Animal 91-389-2361748
Husbandry, Selesih, Aizawl - cvsc_dean@yahoo.co.in 
796014, Mizoram, India  dean@cvsccauaizawl.org

Dean(i/c) College of Veterinary Sciences & covcsccaujalukie@yahoo.co

AH, Jalukie-797110. Peren. Dist. m
Nagaland
State Institutes

Vice- Assam Agricultural University Jorhat 0376-2340001/ 2340013

Chancellor -785013, Assam, India vc@aau.ac.in

Director of Assam Agricultural 0361-2364941

Research(Ve University, Khanapara, drvetyaau@gmail.com
ty) Guwahati-781022   
 
Dean College of Veterinary Science, 0361-2337700
Khanapara, Guwahati-781022. dean.fvsc.aau@gmail.com
Associate Lakhimpur College of Veterinary 8011324088/7896376839
Dean Science, Saboti Rd, 9/6 Koilamari,
Assam 787051
Principal College of Veterinary Science & A.H, 0381 2391005/2391004
Agartala, Bodhjung Nagar, Agartala, cvscrknagar@gmail.com
Tripura 799008
State Department, Assam

Director Directorate of AH & Veterinary 0361-2668609

  Chenikuthi, Guwahati, assamvety@gmail.com 
Assam-781003
Deputy North Eastern Regional Disease 0361-2334177
Director Diagnostic Laboratory nerddlguwahati@gmail.co
Animal Husbandry & Veterinary m
Department
Khanapara, Guwahati-781022
 115 
 
Disease North Eastern Regional Disease 0361-2334177
Investigation Diagnostic Laboratory nerddlguwahati@gmail.co
Officer Animal Husbandry & Veterinary m
(DIO) Department
Khanapara, Guwahati-781022

Director Directorate Of Forensic Science 0361 238 1385

Kahilipara, Guwahati, Assam 781019

State Department, Meghalaya

Director Directorate of Animal Husbandry 91-364-2548388

  and Veterinary, Govt. of Meghalaya, ahvt-meg@nic.in/
  Lumdiengjri, ahvtmeg@nic.in 
  Shillong – 793002, East Khasi Hills  
  District,Meghalaya  
   
Disease State Disease Diagnostic Laboratory, dioahvety@bsnl.in 
Investigation East Khasi Hills District, Shillong
Officer
(DIO)
State Department, Tripura

Director  Animal Resource Development 0381-2323611 

Department, Prani ardd.tripura@gmail.com 
Sampad Bikash Bhawan,
Pandit Nehru Complex
Gurkhabusti, P.o. –Kunjaban, PIN-
799006, West Tripura.

Disease State Disease Investigation. 0381-232263

Investigation Laboratory, Abhoynagar,
Officer West Tripura.
(DIO)
State Department, Nagaland
Director  Directorate of Veterinary and Animal 0370 – 2221320  
Husbandry  
Kohima-797001
Nagaland.
Disease Disease Investigation. Laboratory, 9862997615/9774292842
Investigation Directorate of Veterinary and Animal kedultu@yahoo.co.in
Officer Husbandry,Kohima-797001,
(DIO) Nagaland.
 116 
 
State Department, Manipur
Director  Veterinary & A.H. Services, Manipur, 91-385-2450224
Sanjenthong  
Imphal East-795001
Manipur 
Disease Ibotombi7@gmail.com
Investigation Disease Investigation. Laboratory, 09436890396
Officer Directorate of Vety.AH Services
(DIO) Manipur, Imphal - Pin No. 795001.

State Department, Arunachal Pradesh

Director  Directorate of (0360)2257576

AH & Veterinary Department. directorahvarunachal2016@
Govt. of Arunachal Pradesh. gmail.com 
 
 
Disease Disease Investigation. Laboratory, 08729891694
Investigation Directorate of AH & Veterinary yangtapir@gmail.com
Officer Department, Nirjuli, Papumpare,
(DIO) Arunachal Pradesh.
State Department, Mizoram

Director Directorate of AH & Vety. 0389-2333647

Government of Mizoram
Aizawl, Mizoram – 796001

Disease Disease Investigation Laboratory 0389-2333647

Investigation Directorate of AH & Vety.
Officer Government of Mizoram
(DIO) Aizawl, Mizoram – 796001

State Department, Sikkim

Director Department Of Animal Husbandry 09434110002

Livestock, Fisheries Services
Government Of Sikkim
Krishi Bhawan, Tadong
Disease Dy. Director,DIC, Gangtok zeruiah02@yahoo.co.in
Investigation
Officer
(DIO)
 117 
 
20. CONTACT DETAILS OF COMPANIES/SUPPLIERS
OF LABORATORY ITEMS AND REAGENTS

Items Company Phone No/ Email

Tarson Products Pvt.Ltd. 011 43542761
info@tarsons.in
Axygen Scientific Pvt. Ltd. 91-11-45501541/
Plastic wares 45501542/
25534161/2553462
Genexy Scientific Pvt. Ltd 01792-222 291
sales@genaxy.com
Borosil Glasswares Ltd. 91 (033) 2229 9166 /
2249 5574
calcutta@borosil.com
Glasswares
Riviera Glass Pvt. Ltd. +91-22-28475228
+91-22-28473297
HiMedia Laboratories Pvt. Ltd. 91-22-6147 1919,
2500 3747, 2500 0970,
2500 0278
Media (Chemicals) info@himedialabs.com
Merck Pvt. Ltd. +91-22-6210 9000
Sisco Research Laboratories +91-22-4268 5800
Pvt.Ltd
Genetix Biotech Asia Pvt. Ltd. 011 4502 7000
Bangalore Genei Pvt. Ltd. 098452 64202
ELISA Plate Nunc (Thermo Fisher
Scientific)
D Vacutainer Becton Dickinson Phone No. 0361-
2545282/2631752

Liquid Handling Eppendorf +91 44 66 312 222

Pipette info@eppendorf.co.in
Finpipette (Thermo Fisher
Scientific Ltd.)
Nichipet (Axygen Scientific) 91-11-45501541/
45501542/
25534161/2553462
Tarson Products Pvt.Ltd. 011 43542761
info@tarsons.in