Table of Contents

Preface

iv

Laboratory Safety Procedures

Glassware and Disposal of Waste

vi

Cultivation of Viruses in Chicken Embryos

viii

I. Chicken Embryo Inoculation

Chorioallantoic Sac (CAS) Route

Yolk Sac (YS) Route

Chorioallantoic Membrane (CAM) Artificial Air Sac Route

Chorioallantoic Membrane (CAM) Top Route

II. Preparation of Cell Culture

Chicken Embryo Fibroblast (CEF)

Chicken Embryo Fibroblast-Secondary Cells from Roller Bottles

Chicken Embryo Kidney Cells (CEKC)

Chicken Embryo Liver Cells (CELiC)

Chicken Embryo Tracheal Rings

Chicken Embryo Chorioallantoic Membrane Cells (CAM)

10

III. Cell Culture Techniques

Cell Counting

11

Inoculating Preformed Monolayers

17

Simultaneous Inoculation

17

Monolayer Plaque Assay

18

Modified Agar Overlay Method

20

Preservation and Storage of Chicken Embryo Fibroblast Cells

23

Recovery of Fibroblast Cells from Preservation and Storage

22

Plating of C/E Fibroblast Cells in 24-well Plates from Preservation & Storage

23

Working Solution for ALV-J Propagation in C/E Fibroblasts

24

Coverslip Preparation for FA or H&E Staining

25

Staining CKC Monolayers in Microtiter Dishes

26

May-Grunwald Giemsa Stain in Tissue Culture

27

Hematoxylin and Eosin Staining

28

IV. Hemagglutination Tests

Collection and Preparation of Chicken Red Blood Cells (RBC's)

29

Concentration of Chicken Red Blood Cells for Lab Use

29

Rapid Plate Hemagglutination (HA) Test

30

Quantitative Micro Hemagglutination Test (HA)

31

Hemagglutination Inhibition (HI) Test for Newcastle Disease Virus (NDV)

32

HI Test with Systems Requiring 8 Units of Antigen

33

Preparation of EDS Antigen for HI Tests

34

Preparation of NDV Antigen for HI Tests

35

Rapid-plate hemagglutination assay for IBV

37

Preparation of Infectious Bronchitis Antigen for HI Tests

37

Antigen Production For The Hemagglutination

38

V. Neutralization Techniques
Virus Neutralization Test in Embryos

39

Plaque Reduction Test (Alpha Procedure)

40

Neutralization Test (Beta Procedure)

41

Microneutralization Test for Infectious Bursal Disease Virus

42

VI. Titration Techniques

NDV Vaccine Titration

43

Supplemental Assay Method for Titration of Herpesvirus of Turkeys

(Strain FC-126) or Chicken Herpesvirus (Strain SB-1)

44

Titration of the Herpesvirus of Turkeys (HVT) Vaccine

50

ii

VII. Miscellaneous Virology Techniques

Ether or Chloroform Sensitivity

52

5'-IODO Deoxyuridine (IUDR) Inhibition Test

53

Immunodiffusion Test

54

Protocol for Preparation of a Conjugate

55

Direct Fluorescent Antibody Test (FA)

58

Sample Preparation for Virus Isolation

59

Virus Concentration (For Electron Microscopy Examination)

60

VIII. Enzyme-Linked Immunosorbent Assay (ELISA)

61

IX. Avian Mycoplasma Laboratory Techniques

73

Procedures for Isolation and Identification

74

Frey's Medium

75

PPLO Broth

76

Characteristics of Avian Mycoplasma Species

77

FA for Rapid Identification of Avian Mycoplasmas

78

Mycoplasma HA Test

79

Mycoplasma HI Test

80

Mycoplasma HI Antigen Control

82

Mycoplasma Antigen Production

84

Preparation of Hyperimmune Serum

85

X.

Molecular Techniques

88

Polymerase Chain Reaction

89

Polymerase Chain Reaction (Figure 1)

91

Electrophoresis

92

Restriction Endonucleases (Figure)

94

RNA Isolation (Trizol Method)

95

IBDV Trizol Extraction

95

RT-PCR

97

Fail Safe PCR

98

iii

XI. Appendix

99

F-10/M199 Medium

100

Tracheal Ring Medium

100

Alsevers Solution

101

Tryptose Phosphate Broth (TPB)

101

0.2% EDTA (10X Stock) in PBS

101

PBS with 0.02% EDTA

101

Trypan Blue

101

Trypsin Solution

102

PBS without Calcium or Magnesium

102

HBSS with Antibiotics

102

HI Buffer (20X Stock)

102

Crystal Violet Solution

103

Antibiotics

103

Erythrosin B Stain

104

iv

The primary objective of the Avian Virus Diseases Laboratory Manual has been to
serve as a guide for students taking the Avian Virus Diseases (AVMD 8050) graduate course.
The techniques and procedures described are those used in the virology section of the Poultry
Diagnostic and Research Center of the University of Georgia.
Although numerous techniques have been described to detect the presence of avian
viruses, basic procedures for virus isolation still involve the use of chicken embryos, primary
chicken cells, organ culture and chickens. This manual attempts to describe the different
steps needed to succeed in the isolation and identification of avian viruses.
This manual has been kept updated thanks to the contribution and criticism of graduate
students and technicians.
Important information regarding standard requirements for avian vaccines can be
accessed in the United States Code of Federal Regulations (Title 9, Part 113) at the following
Internet address:
http://www.access.gpo.gov/nara/cfr/cfr-table-search.html
2006

LABORATORY SAFETY PROCEDURES

Microbiological laboratories have certain inherent dangers and hazards which all should
learn and appreciate. The laboratory can be a safe area to learn and work, depending upon the
practices and attitudes of each individual.
1. Give strict attention to all instructions and if not clear, check with your instructor or the
technical staff before undertaking any experiment.
2. Notice the location, purpose, and use of emergency safety equipment in the lab.
3. Wash your hands before leaving the laboratory, even before using the bathroom.
4. Do not eat in the laboratory. Never place pencils, pens, or any other object in your mouth
while in the laboratory.
5. Never take viruses out of the lab, including vaccine strains, without permission.
6. Notify the technical staff or the instructor immediately of all spills or broken tubes, etc.
7. Seek immediate aid from the technical staff or instructor should you hurt yourself.
Report all accidents no matter how small.
8. When using gas burners, be careful not to ignite your clothes, your lab partner's hair, the
lab, or anything else that will burn.
9. Bring only necessary items to lab with you (no one likes fomites).
10. Wear a lab coat or lab apron while working in the lab. Both will be provided. When
you leave the lab, leave your lab coat behind.
11. Clean your laboratory area with the disinfectant provided (in spray bottles) before and
after use.
12. Use proper sterile techniques when working in the lab. Consider all viruses as potential
pathogens, including attenuated strains.
13. Use the hoods whenever you work with "live" viruses. Do not look at the UV light or
work under the hood if the light is on. Switch the light from UV to fluorescent and turn the blower
on.
14. Do not mouth pipet. Pipetting devices are available.

vi

GLASSWARE AND DISPOSAL OF WASTES

1. Uncontaminated glassware should be rinsed and placed in the plastic tray on the cart by
the sink next to the emergency eyewash station.
2. Contaminated glassware or other equipment should be put in the stainless steel tray on
the same cart for autoclaving before cleaning.
3. Disposable equipment and biological wastes should be put into the plastic bags provided,
taped shut, and then put into the autoclave bag for ultimate disposal.
4. Put the needles and barrels of syringes with the device provided. Contaminated syringes
go into the autoclave bag.
5. There is a container in the lab and under each hood for broken glass. Pasteur pipets also
go into these containers, not the garbage.
6. Serological pipets should be put into the disinfectant buckets under the hoods.
7. There is a bucket of disinfectant for disposing of contaminated supernates on the sink by
the eyewash station.
MAM students and researchers:
In the past MAM students have made some field trips in the afternoon after AVMD 8050
lab. On those days when we work with virulent viruses it would be best not to make such trips (or
to work with research animals; virulent viruses can kill your research project, even cross
contamination with vaccine strains can provide you with confounding results at the end of a trial).
However, this is not always practical. So, it is strongly recommended that on these days you
shower and change clothes before going. On those days when we work with attenuated viruses
MAM students should be safe if you follow the aforementioned safety rules (like wearing a lab coat
or apron, using the hoods, and washing your hands).

vii

The next 4 pages are taken from "A Laboratory Guide in Virology" by
Charles H. Cunningham. 7th. ed. Burgess Publishing Co., Minneapolis,
Minnesota, 1973.
CULTIVATION OF VIRUSES IN CHICKEN EMBRYOS
The avian embryo, especially the chicken embryo, is a valuable and widely used medium for
the initial isolation and subsequent passage of many viruses for stock cultures and the production of
vaccines. Chicken embryos are used almost exclusively because of their (1) availability, (2)
economy, (3) convenient size, (4) relative freedom from latent infection and extraneous
contamination, and (5) lack of production of antibodies against the viral inoculum. Eggs only from
healthy, disease-free flocks should be used. It is desirable to have one source of supply for reasons
of uniformity of production and management of the breeder flock.
Commercial egg incubators are recommended. Preliminary incubation may be at 100.4102.2 F (38.0-39.0 C) with incubation of inoculated embryos at 98.9-99.5 F (37.1-37.5 C).
Incubation may also be at 98.9-99.5 F throughout the entire period. Lower temperatures may be
required under certain circumstances.
AVIAN EMBRYOLOGY
Knowledge of the development and physiology of the avian embryo is necessary for
adequate utilization of this medium for cultivation of viruses. The embryo commences
development as a sheet of cells overlying the upper pole of the yolk. The embryo is recognized
only with difficulty during the first few days, but at 4- or 5-days incubation it may be readily
detected by candling. Occasional swallowing movements are made from the 9th day onward. From
the 10th day the embryo rapidly increases in size and feathers appear. Development of the
respiratory tract occurs between the 12th and 15th day. As the embryo increases in size, there is an
accompanying decrease in the volume of the extraembryonic fluids. At the time of hatching there is
no free fluid in any of the extraembryonic cavities. Throughout incubation there is a steady loss of
water by transpiration through the shell.
The amnion and chorion arise by a process of folding and overgrowth of the somatopleure.
The amnion develops first over the head and then the caudal region. By fusion of the lateral folds,
the amnion completely envelops the embryo, except for the yolk sac, from the 5th day of
incubation. From the 6th to 13th days there is an average of about 1 ml of amnionic fluid. By the
10th day, the chorion almost completely surrounds the entire egg contents and is in immediate
contact with the shell membrane.
The allantois appears on the 3rd day as a diverticulum from the ventral wall of the hind gut
into the extraembryonic cavity and rapidly enlarges up to the 11th or 13th day. During the process
of enlargement, the outer layer of the allantois fuses with the outer layer of the amnion and the

inner layer of the chorion to form the allantoic cavity. The amount of allantoic fluid varies
from about 1 ml on the 6th day to possibly 6 to 10 ml on the 13th day. The fused chorion and
allantois is known as the chorioallantoic membrane which is highly vascular and constitutes the
respiratory organ of the embryo.

viii

In the early stages of development, the amnionic and allantoic fluids are essentially solutions
of physiologic salts. After about the 12th day, the protein content and viscosity of the amnionic
fluid increases. The allantoic cavity receives the output of the kidneys, and after the 12th or 13th
day the allantoic fluid becomes turbid because of the presence of urates. The allantoic fluid is
slightly alkaline during the 7th to 12th days but toward the end of incubation the fluid may be at pH
6.
The yolk sac consists of a steadily enlarging sheet of cells. From the 12th day on, the yolk material
becomes progressively drier and the yolk sac more fragile. During the last 24 to 48 hours of incubation
the yolk sac is drawn into the abdominal cavity.
ROUTES OF INOCULATION AND COLLECTION OF
SPECIMENS FROM CHICKEN EMBRYOS
The various procedures outlined herein for inoculation of chicken embryos and for
collection of specimens are a compilation of methods found workable in the laboratory. Certain
modifications of these procedures are required for mass production of viral vaccines to minimize
operational expenses by reducing as much as possible individual handling of eggs.
Some of the factors influencing the growth of viruses in chicken embryos are (1) age of the
embryo, (2) route of inoculation, (3) concentration of virus and volume of inoculum, (4)
temperature of incubation, and (5) time of incubation following inoculation. The presence of
maternal antibodies in the yolk of hens immunized against or recovered from certain viral
infections, among which are Newcastle disease and avian infectious bronchitis, precludes the use of
the yolk sac route for initial isolation and subsequent passage of these viruses.

ix

ROUTES OF INOCULATION OF CHICKEN EMBRYOS

Allantoic Cavity
METHOD 1 Figure 2
1. Candle the egg and select an area of the chorioallantoic membrane distant from the
embryo and amnionic cavity and free of large blood vessels about 3 mm below the base
of the air cell. In this area make a pencil mark at the point of inoculation.
2. Make a similar mark at the upper extremity of the shell over the air cell.
3. Drill a small hole through the shell at each mark but do not pierce the shell membrane.
4. Apply tincture of metaphen or another suitable disinfectant to the holes and allow to dry.

ALLANTOIC CAVITY inoculation employs embryos of 9- to 12-days incubation. The

inoculum is generally 0.1-0.2 cc. Some of the viruses which grow well in the allantoic entoderm
are those of fowl plague, Newcastle disease, infectious bronchitis, influenza, mumps, and Eastern,
Western, and Venezuelan encephalitis. This route has the advantage of simplicity of inoculation
and collection of specimens when large quantities of virus-infected fluid are to be obtained for use
in chemical analysis, vaccine production, and preparation of antigen for serologic tests.

For AMNIONIC CAVITY inoculation, embryos from 7- to 15- days incubation, inoculum
01.-0.2 cc, may be used. The age chosen is largely determined by the virus used or the study to be
undertaken. Slow-growing viruses are benefited by the longer incubation period. The inner
epithelial lining of the amnion and the epidermal epithelium of the embryo are exposed to infection.
Swallowing and respiratory movements of older embryos further serve to bring the infectious agent
into contact with the mucous membranes of the upper respiratory and gastrointestinal tracts. This
route is particularly effective for primary isolation of influenza and mumps viruses from throat
washings.
CHORIOALLANTOIC MEMBRANE inoculation employs 10- to 12-day-old embryos and
inoculum of 0.1-0.5 cc. This route is particularly effective for primary isolation and cultivation of
the viruses of vaccinia, variola, fowl pox, laryngotracheitis of chickens, and pseudorabies which
produce easily visible foci or "pocks." The chorioallantoic membrane is a suitable site for study of
the development of pathologic alterations and inclusion bodies, and titration of viruses by the pockcounting technic.
YOLK SAC inoculation is performed with 5- to 8-day-old embryos and inoculum of 0.2-1.0
cc. This route may be used for initial isolation of mumps virus.
INTRAVENOUS inoculation does not have wide practical application for study of
experimental infections of the avian embryo. The procedure is generally employed for hematologic
studies. Embryos of 10- to 15-days incubation are most suitable for this route. The amount of
inoculum may vary from 0.02 to 0.05 cc.
INTRACEREBRAL inoculation can be performed with 8- to 14-day-old embryos and
inoculum of 0.01-0.02 cc. This route may be employed in studies of pathologic alterations of the
brain following infection. The viruses of herpes simplex and rabies may be cultivated by this route.
Embryos are incubated after inoculation for a period appropriate for the virus employed and
they are examined at least once daily. Death of the embryo within the first 24 hours after
inoculation is generally considered to be due to nonspecific causes such as trauma. Some viruses
kill all embryos and mortality is the criterion of infection. Newcastle disease virus is an example in
which embryos are killed in two to four days depending upon the strain of the virus. With some
viruses such as influenza virus the mortality rate varies on initial passage but may increase with
subsequent passage. The criterion of infection with herpes and pox viruses is the formation of pock
lesions on the chorioallantoic membrane. Other gross pathologic manifestations of infection of the
embryo may be curling and dwarfing of the embryo, fibrosis of the amnionic membrane, edema of
the chorioallantoic membrane, and urates in the kidney and mesonephros such as produced by avian
coronaviruses on initial and low passage in the embryo. Various types of cytologic changes,
including inclusion bodies with certain viruses, may be detected by microscopy.
The embryo should be examined soon after death so that postmortem changes do not
obscure any specific pathologic alterations. Chilling of the embryos for several hours or for
overnight before collection of extraembryonic fluids is recommended to reduce hemorrhage into the
fluids.
Replication of a virus in embryos may be determined by several methods such as
(1) sampling of the virus in the extraembryonic fluids and membranes or in the embryo proper for
quantitative assay of infectivity, (2) pathologic alterations, (3) serologic tests, (4) hemagglutination,
(5) antigenicity, and (6) immunogenicity.

xi

CHICKEN EMBRYO INOCULATION ROUTES

CHORIOALLANTOIC SAC (CAS) ROUTE

1.

Embryos of 9 to 11 days of age.

2.

Candle the embryos for viability. Mark an area on the side of the egg about 1/8 inch
below the air cell in the chorioallantoic membrane that is unoccupied by blood vessels.

3.

Disinfect using Bioguard, punch a hole directly in the top of the air cell (optional).

4.

With egg puncher, make a hole where you marked. (Using sterile technique.)

5.

Use a 25-gauge needle, 7/8 in. length. Insert the needle at a 45 degree angle into the
allantoic cavity about 1/8 in. in depth and inoculate.

6.

Use Elmer's glue to close holes.

This route of inoculation is used mainly to isolate Newcastle disease, infectious bronchitis and
adenovirus.

YOLK SAC (YS) ROUTE

1.

Embryos of 5 to 6 days of age.

2.

Rotate the egg until blood vessels can be seen close to the margin of the air cell. These
vessels may appear as nothing more than an array of faint lines, orange in color, extending
from a clear halo. The embryo is within the area of the halo.

3.

With an egg punch, make a hole in the top of the shell.

4.

Use a 25-27 gauge, 1 1/2-in. length needle. Insert the needle straight down into the yolk
sac until its point is one-third to one-half the depth of the egg.

This route is mainly used to isolate avian enceplomylitis.

CHORIOALLANTOIC MEMBRANE (CAM)

ARTIFICIAL AIR SAC ROUTE
1.

Embryos of 9-11 days of age.

2.

Candle embryos for viability.

3.

Mark an area about 1/4 inch below and parallel to the base of the air cell. Disinfect with
Bioguard.

4.

Drill or punch a hole at this mark being very careful not to tear the shell membrane.
Punch a hole directly at the top of the air cell.

5.

Place the embryo horizontally in the tray, with the hole facing up.

6.

Holding the embryo in the same position and using a rubber bulb, draw air out of the air
cell by placing the bulb over the hole at the top of the embryo. This negative pressure
creates the artificial air cell by pulling the CAM down.
7. Using a 25-27 gauge needle, insert it into the artificial air sac about 1/8 inch and release
the inoculum. Make sure the embryo is laying horizontally for 24 hours then return to
upright position.
This route is used mainly for fowl pox and IBDV.
CHORIOALLANTOIC MEMBRANE (CAM) TOP ROUTE

1.

Embryos of 9 to 11 days of age.

2.

Candle the embryos for viability. Disinfect with Bioguard and punch a hole directly in the
top of the air cell.

3.

Use a 26 or 28-gauge, 1/2 in. needle. Insert the needle straight down the top of the egg the
full length of the needle. Pull the needle back out about 1/4 in. and release the inoculum.

This procedure as well as the artificial air cell route (dropped CAM) are used mainly for isolation
of pox and laryngotracheitis virus. Usually, the titer will not be as high as if the dropped CAM or
artificial air sac method is used.

PREPARATION OF CHICKEN EMBRYO FIBROBLAST (CEF) CELL CULTURES

Use 9-11 day old embryos. The technique described here is for 3-5 embryos.
1.

Spray eggs with Bioguard disinfectant place in hood. Using sterile technique, open shell
and remove embryo with blunt ended curved forceps.
Place all media and typsin in 37c waterbath

2.

Place embryos in petri dish and cut off heads. Removal of limbs and viscera is optional.

3.

Transfer bodies to new petri dish or beaker containing PBS without calcium or
magnesium.
In the beaker, the bodies can be fragmented by carefully chopping them with sterile
scissors. Another procedure that can be used when large number of embryos are to be
processed is as follows:
Attach a cannula to a 35 or 50 cc syringe, remove plunger, pour tissue chunks into barrel
and force through cannula with the plunger into a 30 ml beaker. Keep the cannula and
syringe sterile and use it to draw off supernatant from above settled tissue chunks during
PBS washes.

4.

Wash with PBS 3-4 times to remove red blood cells.

5.

Pour tissue fragments into trypsinization flask containing magnetic stirring bar. Add
about 50 ml pre-warmed (37 C) trypsin solution (0.25%) or Tryple Express (Invitrogen)
to flask and put on stir plate at slow speed into 37 C incubator for 10-15 minutes. Pour
off supernatant into centrifuge tube with calf serum. Add 50 ml trypsin solution and stir
slowly in 37 C incubator for 8 minutes. (Total trypsinization time: 30-35 minutes at
37 C.) This may be repeated 1 more time for a total of 3 trypsinizations.

6.

Centrifuge 10 min. at 1000 rpm. Note the amount of pelleted cells obtained. Pour off
trypsin solution and resuspend cells in 3-5 ml MEM or F-10 (EBSS). The cells may be
counted or diluted 1:200 in F-10.

PREPARATION OF CHICKEN EMBRYO FIBROBLAST SECONDARY CELLS

FROM ROLLER BOTTLES
1.

Trypsin (See Appendix p. 103). We use a pre-made formula called tryple express from
Invitrogen.

2.

Using good sterile technique, carefully discard the medium in the roller bottle.

3.

Add 25-30 ml of the trypsin solution to the bottle. Roll the bottle for approximately two
minutes, or until the cell monolayer becomes cloudy and begins to detach.

4.

Pour the resulting cell suspension through a sterile gauze funnel into a graduated
centrifuge tube containing 5 to 10 ml cold calf serum. This tube should be swirled to mix
the contents and then placed in an ice bath.

5.

If a second trypsinization is required add approximately 10 ml of the trypsin solution to

the roller bottle and follow steps three and four above.

6.

Centrifuge the tubes at 1500 rpm for ten minutes at a 4 C.

7.

Read the packed cell volume from the centrifuge tube. Dilute the cells 0.8:200 with F-10
media (Appendix p. 101) containing 2-5% calf serum or if cell growth is desired use fetal
calf serum. 60 mm plates require 5.0 ml and 35 mm plates require 2.0 ml.

NOTE:

Secondary cells may be made from CEF. Dilute trypsin solution 1:2 with
Hank's Balanced Salt Solution (HBSS). Pour off media on CEF plates, wash
plates with 1 ml trypsin solution (for 60 mm size dish) and pour off
immediately. Add 2 ml trypsin solution to each plate and incubate in 37 C
CO2 incubator for 2- 5 minutes. Remove trypsinized cells from dish with a
pipet and put into centrifuge tube with 1 ml serum to stop reaction.
Centrifuge 10 min. at 1500 rpm. Secondary cells may be plated 1/3 as heavy
as the primary culture.

PREPARATION OF CHICKEN EMBRYO KIDNEY CELL CULTURES (CEKC)

We use 17-20 day old embryos. Kidney cells can also be prepared from day-old or older birds.
The amounts indicated here are for preparing kidney cells from 10-15 embryos.
1.

Prepare media and trypsin solution (Appendix pgs 101 and 103) and set in 37
bath.

C water

2.

Spray eggs with Bioguard disinfectant and allow to dry.

3.

Using sterile technique remove embryos with blunt ended curved forceps and put into
tray.

4.

Either "skin" the embryos (which is the easiest way to get rid of feathers), use regular
dissection methods or cut the backbone right above wing joint and separate. This exposes
the kidneys without having to touch the intestines and viscera.

5.

Remove kidneys and put into glass beaker containing phosphate buffer solution (PBS)
without calcium or magnesium (Modified PBS) or Hank's balanced salt solution (HBSS)
with antibiotics, but without calcium, or magnesium.

6.

Pour off supernatant and clean kidneys. If there are any large chunks, mince lightly with
scissors or squeeze gently with forceps. Wash 3-4 times with modified PBS or HBSS
without calcium, magnesium. Use 75-100 ml PBS total.

7.

Drain off the last wash and pour the tissue fragments into a trypsinization flask containing
a magnetic stir bar. Add 50-100 ml prewarmed (37 C) trypsin-EDTA solution.

8.

Put the flask on a stirrer base in 37

9.

When the supernatant is cloudy, shake flask, then set it down for several minutes to let the
clumps settle out. Take out 1 drop of supernatant and put it on a glass slide and observe.
If there are many single cells and small clumps (2 to 10 cells) with few very large clumps
then it is time to pour off the supernatant. Have ready a sterile graduated centrifuge tube
with 5 ml of cold heat-inactivated calf serum in it. (Set in a pan of ice.) Pour supernatant
through gauze covered funnel into this tube. (The calf serum stops the trypsin action.)
With fresh trypsin repeat process 1-2 times (10 min. ea.) more. Do not extend
trypsinization time past 1 hr. Centrifuge at 1500 RPM for 10 minutes.

C incubator and stir very slowly for 15-20 minutes.

10.

The kidney cells (and RBC's) will pellet. Note the amount of cells obtained. Pour off
trypsin solution. Do a sterility test of it and then discard it. Resuspend cells in 3-5 mls of
minimal essential medium (MEM) or Hams F-10 with Earle's balanced salt solution
(EBSS).
Add the cells to the appropriate amount of MEM (EBSS) with 10% heat-inactivated fetal
calf serum. [One ml of cell pack can be resuspended in approximately 180 ml of MEM
(EBSS)]. Cells can be counted in a hemacytometer by resuspending in a known amount
of media. Make 1:10 dilution of cells in trypan blue. You will want approximately 2.5 x
106 cells/ml of media to plate out the cells. 35 mm2 plates require 2 ml, 60 mm2 plates
require 5 ml. Do one plate first and observe after the cells are allowed to settle for a few
minutes.

The cells should form a monolayer in 1-2 days. Chicken embryo liver cells (CELIC) usually 2
days, chicken embryo fibroblasts (CEF) usually 1 day. When monolayer is formed they may be
inoculated or if it is desirable they may be inoculated simultaneously.

PROCEDURE FOR PRIMARY CHICKEN EMBRYO LIVER CELLS (CELIC)

IN TISSUE CULTURE
Use 13-15 day-old embryos.
1.

Spray eggs with Bioguard disinfectant and place media in 37c.

2.

Using sterile technique, remove embryos from eggs, open embryos to expose livers.

3.

Remove the livers with curved, blunt ended forceps and put them into a beaker containing
sterile buffer solution. Be sure to cut out the gall bladder before putting livers into the
buffer.

4.

Trim off any visible connective tissue or pieces of attached intestine. Mince tissue lightly
with scissors or forceps.

5.

Allow the liver pieces to settle to bottom of beaker. Decant and discard buffer containing
RBC's. Wash 3 times or until the buffer is clear. (Usually 100 ml of buffer is enough for
the collection and washes.)

6.

Drain off the last wash and pour the tissue fragments into a trypsinization flask, rinsing
the beaker out with the trypsin solution. Add 50 ml prewarmed (37 C) trypsin solution
to the flask which already has a magnetic stirrer bar in it.

7.

Put flask into 37

CEKC step #9.

8.

Follow CEKC procedure for remaining steps.

9.

Dilute liver cells 1:150 in F-10 or MEM.

C incubator and stir gently for 15-20 minutes. Check cells as for

CHICK EMBRYO TRACHEAL RINGS

In our laboratory we use either embryos (19-20 day-old) or 1 day-old chickens. Tracheal rings
can also be prepared from older chickens.
PROCEDURE
1.

Open shell and remove embryo cutting away the yolk sac.

2.

Cut skin until trachea is completely exposed.

3.

Carefully remove the trachea with forceps and remove all fatty tissue surrounding it.

4.

Place trachea in glass petri dish containing approximately 5 mls of Hanks Balanced Salt
Solution (HBSS).

5.

Lay tracheas on sterile filter paper and place on tissue chopper. Use sterile razor blade
and cut trachea into rings at medium speed.

6.

Place rings in a separate petri dish containing HBSS.

7.

With small forceps, place rings into multiwell plates. Rings can also be placed in
individual tubes. Cover with 0.5-1.0 ml of media. Be sure rings are immersed in solution.

8.

Place the multiwell plates at 37

9.

At the end of 24 hours, check for ciliary movement under the microscope (use either the
4X or 10X objective).

10.

Score the ciliary movement as follows:

If half the ring has movement, the ring would be assigned a 2.
If 3/4 of the ring has movement, the ring would be assigned a 3.
If the entire ring has movement, the ring would be assigned a 4.
Rings with reading lower than 2 are not used.
The rings are now ready to be inoculated. The ciliary movement should be read after 3-5
or 7 days, depending upon the virus being studied.

11.

C for 24 hours.

Tracheal rings can be used to detect the presence of infectious bronchitis virus (IBV),
Newcastle disease virus (NDV) and laryngotracheitis virus (LT). They can also be used to
run Serum Neutralization Test for IBV.
Tracheal rings can also be used to evaluate ciliary activity after challenge with field
isolate and IBV. Rings are prepared from adult birds 4 days after challenge. The ciliary
activity is evaluated as described.
REFERENCE:
Andrade, Luis F., P. Villegas, and O.J. Fletcher. Vaccination of Day-Old Broilers
against Infectious Bronchitis: Effect of Vaccine Strain and Route of Administration.
Avian Dis., Vol. 27 (1), pp. 178-187, 1983.

10

PREPARATION OF CHORIOALLANTOIC MEMBRANE (CAM) CELLS

1.

Aseptically remove the chorioallantoic membranes from 9-11 day-old chicken

embryos and put them in warm PBS without calcium and magnesium.

2.

Wash the CAM's in PBS until the supernatant is clear.

3.

Refrigerate the CAM's in PBS for 2-3 hours at 4 C. Place the CAM's in fresh
PBS and finely mince them. Allow the tissue to settle and pour off and discard
the supernatant.

4.

Resuspend the tissue in prewarmed HBSS with antibiotics containing 0.01%

hyaluronidase and 0.1% collagenase. Gently stir on a magnetic stirrer for 10
minutes at room temperature. Allow the tissue to settle and discard the
supernatant.

5.

Resuspend the tissue in prewarmed 0.2% trypsin and 0.2% EDTA in PBS, stir
for 15 minutes, allow the tissue to settle and discard the supernatant.

6.

Resuspend the tissue in prewarmed 0.2% EDTA in PBS and stir for 15 minutes.
Filter the supernatant through sterile cheesecloth into cold heat-inactivated calf
serum.

7.

Centrifuge the suspension at 1200 rpm for 5 minutes. Resuspend the pellet in
M 199 growth media containing antibiotics and 10% heat-inactivated calf sera.
Count the cells and bring them to a concentration of approximately 2.3 x 106
cells/ml. with M 199 media. For 35 mm x 10 mm plates add 3 ml of cells per
plate for a final concentration of about 6.9 x 106 cells/plate.

8.

Incubate the plates at 37 C with 5% CO2. The CAM cells should be confluent
in 48 hours. When confluent, change the media to M 199 with 1% calf serum.
The medium should be changed every two days after that.
REFERENCE:
Cursiefen, D. and H. Becht. In Vitro cultivation of cells from the
chorioallantoic membrane of chick embryos. Micro. Immunol. 161: 3-10.
1976.

11

CELL COUNTING PROCEDURE

(1)

Place the cover glass over the ruled area of a counting chamber. These have
special cover slips which allow correct depth of the chamber beneath. Do not
use ordinary cover slips for this purpose.

(2)

With sterile technique and a sterile 1.0-ml pipet, remove 0.5 ml of wellsuspended cells from the graduate cylinder and place in a small test tube.

(3)

With a fresh pipet, remove 1.0 ml of trypan blue stain (Appendix p. 102) from
its bottle, wipe the outside tip of the pipet with a Kimwipe tissue, and add the
stain to the cells in the tube.

(4)

Mix the contents of the tube thoroughly by gently aspirating with a sterile pipet.
Remove a 0.5-ml sample.

(5)

Quickly wipe the outside tip end of the pipet with a Kimwipe and place the tip
of the pipet to the edge of the cover slip on the counting chamber. Release the
pressure slightly on the mouth of the pipet, and allow the fluid to run into the
counting chamber. It may take a bit of practice to do this. You must allow the
fluid to fill only one side of the chamber. Do not add so much fluid that it flows
into the channels on each side of the counting area. It might be good practice to
try using colored water until you get the technique down to a point where you
feel comfortable doing it (Fig. 3-14).

(6)

Allow the cells to settle for 2 min. Carefully lift the chamber and place it on the
microscope stage.

(7)

With the low power objective in place, focus on the ruled area of the chamber.
The counting chamber actually has two ruled areas, one on each side of a
central trough (Fig. 3-14). One ruled area will be found to be sectioned like
this: (Figures 3-15)

12

Fig. 3-14. Filling counting chamber.

Fig. 3-15. Counting chamber grid.

Focus your attention on the four corner blocks (marked 1, 2, 3, 4, above).
Each of these square millimeter areas is divided into 16 squares. With the
cover slip in place, the volume over one large square is 0.1 c. mm.
8) You will be concerned first with finding the average number of cells
per milliliter of your concentrate.
(a) Count the cells in several squares and obtain an average number
of cells per square. You may count only the four corner squares
on both ruled areas or, to increase your accuracy, count the
center squares also.

13

EXAMPLE
Square No.
Count (No. of
cells per
square):

10

36

40

38

41

39

43

41

34

* Center square of each side not counted.

(b)

Determine the average number of cells per square. From the example above
where eight squares were counted,
39 cells per square
8)312

(c)

Adjust for the dilution. In preparing the materials for counting, you combined
0.5 ml of cell concentrate (one volume) with 1.0 ml (twice as much, or two
volumes) of stain diluent. You have, therefore, made a 1 + 2 (or I in 3, or 3X)
dilution of the concentrate. From step b above, you know you have 39 cells per
square of the stain-diluted concentrate. You must now compute how many
more cells there would have been had the material been counted undiluted. To
do this
Take the average number of cells per square:
Times the dilution of concentrate:
To get the total number of cells per 0.1 cubic
mm (millimeter) of concentrate

39
3
___
117

(d) Now, to correct this value to a count per milliliter of concentrate, you must
multiply by 10,000. The rationale is as follows:
1 ml = 1 cc (cubic centimeter).
1 cc is represented by a cube which is 1 cm (or 10 mm) on each edge.
1 cc has 10 x 10 x 10mm = 1000 cubic mm.
Our count of the average number of cells per square was based
on the volume of that square, i.e., 0.1 cubic mm.
To bring this to a 1-cm value, multiply by 10.
To bring the 1-cm value to I-mi value, multiply by 1000 or, combining the two,
multiply the average number of cells per square by 10,000. In brief, average
number of cells per square x dilution factor x 10,000 = number of cells per
milliliter of concentrate.

14

(9) Enumerate all the cells with clear-cut nuclei and surrounding cytoplasm which appear in the
white cell (four corner squares) areas:
(a) Count single cells as one cell [Fig. 3-16(a)].
(b) Count clumps in which individual nuclei and cytoplasm are easily visible as clumps
of single cells, and count each cell [Fig. 3-16(b)].
(c) When individual cells are not easily discernible as such, clumps should be counted
as a single cell [Fig. 3-16(c)].

Fig. 3-16. Criteria for cell count.

Fig. 3-17. Path of cell counting.

(d) In counting the cells, develop the technique of counting from left to right on the
first row, right to left on the second row, left to right on the third row, right to
left on the fourth row (Fig. 3-17). Make it a matter of technique to include in
the count those cells which touch the inner line on the top and right-hand side
of the first and third rows and the top and left-hand side of the second and
fourth rows (Fig. 3-18).

Fig. 3-18. Cell Count.

15

(10) Divide the total number of cells in all four corner squares by four to find the
average number of cells per square.
(11) The average number of cells per square times 10,000 (correction factor) times 3
(dilution factors) will give the cell count per milliliter, i.e., the number of cells in
each milliliter of fluid in the graduate cylinder.

J. DILUTION OF CELL SUSPENSION

Prepare several sets of tubes with cells varying in number from 150,000 per milliliter to
300,000 per milliliter. Individuals will vary in their counting technique, and that number of
cells which will produce a good cell sheet for one person will not be same for another. The type
of tissue will also determine the dilution. Cell concentration may vary between 2 and 8 x 105.
1. To determine the dilution factor (i.e., how much to dilute the concentrated cells in
order to obtain 150,000 or 300,000 cells per milliliter), divide the cell count per
milliliter determined above by the number of cells per milliliter desired.
EXAMPLE

Cells per milliliter of concentrate:

Cells per milliliter desired:
1,000,000
200,000

1,000,000
200,000

=5

This means that you must dilute the concentrated cells five times in order to

Fig. 3-19. Dilution of cell suspension.

16

obtain 200,000 cells per milliliter; i.e., you must dilute one part of cell concentrate with
four parts of diluent.
Let us say that you have 50 ml of concentrated cells which you have determined
must be diluted five times. This means you will have a final volume of 250 ml of cells
at a concentration of 200,000 cells per milliliter. However, 10 to 20% of this final
volume must be calf serum; therefore, if you use 15% serum, 15% of 250 is 37.5 ml.
2. With sterile technique, add 37.5 ml of calf serum to the graduate cylinder
containing the 50 ml of concentrated cells (Fig. 3-19).
3. Add Hanks growth medium up to the 250-ml mark. Cover with foil.
4. Rinse the cover glass and chamber in running water, wipe dry with soft tissue,
and put away.

REFERENCE:
Rovozzo C. Grace and Burke N. Carrol. A Manual of Basic Virological
Techniques. Prentice Hall Biological Techniques Series. Pp. 50-55. 1973.

17

PROCEDURE FOR INOCULATING PREFORMED MONOLAYERS

1.

Swirl plate to resuspend as many RBC's and debris as possible and then decant and
discard growth medium.

2.

Wash monolayer gently with 2-3 mls of prewarmed PBS and discard. (Optional)

3.

Add 0.1 ml sample inoculum to the small 10 x 35 mm plates or 0.2 ml for the larger size
(60 mm). Rock each plate gently to distribute inoculum evenly over the cell monolayer.

4.

Incubate inoculated cultures in 37 C incubator for 45 minutes to allow virus to adsorb.

Rock tray once or twice during incubation if possible.

5.

Add 2 ml maintenance medium to each 35 mm plate (or 5 ml for 60 mm plates).

NOTE: Maintenance media -- 1%-3% heat-inactivated calf serum.

6.

Incubate at 37
cells.

C. Check plates daily for cytopathogenic effect (CPE) and condition of

7.

To harvest samples, freeze-thaw 2-3 times and collect.

PROCEDURE FOR SIMULTANEOUS INOCULATION

Up to 12 hours after plating the cells inoculate 0.1 ml (or more) of inoculum in each plate. Do
not discard media. When monolayer has formed, the media can be discarded and maintenance
media added.

18

MONOLAYER PLAQUE ASSAY (AGAR OVERLAY METHOD)

MATERIALS
Virus suspension
Hanks' 10X medium without phenol red, sterile
Lactalbumin hydrolysate, 5.0% sterile
Calf serum, sterile (heat-inactivated)
Neutral red, 1:1000, sterile
Sodium bicarbonate (NaHCO3) 7.5%, sterile
Antibiotic mixture, (Pen/Strept and Mycostatin)
Noble agar, sterile
Distilled water, sterile
Erlenmeyer flasks, 100 and 200 ml, sterile
Cell cultures, three per dilution
Pipets, 10.0 ml and 1.0 ml, sterile
Cornwall syringe and 18-gauge needle, sterile
Bunsen burner
Flat storage trays
Water baths, 37 and 45 C.
METHOD
(1) Prepare medium A:
Hanks' 10X medium without phenol
Lactalbumin hydrolysate 5%
Calf serum
Neutral red, 1:1000
NaHCO3, 7.5%
Penicillin
Streptomycin
Mycostatin
Distilled water to bring volume to
Place in a 37

C. water bath,

10.0 ml
10.0 ml
amount desired
2.4 ml
0.7 ml
100 IU/ml
100 mcg/ml
2.5 mcg/ml
60.0 ml

19

(2) Prepare medium B:

Noble agar
Distilled water to bring volume to

1.5 g
40.0 ml

Place in a 100 ml flask and cover with foil. Autoclave at 10 lb. pressure, 10 min.
Put the flask in a 45 to 48 C water bath and allow the contents to cool to that
temperature for 1 hr (do not use a lower temperature because the agar will
solidify).
(3) In the meantime, thaw virus rapidly and make serial dilutions in Hanks' solution.
(4) Inoculate the cell cultures as follows:
(a)
(b)

Discard the supernatant using sterile technique.

Add 0.2 ml of each virus dilution to replicate plates. Manually rotate the
inoculum over the surface of the cell sheet and allow the inoculum to
remain in contact with the monolayer for 45-60 minutes at the appropriate
temperature for the virus (37 C).

(5)

At the end of the incubation period, set up your work area with the plates of
cultures, bunsen burner and water bath.

(6)

Combine Medium A and Medium B, and place the container in the 45 to 48 C

water bath. Working quickly, and with sterile technique, add the appropriate
amount of agar- media mixture to each plate, rotating gently over the cell sheet and
avoiding bubbles.

(7)
(8)

Incubate at 37

C.

Examine daily for plaques. These will appear as "holes" in the agar. Some will be
clear, others opaque or translucent. Some will have smoothly defined edges,
others, will have an irregular outline. Some will be large, others small. A
particular virus will produce a particular plaque type.

REFERENCE:
Rovozzo, G. C. and C. N. Burke. A Manual of Basic Virological Techniques. PrenticeHall, Inc. 1973.

20

MODIFIED AGAR OVERLAY METHOD

Use 15 x 60 mm petri dishes.
MEDIA:

2X Agar
Put bottle containing at least 50 ml 3.0% agar in a beaker of water over large
bunsen burner. Bring to boil and let agar melt, then put it in a 46 C water bath.
2X Medium (For 500 ml)
100 ml
10X Earle's balanced salt solution (EBSS)
400 ml
2.5% Lactalbumin Hydrolysate (LAH)
20 ml
Heat-inactivated Fetal Calf Serum
0.5 ml
Gentamycin Sulphate (Stock Solution: 50mg/ml)
0.5 ml
Mycostatin (10,000 l/ml stock)
(100 l/ml final)
Adjust to pH 7.2 with NaHCO3 (7.5% stock)
Prewarm to 46

C.

PROCEDURE:
1.

Make dilutions 10-1-10-6 of virus in 1X Hank's balance salt solution (HBSS).

2.

Discard growth medium and inoculate cell cultures with 0.2 cc diluted virus per petri dish. 3
plates/dilution.

3.

Adsorb for 45 minutes in CO2 incubator with frequent individual rocking of plates to evenly
distribute the virus over the entire cell sheet.

4.

Measure 50 ml melted agar in graduated cylinder and pour into the prewarmed 2X medium
solution.

5.

Keep flask in beaker containing 46

6.

Discard the inoculum.

7.

Quickly, but carefully so as to avoid bubbles, add 5 ml agar-medium solution to each plate.

8.

Incubate at 37

9.

After 4 days, add enough neutral red to cover agar overlay (1-2 ml with Pasteur capillary pipet sterile). Return to incubator.
(Neutral red: 2 ml stock (GIBCO) + 98 ml PBS).

10.

45 minutes later discard excess neutral red.

11.

Wait 2-3 hours and then count the plaques and determine plaque forming units (pfu)/ml.

C water to keep agar from solidifying.

C in CO2 incubator. Examine daily for plaques.

21

PRESERVATION AND STORAGE OF CHICKEN EMBRYO

FIBROBLAST CELLS
1.

From roller bottles with cells in logarithmic growth (approx. 24 hrs), add 25-30 ml of
warmed (37C) trypsin solution to the bottle. Roll the bottle for approximately two to
three minutes or until monolayer becomes cloudy and begins to detach.

2.

Pour resulting suspension into a graduated centrifuge tube containing 5-10 ml of cold
(4C) calf serum.

3.

Centrifuge tubes at 1500 rpm for 10 minutes.

4.

Carefully remove supernatant from the packed cells.

5.

Assess packed cell volume and re-suspend cells in warmed F-10/M-199 media containing
10%-15% Fetal Calf Serum.
** A routine re-suspension volume of cells for cryo-preservation is 2-4 X 106 or
20-40 ml of supplemented media to 1 ml of packed cells.**

6.

Very slowly add 7.0% B 7.5% of cell culture grade DMSO (Sigma D-2650) to the resuspended cells in supplemented media.

7.

Dispense re-suspended cells into 2.0 ml cryovials and place cryovials into a covered
styrofoam container.

8.

Place the container containing the cryovials at B20C for 2-3 hours, then at B80C
overnight (12 hours), then immediately place cryovials in liquid nitrogen for preservation.

An inexpensive and efficient controlled rate freezing apparatus can be

obtained from Biotech Research Laboratories, Inc. Rockville, MD called the
9001 BTRL Cell Freezer

22

RECOVERY OF FIBROBLAST CELLS FROM PRESERVATION AND

STORAGE
1. Remove cryovials containing the appropriate cells.
2. Immediately place the cryovials in a circulating 37 C waterbath.
3. Disinfect outside of cryovials and aseptically dispense the contents into a centrifuge tube.
4. Centrifuge tubes at 1500 rpm for 8-10 minutes.
5. Carefully remove supernatant, which contains the freezing media from the packed cells.
6. Re-suspend cells in F-10 / M-199 media supplemented with 10% Fetal Calf Serum.
**Generally, a vial containing 2-4 x 106 cells ml can be diluted at a ratio of 1:10 to
1:20.**
7. Plate suspended cells accordingly, approximately 400,000 cells ml.
**Simultaneously inoculate cells or inoculate after formation of monolayer.**
8. After 24 hours, remove media and replace with F-10 / M-199 media supplemented with
maintenance media.
9. Incubate cells at 37 C and 5% CO2 for 5-7 days.

10. Freeze and thaw plates a total of three times at 20 C.

23

PLATING OF C/E FIBROBLAST CELLS IN 24-WELL PLATES FROM

PRESERVATION AND STORAGE

1. Remove cryovials containing the appropriate cells.

2. Immediately place the cryovials in a circulating 37C waterbath.
3. Disinfect outside of cryovials and aseptically dispense the contents into a centrifuge tube.
4. Centrifuge tubes at 1500 rpm for 8-10 minutes.
5. Carefully remove supernatant, which contains the freezing media from the packed cells.
6. Re-suspend cells in F-10 / M-199 media supplemented with 1% Calf Serum DEAE Dextran
and Heparin
**Generally, a vial containing 2-4 x 106 cells ml can be diluted at a ratio of 1:10 to
1:20.**
7. Apply 35 l of each serum or plasma sample to each well.
8. Add 985 l of suspended cells into each well.
9. After 24 hours, remove media and replace with F-10 / M-199 media supplemented with 1%
Calf Serum.
10. Incubate cells for 7 9 days.
11. Freeze and thaw plates a total of three times at 20 C. For AC-ELISA add 35 l of 5%
Tween-80 to each well before freezing.

24

WORKING SOLUTIONS FOR AVIAN LEUKOSIS VIRUS SUBTYPE J (ALV-J)

PROPAGATION IN C/E FIBROBLASTS
DEAE-Dextran
Dextran
DD H2O

2 grams
100 ml

Filter to sterilize and store at 4 C.

Use 1 ul of solution per 1 ml of media.
Heparin
Heparin (10,000 units/ml)
DD H2O

1 ml
10 ml

Filter to sterilize and store at 4 C.

Use 4 l per 1 ml of media.
Tween 80
Tween 80
DD H2O
Filter to sterilize and store at 4 C.
Use 35 l per each well in a 24-well plate.

5 ml
100 ml

25

COVERSLIP PREPARATION FOR CELL CULTURE USE IN

FA OR H&E STAINING
1.

Remove coverslip from container with flat-tipped forceps.

2.

Dip coverslip in 95% ethanol. (Keep away from flame!)

3.

Flame coverslip dry (not too hot).

4.

Dip in sterile PBS and place in sterile tissue culture dish. Cells may then be added to the
dish.

26

STAINING CKC MONOLAYERS IN MICROTITER DISHES

1.

Pour the media out of the wells of the microtiter dish. If the wells contain virus, pour the
media into disinfectant solution in the hood.

2.

Wash the cells once with PBS (pH 7.2 and at room temperature). Optional.

3.

Fill the wells with 95% ethanol and allow the cells to fix for 3-5 minutes.

4.

Pour off the ethanol.

5.

Add 1% crystal violet staining solution to the wells and allow the cells to stain for 3-5
minutes.

6.

Pour off the stain and wash the dish with tap water until all excess stain has been removed
from the wells (generally 3 to 4 washes).

7.

Allow the wells to drain and dry the dish.

8.

Read the plates as follows:

VIRUS CONTROL
The wells where the virus has produced cytopathogenic effect (CPE) will be clear. The titer of
the virus will be the reciprocal of the highest dilution where there is CPE.
CELL CONTROL
The cell control should be stained dark (purple or blue) since there is no CPE.
POSITIVE SERUM CONTROL
Should appear similar to the cell control.
NEGATIVE SERUM CONTROL
Should look like the virus control.

27

MAY-GRUNWALD GIEMSA STAIN IN TISSUE CULTURE DISH

1.

Remove medium from monolayer.

2.

*Wash 2 times with warm PBS.

3.

Fix monolayer with methanol for 5 minutes.

4.

Discard Methanol and stain with undiluted May-Grunwald solution for 5 minutes. (Time
may be lengthened to 10 minutes for heavy monolayer.)

5.

Discard May-Grunwald solution and apply Giemsa stock diluted 1:10 with distilled water.
Stain for 15 minutes.

6.

Wash stained monolayer with tap water and air dry.

7.

For tissue culture dishes, add 1 drop immersion oil and spread over stained monolayer.

*NOTE:

For routine staining Step #2 can be deleted. For some studies this is an
important step since it removes all medium components rather than having
them precipitated on the monolayer during fixation.

28

HEMATOXYLIN AND EOSIN (H&E) STAINING PROCEDURE FOR

CELL MONOLAYER IN COVERSLIPS
1.

Carefully remove coverslip from tissue culture (TC) dish using small curved forceps.

2.

Fix in cold acetone 15 minutes or absolute methanol 3 minutes (Note: acetone may shrink
cells).
Transfer to PBS.

3.

Place coverslip in slide carrier and stain using the following order and time required.
TIME

SOLUTION
Hematoxylin

10-20 sec. - 1 minute till dark red

Running tap water

Until runs clear

NH4-H2O

3 dips

Running tap water

4.

Eosin

3 dips

95% Ethanol

1 dip

95% Ethanol

1 dip washes off excess stain

95% Ethanol

1 dip

Absolute Ethanol

1 dip

Xylene

1 dip

Xylene

1 dip

Xylene

1 dip

Mount coverslip on glass slide (cell side down) using permount.

29

COLLECTION AND PREPARATION OF CHICKEN RED BLOOD CELLS

(RBC'S)
1.

Use sterile syringe and needle. For chickens older than 5 weeks, it is advisable to use a 20
gauge, 1 1/2" needle.

2.

We use Alsever's solution as anticoagulant. Draw the Alsever's solution to approximately

half the total syringe volume.

3.

Using 3-5 known SPAFAS birds which have never been exposed to or vaccinated with the
virus you are testing for, draw blood to fill the syringe. Mix well but gently.

4.

Dispense into clean graduated centrifuge tube. Centrifuge at about 1,200 RPM's
(revolutions per minute) for 4 minutes. Remove and discard the supernate.

5.

Resuspend the RBC's to the original volume using HI buffer solution. Mix well and
centrifuge again. Repeat this procedure approximately 3 times.

6.

After the last washing, resuspend the RBC's in buffer solution (in our case HI buffer) to
make a 25% stock solution.

7.

Refrigerate. RBC's should be good for approximately one week.

CONCENTRATION OF THE RED BLOOD CELLS (RBC'S)
FOR USE IN THE LAB

For the hemagglutination (HA) and hemagglutination-inhibition test (HI) using the rigid styrene
or the flex vinyl "U" or "V" button microplates, we use a 0.5% concentration of RBC's. A 0.75%
concentration can also be used.
For the plate test (both for HA and HI), we use a 5% concentration of RBC's.

30

RAPID PLATE HEMAGGLUTINATION (HA) TEST

Used for the rapid detection of Newcastle disease, influenza and hemagglutinating adenovirus.
Also some strains of bronchitis virus will hemagglutinate after concentration.
1.

Start with clean white ceramic plate.

2.

Using a Pasteur capillary pipet, put 1 drop of the allautoic fluid on the plate.

3.

Put 1 drop 5% chicken red blood cells (RBC's) on each sample. Put one drop by itself for
a control.

4.

Stir each pool with a stick.

5.

Gently rock plate to swirl mixtures of samples and RBC's for about 1 minute.

6.

Positive HA: RBC's will agglutinate and give sandy appearance.

NOTE:

Use only fluid which is CLEAN (no urates, yolk material, bacteria, etc.).

31

QUANTATIVE MICRO HEMAGGLUTINATION TEST (HA)

1. Add 50 l of HI buffer to all wells of a 96 well microtiter plate (See Appendix p. 103 for HI
buffer formula).
2. Add an additional 50 l of HI buffer to COLUMN 1, ROWS D-E-F.
3. Add 50 l of virus sample to COLUMN 1, ROWS A-B-C (1:2 dilution).
4. Add 25 l of virus sample to COLUMN 1, ROWS D-E-F (1:5 dilution).
5. Mix COLUMN 1, ROWS A-B-C-D-E-F, thoroughly with an 8 channel 50 l multiwell
pipettor and dilute serially by passing 50 l of from each well for COLUMNS 1-12. Discard
the excess 50 l after the final dilution for COLUMN 12.
6. Add 50 l of 0.8% chicken red blood cells (RBC) to all wells, without touching fluid in wells.
7. The chicken RBC control will be ROWS G-H.
8. Agitate plate gently and let stand for 45 minutes at room temperature. For bronchitis incubate
at 4 C.
9. Read plate and determine titer of virus. The end point is the well with the highest dilution
where there is hemagglutination (No button).

1:2
1:2
1:2
1:5
1:5
1:5
RBC
RBC

A
B
C
D
E
F
G
H

1
O
O
O
O
O
O
O
O

2
O
O
O
O
O
O
O
O

3
O
O
O
O
O
O
O
O

4
O
O
O
O
O
O
O
O

5
O
O
O
O
O
O
O
O

6
O
O
O
O
O
O
O
O

7
O
O
O
O
O
O
O
O

8
O
O
O
O
O
O
O
O

9 10 11 12
O O O O
O O O O
O O O O
O O O O
O O O O
O O O O
O O O O
O O O O

32

HEMAGGLUTINATION INHIBITION TEST (HI)

1. Add 25 l of each individual serum samples (1-4) to COLUMN 1, ROWS A-B-C-D.
2. Add 25 l of the positive serum in COLUMN 1, ROW E and 25 l of negative serum in
COLUMN 1, ROW F.
3. Dilute antigen in HI buffer to contain 10 Hemagglutinating units (HA) in 50 l.
4. Add 50 l of antigen in all COLUMNS for ROWS A-B-C-D-E-F.
5. Add an additional 50 l of antigen (100 l total) to COLUMN 1 for ROWS A-B-C-D-E-F.
6. Add 100 l of antigen to COLUMN 1, ROW G.
7. Add 50 l of HI buffer (without antigen) to COLUMNS 2 through 12 for ROW G.
8. Add 50 l of HI buffer (without antigen) to all COLUMNS through ROW H.
9. Mix ROWS A-B-C-D-E-F-G, COLUMN 1 thoroughly with an 8 channel 50 l multiwell
pipetor and dilute serially by passing 50 l from each well for COLUMNS 1-12. Discard the
excess 50 l after the final dilution for COLUMN 12.
10. Let incubate 15-30 minutes at room temperature.
11. Add 50 l of 0.8% chicken red blood cells (RBC) to all wells.
12. The antigen control (AGC) will be ROW G and the chicken RBC control will be ROW H.
13. Agitate plate gently and let stand for 45 minutes at room temperature. (For bronchitis
incubate at 4C).
14. Read plate no later than 45 minutes, preferably when buttons are observed for the chicken
RBC control. Determine the HI titer of serum sample by observing the highest serum dilution
where a CLEAR BUTTON is observed.

S-1
S-2
S-3
S-4
+
AGC
RBC

A
B
C
D
E
F
G
H

1
O
O
O
O
O
O
O
O

2
O
O
O
O
O
O
O
O

3
O
O
O
O
O
O
O
O

4
O
O
O
O
O
O
O
O

5
O
O
O
O
O
O
O
O

6
O
O
O
O
O
O
O
O

7
O
O
O
O
O
O
O
O

8
O
O
O
O
O
O
O
O

9 10 11 12
O O O O
O O O O
O O O O
O O O O
O O O O
O O O O
O O O O
O O O O

33

HEMAGGLUTINATION INHIBITION TEST (HI) WITH SYSTEMS REQUIRING

8 UNITS OF ANTIGEN IN THE TEST
The test is routinely used in our laboratory to quantify antibodies against infectious
bronchitis and egg drop syndrome.

NOTE:

To save antigen, the test may be run using 25 l amounts.

1.

Add 50 l HI buffer to all wells.

2.

Place 50 l of serum to be tested in the first well of each row (including the 1st well,
making a 1:2 initial dilution)

3.

Dilute the serum serially by passing 50 l from each well using a 50 l multi-channel
pipette. Dilute only through well #11. (Well #12 will be a RBC control.)

4.

Prepare required volume of 8 HAU antigen by diluting in HI buffer. Add 50 l diluted

antigen per well in rows 1-11.

5.

For bronchitis, incubate serum-antigen at 4

temperature.

6.

Add 50 l of .5% CRBC to each well and incubate as above for 45-60 min.

7.

Run known positive and negative sera the same as for test samples. Run antigen control
as in NDV.

8.

Test is read when RBC control forms a button. Tilt the plate 60-70
dilution where button runs.

C for 15-20 min. For EDS, incubate at room

and read last

34

PREPARATION OF EDS ANTIGEN FOR HI TESTS

1.

Prepare CELIC. Inoculate monolayer at 24 hr. incubation. We use AP5 EDS.

2.

Incubate 45 min. at 37

3.

Add maintenance media and incubate.

4.

Observe for adenovirus-like CPE. Freeze when CPE is 75-80% of monolayer. Freezethaw 2-3 times. Collect virus in centrifuge tube. Centrifuge 30 min. at 1500 rpm to
remove cell debris.

5.

Note the amount of supernate and treat with 0.1% BPL. Add stir bar and stir at room
temperature for 4 hours, then chill overnight.

6.

Adjust pH to 7.2-7.4 with NaOH.

7.

Antigen must be titered before using.

in CO2 incubator.

35

PREPARATION OF NEWCASTLE DISEASE VIRUS (NDV) ANTIGEN

FOR HI TESTS*
1.

Inoculate 9-day-old embryonating chicken eggs via the allantoic cavity with 103 to 105
ELD50's of NDV LaSota, and incubate at 37 C.

2.

Discard embryos dead at 24 hours post-inoculation.

3.

Chill eggs when embryo mortality reaches 10 to 30%. Generally this will be around 72
hours after inoculation.

4.

Harvest and pool the allantoic fluids (AF). Fluids inadvertently contaminated with RBC
should be cleared by centrifugation.

5.

Add 0.1% beta propiolactone (BPL) (final concentration) to constantly stirred AF (around
25 C) and, after mixing is complete, transfer fluids to a sterile container. Continue
stirring for 4 hours at ambient temperature (around 25 C) and then chill overnight in
refrigerator (around 4 C).

6.

Clarify AF by centrifugation or filtration. If you centrifuge, the speed and time should
equal that used to sediment the PEG precipitate (described below). 3.900 rpm x 90'.

7.

Dissolve 10% granular PEG 8000 and 2% NaCl (wt/v) in chilled AF by continuous
stirring at 25 C and then place mixture at 4 C for 3 to 24 hours.

8.

Centrifuge the polythylenglycol, PEG-NaCl-AF mixture at 5000 rpm for 60 minutes at 4

C in a GS-3 Sorvall head, or at 3900 rpm for 90 minutes in a #850 International head.

9.

Resuspend pellet in a volume of PBS** equal to 1/10 the original volume of AF (before
PEG and NaCl).

10.

Chill the resuspended 10X virus concentrate (#9) in an ice bath and sonic treat for 2
minutes. Use 3/8 inch diameter probe and 60 watt power setting on Branson Sonifier
model W-200P, or equivalent.

11.

Dilute the sonic-treated antigen with a volume of PBS containing 25% glycerol. The
resulting concentrate contains 5X virus in 12.5% glycerine, and is the final antigen
preparation. Store at 4 C.

36

12.

The titer of the antigen may increase slightly during the first 2-3 weeks of storage,
probably as a result of aggregate dispersion. We do not hesitate to prepare a 2-year supply
and have not experienced problems of instability.

____________________
*

**

Procedure originally described by Beard, Hopkins, and Hammond in Avian Diseases 19

(1975): 692-699. Minor modifications have since been made.
Dulbecco's PBS without Ca++ and Mg++.

37

RAPID-PLATE HEMAGGLUTINATION ASSAY FOR THE DETECTION OF

INFECTIOUS BRONCHITIS VIRUS (IBV)
Used for the rapid detection of infectious bronchitis virus (IBV) in allantoic fluid 48 hours after
inoculation of SPF embryonated eggs with the suspected IBV infected sample.

1. Mix 0.25 ml of allantoic fluid from SPF chicken embryos, obtained 48 hours after
inoculation, with 25 ul of neuraminidase (type V from Clostridium perfringens) at a
concentration of 2 U/ml.
2. Incubate the neuraminidase-treated allantoic fluid at 37 C for 30 minutes.
3. Refrigerate the neuraminidase-treated allantoic fluid at 4 C for 5 minutes
4. Mix 50 l of the neuraminidase-treated allantoic fluid with 50 l of a 5% solution of chicken
red blood cells (CRBCs) on a clean ceramic plate
5. Direct hemagglutination of CRBCs will be observed within 1 minute with a sandy
appearance.

Reference: Ruano, M., J. El-Attrache, and P. Villegas. A Rapid-Plate Hemagglutination

Assay for the Detection of Infectious Bronchitis virus. Avian Dis. 44:99-104. 2000.

38

ANTIGEN PRODUCTION FOR THE HEMAGGLUTINATION

INHIBITION (HI) TEST IN INFECTIOUS BRONCHITIS
1.

Dilute the strain of infectious bronchitis virus (IBV) appropriately according to its titer in
HBSS (Hanks Balanced Salt Solution).

2.

Inoculate 9-11 day chick embryos with 0.1 ml diluted virus.

3.

Incubate inoculated embryos, candle and discard dead at 24 hours.

4.

Remove embryos from incubator 30-48 hours post-inoculation and place in refrigerator
(4 C) overnight or a minimum of 3 hours.
From this point on, handle allantoic fluid or antigen preparation on ice.

5.

Collect allantoic fluid (AF) as free from red blood cells as possible.

6.

Centrifuge AF at 1500 rpm for 30 minutes to remove red blood cells and debris, decant
and save supernate. Note how many ml of AF you have.

7.

Concentrate virus by pelleting at 30,000 G for 90 minutes and discard supernate.

8.

Resuspend virus pellet in HEPES buffer at pH 6.5. Add 1 ml of buffer per each 100 ml
of the allantoic fluid obtained prior to centrifugation. (100 x concentration.)

9.

Add an equal volume of phospholipase C (Sigma Chemical) with 2 unit/ml (final

concentration) diluted from concentrate in HEPES buffer.

10.

Mix until the fluid is in a uniform suspension.

11.

Incubate 2 hours at 37

12.

Storage: the antigen was found to be stable at 4 C for 2 months. Frozen, the antigen
was stable for a similar amount of time. Repeated freeze-thawing adversely affects
antigen titer.

C. Vortexing every 30 min.

The antigen must be titered before use.

HEPES BUFFER:

1.0 L

500 ml

5.96 g Hepes

2.95 gm

8.19 g NaCl

4.09

.15 g CaCl2

0.08 gm

dd H2O to 1L

dd H2O to 500 ml

pH with 1N NaOH

39

VIRUS NEUTRALIZATION TEST IN EMBRYOS

This technique can be used to quantify antibodies against avian infectious bronchitis virus
and avian encephalomyelitis.
There are 2 procedures generally used: the Alpha and the Beta methods.
Alpha - constant amounts of serum and variable amounts of virus.
Beta - constant amounts of virus and variable amounts of serum.
The Alpha method is described here.
1.

Heat inactivate serum sample for about 45 minutes at 56 C in water bath. Make 1:2
dilution if necessary. You will need at least 2.0 ml of serum for test. Use buffer,
tryptose phosphate broth or HBSS for diluting. Make the lowest dilution possible.

2.

Make virus dilutions. Use MASS 41 Infectious Bronchitis Virus (IBV) (Beaudette
Strain) or AE virus (Van Roekel Strain). Make dilutions from 10-1 to 10-6.

3.

Serum dilutions:
Tube 1 = 0.4 ml serum + 0.4 ml virus 10-6
Tube 2 = 0.4 ml serum + 0.4 ml virus 10-5
Tube 3 = 0.4 ml serum + 0.4 ml virus 10-4
Tube 4 = 0.4 ml serum + 0.4 ml virus 10-3
Tube 5 = 0.4 ml serum + 0.4 ml virus 10-2
Incubate for 1 hour at Room Temperature.

4.

Inoculate five 9 to 11 day old embryos per dilution with 0.1 ml inoculum per embryo.
Inoculate the embryos starting with the highest dilution.

5.

Use dilutions 10-2 to 10-6 for inoculation. Total number of embryos required is 30.

6.

Leave at least 5 embryos uninoculated for controls.

7.

Record deaths each day for 7 days. On the 7th day, open all embryos and check for
stunting of embryo which is characteristic of IBV virus. Record results.

8.

Find neutralization index.

40

PLAQUE REDUCTION TEST

(ALPHA PROCEDURE)
1.

Prepare virus dilutions 10-1-10-6.

2.

Heat inactivate (56 C, 30 minutes) an aliquot of all serum samples:

Virus-specific (positive)
Normal (expected negative)
Unknowns (those you are testing)

3.

Add an equal volume of serum to be tested to an equal volume of the selected virus
dilutions.

4.

Incubate the virus-serum mixtures at room temperature for 45 minutes.

5.

Inoculate 0.2 ml of the virus-serum mixtures into replicate cultures.

6.

Inoculate 0.1 ml of virus control dilutions in replicate cultures.

7.

Continue as per procedure for modified agar overlay. #3

REFERENCE:
Rovozzo, G. C. and C. N. Burke. A Manual of Basic Virological Techniques. PrenticeHall, Inc. 1973.

41

NEUTRALIZATION TEST
(BETA PROCEDURE)
1.

Used for infectious bronchitis (in CEKC) and for reovirus and Gumboro (in CEF).

2.

Dilute virus to obtain the appropriate amount of virus to be used in the test.

3.

Add 100 l of virus to well 1 from A to H and 50 l to all other wells except to wells in
column 12 which will be the CELL CONTROL.

4.

In the first well (column 1, A) add 25 l of the heat- inactivated serum sample using a 25
l microdiluter. All samples are placed in well 1 from A to H (8 samples can be tested
per plate).

5.

Using the multimicrodiluter handle fitted with the 50 l microdiluters, transfer 50 l of

virus-serum mixture from well 1 to well 2 and continue to well #10. Discard the content
of the microdiluters by using sterile blotted paper. Column 11 will serve as VIRUS
CONTROL.

6.

Incubate the plates for approximately 30-45 minutes at 37

7.

Add 0.2 ml of freshly prepared chicken embryo kidney cells or CEF diluted to contain
approximately 5 x 104 cells per well.

8.

Incubate for approximately 72 hours.

9.

Fix and stain (see staining technique).

10.

The end-point of any serum sample will be the dilution where the virus has been
neutralized by the diluted serum (it should look like the cell control).

11.

Positive and negative controls should ALWAYS be included.

C.

42

MICRONEUTRALIZATION TEST FOR INFECTIOUS BURSAL DISEASE VIRUS

1.

Prepare the chicken embryo fibroblast (CEF) adapted strain of infectious bursal disease
virus (IBDV) so that approximately 300 infectious units (IU) will be present in 0.05 ml
(50 l).

2.

Using the 50 l pipette, add one drop of the diluted IBDV to all wells (from 1 to 11)
except in well #12. This well will be the cell control.

3.

Add 50 l of the serum sample in the first well (well 1, row A). Follow the same
procedure for each one of the serum samples (sample 2 will be located in well 1, row B;
sample 3 in well 1, row C; and so on).

4.

Using the 50 l microdiluter fitted with the handle or a multiwell pipettor, dilute all
samples in the first well and transfer 50 l to the second well. Repeat the same
procedure up to well #10 and discard the 50 l left.

5.

Allow the preparation to incubate at room temperature for 30-45 minutes.

6.

Add 190 l of CEF that has been prepared and diluted to be used in microtiter plates
(each well should receive 190 l of cells).

7.

Cover the plates with sterile polystyrene covers or with sterile tape.

8.

Incubate for approximately 72-96 hours.

9.

Fix and stain.

Controls:
1.
2.
3.

Always include a known positive and negative antiserum.

Wells 12 will be cell control.
Wells 11 will be virus control.

43

NEWCASTLE DISEASE VIRUS VACCINE TITRATION

1.

Reconstitute lyophilized vaccine (1,000 dose vial) with 30 ml sterile PBS or vaccine
diluent.

2.

Make serial ten-fold dilutions of the vaccine in Tryptose Phosphate Broth (TPB) with
antibiotics or with HBSS (10-1 to 10-8).

3.

Inoculate via chorioallantoic sac (CAS) route. Use 9-11 day-old embryos. Five
embryos/dilution 0.1 cc inoculum. Inoculate 10-3 to 10-8 dilutions.

4.

Incubate at 37

5.

Candle embryos for viability every day. Discard embryos dead in first 24 hours. After
that, for dead embryos, open and do plate HA test, use 5% chicken red blood cells.
Record results of HA test.

6.

At the end of 5-7 days, open all remaining embryos. Do plate HA test and record results.

7.

Find EID50 (Embryo Infection Dose to 50) and calculate titer.

C for 5-7 days.

44

SUPPLEMENTAL ASSAY METHOD FOR TITRATION OF HERPESVIRUS OF

TURKEYS (STRAIN FC-126) OR CHICKEN HERPESVIRUS STRAIN SB-1
(AS RECOMMENDED BY THE USDA)
I.

PURPOSE

This method describes in detail a procedure using chick embryo fibroblast cell cultures
for titrating the herpesvirus of turkeys (Strain FC-126) or chicken herpesvirus strain SB-1 used
as vaccines against Marek's disease. The vaccine is composed of a suspension of chick embryo
fibroblast (CEF) cells infected with the virus.

II.

MATERIALS AND METHODS

A.

Cell Cultures: Secondary CEF cultures are used for the titration.
1.

Preparing Primary Cultures:

Prepare primary chick embryo cell cultures from 9 to 11 day old embryos
(derived from specific pathogen-free flocks*) in the following manner: Swab the
air cell end of the egg with 70% ethanol, flame, and break open the shell with
sterile blunt thumb forceps. Use the forceps to open the membranes, lift out the
embryo, and place it in a sterile disposable petri dish. Four to six embryos may
be prepared together. Remove (and discard) the heads of the embryos with sterile
scissors. Wash the embryos by adding 0.25% trypsin solution (see Solutions, II
B1) to the petri dish. Open the body cavity of the embryos with the sterile
forceps and remove the liver and the bulk of the other viscera. Gently squeeze
the remainder of the embryos with the forceps to remove as much blood as
possible. Pick the washed embryos out of the wash solution with the forceps,
drain them momentarily, and place them in a sterile petri dish. Mince the
embryos thoroughly by cutting with sharp sterile scissors.
Place the minced tissue in a 250 ml sterile trypsinizing flask with a
magnetic stirring bar, add 30 ml of 0.25% trypsin solution (prewarmed to
approximately 35 C), trypsinize for 20 minutes at room temperature. Carefully
decant the supernatant suspension through a sterile funnel with four layers of
gauze into a sterile centrifuge bottle.
____________________
*

SPAFAS, Inc. -- No endorsement expressed or implied.

Approximately 30 ml of growth medium (see Solutions, II B2) should be
added to the centrifuge bottle to stop the action of the trypsin on the cells. To the
remaining fragments in the trypsinizing flask, add another 30 ml of 0.25% trypsin
and repeat the process for another 20 minutes. Add this cell suspension to the first

45

collection. Centrifuge at approximately 250 x g for 10 minutes. Observe the volume

of packed cells, then remove the supernatant.
Dilute the cells approximately 1:300 with growth medium and plant in
suitable culture vessels. Incubate three to five days at 37 to 37.5 C in a high
humidity atmosphere containing approximately 5% CO2. At this time, the cell
monolayers should be confluent.
2.

Preparing Secondary Cultures:

Remove the medium from the primary culture vessel and add an appropriate
volume of 0.25% trypsin solution to each vessel. Let the trypsin solution remain
in contact with the cell sheet for 60 to 90 seconds, then remove it.
Place the vessels in a horizontal position with the cell sheet down and
incubate at 37 to 37.5 C for an additional 10 to 20 minutes, or until the cell
sheet appears to be well separated. The proper length of time will be learned by
experience; too short a time will result in large clumps of cells in the new
suspension. To each vessel, add an appropriate amount of fresh growth medium
and shake or pipette to loosen and break up the cell clumps. Pour the cell
suspensions into an Erlenmeyer flask with a stirring bar. After thorough mixing
make a cell count with a hemocytometer. Adjust the volume so that the cell
concentration is approximately 375,000 per ml.
Plant the secondary cell suspension into 60 mm tissue culture dishes
(gridded plastic dishes or plain dishes if a grid-adapted stage is to be used in
microscopic observation for counting). Add 4 ml cell suspension per plate
(approximately 1.5 million cells).
Incubate the cultures at 37 to 37.5 C in a high humidity atmosphere
containing approximately 5% CO2. When the cultures have reached confluency
(24 hours or less), they are ready for inoculation (virus titration).

46

B.

Solutions: All solutions are filter sterilized.

1. Trypsin Solution (0.25%):
NaCl

8.0 g

KCl

0.4 g

Glucose

1.0 ml

Phenol Red (0.5% solution)

1.0 ml

Trypsin (1:250)

2.5 g

NaHCO3

0.35 g

Purified H20 q.s.

1 liter

Adjust pH to 7.4 with NaHCO3 solution.

2. Growth Medium:
Medium 199 (with Earle's salts)
(powdered)

10 g

Nutrient Mixture F10 (powdered)

10 g

Bacto Tryptose Phosphate Broth

(dry powder)
NaHCO3
Penicillin (potassium G)
Streptomycin
Fetal Bovine Serum* (inactivated)
Purified water q.s.

2.95 g
2.5 g
200,000 units
200 mg
85 ml
2185 ml

Adjust pH to 7.35 to 7.4 by adding NaHCO3 solution.

Before use, add 1.0 ml of a 200 mM concentration of L- glutamine per
100 ml medium.

47

3. Maintenance Medium:
Medium 199 (with Earle's salts)
(powdered)

10 g

Nutrient Mixture F10 (powdered)

10 g

Bacto Tryptose Phosphate Broth

(dry powder)

2.95 g

NaHCO3

2.75 g

Penicillin (potassium G)
Streptomycin
Fetal Bovine Serum* (inactivated)
Purified water q.s.

200,000 units
200 mg
42 ml
2142 ml

Adjust pH to 7.5 by adding NaHCO3 solution.

Before use, add 1.0 ml of a 200 mM concentration of L-glutamine per
100 ml medium.
C.

Preparation of Vaccine for Titration:

Remove one ampule of vaccine from the ultra-cold storage container and thaw
quickly by immersing in a water bath approximately 25 C. Add the
concentrated vaccine immediately to the appropriate amount (according to the
indicated dosage) of the manufacturer's diluent (at room temperature). Do this by
withdrawing the vaccine into a 10 ml syringe through an 18 gauge (or larger)
needle, then withdraw approximately 5 ml of the diluent into the same syringe and
mix gently. Slowly force the contents of the syringe into the bottle of diluent
keeping the end of the needle in the liquid by tipping the bottle. Withdraw 2 ml
from the diluted vaccine, use to rinse the ampule once, then add this back to the
diluted vaccine. Withdrawal and expulsion must be done slowly to prevent
rupturing of the cells. This mixture constitutes "field strength" vaccine.

D.

Holding Period:
Place the vaccine bottle in an ice bath for two hours (gently mix every 30
minutes) prior to proceeding with the titration. Shortly before the end of the two
hour holding period, place 8.0 ml of growth medium (at 4 C) in two sterile test
tubes and 9.0 ml of growth medium in one sterile test tube (make a set of three

48

tubes for each vaccine sample). Use these to make further dilutions in the
titration procedure. These dilution blanks are not held in an ice bath.
E.

Preparing Dilutions and Inoculating Plates:

Mix the vaccine by inverting the bottle 10 to 15 times. Withdraw a sample using
a 2-1/2 ml syringe fitted with an 18 gauge needle and add 2.0 ml to the first tube
with 8 ml diluent (1:5). Mix with a sterile 10 ml pipette and transfer 2.0 ml (of
the 1:5) to a second tube with 8 ml diluent (1:25). Use a clean sterile 10 ml
pipette; mix the 1:25 dilution; then transfer 1 ml (of the 1:25) to the third tube
with 9 ml diluent (1:250). With SB-1 virus, the third tube may contain 8 ml
diluent, in which case 2 ml of vaccine is added making a final dilution of 1:125.
Use a clean sterile pipette, mix the final dilution and inoculate 1.0 ml per plate
into 5 test plates. Do this by drawing a large sample into a 10 ml pipette and
distribute the inoculum into the 5 plates using the graduations between 2 and 7 ml
(this inoculum is in addition to the 4 ml medium already in each plate). Mixing
must be done thoroughly but gently to prevent rupturing of the cells.
Do this procedure of dilution and inoculation as rapidly as possible to prevent
cells from attaching to the surface of the dilution tubes (less than two minutes
should elapse between addition of the cell suspension to a particular dilution
blank and the removal of a sample for further dilution or inoculation).
Swirl the test plates as soon as each vaccine sample has been inoculated.
Incubate the plates at 37 to 37.5 C in a high humidity atmosphere containing 5%
CO2. Twenty-four hours postinoculation (PI), remove the medium from the plates
and replace with 5 ml maintenance medium (see Solutions, II B3). The
maintenance medium may be replaced after two or three days if the pH of the
culture fluids becomes too acid.

F.

Controls:
Titrate a positive control sample with each group of titrations. This positive
control is a specially prepared lot of virus on which several titrations have been
done to ascertain that vial-to-vial variation is minimal. If the positive control
titration result is abnormally high or low in any particular test series, all tests in
that series are inconclusive (No Tests).
Uninoculated (negative) controls may be run to check the integrity of the cell
culture system.

49

G.

Making Foci Counts and Calculating Titer:

Incubate the plates at 37 to 37.5 C until the time for counting foci. With strain
FC126, count the foci on day 5 PI. With strain SB-1 count the foci on day 7 PI.
Use an inverted microscope (and a grid adapted stage if plain plates have been
used) to make the counts.
Count all the foci on each of the plates of the titration series. A focus is counted
as one regardless of size unless it has apparently arisen from two distinct centers.
Calculate the average number of foci per plate and multiply this value by 25** or
50*** depending on the dilution used in inoculation; this result will be the focusforming units (FFU) per bird dose (assuming the volume of one bird dose is 0.2
ml).

____________________
*

Previously tested for freedom from extraneous agents.

**

125 (dilution factor) divided by 5 (number of doses per ml) equals 25.

***

250 (dilution factor) divided by 5 (number of doses per ml) equals 50.

50

TITRATION OF THE HERPESVIRUS OF TURKEYS (HVT) VACCINE

I.

Cell-Associated Virus Vaccine

1.

Dilute one vial of vaccine in 200 ml of diluent or HBSS or Media. (Stock

Solution). Hold vaccine-diluent mixture on ice for no more than 2 hrs.

2.

Add 8 ml of HBSS to 2 sterile tubes, 9 ml to a third one and 4 ml to a fourth.

3.

Add 2 ml of the stock solution to one of the tubes containing the 8 ml of TPB.
Gently mix and transfer 2 ml of this solution to the second tube containing the 8
ml of HBSS. Mix and transfer 1 ml from this solution to the tube containing 9 ml
of HBSS. Mix and transfer 4 ml from this solution to the tube containing 4 ml. In
this way the first tube contains a 1:5 dilution of the stock; the second 1:25; the
third 1:250 and the fourth 1:500.

4.

Complete CEF monolayers prepared in 60 x 15 mm gridded plates are inoculated

with 1 ml of each dilution. Three plates per dilution are inoculated.

5.

During the 30-45 minutes incubation period at 37 C the plates should be rocked
several times to obtain a uniform distribution of the inoculum.

6.

Add 5 ml of maintenance medium.

7.

Incubate for approximately 5 days in CO2 incubator 37

8.

Count the foci produced by the virus. The highest dilutions of the stock (1:500)
are usually the plates where distinct foci are easily observed and counted. Obtain
the average of 3 plates.

9.

To obtain the titer of the vaccine per bird dose, the following procedure is used:

C.

Average number focus counted X dilution factor

PFU per bird dose = _____________________________________________________________

5*
*

5 represents 1/5 of the dose used to inoculate chickens; (0.2 ml) since 1 ml was inoculated in
each plate, the average number of PFU should be divided by 5 to obtain the titer per bird dose.

51

TITRATION OF THE HERPESVIRUS OF TURKEYS (HVT) VACCINE

(CONTINUED)
II.

Cell-Free Vaccine
1.

Dilute vaccine in 200 ml of diluent or use SPNZ-amine (sucrose 0.15 M;

monopotassium phosphate 0.0038 M; dipotassium phosphate 0.0072 M; N-Z
amine 1.5%).

2.

From this stock dilution prepare a 1:20 dilution.

3.

Inoculate 0.1 ml of this dilution to complete CEF monolayers prepared in 60 x 15

mm petri dishes.

4.

Follow steps 5, 6, 7 and 8 of the procedure described for the cell-associated

vaccine.

5.

To determine the number of PFU per bird dose; calculate the average number of
plaques and multiply it by 20 (1:20 dilution factor) and by 2 (0.1 ml was
inoculated).
PFU = Average number of plaques X 40.

52

ETHER OR CHLOROFORM (CHC13) SENSITIVITY

1.

Dilute virus stock or sample to be tested 1:10 and divide into two aliquots.

2.

Add 0.2 ml of CHC13 to 2 ml of one aliquot in a 15 ml centrifuge tube. Dispense 2 ml of

the other aliquot into another 15 ml centrifuge tube.

3.

Mix both tubes on Vortex for 10 minutes, keeping the tubes in an ice bath between mixes.

4.

Allow CHC13 to sediment in refrigerator overnight or by centrifuging (1500 rpm for 30

minutes). Set at room temperature without disturbing for 10 min.

5.

Using a long sterile Pasteur pipet, collect the clear layer on the top being careful not to
pick up any CHC13 which will appear cloudy. The top layer which has been collected
may be left in an opened vial under the hood for 10-15 minutes to allow any CHC13
present to evaporate prior to inoculation. Cap the vial and refrigerate overnight.

6.

Make 10-1 and 10-2 dilutions and inoculate undiluted and diluted samples (CHC13 treated
and non-CHC13 treated) in macro or micro dishes or in embryos.

NOTE:

Glass equipment should be used because the chloroform reacts with plastic.

53

5' IODO DEOXYURIDINE (IUDR) INHIBITION TEST

1.

Prepare maintenance medium with IUDR at the following concentrations: 10-2 M; 10-3
M; 10-4 M. The media must be homogenized and sterilized by filtration (run sterility
check on each concentration).

NOTE:

2.

IUDR goes into solution faster if the pH of the medium is increased. Tighten
the cap of the bottle and place it on a magnetic stirrer preferable at 37 C
for approximately 15 minutes.

One group will work with an adenovirus and the second group with a reovirus. Two
dilutions of each virus will be tested, 100 and 10-1. The total number of plates per group
should be 18-20. The dilutions of virus and IUDR are summarized in the following table.
IUDR MOLAR DILUTIONS

Virus

Regular Medium

Adeno dil 1
Adeno dil 2

2*
2

2
2

2
2

2
2

Reo dil 1
Reo dil 2

2
2

2
2

2
2

2
2

No Virus

Number of plates (35 mm)

3.

Inoculate preformed CELIC monolayers with the appropriate dilutions of the virus being
tested. Use 2-3 plates per dilution of IUDR.

4.

Adsorb virus at 37 C for 45 minutes and discard excess fluid into a beaker containing
disinfectant solution.

5.

Add maintenance medium with IUDR to the dishes.

6.

Harvest dishes when cytopathogenic effect is observed in controls.

7.

Freeze-thaw the dishes 3 times. (The cells can also be sonicated to speed up the process.)

8.

Titrate pooled controls and all plates where IUDR was used.

NOTE:

When testing unknown viruses, both DNA and RNA should be included as
controls.

54

IMMUNODIFFUSION TEST
Used routinely to demonstrate presence of antibodies for Gumboro, viral arthritis,
adenovirus, influenza and Marek's disease.
1.

Prepare agar gel plates:

100 ml

50 ml

8% (8 gm)

4 gm

0.7% (0.7 gm)

0.35 gm

0.1 M PBS pH 7.2

10 ml

5.0 ml

1% Thimerosal

1 ml

0.5 ml

Polyethylene Glycol
(8000 MW)

2 gm

1.0 gm

Distilled Water

89 ml

44.5 ml

NaCl
Noble Agar*

Autoclave for 10 minutes and pour into small (35 mm) tissue culture plates (2 ml per
plate). After agar has cooled, punch holes.
2.

Place antigen in the center well and serum samples in outer wells. Always include a
known positive control. Do not overfill the wells.

3.

Place in moisture chamber at room temperature and check for precipitation daily.

4.

The antigen and specific antiserum should form a band of precipitation. If the unknown
sera samples contain antibodies specific for the antigen in center well, a band should also
be present.

NOTE:

The test can also be run using regular glass slides. The agar is poured on the
slide and the holes are punched. Humidity must be high in the chamber to
avoid desiccation of the agar.

The agar can also be prepared using purified agar or Ionagar #2.

55

PROTOCOL FOR PREPARATION OF A CONJUGATE

1.

Collect 10-25 ml high titered specific antisera.

2.

Prepare saturated solution of ammonium sulfate (NH4)2SO4 by adding 55 g of (NH4)2SO4

to volumetric flask and bring volume to 100 ml with distilled water. Store at room
temperature for several days, stirring at least once a day. Filter and store solution at room
temperature.

3.

Prepare 70% saturation working solution by mixing 7 parts saturated (NH4)2SO4 with 3
parts distilled water.

NOTE:

Different animal species require different working concentrations of

(NH4)2SO4 and different number of precipitations. See reference.

FIRST PRECIPITATION
1.

Gently stir serum and dropwise slowly add an equal volume of (NH4)2SO4 working
solution. (Final (NH4)2SO4 concentration is 35%.)

2.

Incubate mixture at 4

3.

Centrifuge at 1,500 rpm (1570 x g) for 30 min. at 4

4.

Remove and discard supernatant fluid.

5.

Add distilled water to precipitate until volume is equal to that of the original serum.

C for 4 hours, stirring slowly.

C.

SECOND PRECIPITATION
1.

Gently stir protein solution (gamma globulins) and slowly add an equal volume of
(NH4)2SO4 working solution.

2.

Centrifuge at 1500 rpm for 30 min. at 4

3.

Remove and discard supernatant fluid.

4.

Add distilled water to the precipitate until volume of the solution is equal to that of the
original serum.

C.

THIRD PRECIPITATION
1.
4.
5.

Repeat steps 1, 2 and 3 of SECOND PRECIPITATION.

Dissolve globulin in a small amount of distilled water.
Transfer globulins to dialysis tubing and dialyze at 4 C against PBS (pH 8.0). Change
NaCl solution frequently until it is free of sulfate ions (18-24 hrs.).

56

NOTE:

Sulfate ions can be detected by adding equal (small) volumes of saturated

barium chloride solution (20 g BaCl2 in 50 ml distilled water) and the
NaCl dialysate. If sulfate ions are present the mixture becomes cloudy.

PROTEIN DETERMINATION
1. Analysis of protein concentration by Bradford1 Test. Prepare standard curve using 10
mg/ml of Bovine Serum Albumin (BSA) as stock solution.
Tube
1
2
3
4
5
6

Protein (mgs)
0
2
4
6
8
10

BSA
0
.2
.4
.6
.8
1.0

.85% NaCl
1.0
.8
.6
.4
.2
0

2.
3.
4.
5.

Transfer .8 ml of each dilution to separate tube.

Add .2 ml Bradford.
Read at 595 nm. (Blank to 100% transmittance with .85% NaCl (tube #1).)
Dilute unknown globulin mixture 10-1-10-5 and run same procedure.

6.

Calculate amount of protein in globulin mixture. Adjust volume with 0.85% NaCl to
achieve 5-10 mg protein/ml.

NOTE:

7.

Many commercial kits are readily available for protein determination.

Adjust pH to 9.2-9.3 using 0.5M carbonate-bicarbonate buffer (pH 9.5).

A.

7.3 g anhydrous sodium carbonate in 100 ml water.

B.

4.2 g sodium bicarbonate in 100 ml H20.

Mix 26 ml solution A with 74 ml solution B.

M. M. Bradford. Analytical Biochemistry 72:248-254 (1976).

57

CONJUGATION
1.

Place protein solution in a beaker with stir bar. Calculate amount of fluorescein needed
(.035 to .05 mg fluorescein/mg protein). At 4 C with brisk stirring (no foaming) add
fluorescein to beaker.

2.

Stir at moderate speed 12-24 hours.

COLUMN PREPARATION
1.

Assemble Sephadex column in a vertical position. Level the column carefully.

2.

Column K25/45 (diam. 2.5 cm) (length 45 cm). Sephadex grade G25-150. (*See
Reference for other gels and column sizes.) Put 40 gm Sephadex in 1 liter PBS. Stir very
slowly and continuously for 15 min. Set aside for six hours at room temperature. Pipet
off supernatant (fines = broken beads) and resuspend to 1 liter. Mix and allow to settle
again. Pipet off supernatant. Repeat 3rd time.

3.

Mix Sephadex in 500 ml PBS and place in aspirator bottle with stirring bar and clamped
hose attached at base. Place bottle with Sephadex mixture on stir plate at a height above
the column. Mix at a very slow rate.

4.

Clamp column outlet and fill with PBS. Attach hose from bottle to top of column and
open hose. (Make sure there are no bends in the hose.) Open outlet slowly so that the
column will fill with Sephadex slowly. Fill column to within 3-4 in. of the top. Allow
another 1-2 liters of PBS to run through the column. (Do not allow column to run dry at
any time!)

5.

Remove PBS from top of column and allow PBS to drain until Sephadex is almost
exposed. Add fluorescein conjugate to top of column slowly and carefully as not to
disturb surface. Open bottom of column to allow conjugate to flow through column.
When the conjugate is almost all into the column add PBS to fill the top of the column
and reattach column top and PBS supply.

6.

Collect the first yellow band that passes through the column. Collect the conjugate in
several small fractions.

7.

Titer conjugate to determine proper working concentration.

REFERENCES:
H. C. Lyerla and F. T. Forrester. Immunofluorescence methods in Virology Course No.
8231-C. U.S. Department of Health and Human Services. Center for Disease Control. May
1980.

58

DIRECT FLUORESCENT ANTIBODY (FA) TECHNIQUE

FOR ALLANTOIC FLUIDS:
1.

Place embryos in refrigerator for approximately 2 hrs.

2.

Open shell at top and draw choriallantoic fluid. If an excessive amount of blood is mixed
with fluid, discard and place embryos back in refrigerator for an additional 2 hrs. Red
blood cells (RBC's) can give false positive results. If fluid is clean of blood, put into
centrifuge tubes and proceed with test.

3.

Centrifuge for 15 minutes at 1,200 rpm. Remove and discard the supernatant.

4.

With a diamond knife, etch a box on slide to prevent conjugate from running off or use
nail polish and create a square "well".

5.

With a capillary pipet, remove part of the pellet from the bottom of tube and place on
glass slide. Spread a thin layer with cover slip and let air dry.

6.

Place slide in slide jar containing COLD acetone for 15 minutes and rinse in PBS, 3 dips
in each of 3 beakers and then air dry. (This is to fix the preparation.)

7.

Add specific conjugate to cover smear and place slide in high humidity container.
Incubate for 15 minutes preferably in CO2 incubator.

8.

Wash slide with buffer solution. Read slide with fluorescent microscope. Bright green
fluorescence in the cells is indicative of the presence of the virus. Positive and negative
controls should also be run for comparison.
FOR CELL MONOLAYER COVERSLIPS

1.

For FA on cell monolayer coverslips, start with Step #6. At step #9, when coverslip has
dried partially, place drop of glycerine solution (mounting fluid) on glass slide and put
coverslip on top of glycerine (cell side down). Observe through FA scope.

59

SAMPLE PREPARATION FOR VIRUS ISOLATION

Sample received should be either fresh or frozen. If frozen, allow to thaw.
1.

Completely freeze and thaw sample(s).

2.

Take small pieces of submitted sample and mince in Tryptose Phosphate Broth (TPB)
with antibiotics. (If intestine is submitted, express contents before adding to TPB.)

3.

Filter through a sterile 0.45 m syringe filter into a sterile vial. Label and use to
inoculate embryos or cell cultures. (If bacterial contamination is a problem, the sample
may be further filtered through a sterile 0.22 m syringe filter.)

4,

Long term storage of sample should be placed into a -80

nitrogen.

C freezer or into liquid

60

VIRUS CONCENTRATION PROCEDURE

(FOR USE IN ELECTRON MICROSCOPY EXAMINATION)
Virus in either cell culture fluid or in allantoic fluid may be used. If cell culture is
used, freeze-thaw 3 times.
1.

Collect sample in centrifuge tubes. Centrifuge at 1500 rpm for 30 min. at

4 C.

2.

Collect supernatant into fresh centrifuge tubes and centrifuge at 7000 rpm for 45 min. at 4
C.

3.

Collect supernatant into fresh centrifuge tubes and centrifuge 30,000 x g for 90-120 min. at
4 C.

4.

Discard supernatant and resuspend virus pellet in a small amount of PBS (0.1 ml or less).

61

THE ENZYME-LINKED IMMUNOSORBENT ASSAY

S. G. Thayer
INTRODUCTION
The Enzyme-linked Immunosorbent Assay (ELISA) was designed in the 1960's but came into practical
use in 1971. Engvall and Perlman designed an ELISA for use in quantitating IgG for use in human medical
diagnostics.
ELISA was designed as a replacement for RadioImmunoAssay (RIA) which had the disadvantages of:
1. Complex Procedure
2. Handling of Radioactive Materials
3. Disposal of Radioactive Materials
4. Federal Regulations
ELISA had none of these disadvantages and still had the sensitivity of the radioimmunoassay.
SEROLOGICAL TESTS
Serological tests have several components in common.
1. Antigen - Disease agent or related organism.
2. Antibody - Specific serum proteins produced by the animal in response to exposure to the
foreign agent.
3. Indicator - Some means of detection and/or quantitation of the antigen-antibody
reaction.
Hemagglutination-inhibition assays - Chicken erythrocyte.
Virus Neutralization Assays - Cell monolayer destroyed by virus (Ag) or protected
against destruction by the antibody in the test serum.
Agar Gel Precipitin Tests - Antigen and antibody combine to form a line of
precipitation.
ELISA - Color development as a product of an enzyme-substrate reaction.
ENZYME-LABELED ANTIGLOBULIN CONJUGATE
Conjugate: Goat Anti-Chicken IgG - Enzyme
Enzymes: Proteins produced by living cells which promote chemical reactions without undergoing
degradation themselves. They are recycled.
1. Enzymes most commonly used:
A. Horseradish peroxidase
B. Alkaline phosphatase
C. Beta Galactosidase
D. Glucose oxidase
2. Parameters affecting enzyme activity
A. Temperature
B. pH
C. Ionic strength

62

D. Buffer composition
E. Other factors
1. Cofactor concentration
2. Substrate depletion
3. Build-up of product inhibitors
4. Increased back reaction as product concentration increases
5. Adsorption of enzyme to vessel surfaces
6. Denaturation or mechanical disruption
a. Foaming
b. Hydrodynamic shear
c. Microbial contamination
d. Improper storage
3. Characteristics of a good enzyme for ELISA
A. The enzyme should be stable at 25oC and 37oC, and have a shelf life of at least 6 months
at 4oC.
B. The purified enzyme should be commercially available and relatively inexpensive.
C. The enzyme activity should be easily measurable using
colorimetric or fluorimetric methods.
D. Small amounts of the enzyme should be detectable.
The enzyme should have a high substrate turnover number and the reaction product
should have a high molar extinction or a high molarfluorescence.
E. For use in a competitive ELISA the enzyme should not be affected by biological
components of the test sample.
SUBSTRATES
Chemical compounds which are modified by the enzyme and in ELISA applications are selected for
their colored by-products
ELISA FORMATS
1. Direct ELISA
Uses a single species monospecific antiserum coupled with an enzyme for direct
detection of antigen or other protein in histopathological specimens, smears, infected
cell cultures etc.
2. Indirect ELISA
The enzyme label is attached to a second antibody which is a test component rather than to the
detecting antibody. This format uses a single species antibody directed against serum proteins
(IgG) so that it may be used to detect multiple antigens or antibodies.
3. Competitive ELISA
These can be set up for detecting antigen or antibody. This assay incubates a known
concentration of antigen (or antibody) along with the test material. The presence of unknown
antigen (or antibody) competes with the known antigen (or antibody) for the available binding
sites and therefore "blocks" these sites. Maximal color development is a negative test and
minimal color development is a positive test.

63

ELISA TEST COMPONENTS

1. Solid Phase - Strips, beads, tubes, microwell plates.
2. Coating material - Antigen or antibody.
3. Test material - Serum (antibody) or fluid containing antigen.
4. Conjugate - Antibody against antibodies or antigens coupled to an enzyme.
5. Substrate - Source of reactive material which reacts with the enzyme to yield a colored
by-product.
ANTIGEN SPECIFIC BINDING
1. Antibodies bound in the ELISA test represent antibodies that are mostly antigen-specific within the
entire spectrum of the IgG's produced in response to the antigen in question. These may
include VN, HI and precipitating type of antibodies.
2. The design of a serological test determines what is detected by the test.
3. The ELISA selects for antibodies that tend to stick most tenaciously to the antigen because of the
assay design.
A. High working dilutions favor high affinity and high avidity antibodies.
B. Short incubation times select for antibodies that bind most efficiently.
C. Mechanical factors such as the vigorous washes favor the most tenacious of
antibodies.
Antibody Affinity - Reflects the fit of the IgG combining site for the antigen. (Improves with time of
exposure)
A. Early antibodies - Poor fit (IgM, early IgG)
B. Late antibodies - Excellent fit (IgG)
Antibody Avidity - Relative assessment by dilution of antigen-antibody complexes and measuring the
proportion of bound vs free antibody. The more avid the antibody the less free
antibody that is present at high dilution, that is, the antibodies tend to remain
bound in spite of high levels of dilution.
CHARACTERISTICS OF ELISA COMPARED TO THE VN AND HI
1. ELISA tends to measure IgG as defined by the conjugate which is a goat anti-chicken (turkey) IgG.
2. Does detect some IgM but only that of the most avid variety.
3. Test is group antigen specific - High degree of cross reactivity between standard vaccine strains of
IBV, IBDV, and Reovirus. The ability to pick up antibodies to some variant strains of IBDV is
questionable. VN's are far more specific and can be used to a limited degree to separate different
serotypes of viruses. Hemagglutination-inhibition tests can be used on IBV positive sera collected
within 3-4 weeks of exposure to determine which serotype of IBV to which the virus in question
is related.
4. Processing of the antigen for coating ELISA plates apparently cause denaturation or at least
alteration of the antigenic determinants unique to the serotypes.

64

ELISA TEST COMPONENTS

-- Reproducibility-Is based on control of reaction rate variables (Important if the reaction is time
dependent).

1. Quality of the Solid Phase Material

A. Defined by:
1. Antigen binding properties
2. Uptake of non-specific proteins
3. Uniformity of binding
4. Batch variation
B. Most suitable: Polystyrene (Immulon I, II, III, and IV)
These differ in binding strength and background considerations.
2. Antigen
A. Adsorption of antigen to the solid phase is the major factor governing the sensitivity
and the precision of the ELISA.
B. Optimum antigen concentration is the one above which there is little or no increase
in antibody binding. ( 1-10mg/ml )
C. Overcoating - Disadvantageous
1. Antigen monolayer cannot be maintained.
2. Antigen may detach during processing.
a. Decreases sensitivity
b. Reproducibility declines
D. Checkerboard titration used to optimize antigen concentration.
3. Serum
A. Optimum serum dilution in single dilution systems.
1. Important to select dilutions on the linear portions of the dilution curve.
a. Too low - Many strong antibodies will be in excess with
insufficient sites on the solid phase.
b. Too high - Antibody too sparsely distributed for the conjugate to
bind effectively.
2. Predetermined by checkerboard titration.
B. Working dilution of the antibody or test sample containing antigen used to:
1. Minimize non-specific binding (Background)
2. Conserve reagents
4. Conjugate
A. Optimum dilution
1. Determined by serial titration against a standard concentration of IgG.
2. Confirmed against reference positive and negative sera.
B. Incubation time - follows same parameters as mentioned previously for antibody.

65

C. The enzyme molecule:

1. Possesses one or more active sites
to which the substrate attaches, breaks down, yields reaction products,
and regenerates free enzyme.
2. Amplification of the test sensitivity results from the regeneration of free
enzyme.
5. Washing
A. Required after coating or after incubation with other reagents.
1. Removes unbound reagents
2. Removes weakly adsorbed materials
6. Substrate
A. Enzyme specific
B. Chosen for its chromogenic properties
C. Measured photometrically
D. Must have excess
1. Limiting factor becomes the enzyme
2. Amount of color is proportional to the amount of bound conjugate which
in turn is proportional to the amount of antibody bound from the
original sample.
7. Stop reagent
A. Stops enzyme activity by:
1. Altering the pH
2. Denaturing the enzyme
B. End product of substrate degredation is assayed on the linear portion of the reaction
curve.
- assured by using the stop reagent at a predetermined time or at a
predetermined color intensity of the controls.
C. May intensify or change the color of the product.
8. Time of incubation
A. Short ( <30 minutes ) Timing becomes critical
small variations in incubation time greatly affects assay precision.
Antibody binding - variable
B. Long ( >60 minutes ) Non-specific binding becomes a significant problem.
9. Temperature of Incubation
A. 4oC - Slow, inconvenient. Requires more time. Temperature gradients are a problem.
B. Room Temperature - Adequate binding in 30-45 minutes.
-Convenient
-No temperature gradients
o
C. 37 C - Rapid binding
-less incubation time
-inconvenient
-more non-specific binding
-higher background

66

SOURCES OF ERROR IN THE INDIRECT ELISA

1. Poor Assay Specificity
A. Quality of antigen (purity)
B. Choice of solid phase (high or low binding plastic)
C. Use of unpurified conjugates containing free enzyme
D. Extended incubation periods
2. Low Assay Sensitivity
A. Choice of solid phase
B. Antigen concentration (overcoating)
C. Use of low titer conjugates
D. Use of substrate with poor solubility
E. Presence of enzyme inhibitors in buffers (i.e. azides for peroxidases, phosphates for
phosphatases, metal equipment)
F. Inadequate mixing of highly diluted samples (serum and conjugate)
3. Low Assay Precision
A. Volumetric pipetting errors
B. Inadequate incubation periods
C. Poor endpoint standardization of reaction rate variables (i.e. time, temp, concentration)
D. Operator errors (Work becoming too routine)
COMMERCIAL ELISA's FOR AVIAN SEROLOGY
1. ImmunoComb
A. Less expensive than major commercial systems since it doesn't require a reader and
microcomputer.
B. Self-contained system that can be used on-site. It requires only running tap water to perform
the test.
C. Cost per test is about 2x that of other commercial ELISA's.
D. Good correlation with HI tests for NDV and IBV in our laboratory. IBDV correlation not
done in our lab.
2. IDEXX Laboratories, Inc.
A. Good commercial system that provides precoated and quality controlled plates. All reagents
are ready to use and quality controlled.
B. No dilutions of any reagents are required.
C. Uses TMB as its substrate which replaced the OPD used in previous materials. This substrate
is much less sensitive to temperature than OPD.
D. Large selection of tests (14) provided in the 96-well microtiter plate format.
E. Aflatoxin tests available in the microtiter format and in single test format.
3. Kirkegaard and Perry (KPL) Laboratories
A. Good commercial system that provides precoated and quality controlled plates.
B. All reagents except for the substrate require dilution before use. (This requires about 1
hour of additional time. Many laboratories that have predictable workloads dilute
enough reagents for one weeks work on Monday).
C. Selection of test agents. Not as extensive as IDEXX at the present time.
ELISA TESTS AVAILABLE FOR THE AVIAN (KPL and IDEXX)

67

Newcastle Disease Virus (NDV) Chickens and Turkeys

Infectious Bronchitis Virus (IBV)
Infectious Bursal Disease Virus (IBDV) + Variant
Reovirus (detects both VA and Malabsorption) (REO)
Avian Encephalomyelitis (AE)
Pasteurella (Fowl Cholera) Chickens and Turkeys
Infectious Laryngotracheitis virus (ILT)
Lymphoid Leukosis (Antigen and Antibody)
Mycoplasma gallisepticum (Mg)
Mycoplasma synoviae (Ms)
Mg and Ms Combination Plate (Mg and MS)
Chicken Anemia Agent (CAA)
Avian Influenza (AI)
Aflatoxin B1
Ochratoxin
Rubratoxin
Vomitoxin
Zearelanone
T-2
Gentamicin
Sulfas
Notes:

1. The Turkey Pasteurella ELISA does not correlate with resistance to challenge.
2. The Mg and Ms assays tend to cross react with a variety of bacterial antigens and other nonspecific factors produced as a result of vaccination with oil emulsion vaccines.

APPLICATION
Advantages of the ELISA
1. Rapid (2-3 hrs.)
2. Time Efficient (1 protocol for multiple antigens)
3. Computer controlled
A. Plate reading
B. Titer calculations
C. Report generation
D. Record maintenance
E. Data manipulation
- Move data into other programs * Crosstabulation
* Statistical analysis
4. Reproducible
A. <15% same sample different days
B. <10% same sample same day
5. Sensitive
A. Detects antibody titers earlier than VN or HI.
B. Detects antigens in very low concentrations.
Disadvantages of ELISA

68

1. Test is group antigen specific and not type specific.

(Will not differentiate IBV, IBDV, or REO serotypes)
2. In actual disease management does not provide any better information than the VN or HI tests.
3. Will detect titers earlier but this feature is of no real advantage in most practical application of
serological data.
4. Expensive to set up. Equipment costs range about $14,000 for an automatic reader, washer, and
computer.
MATERIALS COST PER TEST
AGP
$2.25
HI
0.50
VN
2.00
ELISA
0.75
CHARACTERISTICS OF THE ELISA COMPARED TO THE VN OR HI
A. Working Dilution
1. VN and HI use low to moderate working dilution of the test sample.
2. ELISA uses a 1:100 - 1:500 working dilution.
B. Titer range
1. VN and HI use + or - 100% relationships
2. ELISA yields titers that can fall anywhere on a continuum.
C. Reasons for only general correlation of ELISA, VN, and HI.
1. Note graph (Miers Bankowski and Zee (Av.Dis.1983)
2. Adequate sampling necessary to establish correlations.
3. There is no individual sample-to-sample correlation of VN or HI titers with ELISA.
D. Correlation of VN and HI titers with the ELISA
1. Sera segregated on the basis of VN or HI titer.
2. Sera tested by ELISA and mean ELISA titers calculated for each titer level of VN
or HI.
3. Performed Linear regression analysis on titers.
4. Line of Best Fit assigned to data points.
5. Coefficients of correlation determined.
6. NDV-HI, IBV-HI, IBDV-VN, VA-VN all correlated with the ELISA. IBDV was the
best. Coefficients of Correlation all >0.95

Coefficients of Correlation
ELISA and
-------------NDV-HI
IBV-HI
IBDV-VN
REO-VN
CONTROL OF COMMERCIAL ELISA'S

Based on Mean
ELISA Titer
------------------R = 0.98
R = 0.99
R = 0.99
R = 0.97

Based on
Titer Group
----------------R = 0.93
R = 0.97
R = 0.96
R = 0.95

69

1. Plate quality assurance.

A. Intra Assay variation - Same serum sample, same day,
same plate.
-- <8% coefficient of variation in any direction.
B. Inter Assay Variation - Same serum sample, different
day, different plate.
-- <12% coefficient of variation in any direction.
C. Commercial controls
-- Must fall within specifications.
-- Good to include in-house control sera on at least
one plate of each antigen type on any one day.
-- If variation in any one of the commercial
controls then the in-house controls will
confirm or negate a problem.
END POINT STANDARDIZATION AND REPRODUCIBILITY
1. Standardization of End Points ( "Titers" )
A. Stop at preselected absorbance of reference positive
serum.
B. Standardize reaction time (Most commercial ELISA's).
2. Titer calculation
A. Subtractive Method
1. Correct sample readings for background
or negative serum values by subtracting
negative serum absorbance value from each.
B. Sample to Positive Ratio ( S/P )
1. Correct positive control and test sample
absorbances by subtracting the negative control
value from each.
2. Set up ratio of the corrected sample absorbance
value and the corrected positive control value.
EXAMPLE
Sample Abs. - Negative Control Mean
S/P =
----------------------------------------------------Positive Control - Negative Control Mean
Note: This is the simplest method for corrected
titer calculation without resorting
to regression analysis.

70

C. "Titer" Calculation using Regression Formula

-- used by both commercial ELISA manufacturers.
1. Set up sample to positive ratio as above.
2. Insert S/P ratio into regression formula for final "titer" calculation.
EXAMPLE
IDEXX Laboratories
Negative Controls = Wells A1 A2
Positive Controls = Wells A3 A4
Sample Abs. - Mean Neg. Control Abs.
S/P = ------------------------------------------------------------------Mean Pos. Control Abs. - Mean Neg. Control Abs.
Log10 Titer = 1.09 (Log10 S/P) + 3.36
If S/P = 2.42 then:
Log10 Titer = 1.09 (Log10 2.42) +3.36 = 3.78
Titer= Antilog 3.78 = 6003
Note : The factors involved in the "titer" calculations are unique for each and every ELISA system.
Materials from one system cannot be interchanged with another.
PRACTICAL CONSIDERATIONS
1. "Profiling"
A. Single Bleed
B. Flock over time
2. Assessing Mean Titers and Coefficients of Variation.
A. Typical ranges of Coefficients of Variation
Coefficients of Variation
Early
Late
NDV
40-70% 80-150%
IBV
40-70
80-150
IBDV
15-50
45-90
IBDV*40-70
70-120
REO
60-100
80-250
* IBDV Tested at 1:5000
Early - Early secondary response

Late - Waning responses

B. Considerations in Interpreting ELISA Serology.

1. Type of vaccine and combinations used to date.
2. Elapsed time since last vaccination.
3. Age of birds
4. Status of field challenge

71

C. Baseline Data.
1. Essential for accurate interpretation.
2. Data points must be at regular or strategic intervals in the vaccination program.
3. Must consider that data may vary even between houses on the same farm using the
same vaccination program.
D. Relationship of Maternal Antibody Decay
1. Excellent correlation of ELISA and VN or HI titer regression.
E. Sample Size
1. Broilers - 15-20 sera
2. Breeders, Layers - 23-30 sera
3. No less than 15 sera for any purpose

# Sera
--------50
30
15
6

ILLUSTRATION
Mean ELISA Titers of Different Size Samplings
Randomly Selected From a Group of 50 Sera
Mean
% Variation
Variation
Titer
of Group Mean
Indiv. Sample Mean
------------------------------------------------------2427
0%
0%
2838
17%
19%
3109
28%
15%
3366
38%
76%

F. Challenge Studies
-- Challenge studies indicate resistance to
challenge for most IDEXX ELISA tests to occur
in the titer groups 3-4.
-- Repeat of some challenge studies (1990) confirms work of 1986.
CHECKERBOARD TITRATION
The procedure of setting up a 2-way titration from the highest concentration to the lowest
concentration beginning in the upper left hand corner of a microtiter plate and proceeding to the
lower right hand corner. That is, dilution of one reagent occurs from left to right and the second
reagent occurs from top to bottom.

72

QUICK REFERENCE
IDEXX Flockchek ELISA Serology
Protocol
1. Complete worksheet with location and identification of specimens.
2. Remove all test materials from refrigeration and allow to warm to room temperature.
3. Dispense 245 l of Sample Diluent into each well of a non-sterile flat bottom microtiter plate except
for wells A1 - A4. These are control wells.
4. Dispense 5 l of each serum sample into the appropriate wells per the worksheet and mix thoroughly
using the Dynatech plate mixer for 30 seconds. This is a 1:50 dilution. (1st Dilution Plate)
5. Dispense 90 l of Sample Diluent into each well of the appropriate coated plates (Working plate)
except for wells A1 - A4.
6. Transfer 10 l of diluted sample from the 1st dilution plate into the appropriate wells of the coated
(Working Plate) plate except for wells A1-A4 per the worksheet. Be sure to avoid contamination
of wells A1-A4.
7. Mix working plate thoroughly using the Dynatech plate mixer for 30 seconds. Be careful not to
aerosolize the diluted samples. Add 100 l of negative control serum to wells A1 and A2. Add
100 l of positive control serum to wells A3 and A4.
8. Incubate plate 30 minutes at room temperature.
9. Aspirate the contents of all wells or dump into a decontamination bucket. Fill all wells with distilled
water and then aspirate or dump into decontamination bucket. Repeat these wash steps 3 more
times.
10. To all the wells add 100 l of Conjugate (Blue) and incubate 30 minutes at room temperature.
11. While the plates are incubating mix 1 part TMB Diluent with 1 part TMB concentrate.
12. Proceed with 4 washes as in Step 9.
13. Dispense 100 l of prepared TMB solution into each well. Incubate 15 minutes at room
temperature.
14. Add 100 l of Stop reagent (HFl Acid) to each well and read plates at 630 nm.

73

LABORATORY TECHNIQUES FOR

AVIAN MYCOPLASMAS

S. H. Kleven
University of Georgia
Department of Avian Medicine
953 College Station Road
Athens, GA 30602-4875

74

Procedures for Isolation and Identification

Freys medium with swine serum (FMS) is perhaps the most commonly used medium for
avian mycoplasmas, but PPLO medium is also commonly used. (Mammalian mycoplasmas may
or may not do well on these media). Cotton swabs are used to sample the tissue, and the broth
culture and/or agar plate are inoculated. Inoculate the broth tubes vigorously, squeeze out the
excess liquid against the sides of the tube, and discard the swab to avoid bacterial contamination.
Broth cultures are considered to be the most sensitive for most species, but direct inoculation of
plates is often successful, thus removing several days from the isolation and identification
procedure.
Tracheal swabs are generally obtained from live birds, but for M. meleagridis cloaca, phallus, or
oviduct may also be cultured. At necropsy, sinus, air sac lesions, or joint lesions may also be
sampled. For M. iowae, the most reliable specimens are oral or esophageal swabs obtained from
late dead turkey embryos; for older birds cloacal swabs are the preferred specimen, but isolation
after the first few days of age is unreliable.
Incubation is at 37 C; some laboratories prefer to incubate under CO2, but it is probably
of little value. Transfer broth culture to agar medium after evidence of turbidity or pH change to
orange or yellow. With M. gallisepticum and M. synoviae the earliest color change occurs at 3-4
days, but it may occur as late as 14 or more days of incubation; do not discard cultures for 1
month. Do not allow cultures to incubate after they become acid because they may be
inactivated at low pH (especially M. synoviae ). After plating, the earliest that colonies of the
pathogenic species can be observed is 3 days; 4 to 5 days is more common. Colonies are
detected by microscopic examination at low power for characteristic colonies. All cultures are
plated after one week of incubation even if no color change occurs. Incubate agar plates in a
closed container containing a moist paper towel to avoid dehydration.
One of the difficulties in isolating the pathogenic avian mycoplasmas is the common
occurrence of M. gallinaceum (a fermenter) and/or M. gallinarum (a nonfermenter). Broth
cultures which exhibit an acid color change within 24 hours often contain M. gallinaceum or
contaminating bacteria. If colonies develop as early as 24 hours incubation M. gallinaceum
and/or M. gallinarum may be present. Either of these species will rapidly overgrow the
pathogenic species in broth culture if allowed to incubate for 24 hours or more. If either of these
organisms interferes with isolation of the pathogenic mycoplasmas, direct plating on agar may be
successful. The colonies which appear after 24 hours are nonpathogens; do not do
immunofluorescence identification of the culture until at least 3-5 days of incubation to allow
colonies of the pathogenic species to develop. Hyperimmune rabbit serum against these
nonpathogenic species may retard growth in broth long enough to allow growth of the
pathogenic species to develop.
Morphology of colonies is unreliable for identification of mycoplasmas. Colony size and
morphology are variable, even in cloned cultures. Generally, the most rapid method of
identifying isolates is immunofluorescence of colonies. Growth inhibition using filter paper
discs impregnated with hyperimmune serum or immunodiffusion of concentrated cultures against
hyperimmune serum are also reliable, but they are slower and do not work well with mixed
cultures, which are common. If conjugates or hyperimmune sera are not available, biochemical
characterization may allow tentative identification.

75

Frey's Medium
________________________________________________________________________
Mycoplasma broth baseA
22.5 g
Glucose
Swine serum

3g
120 ml

Cysteine hydrochlorideB

0.1 g

Nicotinamide Adenine Dinucleotide (NAD)B

0.1 g

Phenol red (1%)

Thallium acetate (10%)C
Penicillin G potassiumD
Distilled water

2.5 ml
5 ml
1,000,000 units
q.s. 1000 ml

Adjust pH to 7.8 with 20% NaOH and filter sterilize

________________________________________________________________________
A Gibco Diagnostics, Madison, Wisconsin.
B Reduced NAD is required for M. synoviae. A 1 % solution of each is mixed in equal parts and
20 ml is added per liter of medium.
C For potentially contaminated specimens add an extra 20 ml of 1% thallium acetate per liter of
medium to bring total concentration to 1:1500.
D For potentially contaminated material, an extra 2 million units may be added per liter of
medium. 1 g of ampicillin per liter of medium will substitute.
For agar medium use 1% of a purified agar such as ionagar #2, Noble agar, or Difco
purified agar. All components except cysteine/NAD, serum, and penicillin are sterilized by
autoclaving at 121 C for 15 min. Cool to 50 C and aseptically add the above components which
have been presterilized by filtration and warmed to 50 C. Mix and pour plates to a depth of
approximately 5 mm (15 ml for 100 x 15 mm plates).

76

PPLO Broth
PPLO broth without crystal violet (Difco)
Glucose
Swine serum
Fresh yeast extractE
Cysteine hydrochlorideA
Nicotinamide adenine dinucleotide (NAD)A
Phenol red (1%)
Thallium acetate (10%)B
Penicillin G potassiumC
Distilled water
Adjust pH to 7.8 with 20% NaOH and filter sterilizeD,F

14.7 g
10 g
150 ml
100 ml
0.1 g
0.1 g
2.5 ml
2.5 to 5 ml
106 units
q.s. 1000 ml

A Reduced NAD is required for M. synoviae. A 1% solution each of NAD and cysteine is mixed
in equal parts, and 20 ml is added per liter of medium.
B For potentially contaminated specimens, use 5 ml of 10% thallium acetate per liter, and add an
extra 20 ml of 1% thallium acetate per liter of medium to bring total concentration to 1:1500.
Add thallium acetate to the distilled H2O before the other ingredients to prevent precipitation of
protein.
C For potentially contaminated material, an extra 2 x 106 units of penicillin may be added per
liter of medium; 200 mg to 1 g of ampicillin per liter of medium will substitute.
D Alternatively, all ingredients except cysteine/NAD, serum, penicillin, and yeast extract may be
autoclaved at 121 C for 15 min, and the remaining ingredients are added aseptically after
sterilization by filtration.
E Fresh yeast extract (also available commercially) is made by placing 250 g dry bakers' or
brewers' yeast in 1 liter distilled H2O and allowing to soak 1 hr. Heat to boiling, allow to cool,
and centrifuge at 3000 g for 20 min. Decant the supernate and adjust the pH of the fluid to 8.0
with 0.1 M NaOH. Clarify by filtration through coarse filter paper and sterilize by filtration.
Dispense in aliquots and store at -20 C.
F For agar medium, use 1% of a purified agar such as ionagar #2, Noble agar, or Difco purified
agar. All components except cysteine/NAD, serum, and penicillin are sterilized by autoclaving at
121 C for 15 min. Cool to 50 C and aseptically add the above components that have been
sterilized by filtration and warmed to 50 C. Mix and pour plates to a depth of approximately 5
mm.

77

Characteristics of Avian Mycoplasma Species

Mycoplasma
Usual
Glucose
Arginine
Phosphatase
Species
host
fermentation
hydrolysis
activity
_________________________________________________________________________________
A. laidlawii
Various
+
+ or M. anatis
Duck
+
+
M. anseris
Goose
+
M. buteonis
Buzzard
+
M. cloacale
Various
+
M. columbinasale
Pigeon
+
+
M. columbinum
Pigeon
+
M. columborale
Pigeon
+
M. corogypsi
Black Vulture
+
M. falconis
Saker Falcon
+
M. gallinarum
Chicken
+
M. gallinaceum
Chicken
+
M. gallisepticum
Chicken & Turkey
+
M. gallopavonis
Turkey
+
M. glycophilum
Chicken
+
or M. gypis
Griffon Vulture
+
+
M. imitans
Duck, Goose, Partridge
+
M. iners
Chicken
+
M. iowae
Turkey
+
+
M. lipofaciens
Chicken
+
+
M. meleagridis
Turkey only
+
+
M. pullorum
Chicken
+
M. sturni
Starling
+
?
M. synoviaeA
Chicken & Turkey
+
Ureaplasma galloraleB
Chicken
?
_________________________________________________________________________________
A Requires nicotinamide adenine dinucleotide (NAD).
B Splits urea.
1. Use basal medium without glucose or phenol red.
2. Glucose: Use basal medium with phenol red and glucose and adjust pH to 7.8. Positive
reaction is color change from red turning to orange or yellow.
3. Arginine: Add 1% L-arginine monohydrochloride and phenol red and to basal medium
adjust to pH 7.0. Positive reaction is color change from orange turning to deep red.
4. Phosphatase: Prepare agar plates with basal medium incorporating 0.1% sodium
phenolphthalein phosphate. The serum and yeast extracted should be heated at 60 C for 1 hr
to remove any phosphatase activity. After colonies have developed add 5N NaOH and detect
positive reaction by development of a pink-red color.

78

A Fluorescent Antibody Procedure for the Rapid

Identification of Avian Mycoplasmas
1.

Mark an area on an agar plate where colonies are well separated.

2.

Press a plastic cylinder (15 mm long x 13 mm diameter) through the agar so that the
colonies are within a well formed by the cylinder and agar.

3.

Place 2-3 drops of properly diluted, specific conjugate in each well. Replace the petri
dish cover.

4.

Incubate at 37o C for 20-30 minutes.

5.

Gently fill well with PBS and empty with a Pasteur pipette to wash.

6.

Remove cylinder, leaving a circular plug in the agar.

7.

Place a drop of mounting solution (50-90% glycerol in PBS) on a clean slide. Invert agar
plug and place on slide (colonies toward the glass). Up to 4 plugs can be placed on 1
slide. For a microscope with epi- illumination, mount colonies right side up.

8.

Moisten agar plugs with PBS and place a cover slip over the surface.

9.

Examine with 10 x objective, using a u.v. light source with a BG-12 exciter filter and
proper barrier filter (such as 50-41). A dry dark field condenser may also used. This
allows location of colonies by darkfield microscopy before u.v. examination.

10.

Include known positive and negative controls.

REFERENCE:
E. J. Baas and D. E. Jasper. Agar Block Technique for Identification of Mycoplasmas by
Use of Fluorescent Antibody. Applied Microbiology 23:1097- 1100. 1972.

79

HA (Hemagglutination) Test
1.

Using a U-bottom Disposo tray, make initial 1:2 and 1:3 dilutions (for mycoplasma
cultures or allantoic fluid from embryos suspected of being infected with viruses or other
samples, not expected to have high HA activity) or 1:10 and 1:15 dilutions (for HA
antigens or viruses expected to have high HA activity), using HI buffer (phosphate
buffered saline, pH 7.2) as the diluent.

2.

Dispense 50 l of HI buffer in other wells.

3.

Make serial two-fold dilutions, beginning with the first well, using a 50 l microdiluter
(flame after use).

4.

Add 50 l of a 0.5% chicken red blood cell suspension to each well. Mix well.

5.

Cell controls should be included in the test. Add 50 l of a 0.5% RBC suspension to 50
l of HI buffer. Mix.

6.

Incubate for 1 hour at room temperature.

7.

Read results. The end-point will be highest dilution which shows complete
hemagglutination.

EXAMPLES:

16

32

64

128

256

512

1024

1280

2560

5120

NO HA ACTIVITY
10

20

40

80

160

320

640

DILUTION FOR 1 HA UNIT = 1:1280

80

Mycoplasma HI Test
HI tests should include proper controls. If the controls do not behave properly, the test
results should not be read. Controls needed are cell controls, serum controls, antigen control,
and at least one positive and negative control serum.
A.

Setup of test and positive and negative control sera.

1. Using U-bottom Disposo trays, dispense 100 l of HI buffer into first well .
2. Add 10 l of serum (test serum, positive control, negative control) to the first well.
3. Make up 4 units and 8 units of specific mycoplasma HA antigen according to the results
of the HA test or previous HI tests.
4. In the second well dispense 50 l of 8 unit HA antigen.
5. In other wells (third, fourth, etc.) dispense 50 l of 4 unit HA antigen.

1:10

100 l HI buffer + 10 l serum

1:20

50 l of 8 unit HA antigen

1:40
1:80
50 l of 4 unit HA antigen
1:160
1:320

6. Make serial two-fold 50 l dilutions, beginning with the first well. This will result in 4
HA units in each well beginning with the second well. The first well (containing no
antigen) is the serum control.
7. Add 50 l of a 0.5% red blood cell suspension to each well. Use cells from the
homologous species if possible, otherwise use chicken cells. Mix well by gently
scratching the bottom of the microtiter tray.

81

8. Incubate at room temperature for at least 1 hour.

9. Read HI's. The end-point is the highest dilution with complete inhibition of
hemagglutination. For complete inhibition of hemagglutination, the RBC button
should be clear and distinct, the periphery should be clear of all cells, and the edge of
the button should be entire. In some cases where it is difficult to determine the end
point, tilt the tray and observe whether or not at a certain dilution the red blood cell
button runs like the button in the first well (serum control). If this occurs, that is a
positive reaction; the negative reactions will not run.

1:10
1:20
1:40
1:80
1:160
1:320
Titer:
1:80
0
(1:10 dilution is serum control)

No inhibition of HA
and

Partial inhibition of HA

Complete inhibition of HA (HI)

82

B.

Mycoplasma HI antigen Control:

1. Setup:

8u

50 l of 8u HA antigen

4u

50 l of 4u HA antigen

2u

50 l of HI buffer + 50 l of 4u antigen

1u
0.5 u

50 l of HI buffer

0.25 u

2. Make serial two-fold 50 l dilutions, beginning with the third well from the top..
3. Add 50 l of a 0.5% red blood cell suspension (chicken or turkey) to each well. Mix well.
4. Incubate at room temperature for at least 1 hr.
5. Read results, which will tell you whether or not the antigen used was of the correct
strength. You should obtain the following result:

83

8u
4u
2u
1u
.5u
.25u

Somewhat less than 4

units in test (about right).
Exactly 4 units in test.
Complete hemagglutination (HA)
or

Partial HA

No HA

C.

Cell Controls. A cell control is necessary to ensure that the red blood cells used contain
nothing which might cause hemagglutination. Combine 50 l of HI buffer with 50 l of
a 0.5% red blood cell suspension, mix well, and allow to incubate at room temperature
for at least 1 hr. The cell control should show no hemagglutination.

D.

Serum control: The 1:10 dilution of each serum sample contains no antigen; it is the
serum control. There should be no hemagglutination in this well.

Note: Some batches of HA antigen give a background reaction; that is, negative sera may give
the appearance of reacting at titers of 1:20 or 1:40 or even higher. On close examination it will
be noted that inhibition of hemagglutination is not complete; that is, the cells around the button
may not be completely cleared, the edge of the button may not be smooth and entire, and the
buttons will not run when the tray is tipped. In order to avoid such reactions it may be

84

necessary to increase the concentration of the HA antigen, even in excess of 4 units. The
antigen concentration should be strong enough to suppress any tendency of negative sera to
react. This will eliminate false positives, but it is done at the expense of sensitivity.
Increasing the antigen concentration decreases the sensitivity of the HI test; decreasing the
antigen concentration increases the sensitivity. We try to use the lowest concentration of HA
antigen that still gives a solid negative reaction with known negative sera.

Mycoplasma Antigen Production

For plate agglutination or HI antigen, medium prepared with swine or other serum can be used.
However, antigens prepared from medium with artificial liposomes substituting for serum have
improved specificity. If antigen is needed for the production of hyperimmune serum, medium prepared
with calf or other source of serum should be used in order to avoid antibodies against the serum
contained in the growth medium.
After the medium has been prepared and sterilized by filtration it is placed in 3 sterile containers,
each succeeding container containing approximately 10 x the volume of the preceding container. Some
of the medium should be tubed up so that the culture to be used can be started and grown in this media
for adaptation. After the culture has adapted to the medium and is growing well (passing once every 24
hours) the first of the three containers can be inoculated with a 24 hour broth culture of the organism to
o
be used. The amount of inoculum is approximately 1:10 (v/v). Incubate at 37 C. With a rapidly
growing organism you should be able to pass the contents of the first container into the second and the
second into the third at 24 hour intervals. The pH of the last container should not drop below 6.0 before
harvest, but 6.5 is preferable.
The organism can be harvested by continuous flow centrifugation or by centrifuging filled tubes
approximately 30 min at 12,000 x g (equivalent to approximately 9000 rpm on a Sorvall GSA rotor).
Spin, empty, fill and spin until all of the culture has been harvested. The resulting pellet can now be
collected.

Agglutination Antigen:
The pellet is collected and ground up using a "Ten Broeck" tissue grinder, in phenolized
phosphate buffer (pH 6.0). This antigen can be standardized by the packed-cell volume described in the
USDA "Suggested Protocol for Production and Standardization of Plate Agglutination Antigen" or by
the use of a colorimeter with the O.D set on 540 nm. With the colorimeter you need a 1:20 dilution of
the ground up material. By trial make a dilution of your 1:20 dilution that will give you a reading of 7376% transmittance. You now dilute with phenolized phosphate buffer (pH 6.0) your ground up material
by whatever dilution you made of your 1:20 dilution that gave the correct reading. This will be your
antigen, unstained. (A 1:20 dilution of this antigen should read 73-76% transmission). Perhaps a more
accurate method of standardization is to adjust the volume to a 1% packed cell volume by using a
Hopkins tube to centrifuge the antigen at 1500 x g for 90 minutes (equivalent to about 2000 rpm on an
International IEC Model 221 horizontal rotor). Add 1 ml of 1% Rose Bengal per 100 ml antigen (final
concentration = 1:10,000). Store at 4 C. (Do not freeze). Antigens for the production of antisera, for
tube agglutination tests, and for M. meleagridis plate tests, are treated as above except for staining (these
antigens are left unstained). For M. meleagridis or M. synoviae use phenolized phosphate buffer, pH 7.0.

HI Antigen:
The pellet is collected, ground as above, and resuspended in HI buffer (phosphate
buffered saline, pH 7.0 to 7.2). An equal volume of glycerol is added and mixed well. The
amount of buffer used should be such that the antigen will have a sufficient HA titer; generally
the final volume will be less than 1% of the original volume of the broth culture. Do an HA on
the ground up material to determine the HA activity (l unit = the highest dilution in the HA test

85

that gives complete hemagglutination).

The HI antigen should be kept frozen in small
aliquots(3-4 ml) at -70o C.

Preparation of Hyperimmune Serum

Hyperimmune serum is needed for identification of isolates by immunofluorescence,
growth inhibition, or immunodiffusion. Hyperimmune serum against M. gallinarum and M.
gallinaceum is also useful for inhibiting growth of these organisms in broth culture to prevent
overgrowth of the pathogenic mycoplasma species. Rabbits are generally utilized for this
purpose.
One major problem with the preparation of such sera is that medium components
(especially serum) adsorb to the plasma membranes of mycoplasmas in sufficient quantity for
induction of high titers against medium components in the immunized host. This generally
presents little problem for immunodiffusion or growth inhibition tests, but it may be a major
problem for other procedures. One way around this problem is to utilize serum from a species
other than the species of serum used in the growth media. For example, we propagate cultures
for antigen production for producing hyperimmune serum in medium containing calf serum
(adaptation of the cultures to medium containing calf serum is necessary, and growth is
somewhat reduced). Rabbit antibodies against the calf serum do not interfere with procedures
utilizing our regular media, which contain swine serum.
Antigen is then prepared as for agglutination antigen, except no dye is added. The
packed cell volume is adjusted to 5% so that an emulsion containing 1 part aqueous antigen and
4 parts oil will contain a 1% packed cell volume.
The best antibody response is obtained with an emulsion containing complete Freunds
adjuvant. However, the local inflammatory response at the injection site is severe, precluding
use of this adjuvant. We use oils donated by a commercial vaccine manufacturer, but the
formulations described in the literature also work well (see especially publications in Avian
Diseases by Henry Stone).
Rabbits should be pre-bled to obtain a pre-immunization sample. Use at least 2 animals
in case 1 dies or produces unsatisfactory titers. On day 0 inoculate 0.5 ml intramuscularly at 4
different sites (2.0 ml total) and 0.2 ml intradermally at 4 sites (0.8 ml total). Test bleed at 21
days and inoculate 0.5 ml of emulsified antigen intramuscularly at 4 sites. We use a tube
agglutination procedure to evaluate antibody titer, but growth inhibition on agar is also a good
indication of high antibody titer. Rabbits do not generally produce good HI titers or strong plate
agglutination reactions. Test bleed again at 28, 35, and 42 days. When antibody titer is
sufficient the animal may be bled out.
They hyperimmune should be tested for specificity and high titer in whatever test system
will be used. Serum can be stored for long periods at 20 C or colder. Immunofluorescence
conjugates are produced by standard methods.
Reference: Senterfit, L. B. Preparation of Antigens and Antisera. In: Methods in
Mycoplasmology. Vol. 1, S. Razin and J. G. Tully, eds. Chapter F2. Academic Press, New
York. 1983.

86

Mycoplasma PCR Master Mix

(MG-C2, MG-16S; MS-vlhA, MS-16S).
Promega Pre-mix

H20
Promega Mix
R 50 uM
F 50 uM

MG

MS

18 ul
25 ul
1 ul
1 ul

18 ul
25 ul
1 ul
1 ul

Promega Individual Reagents

H20
Buffer 10x
MgCl2 25mM
dNTP 10mM
R 50 uM
F 50 uM
Taq 5U/ul

MG

MS

32 ul
5 ul
4 ul
1 ul
1 ul
1 ul
1 ul

32 ul
5 ul
4 ul
1 ul
1 ul
1 ul
1 ul

Add 5 ul of template to 45 ul of PCR mix.

87

Thermocycler Program
94C

3 min

1x

94C
58C
72C

20 sec
40 sec
1 min

35x

72C
MG13R

5 min
Primer Sequences

= GCT TCC TTG CGG TTA GCA AC

186 bp

MG14F

= GAG CTA ATC TGT AAA GTT GGT C

MSLR

= TCG TCT CCG AAG TTA ACA A

214 bp

MSLF

= GAG AAG CAA AAT AGT GAT ATC

MG-C2-R =TAA ACC CAC CTC CAG CTT TAT TTC C

237-303 bp
MG-C2-F

= CGC AAT TTG GTC CTA ATC CCC AAC A

MS vhla-R

= GCT TCT GTT GTA GTT GCT TC

MS vhla-F

= GAT GCG TAA AAT AAA AGG AT

316-395 bp

88

X. Molecular Techniques

89

Polymerase Chain Reaction

I.

Introduction
-

II.

PCR Reaction
-

III.

Denaturing (~94C)
Annealing (~45-60C)
Extension (~72C)

Essential PCR Components

-

IV.

Conceptualized by Kary B. Mullis in 1984

Multitude of PCR assays derived since 1984
Significant tool in Avian Virology

Primers
15-25 base pairs with a high G/C content (Designed by Computer)
Mg2+
Often controls a Molecular Biologists destiny and will affect primer annealing, product
specificity, primer-dimer artifacts and enzyme activity
Taq Polymerase
Enzyme which makes the elongation reaction possible. Will synthesize a product up to 3kb
(Pfu will synthesize up to 40kb).
dNTPs
deoxynucleotide triphosphates are the building blocks of the reaction (dATP, dTTP, dCTP,
and dGTP)
RT Reverse Transcriptase use in RNA viruses, transcribes RNA CDNA for PCR.

PCR Methodology
-

PCR
Ability to amplify 10 copies DNA into 109 copies of target DNA
RT-PCR
Uses the enzyme reverse transcriptase to make cDNA from a RNA preparation.
Touchdown PCR
Lower annealing temperature in a stepwise gradient (Performed automatically by PCR
machines)
Hot Start PCR

Initiation of the reaction at high temperature ~ 95 C which prevents primer dimers

and other non-specific binding

90

Degenerate PCR

When exact sequence is unknown, primers can be developed with several base pair
degeneracy
-

V.

AP-PCR (Arbitrarily Primed)

Pairs of random non-specific primers are selected which illicit a distinct pattern of target
DNA when ran on a electrophoresis gel
Cloning PCR Products
Vectors have been created to complement and take advantage of Taqs natural terminal
transferase activity, which adds a single 3-A overhang.
Real-Time PCR allows PCR to be run and analyzed using computer software in real time.

PCR and RT-PCR in Avian Virology

Practically every virus mentioned in AVMD 8050
EXAMPLES:
-

Avian Leukosis
Both PCR and RT-PCR assays have been developed for group J specific identification
IBDV
RNA extracted from bursa samples can be amplified by RT-PCR and typed as variants or
classics by RFLP patterns or by sequencing
IBV
RNA extracted from tracheas or allantoic fluid of inoculated embryos is amplified by RTPCR and typed by RFLP patterns, or sequencing
Misc.

"Being a simple little thing PCR tends to work its way into many studies"
Kary B. Mullis
The Polymerase Chain Reaction,
Boston, Birkhauser, 1994.

91

92

ELECTROPHORESIS
Electrophoresis: A standard method of separating, identifying and purifying DNA, RNA,
or polypeptide fragments through an agarose or polyacrylamide gel.
I.

Agarose
A. Molecular Size: The larger the molecule the slower it moves through the gel.
Migration is inversely proportional to the log of its base pairs.
B. Agarose Concentration: The higher the percentage of agarose the slower the
movement of the molecule.
C. DNA Conformation: Ethidium Bromide (an intercalating agent) can differentiate
between superhelical, linear, and circular forms of DNA.
D. Voltage: Migration is proportional to voltage applied (10V per cm).
E. Electric Field: Migration of DNA is kept constant if a constant current is kept.
F. Temperature: Temperature range of 4C to 30C does not effect mobility of DNA.
G. Intercalating Dyes: Ethidium Bromide reduces mobility of DNA by intercalating
between its base pairs. Its fluorescence is needed for identification of DNA molecules.
H. Ionic Strength: Proper ionic strength results in proper electrical conduction and
therefore proper DNA mobility through the gel.

AGAROSE
1. Range of separation.
(approx. 10 to 50kb)
2. Easiness of preparation. Not a
neurotoxin like polyacrylamide in liquid
form.
3. Used in other applications, such as
Northern and Southern blots.
4. One buffer system, that can be
operated at room temperature. Quick and
easy.
II.

PAGE
1. Range of separation.
(approx. 5bp to 500bp)
2. Accommodates much larger quantities of DNA
(Up to 10 g per load).
3. DNA recovered is more pure, although better
agarose is being produced and marketed.
4. Higher resolving power. Has the ability to
separate molecules of DNA whose length differ by
as little as 0.2% (i.e. 1bp in 500bp)

Polyacrylamide
A. Non-denaturing: Separation and purification of fragments of dsDNA.
B. Denaturing: Separation and purification of dsDNA fragments on the basis of thermal
denaturation. dsDNA moves through a gel that contains increasing amounts of
denaturants (urea of formamide). Since the base pair binding is suppressed by the
denaturant, different fragments of DNA denature at different points in the gradient gel.
Therefore, two fragments of identical size can be separated from one another.
C. SDS-PAGE: Sodium dodecyl sulfate binds to proteins that have been denatured by
-Mercaptoethanol and heat. This binding masks the protein with a negative charge.
Electrophoretic mobility of the SDS-protein complex will give the approximate
molecular weight of the protein.

Restriction enzymes or restriction endonucleases are proteins (normally derived from bacteria) that
recognize and bind to specific short nucleotide sequences cutting the double stranded DNA molecule at

93

specific base sequences. The recognition sequence is often a six base pair palindromic (DNA segment
whose 5 to 3 sequence is identical on each DNA strand) sequence, however, recognition sequences
from 4 to 12 base pairs have been observed.
Some restriction enzymes make incisions immediately opposite one another producing blunt ends while
most enzymes make slightly staggered incisions, resulting in DNA products with short, single-stranded
overhands at each end, known as sticky or cohesive ends.
N-N-A-G-C-T-N-N
N-N-T-C-G-A-N-N

----------> N-N-A-G
C-T-N-N
N-N-T-C
G-A-N-N
Alu I
Blunt ends
N-N-G-A-A-T-T-C-N-N ----------> N-N-G
A-A-T-T-C-N-N
N-N-C-T-T-A-A-G-N-N
N-N-C-T-T-A-A G-N-N
Eco RI
Cohesive ends
Restriction enzymes are biochemically classified as type I, II and III. The restriction activities of type I
and II endonucleases are carried out by a single large enzyme complex, which recognize specific DNA
sequences with the restriction sites of actual cleavage located at variable distances from the recognition
sites. The majority of restriction enzymes currently used in molecular biology belong to type II enzymes
with the presence of the cleavage site at very specific sites within or close to the recognition sequence.
A common use for restriction enzymes has been the generation of specific DNA patterns or fingerprint
after digestion of a DNA molecule with one or several restriction enzymes. The digested products are
separated by size and visualized in an agarose gel with ethidium bromide or silver staining after gel
electrophoresis.
Restriction enzyme reaction:
A restriction enzyme reaction contains the targed double stranded DNA molecule (to be digested), a
restriction enzyme and a restriction enzyme buffer. Each restriction enzyme shows a higher digestion
efficiency when used in association with its specific buffer. The restriction enzyme buffers are
commonly supplied at a 10X concentration and contains a buffering agent (usually Tris) to maintain a
constant pH, salt (usually NaCl or KCl) to provide the correct ionic strength, and Mg++ (MgCl2) as a
cofactor for enzyme activity.
Commercially available restriction enzymes usually have activities at 10-20 units/ul. A "unit" is usually
defined as the amount of enzyme needed to digest 1 ug of bacterial virus lambda DNA in 1 hour in a 50
ul reaction. We generally use 10-20 units (1 ul) of restriction enzyme per reaction. This is usually far
more than needed, but this excess assures that complete digestion will occur. Digestion times are
approximately 1.5 hours, but can be lengthened. After the reactions are completed, the digested product
can be stored in the refrigerator until ready to be visualized. Loading dye is added to the DNA digests,
and the samples are loaded onto a gel.

94

Restriction Enzyme Analysis

95

RNA EXTRACTION
(TRIZOL METHOD)
1. In microcentrifuge tube mix 200 :l of infected allantoic fluid or minced tissue with 200
ul of Trizol. Incubate for 5 minutes at room temperature.
2. Add 200 :l of chloroform, vortex and incubate for 5 minutes at room temperature.
3. Microcentrifuge for 5 minutes at 14,000 rpm and transfer upper aqueous solution
containing the RNA into a clean microcentrifuge tube.
4. Add 5 :l of carrier. Vortex.
5. Add 400 :l of isopropyl alcohol (isopropanol), vortex and incubate for 20 minutes at
room temperature or overnight @ -20c.
6. Microcentrifuge for 5 minutes at 14,000 rpm.
7. Decant supernatant and wash precipitate (pellet) with 400 :l of 75% alcohol.
8. Microcentrifuge for 5 minutes at 14,000 rpm.
9. Decant supernatant and resuspend pellet in 50 100 :l of DEP water.
10. Store diluted RNA at -80c until used.

IBDV RNA Kit Extraction Procedure

Kit Needed: Roche High Pure RNA Extraction Kit CAT# 668427
Digest approx. 20mg of tissue overnight in 200ul 2% Sodium dodecyl sulfate (SDS) and 20 ul
of Proteinase K (10mg/ml) in 56 C water bath overnight.
Centrifuge at 3,000 rpm for 5 minutes
Add 400 ul of Lysis buffer to 200 ul of digested sample, vortex
Add entire sample of the provided spin column
Centrifuge 1 min, 13,000 rpm
Discard flow through
For each rxn add: 90ul Dnase I buffer + 10ul Dnase I working soln.
Add to spin column, incubate at room temp (RT), for 15-20 min.
Add 500ul of Wash Buffer I
Centrifuge 30sec, at 10,000g
Discard Flow through

96

Add 500ul of Wash Buffer II

Centrifuge 30 sec at 8,000g
Discard flow through
Add 300ul of Wash Buffer II
Centrifuge 2 min at 13,000 rpm
Discard flow through tube (Dont touch spin column!)
Place spin column in new, labeled 1.5 ml tube
Add 50ul of Elution buffer and let sit one min at RT
Centrifuge at 8,000g for 1 min.
Store RNA at -80C

97

IBDV RT-PCR
One step Method
Using Super Script III One step RT PCR kit (Invitrogen)
1. Thaw specific primers and place on ice. Remove RT-PCR Kit from the -20 C freezer
and place tubes on ice.
2. In a 1.5 ml Eppendorff tube mix the following reaction for each sample you will run:

Dep H20
2x Buffer
SSIII/RT enzyme
Forward Primer
Reverse Primer
Total

3.
4.
5.
6.
7.

6 ul
12.5 ul
0.5 ul
0.5 ul
0.5 ul
20.0 ul

1x

Add 10 ul of your RNA sample 0.2 ml tube.

Place tubes in thermocycler pre-warmed to 98 C for 5 minutes of denaturing.
At end of denaturing, quickly remove samples and place on ice for at least 5 minutes.
Add 20 ul of the above mixture in each reaction tube and mix well.
Run the following program on the thermocycler.

Example of Thermocycler program:

50 C
5:00

94C
3:00

94 C
0:30
50 C
0:30

70 C
0:30

x 40 cycles

70 C
7:00
4C
Hold

98

IBDV Rt-PCR
One step Method
Using Titan One step RT PCR kit (Roche Applied Sciences)
1. Thaw specific primers and place on ice. Remove Titan Kit from the -20 C freezer and
place tubes on ice.
2. In a 1.5 ml Eppendorff tube mix the following reaction for each sample you will run:

Dep H20
RNAse inhibitor
5x Buffer
DTT
DNTP
MgCl2
Primer F (50mM
concentration)
Primer R (50mM
concentration
Titan Enzyme
Total

26ul
1ul
8ul
2ul
1ul
4ul
1ul
1ul
1ul
45ul

3. Label 0.2 ml PCR tubes according to your sample being tested and place 45 ul of the
above mixture in each reaction tube.
4. Add 5 ul of your RNA sample to the appropriate tube and mix well.
5. Run appropriate program on the thermocycler.

Example of Thermocycler program:

95 C
60 C
5:00

5:00

94 C
0:30
54 C
0:30

68 C
2:00

x 35 cycles

68 C
7:00
4C
Hold

99

APPENDIX

100

F-10 - M199 MEDIUM

For 500 ml.
F-10 (1X) Gibco
M199 (10X) Gibco
TPB
Gentamycin (Stock Solution: 50 mg/
gentamycin sulfate/ml)
Fungizone (Amphotericin B)
*Hepes buffer 1M (add last)
DD H20 to

200 ml
20 ml
10 ml
.5 ml
5 ml
6 ml
500 ml

Adjust pH to 7.2 with sodium bicarbonate

TRACHEAL RINGS MEDIUM

(Cook's formula)
For 500 ml
MEM (10X)
Hepes buffer (1.0 M)
Pen/Strept (10.000 units/ml10.000 g/ml respectively)
H20

50 ml
10 ml
435.5 ml

Buffer with NaHCO3 to pH 7.4 for use in a CO2 incubator.

Buffer with 1.0 N NaOH pH 7.2/7.4 for use in a non/CO2 incubator.

101

ALSEVERS SOLUTION
Dextrose
Sodium citrate
Citric acid
NaCl

20.5 g
8.0 g
0.55 g
4.2 g

QS to 1 liter with distilled water. Sterilize by autoclaving at 10 lbs. pressure for 10 minutes.

TRYPTOSE PHOSPHATE BROTH (TPB)

TPB dehydrated (Difco)
Sterile distilled water

14.75 g
500 ml

Mix and sterilize in the autoclave for 15 minutes at 15 lbs. pressure (121
temperature. Store in the refrigerator.

C). Let cool to room

STOCK 0.2% EDTA (10X) IN PBS

(Ethylenedinitrilo)-tetraacetic acid,
Dipotassium salt
PBS
Autoclave
Store at 4 C.

1 gm
500 ml

PBS WITH 0.02% EDTA

PBS
10X EDTA (see above)

450
50

Pour 90 ml aliquots into 100 ml screw cap bottles and store at 4

using.

C. Adjust pH to 7.2 before

TRYPAN BLUE
Physiological saline (.85)
NaC1

0.85 g

Distilled Water

100 ml

Trypan blue (0.4%)

Trypan blue dye

0.4 g

Physiological saline

100 ml

Paper filter before use. Concentrations of 0.1% and 0.2% are also used.

102

TRYPSIN SOLUTION: 0.25% TRYPSIN IN MODIFIED PBS + 0.02% EDTA

PBS with .02% EDTA
2.5% trypsin
NaHCO3 (7.5% solution)
Final pH:

90 ml
10 ml
1.8-2.0 ml
7.4-7.6

PHOSPHATE BUFFERED SALINE (PBS) WITHOUT CALCIUM

OR MAGNESIUM
Sodium Phosphate Dibasic (Na2HPO4)

1.60 gms

Potassium Phosphate

0.51 gms

Sodium Chloride

(KH2PO4)
(NaCl)

7.30 gms

QS to 1 liter with DD water

Autoclave

HBSS WITH ANTIBIOTICS

10X HBSS
Gentamycin (50 mg/ml)
Fungizone(250 g/ml)
DDH2O

50 ml
.5 ml
5 ml
445 ml
500 ml

STOCK HI BUFFER 20 X
KH2PO4
Na2HPO4
NaCL

3.25 g
10.80 g
170.00 g

QS to 1000 ml with distilled water. Dilute 1:20 before using. pH 7.1 to 7.2.

103

CRYSTAL VIOLET SOLUTION

STOCK SOLUTIONS
Solution A:
Crystal violet (90% dye-content)
Ethanol (95%)

2g
20 ml

Solution B:
Ammonium oxalate
Distilled water

0.8 g
80 ml

STAINING SOLUTION
Mix 1 part of solution A and 9 parts of solution B.
ANTIBIOTICS
A. FOR CELL CULTURE
1. Gentamycin (50 mg/ml) - use 1 ml/1000 ml.
Flow's Penicillin-Streptomycin (5,000 I.U./ml
and 5,000 mcg/ml).
USE:
Add 10 ml per 500 ml of medium.
Final concentration: 100 I.U. Penicillin/ml.
100 mcg Streptomycin/ml.
2. Fungizone (Gibco, 250 mcg/ml).
USE:
Add 5 ml per 500 ml of medium.
Final concentration: 2.5 mcg/ml.
B. FOR VIRUS ISOLATION IN EMBRYOS
1. Flow's Pen-Strept.
USE:
Add 4 ml per 100 ml of sample dilution.
Final concentration: 200 I.U./ml.
2. Fungizone (Gibco).
USE:
Add 1 ml per 100 ml of sample dilution.
Final concentration: 2.5 mcg/ml.

104

ERYTHROSIN B STAIN
1.

Prepare the following stock solution:

2.0 gm erythrosin B in 500 ml PBS (0.4% in PBS).

2.

For the working solution prepare a 1:10 dilution of the stock in PBS. No filtering
is necessary.

3.

For counting cells a 1:2 dilution of stain and cells is prepared (1.0 ml of stain and
1.0 ml of cells). Allow four minutes for staining before counting.

4.

For HVT plaque counting stain plates after five days. Use only enough stain to
cover the cell monolayer. After five to ten minutes have passed, pour off the
excess stain or use a Pasteur pipette to remove it and then count plaques. You
may rinse the plates with warm PBS if you desire.

NOTE:

Read HVT plates within one hour of staining. Cell degeneration will
begin after approximately one hour. After cell degeneration has
occurred it is still possible to read the plates, but reading is more
difficult.

Erythrosin B sources:
Sigma Chemical Company
Kodak Laboratory & Research Products