Rajalakshmi Engineering College Department of Biotechnology Notes | EduRev

: Rajalakshmi Engineering College Department of Biotechnology Notes | EduRev

 Page 1


Rajalakshmi Engineering College 
   Department  of Biotechnology 
 
 
STAFF NAME : M.SANKAR(Lecturer) BT33 
SUBJECT CODE : 2408 IMMUNOLOGY LAB   Sub code: BT2408 
Semester : VII SEC A 
 
Experiment No: 1:          Handling of animals 
                    
Date:  
 
Aim:   To study the environmental conditions and methods involved in the 
maintenance of experimental of animals 
 
The animal species like rabbit, rat and mice are the most favorite choices of lab 
animals for immunological experiments. These animals are used in immunological 
experiments because of their genetic variation, different size, high fertility rate, short 
gestation period, easy maintenance under lab conditions and resistance to many 
infections. 
Procedure: 
Environmental Condition for Maintenance of Animals:  
This is studied by visiting the animal house.  
1. Light: 
Experimental animals are kept at 12 hrs of light and 12 hrs of dark cycle. Provide 
cool white fluorescent lamp in the room where the experimental animals are kept that 
approximates natural sunlight. 
2. Temperature: 
Keep the laboratory animals at a temperature of 20 – 25
o
C, because fluctuations in 
temperature can create problem for bleeding stream and therefore experimental 
variations. 
3. Humidity: 
Be sure that the animals are not kept in very dry condition (<40% humidity), 
otherwise it will increase the incident of dust and related upper respiratory tract 
Page 2


Rajalakshmi Engineering College 
   Department  of Biotechnology 
 
 
STAFF NAME : M.SANKAR(Lecturer) BT33 
SUBJECT CODE : 2408 IMMUNOLOGY LAB   Sub code: BT2408 
Semester : VII SEC A 
 
Experiment No: 1:          Handling of animals 
                    
Date:  
 
Aim:   To study the environmental conditions and methods involved in the 
maintenance of experimental of animals 
 
The animal species like rabbit, rat and mice are the most favorite choices of lab 
animals for immunological experiments. These animals are used in immunological 
experiments because of their genetic variation, different size, high fertility rate, short 
gestation period, easy maintenance under lab conditions and resistance to many 
infections. 
Procedure: 
Environmental Condition for Maintenance of Animals:  
This is studied by visiting the animal house.  
1. Light: 
Experimental animals are kept at 12 hrs of light and 12 hrs of dark cycle. Provide 
cool white fluorescent lamp in the room where the experimental animals are kept that 
approximates natural sunlight. 
2. Temperature: 
Keep the laboratory animals at a temperature of 20 – 25
o
C, because fluctuations in 
temperature can create problem for bleeding stream and therefore experimental 
variations. 
3. Humidity: 
Be sure that the animals are not kept in very dry condition (<40% humidity), 
otherwise it will increase the incident of dust and related upper respiratory tract 
infections. High humidity reduces the resistance of animal to airborne infections. The 
suitable humidity is 55 ± 15 % 
4. Air Conditioning: 
Provide proper ventilation sufficiently to remove the products by respiration, odor and 
excess body heat. Also, constantly do air washing to remove dust and other particles 
 
 
 
generated by the movement of workers and animals. Always provide the 
ventilation system not less than 15 complete changes of air every hour. 
5. Cage Maintenance: 
 The standard size of the cage for the mice is 29.5 x 22 x 14 cm which can hold 
upto six adult mice. Sterilize all the cages by wiping with 70% ethanol, or autoclaving 
before use. Provide bedding with paddy husk which should be sterilized before use.  
6. Diet and Water: 
The basic nutritional requirements are diet and water for the animals. The feeding 
bottles in different sizes for different types of animals are available commercially with 
suckling tube. Always change the water every day. Be sure that the diet basically meets 
the basic requirements such as proteins, carbohydrates, fats and vitamins, and minerals. 
Keep pelleted feeds, which are available in the market. The daily feed intake of various 
animals is mice- 5 gms, rats- 15 to 30 gms, and rabbits-100 to 150 gms 
7. Handling and Re-straining of Rat and Mice: 
 Rats/Mice are picked up by the tails. However, care must be taken to hold the tail 
at the base, close to the rats body. Place one hand over the animal back with the thumb 
and forefinger gently by firmly pressing its forelegs towards its head. 
8. Handling and Re-staining of Rabbit: 
 Rabbits are characteristically timid and excitable. Occasionally they resist 
handling. Rabbits can be restrained physically by manual or mechanical technique. 
Gentle but firm manual restrain often causes the rabbit to relax and stop struggling. To 
remove a rabbit from a cage, grasp the scrub the neck with one hand and support the 
hindquarters and back with the other. 
9. Identification and numbering of experimental animals: 
Page 3


Rajalakshmi Engineering College 
   Department  of Biotechnology 
 
 
STAFF NAME : M.SANKAR(Lecturer) BT33 
SUBJECT CODE : 2408 IMMUNOLOGY LAB   Sub code: BT2408 
Semester : VII SEC A 
 
Experiment No: 1:          Handling of animals 
                    
Date:  
 
Aim:   To study the environmental conditions and methods involved in the 
maintenance of experimental of animals 
 
The animal species like rabbit, rat and mice are the most favorite choices of lab 
animals for immunological experiments. These animals are used in immunological 
experiments because of their genetic variation, different size, high fertility rate, short 
gestation period, easy maintenance under lab conditions and resistance to many 
infections. 
Procedure: 
Environmental Condition for Maintenance of Animals:  
This is studied by visiting the animal house.  
1. Light: 
Experimental animals are kept at 12 hrs of light and 12 hrs of dark cycle. Provide 
cool white fluorescent lamp in the room where the experimental animals are kept that 
approximates natural sunlight. 
2. Temperature: 
Keep the laboratory animals at a temperature of 20 – 25
o
C, because fluctuations in 
temperature can create problem for bleeding stream and therefore experimental 
variations. 
3. Humidity: 
Be sure that the animals are not kept in very dry condition (<40% humidity), 
otherwise it will increase the incident of dust and related upper respiratory tract 
infections. High humidity reduces the resistance of animal to airborne infections. The 
suitable humidity is 55 ± 15 % 
4. Air Conditioning: 
Provide proper ventilation sufficiently to remove the products by respiration, odor and 
excess body heat. Also, constantly do air washing to remove dust and other particles 
 
 
 
generated by the movement of workers and animals. Always provide the 
ventilation system not less than 15 complete changes of air every hour. 
5. Cage Maintenance: 
 The standard size of the cage for the mice is 29.5 x 22 x 14 cm which can hold 
upto six adult mice. Sterilize all the cages by wiping with 70% ethanol, or autoclaving 
before use. Provide bedding with paddy husk which should be sterilized before use.  
6. Diet and Water: 
The basic nutritional requirements are diet and water for the animals. The feeding 
bottles in different sizes for different types of animals are available commercially with 
suckling tube. Always change the water every day. Be sure that the diet basically meets 
the basic requirements such as proteins, carbohydrates, fats and vitamins, and minerals. 
Keep pelleted feeds, which are available in the market. The daily feed intake of various 
animals is mice- 5 gms, rats- 15 to 30 gms, and rabbits-100 to 150 gms 
7. Handling and Re-straining of Rat and Mice: 
 Rats/Mice are picked up by the tails. However, care must be taken to hold the tail 
at the base, close to the rats body. Place one hand over the animal back with the thumb 
and forefinger gently by firmly pressing its forelegs towards its head. 
8. Handling and Re-staining of Rabbit: 
 Rabbits are characteristically timid and excitable. Occasionally they resist 
handling. Rabbits can be restrained physically by manual or mechanical technique. 
Gentle but firm manual restrain often causes the rabbit to relax and stop struggling. To 
remove a rabbit from a cage, grasp the scrub the neck with one hand and support the 
hindquarters and back with the other. 
9. Identification and numbering of experimental animals: 
Marking experimental animals for identification is prime importance in laboratory 
studies. Identify the individual animal by cage marking, staining the skin with picric acid 
or Indian ink, saffronin, use of punch holes or notches on the ear, ear tags and IR sensors 
etc. 
 
 
 
 
Experiment No: 2                               Immunization                          
Date:  
Aim:   To study the various routes of immunization to introduce antigens in 
to animal body. 
Procedure:  
Antigen can be introduced into an animal body by a variety of routes. The main 
consideration while selecting route is the nature of antigens. Given below are the 
different routes of administration of the antigens. 
1. Intradermal: 
This route is generally used for injecting viscous and slowly dispersing forms of 
antigen. E.g. Antigen emulsified with Freund?s adjuvant. It provides rapid access to the 
lymphatics. Shave off carefully the area to be inoculated in order to avoid any abrasion 
and clean with 70% alcohol. Fill the syringe with solution to be injected and make sure 
that no air bubble is trapped in the barrel of needle hub. Held a fold of skin between 
forefinger and thumb and insert the point of the needle just below the surface. Release the 
skin fold holding the needle firmly at its point of entry. Inoculate a maximum volume of 
0.05 ml antigen. Withdraw the needle slowly by compressing simultaneously along the 
track of the needle with forefinger and thumb. A hard pea is swelling at the site of 
injection indicates the true indradermal injection. 
2. Subcutaneous: 
 This route is suitable for emulsions, precipitates and viscous materials and the 
antigens spread a little more as compared to intradermal mode of injection. Held the 
mouse against wire grid and restrain it by holding mouse tail with right hand. Grasp a 
fold of skin over the back near the neck region between the palm and 4
th
 and 5
th
 finger of 
Page 4


Rajalakshmi Engineering College 
   Department  of Biotechnology 
 
 
STAFF NAME : M.SANKAR(Lecturer) BT33 
SUBJECT CODE : 2408 IMMUNOLOGY LAB   Sub code: BT2408 
Semester : VII SEC A 
 
Experiment No: 1:          Handling of animals 
                    
Date:  
 
Aim:   To study the environmental conditions and methods involved in the 
maintenance of experimental of animals 
 
The animal species like rabbit, rat and mice are the most favorite choices of lab 
animals for immunological experiments. These animals are used in immunological 
experiments because of their genetic variation, different size, high fertility rate, short 
gestation period, easy maintenance under lab conditions and resistance to many 
infections. 
Procedure: 
Environmental Condition for Maintenance of Animals:  
This is studied by visiting the animal house.  
1. Light: 
Experimental animals are kept at 12 hrs of light and 12 hrs of dark cycle. Provide 
cool white fluorescent lamp in the room where the experimental animals are kept that 
approximates natural sunlight. 
2. Temperature: 
Keep the laboratory animals at a temperature of 20 – 25
o
C, because fluctuations in 
temperature can create problem for bleeding stream and therefore experimental 
variations. 
3. Humidity: 
Be sure that the animals are not kept in very dry condition (<40% humidity), 
otherwise it will increase the incident of dust and related upper respiratory tract 
infections. High humidity reduces the resistance of animal to airborne infections. The 
suitable humidity is 55 ± 15 % 
4. Air Conditioning: 
Provide proper ventilation sufficiently to remove the products by respiration, odor and 
excess body heat. Also, constantly do air washing to remove dust and other particles 
 
 
 
generated by the movement of workers and animals. Always provide the 
ventilation system not less than 15 complete changes of air every hour. 
5. Cage Maintenance: 
 The standard size of the cage for the mice is 29.5 x 22 x 14 cm which can hold 
upto six adult mice. Sterilize all the cages by wiping with 70% ethanol, or autoclaving 
before use. Provide bedding with paddy husk which should be sterilized before use.  
6. Diet and Water: 
The basic nutritional requirements are diet and water for the animals. The feeding 
bottles in different sizes for different types of animals are available commercially with 
suckling tube. Always change the water every day. Be sure that the diet basically meets 
the basic requirements such as proteins, carbohydrates, fats and vitamins, and minerals. 
Keep pelleted feeds, which are available in the market. The daily feed intake of various 
animals is mice- 5 gms, rats- 15 to 30 gms, and rabbits-100 to 150 gms 
7. Handling and Re-straining of Rat and Mice: 
 Rats/Mice are picked up by the tails. However, care must be taken to hold the tail 
at the base, close to the rats body. Place one hand over the animal back with the thumb 
and forefinger gently by firmly pressing its forelegs towards its head. 
8. Handling and Re-staining of Rabbit: 
 Rabbits are characteristically timid and excitable. Occasionally they resist 
handling. Rabbits can be restrained physically by manual or mechanical technique. 
Gentle but firm manual restrain often causes the rabbit to relax and stop struggling. To 
remove a rabbit from a cage, grasp the scrub the neck with one hand and support the 
hindquarters and back with the other. 
9. Identification and numbering of experimental animals: 
Marking experimental animals for identification is prime importance in laboratory 
studies. Identify the individual animal by cage marking, staining the skin with picric acid 
or Indian ink, saffronin, use of punch holes or notches on the ear, ear tags and IR sensors 
etc. 
 
 
 
 
Experiment No: 2                               Immunization                          
Date:  
Aim:   To study the various routes of immunization to introduce antigens in 
to animal body. 
Procedure:  
Antigen can be introduced into an animal body by a variety of routes. The main 
consideration while selecting route is the nature of antigens. Given below are the 
different routes of administration of the antigens. 
1. Intradermal: 
This route is generally used for injecting viscous and slowly dispersing forms of 
antigen. E.g. Antigen emulsified with Freund?s adjuvant. It provides rapid access to the 
lymphatics. Shave off carefully the area to be inoculated in order to avoid any abrasion 
and clean with 70% alcohol. Fill the syringe with solution to be injected and make sure 
that no air bubble is trapped in the barrel of needle hub. Held a fold of skin between 
forefinger and thumb and insert the point of the needle just below the surface. Release the 
skin fold holding the needle firmly at its point of entry. Inoculate a maximum volume of 
0.05 ml antigen. Withdraw the needle slowly by compressing simultaneously along the 
track of the needle with forefinger and thumb. A hard pea is swelling at the site of 
injection indicates the true indradermal injection. 
2. Subcutaneous: 
 This route is suitable for emulsions, precipitates and viscous materials and the 
antigens spread a little more as compared to intradermal mode of injection. Held the 
mouse against wire grid and restrain it by holding mouse tail with right hand. Grasp a 
fold of skin over the back near the neck region between the palm and 4
th
 and 5
th
 finger of 
the right hand. Release the tail and lift the mouse in the left hand, clean the skin of belly 
with 70% alcohol and held the fold of the skin between thumb and finger. Insert the 
needle into the pocket of skin, lying behind the skin fold by taking care not to pierce the 
peritoneum. After the desired volume is injected, withdraw the needle and pinch the 
needle track to avoid antigen loss due to package. 
 
 
 
Intramuscular: 
It is one of the most frequently adopted group of immunization and suitable for 
alum precipitated and adsorbed antigens and normally the antigen is deposited in the 
muscular layer e.g. thigh muscles. Insert a needle in the rear, at right angle to the skin 
surface at the point halfway along the femur, so that its point lies within the muscle. After 
the inoculation is made, withdrawn the needle and massage the site is gently. 
Intraperitonial Route: 
This route is generally used for injecting viscous forms of antigens. Place the 
animal on a wire surface and grasp the tail with little forefinger of the left hand around it. 
The animal will anchor on the wire and pull in opposite direction. Pick up the animal by 
grasping the skin over the nape of neck with the index finger and thumb of the left hand. 
Turn the palm so that the animal is in the supine position. Wet the fur slightly at the 
centre of the abdomen with alcohol and expose the small skin area by brushing aside the 
wet fur. Standing in front of the animal, point the syringe upward and insert the needle in 
the peritonial cavity. Penetration is indicated by the abrupt of the cessation of resistance 
against the needle. Refract the needle slightly inorder to reduce the possibility of injection 
into the intestine. Inject the material and quickly withdraw the syringe. 
Intravenous: 
 It is good for particulate antigens and is not advisable for viscous and non-
aqueous and immiscible antigens. Incase of rodents, tail vein is used for intravenous 
inoculation. Rub the tail vein with cotton soap in xyline for vasodialation of veins. Fill 
the syringe with the antigen to be injected, hel the tail in the left hand and insert the 
needle directly along the line of the vein. Gently press on the plunger after syringe until 
the required volume is released. 0.2 ml is generally recommended. 
Page 5


Rajalakshmi Engineering College 
   Department  of Biotechnology 
 
 
STAFF NAME : M.SANKAR(Lecturer) BT33 
SUBJECT CODE : 2408 IMMUNOLOGY LAB   Sub code: BT2408 
Semester : VII SEC A 
 
Experiment No: 1:          Handling of animals 
                    
Date:  
 
Aim:   To study the environmental conditions and methods involved in the 
maintenance of experimental of animals 
 
The animal species like rabbit, rat and mice are the most favorite choices of lab 
animals for immunological experiments. These animals are used in immunological 
experiments because of their genetic variation, different size, high fertility rate, short 
gestation period, easy maintenance under lab conditions and resistance to many 
infections. 
Procedure: 
Environmental Condition for Maintenance of Animals:  
This is studied by visiting the animal house.  
1. Light: 
Experimental animals are kept at 12 hrs of light and 12 hrs of dark cycle. Provide 
cool white fluorescent lamp in the room where the experimental animals are kept that 
approximates natural sunlight. 
2. Temperature: 
Keep the laboratory animals at a temperature of 20 – 25
o
C, because fluctuations in 
temperature can create problem for bleeding stream and therefore experimental 
variations. 
3. Humidity: 
Be sure that the animals are not kept in very dry condition (<40% humidity), 
otherwise it will increase the incident of dust and related upper respiratory tract 
infections. High humidity reduces the resistance of animal to airborne infections. The 
suitable humidity is 55 ± 15 % 
4. Air Conditioning: 
Provide proper ventilation sufficiently to remove the products by respiration, odor and 
excess body heat. Also, constantly do air washing to remove dust and other particles 
 
 
 
generated by the movement of workers and animals. Always provide the 
ventilation system not less than 15 complete changes of air every hour. 
5. Cage Maintenance: 
 The standard size of the cage for the mice is 29.5 x 22 x 14 cm which can hold 
upto six adult mice. Sterilize all the cages by wiping with 70% ethanol, or autoclaving 
before use. Provide bedding with paddy husk which should be sterilized before use.  
6. Diet and Water: 
The basic nutritional requirements are diet and water for the animals. The feeding 
bottles in different sizes for different types of animals are available commercially with 
suckling tube. Always change the water every day. Be sure that the diet basically meets 
the basic requirements such as proteins, carbohydrates, fats and vitamins, and minerals. 
Keep pelleted feeds, which are available in the market. The daily feed intake of various 
animals is mice- 5 gms, rats- 15 to 30 gms, and rabbits-100 to 150 gms 
7. Handling and Re-straining of Rat and Mice: 
 Rats/Mice are picked up by the tails. However, care must be taken to hold the tail 
at the base, close to the rats body. Place one hand over the animal back with the thumb 
and forefinger gently by firmly pressing its forelegs towards its head. 
8. Handling and Re-staining of Rabbit: 
 Rabbits are characteristically timid and excitable. Occasionally they resist 
handling. Rabbits can be restrained physically by manual or mechanical technique. 
Gentle but firm manual restrain often causes the rabbit to relax and stop struggling. To 
remove a rabbit from a cage, grasp the scrub the neck with one hand and support the 
hindquarters and back with the other. 
9. Identification and numbering of experimental animals: 
Marking experimental animals for identification is prime importance in laboratory 
studies. Identify the individual animal by cage marking, staining the skin with picric acid 
or Indian ink, saffronin, use of punch holes or notches on the ear, ear tags and IR sensors 
etc. 
 
 
 
 
Experiment No: 2                               Immunization                          
Date:  
Aim:   To study the various routes of immunization to introduce antigens in 
to animal body. 
Procedure:  
Antigen can be introduced into an animal body by a variety of routes. The main 
consideration while selecting route is the nature of antigens. Given below are the 
different routes of administration of the antigens. 
1. Intradermal: 
This route is generally used for injecting viscous and slowly dispersing forms of 
antigen. E.g. Antigen emulsified with Freund?s adjuvant. It provides rapid access to the 
lymphatics. Shave off carefully the area to be inoculated in order to avoid any abrasion 
and clean with 70% alcohol. Fill the syringe with solution to be injected and make sure 
that no air bubble is trapped in the barrel of needle hub. Held a fold of skin between 
forefinger and thumb and insert the point of the needle just below the surface. Release the 
skin fold holding the needle firmly at its point of entry. Inoculate a maximum volume of 
0.05 ml antigen. Withdraw the needle slowly by compressing simultaneously along the 
track of the needle with forefinger and thumb. A hard pea is swelling at the site of 
injection indicates the true indradermal injection. 
2. Subcutaneous: 
 This route is suitable for emulsions, precipitates and viscous materials and the 
antigens spread a little more as compared to intradermal mode of injection. Held the 
mouse against wire grid and restrain it by holding mouse tail with right hand. Grasp a 
fold of skin over the back near the neck region between the palm and 4
th
 and 5
th
 finger of 
the right hand. Release the tail and lift the mouse in the left hand, clean the skin of belly 
with 70% alcohol and held the fold of the skin between thumb and finger. Insert the 
needle into the pocket of skin, lying behind the skin fold by taking care not to pierce the 
peritoneum. After the desired volume is injected, withdraw the needle and pinch the 
needle track to avoid antigen loss due to package. 
 
 
 
Intramuscular: 
It is one of the most frequently adopted group of immunization and suitable for 
alum precipitated and adsorbed antigens and normally the antigen is deposited in the 
muscular layer e.g. thigh muscles. Insert a needle in the rear, at right angle to the skin 
surface at the point halfway along the femur, so that its point lies within the muscle. After 
the inoculation is made, withdrawn the needle and massage the site is gently. 
Intraperitonial Route: 
This route is generally used for injecting viscous forms of antigens. Place the 
animal on a wire surface and grasp the tail with little forefinger of the left hand around it. 
The animal will anchor on the wire and pull in opposite direction. Pick up the animal by 
grasping the skin over the nape of neck with the index finger and thumb of the left hand. 
Turn the palm so that the animal is in the supine position. Wet the fur slightly at the 
centre of the abdomen with alcohol and expose the small skin area by brushing aside the 
wet fur. Standing in front of the animal, point the syringe upward and insert the needle in 
the peritonial cavity. Penetration is indicated by the abrupt of the cessation of resistance 
against the needle. Refract the needle slightly inorder to reduce the possibility of injection 
into the intestine. Inject the material and quickly withdraw the syringe. 
Intravenous: 
 It is good for particulate antigens and is not advisable for viscous and non-
aqueous and immiscible antigens. Incase of rodents, tail vein is used for intravenous 
inoculation. Rub the tail vein with cotton soap in xyline for vasodialation of veins. Fill 
the syringe with the antigen to be injected, hel the tail in the left hand and insert the 
needle directly along the line of the vein. Gently press on the plunger after syringe until 
the required volume is released. 0.2 ml is generally recommended. 
Foot Pad: 
This method can be used inoculate particulate and cellular antigens. Clean the 
foot pad with 70% alcohol and insert 26g needle either distal or proximal direction to a 
depth of about 5 mm in to the foot pad. A volume of upto 25µl can be injected by this 
method. 
 
 
 
Experiment No: 3              Raising of antiserum to BSA in Rats 
 
Date:  
 
Aim:   To raise antiserum to bovine serum albumin in rat 
 
Materials required: 
Rat, Adjuvant (peanut oil), BSA (0.5%), 70% Ethanol, 1 ml syringe, 18 gauge 
opaque needle. 
Principle: 
Many soluble, native protein derived animal/plant source as antigenic although 
they vary in their capacity to stimulate antibody production. Antiserum against soluble 
native proteins can be raised by repeated injection of protein dissolved in normal saline or 
antigen adjuvant emulsion. 
 Adjuvant may be described as a substance that when mixed with an antigen prior 
to injection enhances antibody production. Adjuvants are used in the case of antigen of 
low immunogenicity. 
Procedure: 
Prepare an emulsion of 0.5% BSA in equal volume of oil. Inject 0.5 ml of 
emulsion into rats intraperitonially or intramuscularly or intradermaly. Inject the final 
dosage after seven days. Allow the animal to bleed, collect the serum and check for BSA 
antibody. Antiserum prepared in this way is used for immunological experiments such as 
agglutination or immunoelectrophoresis or immunodiffusion etc. 
Result: 
 Rats are immunized by intradermal/intraperitonial/intramuscular injection. 
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