Experiments on collection and preparation of biological materials for laboratory animals
Experiments on collection and preparation of biological materials for laboratory animals
The study of the toxic effects of foreign compounds. It is often necessary to determine the concentrations of compounds or their metabolites in the blood, urine and tissues of animals exposed to foreign compounds, as well as to analyze and measure the biochemical changes caused by the compounds. For this reason, the collection and preparation of biological materials is one of the most important basic techniques in food toxicology.
This experiment focuses on the collection and preparation of biological materials commonly used in food toxicology tests.
Operation method
Experiments on collection and preparation of biological materials for laboratory animals
Materials and Instruments
Adult mice, rats, rabbits Move I. Reagents and materials For more product details, please visit Aladdin Scientific website.
Heparin Saline Solution PBS Buffer
Rabbit box Rabbit fixation frame Mouse fixation plate Large scissors Tweezers Pediatric small bone forceps Centrifuge tubes Glass capillary tubes Syringes Suction tubes Droppers Screeders Petri dishes Culture machines Centrifuges Blenders Mouse metabolic cages
(i) Experimental animals: adult mice, rats and rabbits.
(ii) Equipment
1. Rabbit box, rabbit fixation frame, rat and mouse fixation plate.
2、Anatomical equipment large scissors, forceps, pediatric small bone forceps.
3、Other equipment centrifuge tube (2-10 ml), glass capillary tube (inner diameter 1-1.5 ml), syringe (1, 2, 5 and 10 ml) and the corresponding needle, pipette (5-10 ml), dropper, homogenizer, Petri dish (diameter 5-10 ml).
4. Instrumentation Centrifuge (4000r/min), blender (2000r/min), metabolic cages for mice and rats. Animal weighing, electronic balance.
(III) Reagents
1, Anticoagulant 0.5% heparin saline solution.
2, Saline or PBS buffer.
(IV) Others
Iodine, alcohol cotton balls, filter paper.
II. Procedure
(I) Collection of blood
1. Blood collection methods for rats and mice:
① Blood collection from rat's tail: this method is used when the amount of blood required is very small. Fix the animal and expose the rat's tail, immerse the tail in 45~50℃ warm water for a few minutes to make the tail vein congested, wipe it dry, and then disinfect it by rubbing it with an alcohol cotton ball. The tip of the tail (about 0.2~0.3cm) was cut off and the first drop of blood was swabbed. The tail blood was then quantitatively aspirated with a hemosiderin pipette, or the tail blood was dropped directly into a container. After blood collection, a dry cotton ball was used to stop bleeding. Blood can also be collected by directly puncturing the tail vein with a 7-8-gauge hypodermic needle attached to a syringe without cutting the tail.
② Orbital venous plexus blood collection: this method is used when a moderate amount of blood is needed and the death of the animal is avoided. The thumb and forefinger of the left hand hold the neck of the rat or mouse tightly, and press both sides of the neck to make the retro-orbital venous plexus congested, but the force should be appropriate to prevent the animal from asphyxiation and death. The right hand holds a glass capillary tube from the right eye or left eye canthus at an angle of 45°, the depth of penetration is about 2-3 mm in mice and 4-5 mm in rats, if resistance is encountered, slightly backward to adjust the angle and then penetrate again, such as the appropriate puncture, the blood can naturally flow into the capillary tube.
After obtaining the required blood volume, the pressure on the neck was removed, the capillary tube was pulled out, and a dry cotton ball was used to stop the bleeding.
(iii) Blood collection from severed head: This method is used when a large amount of blood is required and there is no need to continue to preserve the animal's life. Hold the animal in the left hand and scissors in the right hand, quickly cut off the head and neck, and invert the animal to let the blood drip into the container. Care should be taken to prevent broken hairs from falling into the container.
2. Blood collection method for rabbits:
① Blood collection from the vein at the ear margin: this method is one of the most commonly used methods of blood collection and can be repeated many times. Fix the rabbit in the rabbit box, pull out the fine hairs of the ear margin to be collected, gently flick the ear with the fingers or irradiate the rabbit's ear with the electric light to expand the blood vessels in the ear, and then disinfect it. Press the root of the ear with the left hand, puncture the vein with a needle or make a small cut on the blood vessel with a razor blade, and let the blood flow out naturally. Blood can also be drawn directly from the vein at the edge of the ear with a syringe. After blood collection, a dry cotton ball is used to stop the bleeding. If it is not easy to stop the bleeding for a while, a wooden clip can be used to hold the ear shell for 10-20 minutes.
② Cardiac puncture blood collection: fix the rabbit in supine position on the rabbit platform or by an assistant, cut the hair on the 2nd to 4th ribs of the left chest, and routinely disinfect it. The left edge of the sternum of the 3rd to 4th ribs was punctured at the most obvious place of the heartbeat, and the blood gushed into the syringe after the needle was inserted into the heart. The needle was inserted into the heart and blood gushed into the syringe. After blood collection, the needle was quickly withdrawn so that the puncture hole in the myocardium could be closed easily and the eye of the needle was pressed with alcohol cotton balls to stop bleeding. In rabbits weighing 2 kg, 10-20 ml of blood can be collected every 2-3 weeks.
③Femoral artery blood collection: fix the rabbit supine on the rabbit platform, straighten the hind limbs of the animal with the left hand, hold the syringe in the right hand, and stab the needle into the femoral artery with the index of vasomotor movement. If the artery is pierced, bright red blood will flow into the syringe. The needle was quickly withdrawn after the blood was drawn, and the bleeding was stopped by pressing with a dry cotton ball.
3, the dog's blood collection method:
① Blood collection from the lateral small saphenous vein of the hind limb and the subcutaneous cephalic vein of the forelimb: this method is most commonly used and convenient. The lateral small saphenous vein of the hind limb is located in the superficial subcutaneous area of the lower 1/3 of the tibia of the hind limb, from the front side to the upper side of the back, and the subcutaneous cephalic vein of the forelimb is located in the front of the forelimb above the dorsal side of the paw. Before the blood was drawn, the dog was fixed on a dog table or made to lie on its side and was secured by an assistant. The hair at the site of blood draw was clipped and routinely sterilized. One person presses hard on the proximal end of the vein or ties it tightly with a tourniquet to fill the vein, and the other person holds a syringe for venipuncture. After obtaining the required amount of blood, the needle is withdrawn and the bleeding is stopped by compression with a dry cotton ball.
② ear margin vein blood collection: when a small amount of blood or for routine blood tests, the dog's ear margin vein blood collection method. After cutting the hair, the dog's ear shell is heated first, or the ear shell is rubbed with a xylene cotton ball, and then the dilated blood vessels are cut with a razor blade, so that the blood drops into the container.
After blood collection, a dry cotton ball is used to compress the cut to stop bleeding.
4. Points of attention for blood collection:
① Excessive amount of blood collected at one time or too frequent blood collection in experimental animals can affect the health of the animals, resulting in anemia or even death, and the maximum safe amount of blood collection is shown in Table 1.
② selection of blood collection methods, mainly depends on the purpose of the experiment and the amount of blood required, the amount of blood required for less can be punctured to take the blood capillaries, when the amount of blood required for more blood can be used as a venous blood collection, if you need to repeat several times the venous blood collection, it should be distal to the beginning.
If anticoagulation of whole blood is required, anticoagulant should be added in advance in the syringe or test tube, commonly used anticoagulants are:
A. Potassium oxalate: commonly used for anticoagulation of blood samples for testing. Add 2 drops of saturated potassium oxalate solution in the test tube, wet the wall of the tube evenly, put it into the oven (80 ℃) to bake dry, and wrap it for spare. Each tube can make 3~5ml of blood not coagulate, blood samples for potassium and calcium content determination cannot be anticoagulated with potassium oxalate.
B. Heparin: Take 0.1ml of 1% heparin solution in the test tube, moisten the inner wall of the test tube evenly, and put it into the oven (80~100℃) to bake dry. Each tube can make 5~10ml of blood not coagulate. Commercially available heparin injection contains 12.500 U of heparin per ml, which is equivalent to 125 mg of heparin sodium.
C. Sodium citrate: 3.8% sodium citrate solution can make 9 portions of blood non-coagulation, used for erythrocyte sedimentation rate determination.
Because of its weak anticoagulant effect and strong alkalinity, it is not suitable for blood samples for laboratory use.
(ii) Separation of serum from cells
1, the preparation of serum will be placed in the refrigerator at 4 ℃ to save 3-4h, 1500-2000r/min centrifugation 15min. supernatant is light yellow that is serum, suck out the standby; such as the supernatant is light red (or even red), suggesting that there is hemolysis, should be discarded in general.
2, cell separation separation of blood cells, blood collection of all vessels are first anticoagulant treatment. That is, the anticoagulant will first be inhaled into all the apparatus, so that the uniform coating on the wall of the apparatus. Blood collection so that the anticoagulant is dissolved in the blood, and mixed. 2000r/min centrifugation 20nim, suck out the supernatant plasma. Between the plasma and the red blood cells, the white blood cells are transferred to a separate centrifuge tube if needed, or discarded if not needed. The erythrocytes left in the centrifuge tube were mixed gently with an equal volume of saline, centrifuged again, and the supernatant was discarded. Wash 3 times until the supernatant is colorless and transparent, then the erythrocytes are obtained. Leukocytes can be treated in the same way.
(iii) Collection of urine
1, a urine collection method: in experimental research, sometimes for some experimental purposes, require every interval of time to collect a urine, such as every 4 hours to observe the excretion of drugs. In this case, the following methods are commonly used to collect urine.
①Forced urine method: this method is applicable to rabbits and cats. The assistant holds the animal, and the operator gradually presses the bladder with his right hand from the abdominal cavity downward to force out the urine.
② urinary catheterization method: this method is applicable to rabbits, cats, monkeys and dogs and other animals, is one of the more commonly used methods. The animal takes the supine position fixed on the operating table, the urethral opening is routinely sterilized. With the left hand to fully expose the urethral opening and fixed, the right hand holding the catheter (tip coated with disinfected petroleum jelly or liquid paraffin) along the urethra gently and slowly inserted into the rabbit inserted about 8 ~ 12cm, once into the bladder cavity, that is, see the urine outflow. If there is no urine outflow, the catheter can be moved up, down, left and right appropriately until the urine flows out, and then the catheter is fixed to the animal body with adhesive tape.
③ Ureteral intubation method: this method is applicable to rabbits, cats, monkeys and dogs. Take rabbit as an example: fix the rabbit in supine position on the operating table after anesthesia, make a 4~6cm incision along the mid-abdominal white line at the upper edge of pubic symphysis, open the abdominal cavity, and find out the left and right ureters on both sides of the base of the bladder, and then separate them and put on two threads under the two ureters, and ligate the near end of the bladder, and then ligate the line to the side of the kidneys.
2、Method of collecting urine continuously
The method of retaining urine in rats and mice: In toxicology experiments on small animals, urine is often collected for 24 hours or for a specific period of time.
For this reason, commonly used metabolic cage with feces and urine separation funnel to collect urine, this device in addition to the bracket are made of glass or plexiglass, easy to clean. The device mainly includes a round plexiglass cage, round glass chassis with holes, for drinking water and food devices, conical urine collection funnel and fecal-urine separator and so on. The animal is placed in the metabolic cage, and the side port of the fecal-urine separator funnel is connected to a 150~200ml urine collection container to collect urine.
Generally within 5~6 hours, an average of 0.4~0.5ml of urine can be collected from each mouse. If the mouse is given a gavage of 0.02 ml per gram of body weight before urine retention, the amount of urine can be increased to 0.7-0.8 ml. In normal rats without water loading, the amount of urine excreted is about 0.5 ml/100 g of body weight/hour.
The components of continuous urine collection devices for cats and rabbits are basically the same as those for rats. However, metabolic cages are often made of wire and enamel. The container for urine collection should be larger.
3. Points to note for collecting urine
① The urine collector must ensure the separation of feces and urine to prevent fecal contamination of urine. The specimen container must be clean, and its capacity depends on the animal.
② specimen collection, must be fresh when the test, if you need to be placed for a longer period of time, it must be stored in the refrigerator or add appropriate preservatives.
③ analysis of metal ions in the urine, the metabolic cage and other metal materials should be avoided, the best urine collection containers with polyethylene material.
④ In order to satisfy the amount of urine required for the experiment, appropriate amount of water and greens can be fed before collecting the urine.
(iv) Preparation of tissue homogenate for experimental animals
Immediately after the animals are executed, take out the required tissues and put them on dry ice. Or placed on ice, gently remove the surface of the clotted blood and clotted tissues and other appendages, and then washed several times by ice-cold saline, absorb the water with filter paper, weighing a certain weight of the tissue for spare. If there is a special need or short-term preservation, it should be put into liquid nitrogen or frozen in the refrigerator.
The stripped and treated organs are quantitatively placed in the homogenizer, and a certain proportion of solution is added according to the design requirements. Take liver tissue homogenization as an example, weigh 1 g of liver tissue, cut it up in a surface dish, dilute it in the homogenizer at 1 : 9 (1 part of liver tissue plus 9 parts of 0.155 M KCl solution), and grind it with an electric stirrer at 3,000 rpm for 2~3 minutes. The sample was then centrifuged at 3,000 rpm for 10-15 minutes at 4°C. The supernatant was taken for measurement. The enzyme activity (GPT or GOT) of the liver tissue homogenate can be determined from the supernatant.
When preparing tissue homogenates for drug extraction of tissues, the basic method is the same as above, but the homogenization is not necessarily performed under freezing conditions. After the tissue mass is ground into a homogenate with a suitable proportion of non-ionized water, the homogenate does not need to be centrifuged. However, it is sometimes necessary to hydrolyze, so that the bound drug into a free state, and then add the organic solvent for extraction, oscillation, extraction, so that the drug or metabolite extraction into the organic solvent, so as to achieve the purpose of separation from the tissue.