Virus isolation experiments
Virus isolation experiments
Viral diseases are easily detected in the early stages of the disease, so specimens should be collected as early as possible during isolation.
Operation method
the root cause and symptoms of a disease Move Specimen collection: For more product details, please visit Aladdin Scientific website.
Specimens commonly used for virus isolation include blood, cerebrospinal fluid, feces, rectal swabs, throat gargle, pharyngeal swabs, and autopsy tissue. The type of specimen taken varies depending on the nature of the disease. For example, respiratory diseases often take throat rinse, pharyngeal swabs; digestive diseases take feces, rectal swabs; neurological diseases take cerebrospinal fluid, feces and so on. Specimens should be taken to avoid contamination, some specimens such as feces, throat wash, although it contains a large number of bacteria, but in order to maintain good working habits, should be operated in aseptic procedures, and at the same time to avoid contamination of the specimens between each other and to protect the safety of the staff. The specimen should be placed in a sterilized container, with the patient's name and date of taking affixed to the outside, and at the same time, the specimen should be registered in a special form with the specimen number, the patient's name, sex, age, address, clinical diagnosis, date of onset of disease, symptoms and date of taking the specimen.
Specimen collection methods are as follows:
1. Fecal specimen: Pick the feces as big as the tip of the little finger (about 2 grams) and put it into a sterile wide-mouth bottle or cardboard box.
2. Rectal swab: If there is an urgent need to obtain a fecal specimen for virus isolation, a rectal swab can be taken by using a sterile cotton swab soaked in Hank's liquid or saline, inserted into the rectum, slightly rotated, and then stained with intestinal contents, removed and placed in a test tube containing 2 ml of Hank's liquid or saline.
3, pharyngeal gargle: let the patient cough several times before collection, and then use Hank's liquid (or broth, saline, etc.) 10 ~ 15 ml, 2 ~ 3 times gargle, let the patient tilt his head back so that the liquid in the throat repeatedly rinsed, the gargle spit in a sterile wide-mouthed glass bottle.
4, throat swab: if you want to isolate the virus from the throat, and the patient is a child who can not gargle, two sterile cotton swabs can be used to carefully rub the throat, and then the swabs are put into a test tube containing 2 ml of Hank's liquid or saline.
5, cerebrospinal fluid: for patients with viral infections of the central nervous system, l ~ 2 ml of cerebrospinal fluid can be retained during lumbar puncture and placed in a sterile test tube.
6, blood: in the early stage of the disease (2-5 days after the onset of the disease) from the elbow vein aseptic blood 2-5 ml, put in a test tube containing heparin, mix thoroughly and standby (or directly into the test tube to make a clot).
7, autopsy materials: dead patients autopsy, can be aseptically collected from a variety of organs and tissue specimens put in a sterile vial (or add 50% neutral glycerol saline) standby.
Preservation and transportation of specimens:
1, specimen preservation: specimens should be sent for examination as soon as possible after collection. Before sending to the laboratory can be temporarily stored in the refrigerator at 4 ℃ for preservation. After the specimen is sent to the laboratory, it should be inoculated as soon as possible, or stored in a low-temperature refrigerator below -20 ℃.
2, specimen delivery: most of the virus specimens should be refrigerated transport, the specimen can be wrapped in a plastic bag, and then put the specimen transported in the icemaker (inside the ice or dry ice).
Specimen processing:
Virus isolation is to use live tissue cells, such as the material being examined mixed with stray bacteria, the culture will not be successful, so the material should be processed before inoculation. Specific methods are as follows:
1, fecal specimen: the specimen should be processed as early as possible. The method is as follows: weigh 1 gram of feces and add 4 ml of Hank's liquid (adjust pH 7.2-7.4 with NaHCO3 beforehand); stir and mash the feces with a bamboo stick to make a 20% suspension. The suspension should be kept at -20 ℃ in a low-temperature refrigerator for at least one night; take out the fecal suspension one day before virus isolation, precipitate for 30 minutes at 3,000 rpm, absorb the supernatant, add 1,000 units of penicillin and 1,000 micrograms of streptomycin (hereinafter referred to as the double antibody) per milliliter, and put it in a refrigerator at 4 ℃ for 4 hours or overnight, and then it is ready to be used for virus isolation.
In order to remove the toxic substances in the feces, plant activated carbon can be used. The method is as follows: Prepare 5% Hank's suspension of activated charcoal, shake well and mix well, and then divide each tube into 4ml. Add 1g of feces to each tube to make 20% suspension, place it in 4 ℃ for 2~4 hours to adsorb the activated substances, during which it should be shaken several times, and then place it in -20 ℃ low-temperature refrigerator for preservation after adsorption, and the subsequent steps are the same as the previous ones.
2, rectal swabs: cotton swabs in a test tube containing Hank's liquid on the wall of the pressure squeeze so that the specimen material washed out, centrifuged by 3,000 rpm precipitation for 30 minutes, take the supernatant plus double antibody, placed in 4 ℃ role of 4 hours or overnight, used for virus separation.
3, throat swabs and throat gargle: treatment is basically the same as rectal swabs.
4, cerebrospinal fluid and blood: normal sterile, such as suspected bacterial contamination, can be added to the sample of double antibody, 4 ℃ overnight standby. If it is a blood clot, take it out of the refrigerator before use, grind it in the sterile milk bowl (or put glass sand), and add equal amount of Hank's liquid to make emulsion for separation.
Autopsy specimen: after taking out the specimen, wash off glycerol with sterile distilled water, grind it with sterile milk bowl, and then use Hank's liquid to make l0% suspension by 3,000 rpm to separate the precipitation for 30 minutes, take the supernatant and add the double antibody, put it in 4 ℃ overnight, and then separate the virus on the next day.