The preservation of parasite specimens, in addition to making slide specimens for permanent preservation, can also be used for needle insertion method, low temperature preservation and other preservation methods. The preservation of protozoa with liquid nitrogen has the advantage of maintaining the biological characteristics of protozoa and preserving them for a longer time.
Operation method
concentration method
Materials and Instruments
Protozoa Encystment Eggs Worms Insects Protozoa Move I. Preservation of protozoan encystments and eggs For more product details, please visit Aladdin Scientific website.
Mercury, iodine, aldehyde, saline, pasteurization, alcohol, menthol, glycerin.
Slides Coverslips Cotton paper Cotton Waxes Liquid Nitrogen Tanks
Mercury iodine aldehyde solution 10 ml and feces 1 g mixed and sealed in a bottle, can be preserved in which the protozoan cysts and eggs for several months, can also be concentrated in the fecal sludge treated with equal or 1 times the amount of 10 % formaldehyde solution (heated to 70 ℃), shaking, and paraffin sealing the bottle.
II. Preservation of adult worms
1. Nematodes
After washing in saline, fix them in 70 % alcohol or pasteurization solution (3 parts formaldehyde, 97 parts saline) heated to 70-80 °C. After cooling, transfer them to fresh 70 % alcohol or pasteurization solution for storage. Small nematodes (e.g. trichinella, pinworms, hookworms, etc.) should be fixed with glycerol alcohol (95 ml of 70 % alcohol, 5 ml of glycerol) and stored in 80 % alcohol; glacial acetic acid can also be used for fixation for about half an hour and then transferred to 70 % alcohol or glycerol for storage.2. Trematodes
Small trematodes can be placed in a vial with saline, shaken vigorously for a few minutes, poured off the saline and filled with fixative. Larger trematodes should first be placed in menthol alcohol (menthol 24 g, 95 % alcohol 10 ml), so that the muscle relaxation of the body, with a slide flattened and then fixed, or put the washed trematodes between two slides with a fine line tightly tied and flattened and then fixed.Generally, 10 % formaldehyde is used for fixation, and after 24 hours it is transferred to 5 % formaldehyde for storage; or it is fixed in 70 % alcohol for 0.5-3 hours, depending on the size of the worm, and then transferred to new 70 % alcohol for storage.
3. Tapeworms
Large tapeworms (e.g. pig and cow tapeworms) can be washed several times with water, and then after several hours or overnight in saline (4 ℃), the body of the tapeworms can be fully stretched, and then they can be fixed in 3% formalin, and then transferred to 5 % formalin for preservation after 24 hours, or they can be fixed in a large glass plate with a large flattened plate first if necessary. Small tapeworms can be washed and fixed in 3 % formalin for 3 to 5 hours, lightly pressed on a slide, fixed in 5 % formaldehyde for a few hours along the edge of the coverslip, and finally stored in 5 % formalin solution.
III. Preservation of insects
1. Dry specimen preservation is the preservation of adult winged insects.
Special insect needles can be used to insert the body of the insect. For large insects (flies, gadflies, etc.), use a No. 1 to No. 3 insect needle and insert it straight from the back of the body, to the right of the mesothorax (Figure 21-9). Be careful to keep the left side intact for identification. Small insects (mosquitoes, lacewings, gnats, midges, etc.) can be inserted with a short No. 00 needle from the ventral surface of the thorax between the bases of the two mesopods, without piercing the dorsal surface of the thorax, and then inserted from the other end of the cork piece with another long needle. Finally, a piece of cardboard is inserted in each case to record the name, place and time of collection, and inserted on the cork board of the insect box or on the cork of the glass tube. A paper wrapper of camphor powder is sufficient for the insect box. If the number of specimens (Figure 21-9), can be stored in a plastic tube (or glass tube), the bottom of the tube put a small amount of camphor powder, and then lay a layer of cotton, filter paper, insect specimens on the filter paper, soft cotton paper wrapped in cotton, gently stuffed in the top of the specimen, the mouth of the bottle with a cork, and then sealed with wax.
Fig. 1 Pin-insertion method for winged insects
2. Wet specimen preservation is used to preserve the egg and larval stages of winged insects and the developmental stages of wingless insects and ticks.
Live specimens are first fixed in heated 70 % alcohol (60-70 °C) and then stored in 5 % glycerol alcohol (7 %) for one day; they can also be fixed in 5 or 10 % formalin and Bless solution.
All preserved specimens shall be recorded in detail with the name of the specimen, the host, the place of collection, the date of collection and the name of the collector.
Camphor mixture preparation: 6 parts of camphor powder, 1 part of chloroform, 1 part of wood distillate, 4 parts of paraffin oil. Firstly, 1.5 parts of camphor powder and 1 part of chloroform are mixed, then 1.5 parts of camphor and 1 part of wood distillate are added, mixed well with a glass rod, and finally 3 parts of camphor powder and 4 parts of paraffin oil are added, and then stirred well for spare. This mixture is flammable, should be placed in a tight and airtight bottle storage.
Commonly used fixative preparation:
(1) Alcohol: 70 % or 75 % alcohol is usually used as fixative. Dilute with 95 % alcohol from a commercial supply. Take 95 % alcohol and add it to the desired dilution, then add distilled water to 95 ml; alternatively, it can be diluted as follows.
Alcohol concentration required / original alcohol concentration x amount of prepared alcohol = amount of original alcohol required (ml)Amount of prepared alcohol - amount of raw alcohol required = amount of distilled water to be added (ml)
(2) Formalin (37 % to 40 % aqueous solution of formaldehyde)
The concentration of commonly used fixative is 5 % to 10 %. If you need to maintain neutrality, in the preparation process, you can add about 2 cm thick magnesium carbonate to 500 ml of the original solution (37 % to 40 % of aqueous formaldehyde), or put a few small pieces of marble (calcium carbonate) and neutralize it; you can also use the following formula (10 % formalin): 100 ml of the original solution of formalin, 900 ml of distilled water, 4 g of sodium bicarbonate ( Na2H2PO4-H2O ), 0.5 g of sodium bicarbonate (Na2H2PO4) and 0.5 g of sodium bicarbonate (Na2H2PO4) mixed and prepared. Disodium bicarbonate ( Na2H2PO4 ) 0.5 g mixed.
(3) Bless (Bless) liquid: 7 ml of original formalin, 90 ml of alcohol (70 %), glacial acetic acid (added before use) 3 to 5 ml mixed and formulated.
IV. Cryopreservation of protozoa
The preservation of protozoa with liquid nitrogen has the advantages of maintaining the biological characteristics of protozoa and preserving them for a longer period of time. Now we only introduce the cryopreservation methods for Plasmodium, Toxoplasma, Trichomonas and Trichomonas vaginalis.
1. Freezing methods
Plasmodium falciparum: Anticoagulant worm-containing blood or in vitro cultured cultures collected from infected persons should be centrifuged at 1500 rpm for 10 minutes, and 24% dimethylsulfoxide (DMSO) saline solution (24 ml of DMSO in 76 ml of 0.9% saline or 5 % glucose saline) should be added as a protective agent equal to the amount of deposited cells, and the mixture should be mixed well and left at room temperature for 30 minutes, then packed into sterile ampoules (or plastic tubes) in batches of 0.5~1.0 ml. 0.5~1.0 ml was divided into sterile ampoules (or plastic tubes), sealed (or capped tightly) and put into a gauze bag with the batch number labeled, and then put into the lifting tube of the liquid nitrogen tank, and then placed in the neck of the tank, which was at about -70 ℃, and then placed into the liquid nitrogen (-196 ℃) for freezing after 30 minutes.
Plasmodium falciparum: Blood was taken from the heart of mice infected with Plasmodium falciparum on the 3rd to 4th day (or blood was taken from the eyeballs), injected into test tubes, anticoagulated with heparin, and added with equal amounts of 10 or 15 % DMSO and 15 or 20 % calf serum as protective agents; or added with equal volumes of glycerol and sorbitol protective solution (180 ml of 4.2 % sorbitol saline and 70 ml of pure glycerol), and mixed thoroughly. Tube and freeze as above. Alternatively, after anticoagulation with 0.1 mm3 of 3.8 % sodium citrate in 1 mm3 of positive mouse blood, the blood can be packed in liquid nitrogen canisters and immediately stored directly in liquid nitrogen, with the majority of protozoa remaining viable for 2 years.
Toxoplasma gondii: 2 ml of 10 %DMSO was aspirated with a sterile syringe and injected into the peritoneal cavity of mice infected for 4 days, pumped and washed twice, and then injected into sterile plastic tubes (0.5-1 ml/tube) as soon as the extracted liquid was mixed well, and the method of freezing was the same as above.
Trichomonas vaginalis: Vaginal secretions were taken with a sterile swab and incubated in culture medium for 48 hours, then transferred to RPMI-1640 medium for 2 days. The culture medium containing the worms was centrifuged at 1000 rpm/min for 10 minutes, and 2 ml of 10 % DMSO was added to the precipitate, which was divided and frozen as above.
Trichinella humanis: Worm-containing stools from diarrhea patients were cultured in Rockwell's medium for 48 h. The number of worms (up to 7 × 105 ) was counted after 2 days of incubation in Rockwell's medium or RPMI-1640 medium. An equal amount of 10 % DMSO was added and Tween-20 was added to the DMSO. The tubes were mounted as above. However, the tubes should be frozen at -10 °C for 15 min, transferred to the neck of a liquid nitrogen tank for 2 min, and then frozen in liquid nitrogen.
The protective agents (liquids) used above should be autoclaved and stored in a refrigerator at 4 ℃. 50 % DMSO was prepared in RPMI-1640, and then diluted to the desired concentration when used by adding 15 % or 20 % calf serum and adjusting the pH to 7.2.
2. Recovery and observation
After freezing for 1~2 years, take out the seed-preserving tubules from the liquid nitrogen tank when needed, and quickly put them into 37~40 ℃ warm water, which will be dissolved after 4~5 minutes. Take the frozen Plasmodium falciparum and Toxoplasma gondii and inoculate 2 mice through the peritoneal cavity respectively, 0.2 ml for each, and observe the pathogenicity; or take the blood or peritoneal fluid of the mice 4-5 days after inoculation, make a smear, stain with Giehl's solution, and microscopically examine the protozoa. Two kinds of Trichinella can be resuscitated in the same way, but need to be cultured for 3~4 days, and then make smears for microscopic examination of active trophozoites, or staining observation.