Experimental reagent 1. Culture medium: common culture medium 2. Others: 0.5% formaldehyde saline, 0.5% lime salt water, sterile physiological saline Laboratory equipment standard turbidimetric tube, centrifuge, etc. Experimental Material 1. Animal: Rabbit 2. Strains: Typhoid fever H901, Typhoid fever O901 Experimental procedure 1. Preparation of antigen: Selection of standard strains: The strains of typhoid fever H901 and typhoid fever O901 should have typical morphological colonies and biochemical reactions. Self-aggregation does not occur in physiological saline, and those with high specific agglutination with specific serum can be used as strains. 2. Preparation of bacterial solution 1) Stock solution preparation: Preparation of H bacterial solution: Inoculate qualified H. typhi strains in common agar in a Kirschner bottle or large test tube and incubate at 37 ° C for 18-24 hours. Visually inspect for the growth of contaminants and perform microscopic examination if necessary. Wash the moss with sterile formaldehyde physiological saline, put the washed liquid into a sterile test tube, and put it in a 37 ℃ thermostat for 18-24 hours to sterilize. Get the stock solution. For the sterility test, the bacterial solution is inoculated in broth and agar medium and cultured for 4 days. Only those without viable bacteria can use it. Preparation of O bacterial solution: After cultivating qualified strains of typhoid bacillus H according to the above method, wash the moss with sterile 0.5% lime salt water, put the washing solution into a sterile test tube and place in a 37 ° C incubator The stock solution was sterilized in 24 hours. It can be used when the sterility is checked (for example, the O. typhimurium O strain can also be used to prepare the O antigen. The H bacterial solution is washed with 0.5% lime acid physiological saline and placed in a 100 ° C water bath for 60 minutes to destroy its H antigen is O Bacterial fluid. 2) Preparation of application liquid: The qualified stock solution was counted with a standard turbidimetric tube, and then diluted with physiological saline to contain 1 billion bacteria per ml. It is to add appropriate amount of formaldehyde to the application solution to make it 0.25%. The prepared stock solution and application solution are kept in the refrigerator at 2-10 ℃ for use, and the validity period is 1 year. Calculation of bacterial solution concentration and dilution method: The concentration of the bacterial solution can be determined by McFarrent's standard turbidimetry method as follows: a. Prepare 1% sulfuric acid solution and 1% barium chloride solution respectively b. Take 10 test tubes with the same caliber and the same texture, add sulfuric acid and barium chloride solution as shown in the table below, seal the tube mouth, and indicate the number for future use. Tube number 12345678910 1% BaCl2 (ml) 0.10.20.30.40.50.60.70.80. 91.0 1% H2SO4 (ml) 9.99.89.79.69.59.49.39.29.19.0 Equivalent bacteria (100 million / ml) 36912151821242730 c. Place the washed bacterial stock solution in the same test tube as the turbidity tube and dilute it. Compared with the standard turbidimetric tube, the turbidity is equivalent to the number of the turbidimetric tube, and then the number of bacteria in the turbidimetric tube is multiplied by the dilution factor to obtain the number of bacteria contained in each milliliter. If the turbidity of the bacterial stock solution is diluted 1: 5 and the third tube is equivalent, the number of bacteria per milliliter of the stock solution is 9ⅹ5 = 4.5 billion. d. Dilution of bacterial solution 3. Immunization method 1) Choose 2-3 healthy male rabbits weighing 2-3 kg, and collect 1 ml of blood from the ear vein to separate the serum. Do an agglutination test with bacteria for immunization to determine the presence or absence of natural lectins. If there is no or trace, the animal can be used for immunization. 2) Inject the diluted antigen into the rabbit ear vein according to the following dosage and procedure. Date Day 1 Day 5 Day 10 Day 15 Day 20 Injection dose 0.5ml, 1ml1, 5ml, 2ml, 2.5ml 3) Seven days after the last injection, 1 ml of blood was collected from the ear vein and the serum was separated. Use the above-mentioned bacterial solution as a test tube agglutination test to titrate the titer of the antibody in the antibacterial serum. If the titer is not high, the bacterial solution can be injected incrementally again 1-2 times, and then blood test is performed. Carotid artery bleeding can be done if the blood test is qualified (see appendix for methods). Separate the serum, add preservatives (such as mercury sulfide of 1 in 10,000) and store at 4 ° C until use.
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