Determination of viral titer can be applied to measure viral virulence.
Operation method
50 percent endpoint method
Principle
The 50% endpoint method is a series of dilutions of a viral suspension inoculated into animals, chick embryos, or cells cultured into monolayers. The lethal dose or cytopathic lesions in animals or chick embryos at each dilution are curved to find the endpoint dilution that causes 50% of the deaths or cytopathic lesions in the animals or chick embryos. LD50 (50 % Lethal dose) is the amount of virus that causes 50% of animal or chick deaths, ID50 (50 % Infectious dose) is the dose that causes 50% of animal or chick infections, and CCID50 (50 % cell culture infectious dose) is the dose that causes 50% of lesion effects in cell culture. CCID50 (50 % cell culture infectious dose) is the dose that causes 50 % of cell culture lesions.
Materials and Instruments
Virus Move 1. Preparation of cells. Prepare a monolayer of cell culture, observe it under a low-power microscope, select well-grown cells, and proceed according to the "passaged cell culture" method, wash, digest and count the cell monolayer. Prepare cell suspension of desired concentration, the number of cells may vary depending on the cell type, usually 200,000 -300,000/ml. pre-design and label the micro cell culture plate; add cell suspension into micro wells with 200µl per well using a micro spiker. 2. Incubate the cells. Incubate the microtiter plate with cell suspension in a 5% CO2 incubator at 37°C until the cells grow into a good monolayer. 3. Inoculate with virus. 3. Inoculate with virus. Use Eagle's Maintenance Solution containing 2% calf serum as diluent to make serial 10-fold dilutions of the virus to be tested, e.g., 10-1, 10-2, 10-3...... 10-10 . Discard all the culture medium in the culture plate, and use a micro-sampler to add the virus solution of each dilution into each micro-well sequentially and in pairs, 100µl per well, 4 wells for each dilution, and at the same time, set up 4 wells of cell control wells, add no virus, only add dilutions, and put them into 37℃ 5% CO2 incubator for incubation. 4. Observe the results. Observe the cell lesions under the inverted microscope at different times after incubation, such as 18 h, 24 h, 36 h, etc. The degree of cell lesions is indicated. Indicate the degree of cellular lesions. The following symbols are used to indicate the degree of cellular lesions when the whole "monolayer zone" is viewed and the overall situation is weighed: -: Indicates no cytopathy. +: Indicates 1% - 25% of the cells are diseased. ++: indicates that 26% - 50% of the cells are diseased. +++: indicates that 51% - 75% of the cells are diseased. ++++: indicates that 76% - 100% of the cells are diseased. 5. Record the results. The titer of virus-induced cytopathic effect is expressed as 50% cell culture infectious dose (CCID50), i.e., the highest viral dilution that causes lesions in 50% (half) of the cells is 50% infectious units, or CCID50 for short, or sometimes referred to as half the tissue culture infectious dose (50 %). Sometimes it is also referred to as the 50 % tissue culture infectious dose (TCID50). The time at which the cytopathic effect occurs is not identical for different viruses, and is determined by the fact that the lesion in the infected cell no longer develops, while the control cell remains intact. 6.Calculation. (1) Calculation of CCID50 Calculation of CCID50 by the Reed-Muench method (image insert http://res.dxycdn.com/trademd/upload/userfiles/image/2017/08/B15022615761176i7hmtaq3r.png) Calculation of CCID50 by Karber method Formula: lg CCID50< or LD50 , ID50)=L+d(S- 0. 5) L-Logarithm of the lowest dilution of the virus; d - dilution factor, i.e. group spacing; s - sum of cytopathic ratios (excluding the ratio of the lowest dilution cytopathic). Image insert: http://res.dxycdn.com/trademd/upload/userfiles/image/2017/08/B15022616615769hmgu53nbg.png (2) Calculation of ID50 and LD50 The calculation of ID50 is exactly the same as CCID50, but it depends on whether the inoculated animal or embryo is infected or not, and the criteria for determining whether it is infected or not are as follows: rabbits inoculated with swine fever virus have a heat reaction, and chickens inoculated with Newcastle Disease Virus have clumping of blood cells. The calculation method of LD50 is exactly the same as the previous two, except that the judgment criterion is based on whether the inoculated animal or embryo is dead or not. (3) Conversion of CCID50 and PFUs If the same cell line is used for the CCID50 test and the Plaque Forming Units (PFUs) test, 1 ml of viral solution will produce PFUs equal to about half of the CCID50 titer. This is the amount of infection that will result in at least 1 viral vacuole on a monolayer of cells. In actual measurement, more than one empty spot may be present on a single cell layer, so the number of empty spots is calculated according to the actual number of empty spots measured. If derived from the mathematical formula, the expected PFUs would be more than half of the CCID50 titer. This is because at the time of the CCID50 measurement, negative means that the number of empty spots on a monolayer of cells is 0, and positive means that 1 or more empty spots are present on a monolayer of cells. According to the Poisson distribution formula P(O) =e(-m), where P(O) represents the proportion of negative wells and m represents PFUs/ml, because P(O) = 0.5 for any CCID50, e(-m) =0. 5,m= -ln 0. 5≈0. 7 . So 0. 7 multiplied by the titer of the CCID50 is the expected PFUs. Caveat Because the cells can be affected by a variety of non-specific factors and lesions, so be sure to observe the control group of cells, and then observe the experimental group of cells, at the same time in the observation of the cells with or without lesions, should also pay attention to the following issues： （1） the phenomenon of non-adaptation: the first isolation of the virus or the laboratory cryopreservation of the virus for too long, when it will be infected with the cells, often at the beginning of the lesions do not appear, but continue to blindly pass on that will appear lesions.(2) Lesionlessness: Some viruses are able to multiply in cells but do not show lesions.(3) Cell-carrying phenomenon: Because many animals and chicken embryos often have "latent viruses" that cause cell-carrying, special attention should be paid to this problem when isolating viruses. In addition, mycoplasma contamination is also sometimes encountered, which is a more difficult problem to deal with. A good cell monolayer should have clear cell boundaries, fine and uniform intracytoplasmic particles, and no fused cells. Common Problems Cellular lesions are mainly the following 1. The whole cell is changed, often manifested in two cases, one is, the nucleus and the whole cell are swollen, the cytoplasm is granular changes, the edge of the cell membrane is not neat; the other is, the whole cell is wrinkled, rounded until the fragmentation of the shedding and so on. This kind of lesion is seen in many viruses, such as enterovirus, sore virus, enterovirus, rhinovirus, coxsackievirus and so on; 2. cell aggregation: e.g. adenovirus; 3. Cell fusion to form syncytia, i.e., most of the diseased cells fuse with each other to form "giant cells", but the nuclei of individual cells are still distinguishable, e.g., Paramyxovirus, Scarabella virus; 4. Cells with only minor lesions, e.g., orthomyxoviruses, rabies viruses, coronaviruses, retroviruses, and arboviruses. For more product details, please visit Aladdin Scientific website.
Eagle's Maintenance Solution with 2% calf serum
Cell Culture Chambers, Cell Culture Plates