The separation and purification of phages

, purpose requirements
1.Learn the basic principles and methods
separation, purification
bacterphages and diseases.
2.Observe phage spots.II, Basic Principles
Because phages are specialized parasites, the presence of specific phages can be found everywhere in nature, i.e. phages are distributed along with the distribution of host bacteria. For example, feces and gutter sewage contain a large number of E. coli, so it can be easily isolated to E. coli phage;Because the phage invades the bacterial cells and replicates, causing the cell to cleavage, the phage is released from it, so (a) in the liquid
culture
base can make the turbid bacteria suspension become clear, this phenomenon can indicate the presence of phages, can also be used to add sensitive strains and liquid
culture base
in the sample, so that the phage proliferation release, so that it can be isolated to a specific phage; (b) on a solid
gagide
tablet with host bacteria growth, the phage can crack the bacteria to form a transparent empty spot, called phage plaque (Figure XII.-1), a phage produces a phage spot, using this phenomenon can be isolated to the phage for purification and determination of the phytophage price.this experiment is the separation of E. coli phages from cul-de-sour sewage, newly isolated phages are often not pure, such as the form of phage spots, inconsistent size, and so on, and then further purified., equipment 37 degrees C culture 18 hours of E. coli slope, gutter sewage;This experiment used ordinary paste proteinculture base 500 ml triangle flavott containing three times concentrated liquid culture base 100 ml,
test tube
liquid culture base, agar plate, upper agar culture base (including agar 0.7%, test tube sub-pack, 4 ml per tube), the bottom agar plate (including culture base 10 ml, agar 2%) .sterilization straws, sterilized glass coaters, sterilized Cai's bacterial filters, sterilized filtration bottles,
stration temperature
water bath tanks,
vacuum pumps
etc., the operation step 1.Bacillus separation(1) preparation bacteria suspension to take one of E. coli slope, plus 4 ml of sterile water washed down the moss, made into bacterial suspension.(2) multiply culture in 100 ml triple concentrated meat paste proteinliquid medium triangular flavon, add sewage sample 200 ml with E. coli suspension 2 ml, 37 degrees C culture 12-24 hours.(3) prepare the lysate to centrifuge the above mixed culture fluid 2500 r/min for 15 minutes. installs the sterilized Cai's
Filter
device on the sterile filter bottle, the filter bottle and the safety bottle with a rubber tube, and the safety bottle is then attached to the vacuum pump (Figure XII.-2). The vacuum table on the figure can be connected or not connected. the centrifugal liquid into the filter, open the vacuum pump, filter sterilization. The resulting filter is poured into the sterilized triangular burner and cultured overnight at 37 degrees C for sterile examination. (4) confirmed test the presence of phages by sterile tests without bacteria growing filters. (a) add a drop of E. coli suspension to the paste protein agar plate, and then apply the liquid to a uniform thin layer with a sterilized glass coater. (b) after the plate bacteria liquid dried, scattered droplets plus a few small drops of filter on top of the flat bacteria layer, put 37 degrees C culture overnight. If a transparent phage spot with sterile growth is formed at the drip filter, it is proved that there are E. coli phages in the filter. 2. The purification of phages (1) If the presence of phages has been proved, then the inoculation ring filter is inoculated in the liquid media, and then added 0.1 ml E. coli suspension, so that mixed. (2) Take the upper agar media, dissolve and cool to 48 degrees C (pre-meltable, cooled, put 48 degrees C in the water bath spare), add the above phage and bacteria mixture 0.2 ml, immediately mix. (3) and immediately pour into the underlying culture base and spread well. culture for 24 hours at 37 degrees C. (4) At this time the growth of the separation of a single phage spot, its shape, size is often inconsistent, and then with a vaccination needle in a single phage spot, carefully take the phage, access to the liquid medium containing E. coli, cultured at 37 degrees C. (5) after the liquid in the tube is completely dissolved, filtration of sterilization, that is, the purified phage. (above (1) (2) (3) three steps, the aim is to get a single phage spot on the plate, whether the purpose can be achieved, determine the concentration of the phage filter and the amount of filter added to the isolated bacteriophage, preferably In doing sterile testing at the same time, by the teacher to do preparatory tests, if the plate of phage spots in one piece, you need to reduce the amount of inoculation (less than one ring) or increase the amount of liquid culture base; so as not to redo the whole class). 3. The preparation of high-efficiency bacterphages is often not very effective and needs to be multiplied. the purified phage filter and liquid media in a ratio of 1:10 mixed, and then added E. coli suspension appropriate amount (can be equivalent to phage filter or 1/2 of the amount), culture, so that proliferation, so repeated transfer several times, and finally filtration, can get high-efficiency bacteria system products. friday, the experimental report the bacteriophage spot that appears on the tablet.