Compared with other bacterial biofilm in vitro culture, the microplate method has the advantage of batch treatment, especially in the rapid operation of a large number of samples, so that the test can be maintained, so that the 96-well plate can even detect 384. Confucius. This method is widely used in bacterial biofilms. The microplate quantitative detection method can not only determine if the bacteria can form a biofilm, but also combine different staining methods, and can also quantify the ability of the bacteria to form organisms. This is very beneficial for laboratory biofilm research, and 96-well microplate quantitative detection is a method widely used in the laboratory to quantify the detection of bacterial biofilms.
Main reagents and instruments:
Polystyrene 96-well plate, PBS buffer, methanol, 1% crystalline hydrochromol, 33% glacial acetic acid solution, enzyme symbol or spectrophotometer.
Experimental steps:
(1) 100 μl of the culture solution was added to the respective holes of the 96-well polystyrene microplate culture plate, and 10 μl of the overnight culture solution was inoculated, incubated at 37 ° C for 36 hours.
(2) The culture fluid is sued, and 200 μl of sterile PBS buffer is added to each well, and the flat hole is washed 3 times;
(3) 100 μl of methanol was added to each well for 15 minutes, then the methanol in the culture well was then discharged;
(4) Add 100 μl of 1% crystalline hydrochromol to each well, and it is stained at room temperature for 5 minutes;
(5) After absorbing the crystalline purple colored liquid in the culture medium, the excess dye is flushed with flow water;
(6) Invert the plate on the filter paper to remove the residual water and dried in an oven at 37 ° C or dried at room temperature;
(7) After completely drying, 100 μl of 33% glacial acetic acid solution was added to each well, and 30 minutes were taken for 30 minutes at a thermostat of 37 ° C, and crystalline purple solution.
(8) Measure the OD value of the solution in the culture well by using the enzyme label;
(9) Repeat 3 holes for each strain in each test, and take three mean (D values) as the test value;
(10) The culture solution of the uncharged bacteria was used as a negative control, and the negative value was twice the limit value (DC).
The result is judged:
According to the D value, the strain can be divided into three categories:
(1) Biofilm formation force (D> 2 × DC);
(2) Biofilm formation is weak (DC
(3) No biofilm formation strain (D ≤ DC).