Experiments on bacterial culture in liquid medium
Experiments on bacterial culture in liquid medium
Source of content: compact guide to molecular biology 5th ed.
Operation method
Experiments on bacterial culture in liquid medium
Materials and Instruments
Glass bottles Flasks Move I. Overnight culture For more product details, please visit Aladdin Scientific website.
1. Transfer 5 ml of pre-warmed liquid medium into a sterile 16 mm or 18 mm tube.
2. Pick a single colony with an inoculating ring, submerge it in the culture medium and gently vibrate the ring so that the bacteria to be inoculated are dispersed in the culture medium.
3. Cover the tube and incubate at 37°C on a shaker or rotary drum incubator at 60 r/min until saturation ( 1X109~ 2X109 cells/ml , >6 h).
Second, large-volume culture
1. Dilute overnight cultures with freshly pre-warmed culture medium at a ratio of I1: 100 in a sterile Erlenmeyer or baffle flask, which should be more than 5 times the volume of the culture medium.
If the culture is not shaken, the volume of the flask used should be more than 20 times larger than the volume of the culture solution to ensure adequate aeration.
2. 37°C with vigorous shaking culture (about 300 r/min).
III. Monitoring growth using counting plates
1. Cover a clean counting slide (or hemocytometer) with a clean coverslip.
2. Carefully place a small drop of culture solution onto the edge of the coverslip so that the culture solution can spread under the coverslip.
3. Observe under a phase contrast microscope at 400x and calculate the bacterial concentration on the basis that each bacterium seen in a small square corresponds to approximately 2X107 cells/ml . 
IV. Monitoring growth using a spectrophotometer
Because of the variability of instruments, the spectrophotometer should be calibrated with a known concentration of culture solution.
1. Measure the OD600. To obtain an accurate reading, dilute the culture solution to an OD600 < 1.
2. Calculate the bacterial concentration as approximately 108 cells/ml per 0.1 OD.