Application of Cell Culture Flasks – Lysogenic Bacteria Check

When culturing cells, people required various cell culture consumables, among which cell culture flasks are the most popular ones.

Different types of cell culture flasks:

The adherent cell culture flask is square with a wide neck designed to facilitate cell harvesting.
Cell culture flask sizes are commonly 25cm2, 75cm2, 175cm2, 225cm2, etc.
25cm2 cell culture flask and 75cm2 small format bottles are for cell recovery, and small-scale expansion.
The 175cm2 and 225cm2 larger bottles are for medium-scale cell culture.

Lysogens are bacteria that integrated prophages on their chromosomes and can grow and reproduce normally.

Inoculate Escherichia coli 225(λ) activated by LB slant into a 250ml cell culture flask containing 20ml LB medium
Take 2ml of the bacterial suspension. And insert it into another cell culture flask.
In a 250 ml Erlenmeyer flask containing 20 ml of LB medium, shake the culture for 2 h at 37 °C to log phase.

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If tested the strain is Bacillus, make a spore suspension first, and then treat at 80°C for 10 minutes to kill the phage
If tested the strain is non-Bacillus, washed log-phase lysogens with phage-prepared antiserum or 0.2% sodium citrate solution
Then, after centrifugation at 4000 r/min for 10 min, added supernatant with sensitive indicator bacteria for a double-layer assay
Induction of lysogenic bacteria:
The bacteria collected after washed centrifugation twice with sterile normal saline
And the final concentration was 1011 cells/ml with normal saline or 100 mmol/l Tris-HCL buffer (pH 7.0).
Bacterial suspension, add 5 ml of bacterial suspension to each dish, irradiate 30 cm with a 30W UV lamp for 30 s
Then immediately add 5 ml of double-concentration LB culture solution, and culture at 37°C for 2 hours in the dark after mixing.

Lysogenic bacteria inspection:

Operate according to the double-layer agar method, add a few drops of chloroform to the above induction lysate, and dilute appropriately with the 10-fold dilution method
And select the last 3 dilutions, take 0.3ml for each dilution and 0.2ml for each dilution.
Mixed phase-sensitive Escherichia coli 226 bacterial suspensions
Then LB half medium thawed and cooled to 45°C was added, mixed immediately, and then poured into LB solid medium cell culture flask.
After curing, hold at 37°C for 6 hours. If a large number of plaques appear on the culture plate, it proves that the tested strain is a lysogen.
And to check whether the bacteria are lysogenic bacteria, follow the above steps, pay attention to sterility during operation, and avoid affecting the test results due to improper operation.