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.
Application of Cell Culture Flask in the Detection of Lysogenic Bacteria:
- Lysogens are bacteria that integrated prophages on their chromosomes and can grow and reproduce normally.
Cell Culture Flask in Lysogenic bacteria culture:
- 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.
Exclude free page:
- 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.