Table 2.
Problem | Possible reason | Possible remedy |
---|---|---|
Low or no growth in overnight Vibrio cultures and during regular growth cycle | High inoculum from the frozen glycerol stocks; Contaminated culture tubes; Low ratio of growth flask to culture | Inoculate very low volume from frozen glycerol stocks, make sure to use clean and sterile culture tubes; for growth curves, make sure that the ratio of culture / vessel volume is at least 1:5. |
Contamination in motility agar plates | Moisture accumulation | Use freshly prepared plates, as the swimming plates cannot be stored inverted causing moisture-based contamination issues. Make sure to dry the plates at 37°C prior to inoculation. |
No colonies with the intended genetic change (gene deletion or complementation) after genetic manipulation, only wild type colonies | Lack of homologous recombination | For streaking colonies on to MMM sucrose plates, use a very low initial inoculum from the MMM Cm plates. Giving enough time (24-48h) to obtain well, isolated colonies from low inoculum is crucial thus encouraging double crossover and homologous recombination providing the intended mutant colonies. |
No colonies after gentamicin protection assay/ No intracellular bacteria in invasion assay / No cytotoxicity in LDH assay | T3SS2 not induced | Use freshly prepared bile salts solution and make sure the Vibrio cultures are grown at 37°C which is crucial for T3SS2 induction. It is also important to use fresh MMM plates (use within a month of preparation) as the salts precipitate in the agar after prolonged storage at 4°C. |
Emulsion/ brown coloration in sample buffer after attempted resuspension of precipitated protein | Too much NaOH | Be mindful of 10M NaOH droplets that cling to the outside of the pipette tip before adding NaOH, as the base is highly concentrated and too much can prevent the protein precipitate from properly resuspending. Supplementation of 10M HCl in 0.2μL increments or increasing the volume of SDS sample buffer may help, but often it is better to retry the experiment. Diluting NaOH to 5M in MilliQ deionized water may help prevent this outcome as well. |
Low hit count of proteins on mass-spectrometry analysis of secreted proteins | Collagen contamination, Not enough precipitated protein | Use clean PPE, rinse gloves and all tubes with 70% MeOH before use, and prepare samples in a positive pressure hood; Increase induction culture volume to 75mL or greater as needed |