I can see with bare eyes that my defined medium for bacterial culture has a pretty high opacity, but the OD doesn't exceed 2 at 600nm.I checked the sterility of my glassware and medium, the medium is pretty transparent in the beginning. What else could cause this problem?(I have high glucose concentration and low leucine concentration in my media.)
If you are able to visibly see turbidity in your culture then there is growth. The spectrophotometer calculates the OD based on the blank. If your blank is compomised then your OD readings are not going to be reliable.
If you want to rule out any other possiblities, you can set up a control where you take similar volume of culture media without any bacterial inoculum and run it beside your test culture. If the control is also getting turbid then the problem is with improper autoclaving of the media and the glassware or bad antibiotic selection.
Be sure that you are using sterile medium as your blank for your OD measurements. Having an OD600 of 2 is pretty normal for a dense culture of E. coli, so if you are getting an OD of 2 then it should be fine. Normal overnight cultures are 1.5-2 OD.
Getting an OD600 of 2 is quite high and represents an overly grown culture generally. It is not recommended to measure the cultures OD above 0.9 -1 because that represents overgrowth and after that death phase comes and the detection limit of Spectro is also generally reached. If you still need to measure higher OD then you should dilute your culture and then take OD and then multiply by the dilution factor to get the original OD value. I am assuming you are measuring your samples by normalizing with blank.
- Badges
- Science topic
- Similar topics
- Engineering
- Modeling