Hi everyone,
I have a question regarding diluting overnight bacterial culture to measure OD. I diluted my overnight culture with PBS. When I measured OD using spectrophotometer I used plain PBS as blank or PBS with media (same dilution as measured soloution) but I got either negative or zero readings. Is using PBS to dilute overnight culture a good choice, or should I use media for dilution?
what was the dilution factor you used to dilute bacterial suspension ? Is the PBS yo used is commercially ready to use or you prepared it ?if you prepared it then was it filtered? And at what wavelength did you tried to obtain O.D at ?
You can use PBS to dilute your overnight cultures. Set the blank using only PBS. Make 10 fold dilutions and then measure the O.D. 600nm.
Why do you dilute with PBS ?
You should dilute in the same media you are using for your cultures, and blank your spectrophotometer with the same (sterile) media.
We do that daily in my lab without any problem.
Paradyumna, I prepared PBS by dissolving one tablet in 100ml water but I did not filter. I measured at 600 OD
Tushar, using PBS as blank gave me negative values.
Martin, my supervisor recommended diluting with PBS, probably bec its cheaper that media!
Hi, you should have no issues with absorbance measurements as long as the blank is prepared as samples. For overnight cultures I usually perform a 1/10 dilution using distilled water, and that works fine. If your cells are sensitive, using PBS, 0.85%NaCl, or any other buffer should work too. Based on what you're saying, I don't see you're doing anything wrong. Some things you can check are:
1. If you have never used that spectrophotometer before, myabe you're placing the cuvettes in the wrong angle. Make sure the transparent side is positioned so that the light beam can pass through it.
2. Make sure you're using a volume enough to fill the cuvette section which will be reached by the light beam. This can vary depending on the type of cuvette you're using, and the spectrophotometer. I use to perform 1/10 dilutions with 60 uL cell suspension plus 540 uL water, PBS or whatever you have, but you have space enough to increase volumes up to 200 uL bacterial culture plus 1800 uL water. try using a greater volume and see if you get good readings like that.
3. Make sure you don't have bubbles, they severily affect the readings.
4. Make sure the medium does'n have any pH indicator or compound, which causes color changes read at 600 nm. I have performed readings without issues using NB, KB, LB, Terriffic broth, 2xYT, M9, AB, MBMA, and a long etc. you should face no issues using almost any kind of standard medium.
If none of these things solve your issues, I would say the spectrophotometer is not working well.
Also, try to use an undiluted culture versus the o/n culture directly. Even though the reading will be not real, you definitely should see an increase of absorvance values up to or higher than 1 (it depends on the bacterial species you're working with, but usually it's like that). If you are still getting weird readings, maybe you need a new spec.
Kindly check https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5497461/
Also check https://www.sciencedirect.com/science/article/pii/S2215017X15000454
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