I am culturing an anearobic species in serum vials and am adding resazurin to detect oxygen contamination. To be able to monitor the growth, I wanna read the OD of samples taken from the vial over time. While resazurin is a colourish indicator, what wavelength should I choose to prevent conflict with monitoring the turbidity of culture over time(OD)?
Resazurin has two maxima of absorption: 600 & 572 nm http://nopr.niscair.res.in/bitstream/123456789/19122/1/IJEB%2037%288%29%20782-786.pdf
However, resazurin absorption spectra are very sensitive to medium pH. Thus you will have to adjust medium pH to same level in all samples. Additionally, you will have to prepare a calibration curve. Good luck. IB
I would rather recommend that you skip the indicator. The indicator would always be a pain in the neck - as its absorbance would interfer in an unpredicatble way with OD readings, independent, weather you conduct them at 570 nm, 600 nm or other wavelenghts routninely used.
If you work carefully with serum bottles - we use them also for anaerobic cultures and use several cycles of atmosphere exchange prior to incoluation and also carefully take samples through the septum with bottles upside down - to avoid contact of the atmosphere with the gas phase - then you get reproducible conditions without oxygen.
Thanks so much dear Prof Igor and Christoph for ur thorough advise, so I focus more on careful media preparation without adding indicator. Actually am working on SRB enrichment and coz of low growtb I added resazurin to check if the reason is oxygen contamination?
Hi Dear
In my opinion, to identify the prefect wavelength to determine OD of any culture, one has to follow Lambert law. It means, take a sample without inoculum for blank and routine sample for detecting cell growth. Let us start with a wavelength of 580nm having zero absorbance for blank; the actual sample you can have any reading say 0.96. In the next step, just take absorbance of actual sample while varying the wavelength, you can start from 420 go to 600nm. Plot your absorbance readings versus wavelength. You can find only one point as maximum absorbance; that would define your actual wavelength for your specific culture. Finally, you can use blank for the new determined wavelength and then read all OD of next samples. Incase you use diluted sample, also dilute your blank as well. For additional reading please go to my book "Biochemical Engineering and Biotechnology" 2nd Edition Elsevier 2015.
Wish you good luck
When I was growing SRBs, I had a couple of options. As Christopher Wittmann says above, you could leave resazurin out of the medium when you do an experiment and you want to measure OD. Once you have made the medium many times, you get a feel whether it has oxidised -by the slightly darkish tinge that it has (I assume here that you using the bicarb buffered medium with sulfide as a reducing agent?). If you want an extra reducing agent, you can add some fresh dithionite solution immediately before inoculating. If you really want to keep resazurin in you medium and simply want to track OD over time, and you don't need 3 decimal place precision, what I have done in the past is find myself a good quality single beam spectrophotometer that had an adaptor and you could slide the whole Hungate tube tube in and out of the spec directly. The latter suggestion will depend on the exact need of you measurement of course.
Thanks so much dear prof Ghasem for ur kind advice as always. I do follow it up.
Dear Prof Gavin, i have followed urs and prof Chritoph advice and already skipped resazurin in my media. i will do appreciate if you refer me to any of ur publications about SRB enrichment. actually am enriching the SRB on H2:CO2 gas for further trasfering to my system. i just wonder if i stop carbonate buffer solution in that system and keep adjusting pH by a pH contolling system, it would be fine?
Hi Tahereh
I would say in you system some sources of phosphate is required. Controlling pH is also important by all means. try different sources and see the impact of phosphate and other buffer sources.
Regards
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