If I want to know the cell concentration in a sample (by absorbance), shouldn't I do first a separation by means of centrifugation to get rid of debris? in many protocols it is explained that I should try many dilutions and then do a curve, but what about other proteins and debris that might affect the reading? isn't it necessary to do a separation first? Also if I want to fix cells, should I measure the OD before or after fixation? Thank you.
In most cases, an OD600 measurement. zeroed against fresh medium as the other respondents have pointed out, is entirely sufficient. If you have cell debris around, centrifugation and resuspending won't help much because a good deal of the debris will pellet with the cells. Dilutions are only necessary if the OD goes above 1, in which case your OD measurement may not be accurate any more. If people are telling you to do dilutions, they are probably confusing OD measurements with viable counts, where you spread dilutions of the culture on agar plates and count the colonies. If you have a culture that is full of debris, this may be the only option. I would avoid centrifuging such a culture as the debris may make the pellet 'claggy'' and cause the cells to stick together.
Typically you take the OD straight from cell culture, without washing or spinning down. However don't forget to sero/baln/Baseline with the raw media before you take your OD. you also take your OD before fixing. If you are fixing your cells for microscopy your should Harvest the cells at early Log phase such as OD600=0.1-0.3. This will give you optimum density. Don't forget to wash the cells AFTER the OD and BEFORE the fixing. Phosphate Buffered Saline is usually good for that.
Hi Vanessa,
I agree with Sophia. Inoculate the bacterial strain onto a broth medium and incubate it overnight to get a log phase of the organism. Alternatively, you could do a fresh subculture of the isolate and emulsify in normal saline or nutrient broth and then take the OD (absorbance). Take OD before fixing, wash and then fix.. Best wishes
Any suggestions, if the bacterial culture produces granular turbidity. Is there any alternate to vortex the suspension and take OD as soon as possible.
I agree with the above, take the OD straight from the cell culture without washing or centrifugation.
Bikash, what bacteria/medium are you working with? If you are using E.coli and LB you should not get granules. If you are working with a sifferent species, there are often tricks to prevent granules/clumps forming, such as using baffled flasks when shaking or adding high % sucrose or PEG to prevent clumping. However, it depends on the bacterial strain.
Thank you for all your comments, I'm working with Bordetella pertussis.
Sophia, I am recently working with E. coli and Streptomyces. As you mentioned there is no problem with E. coli.
But if I measure OD from the cell culture and take the raw media as a baseline, isn't it the same if I separate the cells and then do the measurement? I mean, I'd skip the step of taking raw media as a baseline.
Hi Vanessa,
You raised a concern about proteins and debris affecting the reading. I would not be worried about proteins - they don't absorb much at 600 nm. Regarding debris - assuming you mean cellular debris, this would not start appearing significantly until much later in the growth phase of the culture - so again not something to worry about too much. If your cells tend to clump, then you might have to take precautions like Sophia suggested.
Good luck!
Just as what Sule said, you shouldn't worry about proteins because of the range of the absorbance. But you can make a separation by means of centrifugation to get rid of debris firstly. You can find many researchers measure OD after centrifugation and then wash and resuspend using this method. And adjusting baseline is the first and important step before you do this work.
Hi, taking OD600 measurements and get a good reading requires that you perform a growth curve for the organism you use and get the concentration of your bacteria with two different techniques, i.e. by palate counting or counting chambers. The normal OD600 values are standardized for e.coli, meaning that if your organisms is, regarding cell size, different from e.coli, you have to get your own specific values. However, in your case that should not be a problem as b.pertussis is quite similar to e.coli.
If you want to avoid cell clumps, incubate them with glass beads. We typically use 5mm diameter beads. Use around one bead per mL of medium. The beads in the medium will prevent the cells from clumping without the need for PEG or any other chemicals.
Hope that helps.
Greetings
Hi Vanessa,
even if you separate the cells and resuspend them in fresh medium for OD measurement you should zero the photometer with the resuspension medium. Always when you do photometric measurements you should adjust Baseline. Furthermore, it is important to dilute the bacterial sample when you measure in the high concentration range of cells (use for dilution the same medium that you used for resuspension and Baseline adjustment). For E. coli starting from OD 0.5 one should dilute the sample before measurement. Otherwise you will get false results due to the scattering of the cells especially at high cell concentration.
Good luck
In most cases, an OD600 measurement. zeroed against fresh medium as the other respondents have pointed out, is entirely sufficient. If you have cell debris around, centrifugation and resuspending won't help much because a good deal of the debris will pellet with the cells. Dilutions are only necessary if the OD goes above 1, in which case your OD measurement may not be accurate any more. If people are telling you to do dilutions, they are probably confusing OD measurements with viable counts, where you spread dilutions of the culture on agar plates and count the colonies. If you have a culture that is full of debris, this may be the only option. I would avoid centrifuging such a culture as the debris may make the pellet 'claggy'' and cause the cells to stick together.
Hello Vanessa,
I do not know the bacterial culture that you want to measure, but we must be aware that many "traps" are possible when making an optical measurement of suspended particles.
The best is probably that you read my article
"A Method for Exploring Suspended Particles Interactions by Means of Optical Density Measurements: Application to Bacterial Floc and Solid Nutrients"
here.on ResearchGate
Contact me again for further information.
Jacques Thierie
The spectrophotometer measures turbidity directly. It is important to have the cells in known physiological state of growth i.e.as the cell size varies with phase of growth (lag, log, stationery), the approximate relationship between absorbance and CFU will also vary. Finally, it is necessary to blank the spectrophotometer (i.e., adjust the absorbance reading to zero) using the suspending fluid (un-inoculated medium).
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