I am using a Nanodrop machine to measure cell density at 730 ( Cyanobacterial cell). But I have some doubt that sometimes this machine gives varied data in replication. Does anybody have experience to measure cell density in Nanodrop reliability?
In general I would assume that the bigger your sample size is, the more reliable will be your result. Now if you look at not even a millimeter that the nanodrop measures vs the cm that you would have in a normal spectrometer, you definitely expect less reliability. Also, you measure something way bigger so that small fluctuations have a way bigger impact on your result. In conclusion I am pretty sure that the Nanodrop is not a reliable way to measure cell density. Also, if someone wants to check RNA later and maybe wants to reuse the sample (it happens) then they will not appreciate you using the Nanodrop with bacteria ;)
I've found that it is reproducible for absorbance at 260 & 280 nm for protein concentrations - you just have to make sure that you measure the sample as soon as you put it onto the machine, as the droplet can evaporate over time, making the solution more concentrated and biasing the readings in favour of higher concentrations. I've not tried with cells, but I assume similar will apply. As long as you clean the photometer correctly between samples, and re-calibrate it with a blank regularly, it should be fine.
@Mathias Labs, But Nanodrop is higly reliable in counting DNA concentration even with such a small amount, 1.5 microL and nobody raised any question on that. Why does it a matter of sample size, according to you, in the case of measuring OD for cell count? Moreover, what are the problems if someone use nanodrop to count RNA following after measuring OD for cell? Would you please give me details?
The way I approached that question was to explain the inaccuracies he observed. Nanodrop is designed for protein or nucleic acid measurements. Those molecules are small in size (let's say 2nm diameter), whereas e.coli has a diameter of 1microm. So there is space for 1k e.coli theoretically. Since you only measure solution w/ let's assume 1% e.coli, we are talking about ten cells in the beam. That I would say is a small sample size and prone to variations.
@Adam: I was thinking about the replicates with sample size, not the accuracy. Of course spectrometers also vary, but at least 1 bacterium moving out of the beam means 1% change, not 10%.
I am sure there is a smarter way to explain it ;)
@Mathias Labs , your explaination of error in measuring cell density by Nanodrop makes sense. However, why does then there is option in that machine to measure cell?
This document basically suggests that Nanodrop OD600 reads are not accurate for measuring bacterial cell density unless a conversion factor is determined by dividing the cuvette OD to Nanodrop OD, which can then be used in a decently wide range of ODs.
https://tools.thermofisher.com/content/sfs/brochures/T041-T041-Comparing-Microvolume-and-Cuvette-Based-Measurements-of-Microbial-Cell-Cultures.pdf
But that also means you need access to a cuvette spectrometer, at least once every time you need to calculate the conversion factor for a specific application/OD range.
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