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I am hoping to measure growth rates of a bacterial culture in several growth conditions. I am concerned that these growth conditions may cause cell death, which would lead to a decreased correspondence between optical density and number of live cells, so I want to independently measure the number of live cells. I understand that plating serial dilutions of a culture and counting the number of colonies to calculate the Colony Forming Units (CFU) per mL is the gold standard for measuring the number of viable cells. However, I have seven different conditions I want to test in parallel, and making 3+ plates for each condition (to do serial dilutions) and taking measurements at several points in the growth curve leads to a lot of plates. In general this method seems more labor intensive than what I need. I am mostly doing this as a sanity check, so it seems like a lot of work just for a sanity check.

My question is: are there any alternative methods to CFU plating for measuring cell viability, given that I am doing a lot of experiments in parallel? I don't have access to a FACS machine, so cell sorting won't work. I have read this question on measuring live cells in algae. I'm not sure that a hemocytometer is the right alternative for my particular high-throughput application (though you could convince me otherwise). The best answer to this question would compare from experience the precision, accuracy, sensitivity, and ease of use of this alternative method with CFU plating.

Thanks!

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Even small cell counters are available. Stain cells with trypan-blue and you can count viable cells. I think invitrogen supplies this machine. –  WYSIWYG Nov 16 '13 at 12:34

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up vote 5 down vote accepted

I've only used it for mammalian work, but the Cedex HiRes Analyzer from Roche is pretty sweet. From their docs you can analyze particles from 1-90 μm in size (the cells I work with are about 20 μm), and there are a ton of configuration options depending on your needs. As long as your cells can be stained with Trypan Blue (it's been a long time since I've done bacterial work, pardon my ignorance) to look at viability, then this machine should work quite well. When set to Maximum (i.e., the analysis takes as long as possible), I get results in less than 4 minutes per sample - and this includes about a minute for the sample to settle in the flow cell. If you lower the sensitivity, you can get results faster.

Basically the way it works is you take 300 μl of your liquid culture (diluted if you think the concentration is too high), and add it to a sample cup. Put all your cups in the tray, fill out the sample info and analysis options in the MultiRun screen, and hit Start. The machine includes all the reagents onboard, so it mixes the Trypan Blue with your samples, then injects the proper amount into a flow cell. The sample is allowed to settle, then a high-resolution image is taken of the entire length of the flow cell. Then, depending on your sensitivity settings, a certain number of frames are taken from the full-length image and analyzed. An average count is then generated based on all the images, and your final numbers are spit out. You can then print or save a report of the cell counts and viability.

The software comes with some analysis tools as well, so if you keep multiple cultures going over time with different growth conditions, you can aggregate the data for each one and generate graphs of cell counts, viability, cell size, etc. - there are at least 10 or 20 parameters to choose from. All the image analysis options are configurable, so if you look at the results and think the machine is picking too many dead cells, or not enough, you can change the parameters to your liking, and save the options as a custom analysis type.

One option I like that would beat the pants off CFU-based counting (besides the drastically-reduced time) is the fact that the machine can detect aggregation of the cells. One issue with CFUs is that it's hard sometimes to know that a certain colony really arose from just one parent cell - if you let the colonies grow long enough you may be able to tell based on size, but it's a tough call. Since the Cedex is looking at the cells themselves, it doesn't matter if they're aggregated or not, as they'll all get counted (assuming the aggregation particle doesn't contain a huge amount of cells, like 50+, that can't be resolved by the software due to occlusion of the image).

I don't know how much these guys cost, so depending on your budget you may not be in the market for one, but if you're going to be doing a lot of counting then one of these is invaluable. I'm sure other companies make similar instruments, but I haven't used them, so I don't know how they compare to the HiRes. I do know that the person at my company that was responsible for buying the HiRes is very good about researching possible alternatives, so this one is probably as close to the top of the line as we could find, for the needs that we have.

Good luck with your counting!

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Thanks for the detailed, well-explained answer! I'll have to check whether Trypan Blue works for bacteria; from my initial investigation it seems so. I'm a bit leery about buying a whole machine to do this, but at some point if I want to beat tedious (and cheap) CFU counting I'm going to have to get real and buy something; if it could be done better than CFU with similar technology, it would have been done before! –  A. Kennard Nov 17 '13 at 17:04

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