How Can I Accurately Count Bacteria Using Optical Methods?

[SSJVegetto]

Hi all,

i'm currently working on a project but i need to find a way to count bacteria i have to be able to tell the difference between 10^4 and 10^5 bacteria/mL. This should be done optically but I'm open for any suggestions.

I have read something about optical density and flow cytometry but i need to figure out if the second option is a possibility but the first is said it can only see bacteria of minimal 10^7 cells/mL.

Thanks in advance.

Bob

 

[zhermes]

A flow cytometer is the standard answer, and definitely the most precise; but they cost tens of thousands of dollars.
You can do it manually using something like a Petroff-Hausser counting chamber, but that's tedious...
You can also do a 'Turbidimetric Measurement' which essentially is a measure of how 'cloudy' the fluid is; this has to be calibrated carefully, and then you can use a colorimeter which are pretty cheap. This is probably what you were thinking of when you said 'optical density'. If the density is too low, could you take a sample of the bacteria, concentrate it, and then measure it?

 

[SSJVegetto]

Well the bacteria are going to get really concentrated at 1 spot and the size of that spot is dependent on the number of bacteria... and yes i meant the turbidimetric measurement with optical density measurement. Thank you for your reply!

 

[Ygggdrasil]

Plate out serial dilutions of your bacteria onto agar plates then count colonies.

 

[SSJVegetto]

That is not going to be an option! Since then the entire device won't work the way its supposed to..

 

[Moonbear]

That is not going to be an option! Since then the entire device won't work the way its supposed to..

Huh? All of the methods for counting bacteria require removing them from the plate and dispersing them.

Best bet really is to locate a flow cytometer. If this is something you only need to do infrequently, or that you need to test for viability before committing to pursuing the project, you can probably locate a lab that has one or a core facility with a shared one to use. If it's a long term part of a project and needs to be done daily, then it's worth the investment to get the equipment in our own lab.

 

[Curious3141]

One thing about flow cytometry, turbidimetry and other indirect methods is that they don't take cell viability into account. Meaning dead bacterial cells get included in the count. This is an issue avoided with plate count methods.

If you're unconcerned with viability and just want a crude estimate, you can use a nephelometer, which measures turbidity. The commonly used measure in microbiology is multiples of the McFarland standard, where McFarland 1 (one) is generally held to correspond to 3E8 (3*10^8) cfu (colony forming units)/ml of bacteria. We use this device in clinical labs to standardise the inoculum of bacteria for drug susceptibility testing. The "old school" method is to compare the bacterial suspension directly against Barium sulfate standards using a Wickerham's card (a laminate with black lines of various thicknesses drawn on it). Either way, these are just ways to get an estimated bacterial cell density. To actually measure it, the most common and accurate method is to use plate counts at different dilutions, with redundancy (in duplicate or triplicate) at each dilution, count the colony forming units and work out the actual cell density in the original suspension. Confidence interval estimates can be calculated assuming a Poisson distribution.

 

[zhermes]

One thing about flow cytometry, turbidimetry and other indirect methods is that they don't take cell viability into account. Meaning dead bacterial cells get included in the count. This is an issue avoided with plate count methods.

You can exclude dead cells in flow cytometry to high accuracy using many methods---the easiest being PI stains, but not entirely efficacious (but can easily be coupled to other methods).