Hi, as mentioned above there are many posibilities. If you have a fluorescence microscope, I advise you to filter your bacterial suspension obtained from the roots on a 0.2 µm filter, and then stain the bacteria with DAPI (a colorant that will stain DNA/RNA). You can then count the bacteria. If your microscope permits it, you can take a picture, and analyse it with free image analysis software such as ImageJ to count the bacteria automatically. If you have a flow cytometer, wonderful, then I advise you a live-dead stain with SYBR green and propidium Iodide (you may also use comercial kits, that will however cost you 5 times more for only marginally better result).

If you want to stick with dilution plating, I have got excellent experience with drop plating: Basically you make your serial dilution in a microtiter plate, and instead of plating you spot a drop of 5-10 µL of each dilution an agar plate. It is very fast and does not require protocol optimisation or looking for new material. Good luck, let me know if I can help you with more detailled protocols!

There are P. syringae strains (e.g. P. syringae maculicola ES4326) that harbor a luciferase operon encoding for luciferase and luciferin. Some groups commonly employ these strains to perform infection of plants, followed by homogenization of the plant material. Plant extracts are then dispensed into a plate reader and the luminescence is measured. You still need a standard curve of bacteria for proper quantification. I do not think that these strains are impaired in viability, but the system (especially the Ps strains) might not be compatible with Phaseolus vulgaris.

Good luck with your research.

Since you work on leaves, you could use the LUX operon like Fan et al:

Plant J. 2008 Jan;53(2):393-9. Epub 2007 Oct 27.

High-throughput quantitative luminescence assay of the growth in planta of Pseudomonas syringae chromosomally tagged with Photorhabdus luminescens luxCDABE.

Fan J, Crooks C, Lamb C.

Regards

Nemo

Check out this article ...may be helpful

http://aem.asm.org/content/73/8/2653.full

An alternative to gfp-tagging is to inoculate and collect the bacteria as you have been doing, and then stain with the Bacteria Counting Kit (Molecular Probes, Life Sciences) as per the manufacturer's instructions. This then allows enumeration of bacteria by flow cytometry. Will likely take a bit of optimization, and there may be some background noise from the seed, but we have found it to work well for Pseudomonas aeruginosa recovered from human cell cultures.

All the best - David

Flow cytometric method using nucleic acid stain could be a good option for you after some initial optimization. For example, you can use green fluorescence nucleic acid stain SYTO®) 13 (Invitrogen Molecular Probes) for staining bacterial cells and a standard solution containing fluorescent beads as reference. I have used it for total bacterial counts from environmental samples.

try to visualize colonies under UV light at 360 nm and counted colonies that show green fluorescence

Hi, as mentioned above there are many posibilities. If you have a fluorescence microscope, I advise you to filter your bacterial suspension obtained from the roots on a 0.2 µm filter, and then stain the bacteria with DAPI (a colorant that will stain DNA/RNA). You can then count the bacteria. If your microscope permits it, you can take a picture, and analyse it with free image analysis software such as ImageJ to count the bacteria automatically. If you have a flow cytometer, wonderful, then I advise you a live-dead stain with SYBR green and propidium Iodide (you may also use comercial kits, that will however cost you 5 times more for only marginally better result).

If you want to stick with dilution plating, I have got excellent experience with drop plating: Basically you make your serial dilution in a microtiter plate, and instead of plating you spot a drop of 5-10 µL of each dilution an agar plate. It is very fast and does not require protocol optimisation or looking for new material. Good luck, let me know if I can help you with more detailled protocols!

Krishnakumar (CSIR-NIIST)

Simple fluorescent staining of the bacterial suspension with either DAPI or SYBR Green or Acrydine Orange will give you the cell count. More accurately, if you can use Propidium Iodide together with any of these dyes (except AO), with the appropriate filters for the different dyes will give you live/dead cell count in your sample.

good luck

krishnakumar

I agree that DAPI labelling is simple and ready to use. I propose to label directry the seeds to count per surface unit the number of cells. If vability is of concern, the same with te dead/live kit.

Need an epifluorescence microscope.

Good luck

Olivier

I agree with the fluorescence staining advice. Dilute, preserve them (lugol's + buffered formalin + sodium thiosulfate; or any of the numerous preservation methods out there), then stain them with DAPI that binds to DNA and fluoresces blue under UV excitation. DAPI is to bind to living cells. Filter onto a 0.2 um black PC filter, place on a microscope slide with immersion oil and cover slip. Then image count and calculate. Flow cytometry is a great option too, but requires more work than direct counts....but you get a count of more cells. Depends on what equiptment you have and what time you have. Good luck!

If you are really concerned with bacteria-seeds interactions, I recommend to directly observe bacterial populations as "biofilms" on seeds. Just gently rinse the seeds with isotonic medium to count bacteria fixed on them.

Try with DTAF (Molecular Probes) , is an innocuous fluorchrome. I used DTAF to see bacteria attached inside the gut of Artemia and shrimp larvae. The protocol is very simple and the bacteria is viable. See the paper attached

Article Assessment of fluorescent-labeled bacteria for evaluation of...

Michael, We have been able to "mark" DNA viruses with certain fluorescent dyes that do not interfere with replication. I suggest you try several different dyes...many will inhibit replication, but you might get lucky and it's not a big investment.

DTAF do not interfere with bacteria replication, I proved in the paper with vibrios, but I agree with David, you must try with different fluorochomes

This migth help you to get some ideas on your question:

Weinbauer, M. G., Bettarel, Y., Cattaneo, R., Luef, B., Maier, C., Motegi, C., Peduzzi, P., et al. (2009). Viral ecology of organic and inorganic particles in aquatic systems: avenues for further research. Aquatic Microbial Ecology, 57(3), 321–341. doi:10.3354/ame01363

Luef, B., Neu, T. R., & Peduzzi, P. (2009). Imaging and quantifying virus fluorescence signals on aquatic aggregates: a new method and its implication for aquatic microbial ecology. FEMS microbiology ecology, 68(3), 372–80. doi:10.1111/j.1574-6941.2009.00675.x

Thank you all for your suggestions. First I'm going to try and tag our Pseudomonas syringae strain with a lux operon as they did in the Fan et al Paper. As much as I would love to go with the staining flow cyto approach, I do think our lab has the monetary resources that would be required. Again thank you all, these have been some great suggestions.

Hello, following all the answers given preicously, once you find a suitable fluorescent dye, you can always count total numbers of bacteria using flow cytometry. I believe that Molecular Probes has a bacterial counting kit, I have used it for the enumerations of total counts of bacteria with success. Hope it helps.

Hello,

We used the following approach to detect bacteria in urine. But it should work for your application. William J. Palin, Timothy C. Miller, Determination of microbial cells in aqueous samples. U.S. Patent No. 4225783, Sep 30, 1980.