Hi Karoly,

I work with SybrGreen or Syto9 for total counts and population analysis of freshwater and drinking water bacteria and it works fine. I do not fix the samples before. A Partec Cyflow in any case is suitable to detect bacteria from fresh water samples, since on some flow cytometers, the small size of bacteria is below the detection limit.

I use the stock from invitrogen/molecular probes S7563, dilute it 100x in DMSO and another 100x with the sample. With a 488nm laser, counts can be done either with the histogram for green-fluorescence or with a red/green dotblot. Is your cytometer equipped with a 488nm laser? Would you like to do sorting on bacteria? What is the aim/research question?

This document may be useful, it describes a standard method for bacteria counting in drinking water

http://www.iwapublishing.com/pdf/SwissFoodBookFlowCytometry.pdf

many regards, Franziska

I must say, without looking at the scatter plots of your attempt, it is a bit difficult to provide a good clue that can solve the problem. Scatter plots reveal a lot about the sample and staining and other characteristics. Firstly, it is important to concentrate the bacteria by centrifugation of your sample. Second is to label with DAPI and make a smear on a glass slide and have a look in Fluorescence microscope. Can you see clear bacteria? Concentrate and plate on a LB or suitable agar plate to see the nature of the bacteria. After these attempts, it is possible to standardize the flow cytometry based scanning of your samples.

good luck and I hope it is useful.

Krishnasastry

Hey there,

we use the live/dead stain by invitrogen. This viability assay also bases on DNA staining. Here the bacteria become red or green based on their viability. This system works very well with staphylococci and streptococci. We used a FACSCalibur by Becton Dicinson.. For this system is no UV-LED or laser needed. You will find detailed protocols for flowcytometry in the web. For example here: http://www.bdbiosciences.com/documents/Bacterial_Detection_Live_Dead.pdf.

I hope this will help you,

Sylvio

Hi, I see two possible problems: first, it can be possible that your bacteria are very small and have a low rRNA content (which may well be the case if you are working with oligotrophic lakes for example), so that you may miss them using standard protocols developped for lab cultures. Another problem may be the flow cytometer settings (sorry, this is certainly the first thing you checked, but I had myself a lot of fun trying to optimise them in the past). Use a log gain for bacteria, and check also that the threshold is not set to high in order to avoid excluding small cells (cf. first part of the comment). You may also want to try simple alternatives to the commercial kits (SYBR green, or even DAPI if you have a UV led) that will give you similar results for a fraction of the cost.

Good luck!

Alex

We could get satisfactory results by using SYBR green while quantifying bacteria from oligotrophic freshwater lake (formalin-preserved).

Hi Karoly,

I work with SybrGreen or Syto9 for total counts and population analysis of freshwater and drinking water bacteria and it works fine. I do not fix the samples before. A Partec Cyflow in any case is suitable to detect bacteria from fresh water samples, since on some flow cytometers, the small size of bacteria is below the detection limit.

I use the stock from invitrogen/molecular probes S7563, dilute it 100x in DMSO and another 100x with the sample. With a 488nm laser, counts can be done either with the histogram for green-fluorescence or with a red/green dotblot. Is your cytometer equipped with a 488nm laser? Would you like to do sorting on bacteria? What is the aim/research question?

This document may be useful, it describes a standard method for bacteria counting in drinking water

http://www.iwapublishing.com/pdf/SwissFoodBookFlowCytometry.pdf

many regards, Franziska

Hi Karoly

In addition to the suggestions other people have made, I have found it critical to have a positive control for flow cytometry with bacteria to help locate them in the scatter plot and distinguish them from noise or contaminant particles. Could you grow a culture of a species of bacteria you expect to find in the water, stain it and run it through the cytometer? This would a least help you to gate on the relevant part of the scatter plot and thus exclude debris.

I agree with Charlie and others, at first it would be useful to concetrate your samples,identify bacteria in scatter plot and check whether your staining protocol is working using fluorescence microscopy.

Hi, well I've been working only with marine bacteria so in general we re using Syber Green for cell staining. We use 488 nm blue laser, and you can see the population on plots: FL1/SSC or FL1/FL3. In my lab we ve been using Partec flow cytometers for many years, and they have the general problem with high electronic noise and not so good side scatter..but since yours is newer instrument maybe it has better SSC.

So I would suggest to first (as people above suggested) stain your samples with DAPI and check it on epifluorescence microscopy, try to use SyberGreen for flow cytometry and increase FL1 while analysing to get the population at the center of the plot (of course having bacteria culture will be useful to arrange your counting protocol before analysing your samples but its optional).

Good luck

Cheers

I found some dyes are quenched by PBS buffer (pH7.2), so maybe you can try to using other staining buffers (such as Tris, TE, MOPS, etc). Adding detergent (such Tween 20) or EDTA may help dyes to penetrate into cells. Second, keep constant the pH of staining buffer as pH have great impact on fluorescent intensity. Thirdly, as other mentioned, parameters setting is also important, especially threshold setting. Usually, I use FL1 (green fluorescence) when using Sybr Green. This can be done with a positive bacterial control or beads (1.0 um). Remember to check or change the default threshold, which in our Flow cytometer is FSC that only capture large particles and miss most bacterial particles.

We use the BD TM cell viability kit comprising of two dyes Thiozole orange (TO) and Propidium iodide (PI). . PI is used to detect live and dead cells whilst, TO is capable of penetrating both live and dead cells, the former characterized by intact impermeable membranes and the latter by compromised membranes.

The fluorescent signal from TO allows for live cells to be enumerated and gated appropriately since it will only bind to live cells and not to contaminated debris acquired during the cell preparation process. The PI dye penetrates only compromised membranes of dead cells. These two dyes give reliable and rapid results to differentiate between live and dead cells.

Dear All, just a side note, please call those dyes SYBR and not Sybr or Syber, etc - the name SYBR comes from the initials of Steve Yue and Bruce Roth...

I can also recommend to start with a pure culture to establish general working procedures. The setup of the instrument is very important and, as already others recommended before, fluorescent beads can be used to ensure proper alignement. From the signals of the beads you know how well the optical path is aligned and you can compare the setup every day. With a pure culture you can check the successful staining with the microscope and then measure them with the flow cytometer. I made very good experiences with DAPI staining and paraformaldehyd fixation but the procedure needs to be adjusted for every system you are working with. Maybe here you can also find some more ideas on trouble shooting here: http://www.ncbi.nlm.nih.gov/pubmed/23288319.

By the way, what is the intensity of your laser? Depending on that and the DNA content of your cells it can be difficult to distinguish small cells from instrumental noise or particles. Again, testing with pure cultures can help.

Good luck!

 Dear all, e have partec flow cytometer (Cyflow Space), since one  week i am trying to do some settings for autotrophic forms, i tried with the earlier settings for the autotrophs but the clusters are are comming on screen, even if i run blank MiliQ water the blank noise is too high. @Dr Karoly since you are using the same instrument could you also please suggest what should i do.  Is it like there is issue with detectors or laser?. what should we do if background is too high?. if i try to reduce it with setting FSC SSC of ffluorescence, then the sample cluster is not visible.

Please suggest some tips.