4% PFA should work fine (it is the standard fixative). Though I haven't stained mycobacterium before, most staining protocols follow these steps:

1)Fixation (10 min 4% PFA-- wash your cells with PBS first)

2) Blocking step (serum from the same species as your secondary antibodies 5%, or 3% BSA should work)/permeabilization step (for your experiment perhaps try a mild detergent such as .1% saponin ) (1hr at RT)

3) Addition of primary antibody (1hr at RT or overnight at 4C) (I usually added these antibodies into my blocking solution)

4) Washing (3x 5min)

5) Addition of secondary antibody (1 hr at RT)

6) Wash (3x 5min)

7) Mount (I like to use the ProlongDiamond or Gold from Invitrogen with DAPI, Vectorshield is also good)

I hope this helps! The easiest way to find out if your staining procedure works is to try it. The more experience you get, the more you will have a feel for what is necessary and what isn't. Everyone has there own preferences in regards to concentrations of blocking serum etc.--- bottom line: lots of different variations on protocols work. Most of the diversity in protocols is because of personal paranoia or "just the way we do things" and for no other reason.

Hi Renu Bisht

For bacteria fixation, you can use 4% PFA (10 min) as recommended by Elliot Glotfelty and for reducing noise ration, when analyzing your samples, you can wash your bacteria before fixation and after fixation with PBS (3 o more times) and by setting in your microscope. However, significant changes in the protocol are needed according to particular considerations. So, I would like to ask you if you are using transformed-bacteria (GFP as reported) or if you want to stain any particular bacterial protein? if your bacteria is infecting cells or not? if you want to see the protein localization in living bacteria or only in dead ones, the type of confocal microscope, etc.

Regards.

Hi Fabian R Villagomez,

Thanks for your reply. To answer your question, I am actually using transformed bacteria (GFP- with my protein of interest) and interested in studying the cellular localization of the protein. I can check it in dead or live cells both.

Hi Renu Bisht,

I have some experience working with different mycobacterial species and transformed bacteria (DsRed as reported. Here, some considerations for your protocol.

I suppose you are working with bacterial exponential growth phase. So, I would recommend working on ice (for decreasing bacterial metabolism) for harvesting of bacteria: obtaining the bacterial pellet by (4°C) centrifugation (4000-5000 r.p.m./5min) and then washing with cold-PBS (to cover the bacterial pellet). Afterwards, disaggregation of bacteria by vortexing (20-40 sec; if much bacteria clumps you can vortex using 0.05% Tween-PBS, 4°C, followed by washing with cold-PBS; however, you need to consider the effect of Tween on cell wall) prior being put (drop or drops) onto the coverslips (I think that the less number of bacteria you can put, the better). Also, you can homogenize the sample (for example, by putting another coverslip onto your sample, and then take it out). For fixation, you can use 4% PFA (to cover the coverslip) for 10 minutes (PFA recently prepared and/or filtered is better), which can be added to the coverslip when the sample begins to dry. Later, you can discard the PFA and wait until the coverslip is dried (you can blow with a sheet of paper to speed the process of drying).

The coverslips can be mounted onto slides using or not mounting medium and sealed with nail polish (or other). I have used dako, bectashield, and glycerol (as reported https://nic.med.harvard.edu/resources/media/) as mounting medium for my own samples, but all of them decrease both, detected fluorescence intensity, including GFP-tagged proteins, and noise. So, you will define when observing the fluorescence of your own samples, because, in spite that the fluorescence intensity of GFP is good, such intensity is going to be closely related to the protein of your interest.

On the other hand, for living bacteria, the protocol would be as mentioned, but working on ice or fixation would not be necessary. You can put your bacteria in plates (35 x 10mm glass bottom, 0.17mm; or others) containing medium without phenol red or in PBS (temperature and atmosphere according to your needs).

The other very important part is the confocal microscope and the acquisition settings. But it is another issue!

I hope it helps you.

Regards!

Hi Fabian R Villagomez,

Thank you so much for your detailed explanation of the protcol and amazing tips. Yes, I would be working with the log phase bacterial growth. Though I still have some queries, like, is there a need to pre coat the coverslip with poly-L-lysine as I read its required for non adherent cells?

And also can you provide me information regarding the dyes you use to stain the mycobacterial cells? I have phenol auramine with me, would it be fine or is there any other better option available? I find it silly to ask but can you please, also explain which filters and magnification should be used? It would be of great help to me.

Thank you so much in advance.

Have a great day ahead!

Hi Renu Bisht

I have stained non-tuberculous and tuberculous mycobacteria species with ziehl neelsen and kinyoun staining. However, those bacteria were not transformed with a reported gene.

I have worked with bacteria (mycobacteria and others) that attach easily to coverslips. So, I did not use poly-L-lysine. I´ve used this compound for treating coverslips for non-adherent cells, such as THP-1, U-937, and Jurkart (some of them, infected by mycobacteria). This, in order to increase the number of attached cells to the coverslip (according to my experimental needs). So, I think you can work with poly-L-lysine and without it, but probably you will have more number of attached bacteria to your coverslip.

On the other hand, I´m not sure if the cited staining could be interfering with GFP (If yes, you could vary the microscope settings by comparing transformed-stained vs non-transformed-stained bacteria). Also, I think more magnification is better, given that you work with bacteria, and also you need a blue laser for exciting GFP.

In laboratory, I used mainly the Leica TCS SP8 X microscope, which is very easy to use and powerful confocal microscope. So, as an option, you can contact the corresponding responsable.

Don´t worry for asking, my pleasure if I can help.

Regards!

Hi Fabian R Villagomez,

Thanks very much for your valuable guidance. I will surely get back to you, if any further queries.

Have a great day!

Regards

Renu Bisht