Use Immunogold Labelling technique. The technique is now recognized as the method of choice for both transmission electron microscopy and, more recently, scanning electron microscopy.

If cultured on minimal media, fluorescent Pseudomonads will produce compounds that fluoresce blue/green under UV light. King's B medium as well as Pseudomonas Minimal Media will upregulate the production of the fluorescent agents making identification quite easy. This method is extremely rapid and cheap, but relies on isolated organisms. 

Perhaps you can see the fluorescent compounds with the appropriate filtered microscopy set up...

Good luck! 

Use Immunogold Labelling technique. The technique is now recognized as the method of choice for both transmission electron microscopy and, more recently, scanning electron microscopy.

Use Pseudomonas Agar (For Pyocyanin) and Pseudomonas Agar (For Fluorescein) from HiMedia Laboratories. You will get pigmented colonies of Pseudomonas on these media. This is the simplest visualization method one can use.

Good Luck...

As per your query, you wanted to differentiate Pseudomonas from Salmonella by observing in microscope, (especially in SEM or other high level microscopy). Its very easy to differentiate these two bacterium by observing the pattern of flagella in the bacterium. In Pseudomonas flagella is either or in polar or polar lophotrichos (present only in end region in single or cluster.)

But in Salmonella, it is a Peritrichous flagellate, where presence of flagella around the bacterium can be found. 

If your study and the sample belongs to any of these two isolate, then it is easy to differentiate by microscopical observations.

But still identification of Pseudomonas in MHA plate with pigmentation or Cetrimide agar will give you selective growth of pseudomonas. SS Agar for Salmonella selection.

With warm regards

Murugan N

Note please that not all Pseudomonas spp (and certainly not all pseudomonads) produce pigment on Pseudomonas F and P agars and not all will grow in the presence of Cetrimide.    And not all of these (and not all Salmonella) are motile. You're better off using some of the classic enteric media such as MacConkey and EMB.  If you chosen two specific isolates, perhaps you can learn to learn to distinguish their relative morphologies microscopically.

Thank all of you for the idea. However, I still want to use microscopy method such as epifluorescence to visualize the spatial organization of the co-cultured bacteria treated with stress conditions. I notice the PNA-FISH has been used to differentiate two bacteria in situ, however, this method is not easy.

OK.  You have already the right answers. For visual examination, the best solution is combination of classical and pigment analysis plates. And for microscopy methods, you are also right the best, even not easy ,is PNA.FISH.

Salmonella agglutinate in their respective group antisera.

Pseudomonas are oxidase positive while Salmonella are oytochrome oxidase negative.

Additionally, use Salmonella-Shigella agar. It will show Salmoella clearly at least.

Why r u using such expensive  methods when their are very cheap methods are available 

Just use cetrimide agar / psuedomonas agar- it will inhibit the others 

and use bismuth sulphate agar/ wilson blair agar for salmonella- salmonella shows shiny black colony

The easiest and cheapest way is performing the cytochrome oxidase c strip test of typical shining and smelling colonies.

I am not sure of fully understand your request for "spectral organization". If you means to identify by SEM the differences in the 3D structure of the cell wall, then is very difficult and very expensive. But if you means, just to be able to recognize both species when growing jointly, there are many more simple and inexpensive methods, already described by other answers.

Oxidase Test: (Easiest Method)

Pseudomonas - oxidase positive. Salmonella - oxidase negative

On Salmonella Shigella (SS) Agar

H2S producing Salmonella species - colourless colonies with black centre

Pseudomonas - smooth, transparent or translucent, colourless colonies

On Xylose Lysine Deoxycholate (XLD) Aar

Psuedomonas - Red colony

H2S producing Salmonella species - Red colony with black centre

For the identification of your bacterial isolate  u can perform 16srRNA gene sequencing. But it is expensive

In addition to the color of colonies on the XLD medium (for Salmonella) and Pseudomonas agar for Pseudomonas, you can observe the colonies under U.V. light. Pseudomonas colonies are fluorescent under this spectrum  of light.

you can use oxidase test, pseudomonase =+ve and salmonella = -ve. also you can use urea media positive for pseudomonas and negative for salmonella. 

How wonderful all research activities would be if such short -cuts to bacterial identification were made available. Bergey's manual would have become redundant long time back.

I think if you are simply trying to apply a microscopic approach to look at your cultures then atomic force microscopy (AFM) might be a good choice, certainly over SEM since it can image sample under (near) physiological condition (under fluid, or maybe in the culture medium) and obtain images with similar or even better resolution than SEM. AFM sample preparation is relatively simple and it can prevent artifacts caused by some traditional EM sample prep techniques, which are BTW relatively harsh. For the experiment set-up you might want to start with image the two cultures separately to obtain information such as dimension and surface morphology of each cell line. You might even be able to discover some special surface features on each cell lines that can serve as marks in later experiments. Then you can image the co-cultured cells and look for the spectral organization. Of course there is no guarantee that this approach will work but you never know until you try it out.

BTW, for sample preparation, in the literature there are many studies using AFM to image E.coli, you may find some information that could help you in those papers.

smell and oxidase

plus pearlescent surface