What is/are the problem(s) you're having? Can you see phages, just not very well, or not at all? How are you purifying and staining your samples? 

You need to tell me how did you isolate & purify  your phage. what do you see after staining your phage particles?

Have a nice day,

purified phage containing SM buffer centrifuged 30.000 RPM for 2 hr, collected the pellet, and the phage containing pellet droped equal volume of 1% phosphotungstic acid and dropped on carbon coated copper grid and dried at 37 °C for 1 hr. and image analysed. 

here with i have attached my purified phage plaque plates. 

Dear Nattan Stalin,

That photo does not say a lot. What is the dilution that you plated? How high is the titer of the sample you used for TEM and how much did you use?

My first guess would be that the titer is just too low to see with TEM.

Dear experts, 

Thank you for your excellent suggestion, I have taken bacteriophage TEM images by ultracentrifugation with negative stain, the resolution is low and not as clear images. 

A more typical protocol would be something like.

1. Take a continuous carbon film on an EM grid, and glow discharge in air for 30s or so. 

2. Add a 3uL drop of your virus to the grid - wait 30s

3. Add three x 20ul aliquots of stain - I would recommend 2% uranyl acetate rather than phosphotungstic acid. The excess will drip off onto a wad of filter paper you put on the bench for this purpose!

4. Blot away excess fluid with edge of a piece of filter paper at the edge of the grid - NOT face to face.

5. Dry for at least a few minutes. 

6. Stick in TEM.

7. Your EM image is very difficult to interpret - I would guess you have (a) horrible/no staining and (b) are a very long way from focus. You probably only need to be ~500nm under focus to get good contrast of well-stained virus particles, so maybe an EM refresher would be a good idea.

Thanks so much for all responses sent so far, much appreciated

Neil Ranson Dieter Vandenheuvel As my research on Phage resistance mechanism, So I want to see the morphological feature how the phage can not attach with bacteria. can you tell me how many phage I have to add with bacteria sample for TEM analysis ?

Dear Nazmul Islam,

I'm afraid you are looking at the wrong thing.

If the bacterium has become resistant to a certain phage, you are not going to see any change in the structure of the phage. There will be a change in the bacterium. But then the next problem is, this change might be structural, but it can be caused by many other things. There are many ways in which a bacterium can gain resistance against a phage. Furthermore... Even if it is structural, and by that I mean something has changed at the molecular level, TEM is absolutely not the right technique to visualize this. It is far from detailed enough to see structural changes on molecular level.

You should give more details of what you exactly want to visualize.

Respected Dieter Vandenheuvel

Thank you for your suggestion. However, actually I want to see how phage react with phage resistance mutant. Most importantly, I think my mutant cell surface mainly resposible for phage resistance . Can you give me some suggestion how can I get resistance mechanism ? this is my main aim . Yeah, I already read some paper of you. Did you find any phage resistance mutant ? Thanks professor.

I think it is difficult to prove this with 100% certainty, but you can at least get a better idea that the mutant cell surface might be an actor here.

If the phage is not able to bind on the mutant cell surface (receptor is not present anymore or defective), you should be able to visualize this with TEM.

First, infect the wild-type bacterium with the phage, and visualize the cells with TEM. You should be able to detect the bacterial cells, and see the phages attached to the cell surface. This will also help you to determine the settings for the TEM visualization.

Next, infect the mutant bacterium with the phage. You won't see any phage attached to the cell membrane.

Finally, complement the mutant bacterium, so it expresses the functional receptor again. Infect with the phage. Now you should be able to see the phage again attacking the bacterial cells.

This would at least support your statement that the cell surface receptor is a reason for phage resistance.

Respected Dieter Vandenheuvel

As I check in TEM & SEM, I found my mutant had some cell surface change more with wild type. I already attach some file . Can you check if you have time ? however, ABC transporter gene present in my mutant. By TEM analysis I think the cell surface had some Glycoprotein or glycolipid present . Do you think this is phage resistance barrier ?

Dear Nazmul,

Regarding the bacterial resistance, did you performed adsorption test to both wild type and mutantant?

In my case the phage still adsorp and attach to the bacterial surface but at lower rate than the wild type. However, no plaque were formed in AOL and no increase in phage titer also after 24h regarding the mutant.

TEM reveal that phage still attach to the mutant but with no successful infection. Regarding the first step of bacterial resistance , phage (siphophage) need to adsorp to the bacterial surface , passing the bacterial membrane including PG and cytoplasmic membrane , and injecting its genetic material into the bacterial cytoplasm, in order to overcome the first stage of bacterial resistance and continue to successful infection of there are no other means of resistance. Any issue with one or more of this process might lead to BR.

If you interested in this type of bacterial resistance. Please refer to our latest publication which explore that and published lately (attached copy).

Hope that will help.

Dear Nazmul Islam,

Do you have any control? TEM/SEM of the wild-type? Otherwise you can't compare. But even then, if the resistance is due to a small molecular change, you will not see it on the pictures! This is what I mentioned in my previous answer.

Mohammed Al zubidi

Below I attached my phage adsorption curve compare with WT and Mutant.

One step growth curve with WT. Can you check .

Dear Nazmul,

The adsorption (AD) test need more optimization as it did not coincide with one step growth (regarding the wt).

It is clear from the AD that the phage did not adsorp to the mutant (M). That mean it is resistant strain or the strain is a contaminant from other bacteria.

In my opinion, you need to prove that it is a mutant from its parants wild type bacteria and complimant it, so it will return its sensitivity to the same phage. In addition, sequencing for the mutant gene will help a lot and comparison to the sequence of the WT. Furthermore, 16s rRNA, Gram stain, etc will insure that you do not have any contamination.

Hope that will help.

Mohammed Al zubidi

I already done PFGE test. It showed that my mutant and WT is same band profile. So it is no contamination at all. Have any other experiment to prove which is the main reason for resistance ?