Viewing Bacteria With Bright-field Microscopy

[MicroscopyLearning]

You need as much depth of field as possible because bacteria vanish even if they are just slightly off your focal point/ Close your iris as much as you can. Use very bright light and (if using a camera) set your software to as high a contrast as you can. In almost any sample where you see protists, you will see an amazing array of very small organisms, flagellates and bacteria. Cyanobacteria are quite large.

Thank you for the reply. By the iris, you mean the condenser's aperture diaphragm, correct? Do you think the light on the Swift Optical model SW350T-AK-PK would be a sufficiently bright light source. Moreover, would the 40 times objectives be quality enough for the viewing of bacteria in bright microscopy?

[Alexander]

Close your iris as much as you can.

To reduce resolution down to Coke bottle level?

[DonSchaeffer]

Guys. I learn by trial and error. In my system following those steps allows me to see things that would ordinarily be hidden. I also can see structural details that would ordinarily be washed out. I do not have a fancy system. I'm just doing the best I can with what I have.

Check this out if you wish.
https://www.youtube.com/watch?v=PNhFIyWHjHc&t=7s

[dtsh]

Close your iris as much as you can.

To reduce resolution down to Coke bottle level?

It's a trade off between contrast and resolution; too much and you get artifacts, too little and it's hard to see anything.

[Alexander]

Close your iris as much as you can.

To reduce resolution down to Coke bottle level?

It's a trade off between contrast and resolution; too much and you get artifacts, too little and it's hard to see anything.

You are right! What will you get following Don's advice to close the iris "as much as you can"?

There are three proper method to view bacteria with a light microscope: staining, staining and staining.

[dtsh]

It's a trade off between contrast and resolution; too much and you get artifacts, too little and it's hard to see anything.

You are right! What will you get following Don's advice to close the iris "as much as you can"?

There are three proper method to view bacteria with a light microscope: staining, staining and staining.

Not disagreeing! Staining has been mentioned as the defacto method how many times in this thread?

As for the question of what you'll get following Don's advice? You might get to see the rough shapes of bacteria, but with artifacts that aren't really there making it appear as though there is more detail, but those details are just artifacts. OP seems to want to see *something*; so following Don's advice isn't terrible, though it's certainly not a good method. I think Don is aware of that (it's been explained to him before) and I hope OP is aware that staining is the best method as has been pointed out to them several times, but they appear to want to do something else. I think if they follow Don's advice the result will be underwhelming, but it may lead to further inquiry and give them some context of the challenges of viewing specimens which are at the limits of most light microscopes.

[DonSchaeffer]

I made a short demonstration showing the effect of use the aperture.
https://youtu.be/Tb2OZGJElDI

[MicroscopyLearning]

I made a short demonstration showing the effect of use the aperture.
https://youtu.be/Tb2OZGJElDI

What microscope do you use?

[Alexander]

I made a short demonstration showing the effect of use the aperture.
https://youtu.be/Tb2OZGJElDI

Does your video show bacteria? Just asking.

[DonSchaeffer]

Yes. Several shapes. Round, linear, corkscrew. Then there are flagellates of various types.

I have an Amscope 500C. I replaced the 100X oil objective with a 600X plan. I use a Hayear HY500B camera. I never use the eyepiece.

[farnsy]

Would DIC be any more useful in seeing detail?

It depends on why you can't see the detail. DIC, phase contrast, darkfield, and oblique illumination allow you to see things that you couldn't see otherwise because they are **transparent** when there is a super bright light behind them. We can think of these techniques as "optical staining." They are very useful because lack of contrast is often the limiting factor, especially when looking at living microscopic organisms.

On the other hand, if the details are just too small to resolve, then contrast-enhancing techniques don't do much to help. You can move to higher numerical aperture objectives until you get to 1.4, and then you have to move beyond normal microscopy.

In my experience, small bacteria are usually small enough that you can't see much except their general shape (rod, sphere, etc.) even if you have plenty of contrast.

[josmann]

High magnification objectives are designed to image the plane exactly on the bottom of a .17mm (#1.5) coverslip. This is because the primary reason many microscopes are made at all is to image very thin specimens such as blood smears and histological samples. If you want to image bacteria clearly, you want to isolate them in your sample tube from any debris that would increase the thickness below the coverslip. One way to do this is to incite a bacterial bloom (add some beef broth or milk, perhaps) such that bacteria densely populate the debris-free liquid (in fancy microbio terms, this is called the “supernatant”). Sample some of this liquid and prepare a slide with a #1.5 coverslip. Use a tiny drop of liquid and press firmly with a gloved finger to evenly distribute the drop into a very thin layer.

With darkfield or oblique illumination, densely populated bacteria will be visible even at 4x. Depending on the species, you can see shapes pretty well at 40x and more detail at 100x. In brightfield, you normally need to close the condenser iris down a fair bit for good contrast. One feature that is possible to see with advanced techniques is the bacterial flagella of certain rod shaped species. I have personally observed this at my workplace with a high end Olympus PlanApo 100x phase contrast objective. The flagella were actually remarkably clearly resolved. I have not yet attempted the same with my considerably less sophisticated home setup but I think it’s possible given the appropriate equipment and setup.

[DonSchaeffer]

What Josman said!

[Charles]

I would also suggest staining is the way to go. Back in the days when sailors were coming back from shore leave with a dripping pecker and pain on peeing, a few days later, we would gram stain their secretions for diagnosis of N gonorrhea. After the gram stain, we can differentiate between gram positive and gram negative bacteria. We would see gram positive rods and gram negative cocci. Of course we were interested in finding the gram negative diplococci inter and extra cellular bacteria, which showed as pairs. A nice pink pair like two colons (,) facing each other. We used the 100X objective (10X eyepiece) oiled directly to the slide with no coverslips. They presented themselves quite nicely, but we still did not see much detail in the bacteria we saw.

[Dubious]

Researchers have spent their entire careers on this; and given the resolution limits of the optical microscope explained above, we are still using Gram's century-old method of staining to separate bacteria into gram-positive and gram-negative groups. For the amateur observer with an optical microscope, I'm afraid it doesn't get much better than being able to see tiny blots moving about. But there are a few unusually large bacteria for which you would see more:

https://largest.org/animals/bacteria/

[MicroscopyLearning]

You need as much depth of field as possible because bacteria vanish even if they are just slightly off your focal point/ Close your iris as much as you can. Use very bright light and (if using a camera) set your software to as high a contrast as you can. In almost any sample where you see protists, you will see an amazing array of very small organisms, flagellates and bacteria. Cyanobacteria are quite large.

How would you recommend differentiating between sediments and the bacteria? I have been trying to observe a sample of rod-shaped bacteria from a kind of kefir-like drink, how should I differentiate between the sediments and the non-motile rod-shaped bacteria?

[BramHuntingNematodes]

For living bacteria use ectoplasmic contrast to see the tiny bits of their souls

[MicroscopyLearning]

For living bacteria use ectoplasmic contrast to see the tiny bits of their souls

What do you mean by this? I am not familiar with ectoplasmic contrast. Is this an optical technique for viewing them?

[Alexander]

For living bacteria use ectoplasmic contrast to see the tiny bits of their souls

What do you mean by this? I am not familiar with ectoplasmic contrast. Is this an optical technique for viewing them?

This appears to be a joke to comment someones claims about the visibility of bacteria with a light microscope.

[Dubious]

Yeah, we don't yet have electoplasmic contrast in the arsenal, maybe someday But really, even the most humble compound microscope of today is better than the microscopes that early researchers used to make important discoveries. Van Leeuwenhoek's microscope used a tiny glass sphere as the only optical element, and he was able to observe and describe bacteria.

[MicroscopyLearning]

Yeah, we don't yet have electoplasmic contrast in the arsenal, maybe someday :D But really, even the most humble compound microscope of today is better than the microscopes that early researchers used to make important discoveries. Van Leeuwenhoek's microscope used a tiny glass sphere as the only optical element, and he was able to observe and describe bacteria.

What would your general procedure for viewing bacteria be?

[Dubious]

Are you presently observing protists? If so, using Don's technique, you should see bacteria wherever there are protists, with the 40x or higher objective. Depth of field will be shallow so you don't want much water under the slip cover--you may have most luck at the point that the water begins to dry up. Beyond that, see the suggestions above for culturing bacteria, and for staining it to make it easier to see, etc. Bacteria are literally everywhere.

[MicroscopyLearning]

Are you presently observing protists? If so, using Don's technique, you should see bacteria wherever there are protists, with the 40x or higher objective. Depth of field will be shallow so you don't want much water under the slip cover--you may have most luck at the point that the water begins to dry up. Beyond that, see the suggestions above for culturing bacteria, and for staining it to make it easier to see, etc. Bacteria are literally everywhere.

Currently I am using the method of brightfield microscopy for this. I am at the 40 times observing yoghurt samples, but I am having some trouble identifying the bacteria. Would you recommend drying the specimen and then trying to observe it?

[Dubious]

No, with yoghurt (and make sure it is yoghurt that still has live cultures) I would go the other direction, to thin and separate the yoghurt enough to see individual bacteria. That is, put a drop of yoghurt on the slide, then dilute it with a drop of water before placing the slip cover on top. With a 40x objective, you should be able to see something.

Also, see what Apochronaut and others said above, about observing bacteria in milk, etc.

[MicroscopyLearning]

No, with yoghurt (and make sure it is yoghurt that still has live cultures) I would go the other direction, to thin and separate the yoghurt enough to see individual bacteria. That is, put a drop of yoghurt on the slide, then dilute it with a drop of water before placing the slip cover on top. With a 40x objective, you should be able to see something.

Also, see what Apochronaut and others said above, about observing bacteria in milk, etc.

Will diluting the specimen into a glass of water than piping out the water onto the slide work? Also, I saw in a video that Microbehunter did not dilute his sample and rather put it on and looked at the edges of the sample. Which way would you recommend?

[Alexander]

Will diluting the specimen into a glass of water than piping out the water onto the slide work? Also, I saw in a video that Microbehunter did not dilute his sample and rather put it on and looked at the edges of the sample. Which way would you recommend?

The challenge is to clean the specimen from unwanted debris while maintaining a concentration of bacteria as high as possible.

I strongly recommend to start with pure yoghurt culture which is free of unwanted things and very concentrated.

[MicroscopyLearning]

Will diluting the specimen into a glass of water than piping out the water onto the slide work? Also, I saw in a video that Microbehunter did not dilute his sample and rather put it on and looked at the edges of the sample. Which way would you recommend?

The challenge is to clean the specimen from unwanted debris while maintaining a concentration of bacteria as high as possible.

I strongly recommend to start with pure yoghurt culture which is free of unwanted things and very concentrated.

What do you mean by a yoghurt culture? Do you mean the bacteria sample or do you mean a yoghurt starter culture?

[Alexander]

What do you mean by a yoghurt culture? Do you mean the bacteria sample or do you mean a yoghurt starter culture?

A yogurt starter culture. It contains little more than bacteria typically streptococcus thermophilus and sometimes lactobacillus delbrueckii. Activate the culture in warm water with some lactose in it and here you are.

[MicroscopyLearning]

What do you mean by a yoghurt culture? Do you mean the bacteria sample or do you mean a yoghurt starter culture?

A yogurt starter culture. It contains little more than bacteria typically streptococcus thermophilus and sometimes lactobacillus delbrueckii. Activate the culture in warm water with some lactose in it and here you are.

Ok. I see what you mean now, but doesn't a starter culture sometimes contain yeast sometimes though?

[Alexander]

Ok. I see what you mean now, but doesn't a starter culture sometimes contain yeast sometimes though?

Yeast doesn't produce yogurt.