Viewing Bacteria With Bright-field Microscopy

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MicroscopyLearning
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Viewing Bacteria With Bright-field Microscopy

#1 Post by MicroscopyLearning » Tue Jun 08, 2021 4:54 pm

Hello. I have heard that you can get a clear image of bacteria with phase-contrast microscopy, but I would also like to know if it is possible with bright-field microscopy. And if immersion oil is needed for this.

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Re: Viewing Bacteria With Bright-field Microscopy

#2 Post by PeteM » Tue Jun 08, 2021 5:17 pm

Bacteria are usually stained to add contrast and help in identification. Most of them tend to be small - with not much more than their shapes visible even at 400-1000x. Given all that, brightfield viewing works fine.

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Re: Viewing Bacteria With Bright-field Microscopy

#3 Post by apochronaut » Tue Jun 08, 2021 6:52 pm

Bacteria can be difficult because many of them are quite transparent, however there are several techniques that you can use in BF. To get a practice culture, take some vegetable : cabbage, cauliflower, broccoli, turnip, radish, carrot, cucumber, zucchini, beet ; something firm and with a skin or rind that is intact, about 1/2 c. sliced in 1/4" slices and place in a small 250ml.or 8 oz. mason or single lid jar. Fill the jar to within 1/2" of the top with water, then place a plastic lid or small flat stone on the vegetables, anything to weight them and keep them under the water . Pour off excess water to keep 1/2" headspace. You will have around 1/2 c. of water or roughly 125 ml. Add 1 tsp. of salt. Tighten on the lid and shake the salt to dissolve it. Loosen the lid slightly, place in a small overflow bowl and set to ferment in a warm place....30°c or around 80°F . In about 2 days you will start to get enough lactic bacteria to see and experiment with, followed by lots of budding yeasts. Once it is really going and you can smell a sauerkraut aroma, making sure there is still 1/2" headspace, you can almost tighten the lid and put the jar in an open plastic bag or small bowl in the fridge, to guard against overflow . It will continue to ferment for many weeks in the çool, with a very healthy yeast/bacterial colony.
Lots of different mask shapes under the condenser will give you some variations on oblique illumination, which will enhance contrast.
It is best to oil both the condenser, if it has an N.A. over 1.0 and the objective. The yeast and bacteria show up quite well under BF.
You can also try various food colourings or vegetable dyes. The water from boiling, carrot, red and yellow beet, red cabbage, onion skin etc.

Don't forget to eat your fermented vegetables

MicroscopyLearning
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Re: Viewing Bacteria With Bright-field Microscopy

#4 Post by MicroscopyLearning » Tue Jun 08, 2021 8:28 pm

Hello, thank you for the response. Can you clarify what you mean by a mask shape. I am not familiar with this term.

apochronaut
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Re: Viewing Bacteria With Bright-field Microscopy

#5 Post by apochronaut » Tue Jun 08, 2021 9:20 pm

Masks are types of filters that reroute, reshape, reduce or block portions of the illumination beam. They can be used in combination with one another or singly. A type of relief contrast, somewhat like DIC for instance can be achieved using masks. A slit condenser, which is a form of malleable mask is used to produce a type of DIC, in fact being the condenser type in at least one commercially produced DIC system.
They can also be simple cutout shapes too, measured to fit a filter slot , such as these sold on ebay, which can be ganged.
https://www.ebay.ca/itm/274035677903?ha ... SwT6ldlMIv

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Re: Viewing Bacteria With Bright-field Microscopy

#6 Post by DonSchaeffer » Tue Jun 08, 2021 10:58 pm

I find that if you close the condenser all the way, you focus carefully and amplify the image with side lighting using the highest power objective you can get very good images of bacteria. They look like tiny circles and ovals if your focus a little ahead of them and they sharpen to dots and lines--but very clearly visible. You can even get a 3-D like view if your use light carefully.

MicroscopyLearning
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Re: Viewing Bacteria With Bright-field Microscopy

#7 Post by MicroscopyLearning » Wed Jun 09, 2021 4:24 pm

apochronaut wrote:
Tue Jun 08, 2021 9:20 pm
Masks are types of filters that reroute, reshape, reduce or block portions of the illumination beam. They can be used in combination with one another or singly. A type of relief contrast, somewhat like DIC for instance can be achieved using masks. A slit condenser, which is a form of malleable mask is used to produce a type of DIC, in fact being the condenser type in at least one commercially produced DIC system.
They can also be simple cutout shapes too, measured to fit a filter slot , such as these sold on ebay, which can be ganged.
https://www.ebay.ca/itm/274035677903?ha ... SwT6ldlMIv
Ah. This makes sense. Thanks for the explanation. I will investigate more into this.

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Re: Viewing Bacteria With Bright-field Microscopy

#8 Post by MicroscopyLearning » Wed Jun 09, 2021 4:33 pm

DonSchaeffer wrote:
Tue Jun 08, 2021 10:58 pm
I find that if you close the condenser all the way, you focus carefully and amplify the image with side lighting using the highest power objective you can get very good images of bacteria. They look like tiny circles and ovals if your focus a little ahead of them and they sharpen to dots and lines--but very clearly visible. You can even get a 3-D like view if your use light carefully.
What magnification do you recommend that I be on?

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Re: Viewing Bacteria With Bright-field Microscopy

#9 Post by DonSchaeffer » Wed Jun 09, 2021 8:49 pm

At least 40x objective...60X is better

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Re: Viewing Bacteria With Bright-field Microscopy

#10 Post by dtsh » Wed Jun 09, 2021 10:17 pm

Even with the 100x objective bacteria are still very small and it is difficult to see much detail.

Without staining or other techniques, there's usually not a lot to see aside from the basic shape with brightfield; even with phase there's not much to see, though it does make seeing the less visible ones much easier without the stain.

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Re: Viewing Bacteria With Bright-field Microscopy

#11 Post by apochronaut » Thu Jun 10, 2021 2:26 am

Even in the 19th century, bacteriologists were using BF to understand their chosen passion with a surprising degree of accuracy.They applied oblique illumination and with patience they observed behaviour and noted it. Early research microscopes were always equipped with sophisticated oblique condensers.
When the electron microscope was invented, there was a bit of a dark age for microscopy. Everything became structure because the electron microscope only saw that. The poor old light microscope is powerless to see the stuctural detail of the electron microscope but it can reveal process and behaviour, well beyond the capability of the electron microscope. The electron microscope is kind of like a tool for miniature bridge builders.
With better technique, lighting and optics in BF one can see details of bacteria that might not be obvious to the inexperienced . Patient observation will reveal details of behaviour that will complete the details.
Have a look at turn of the century bacteriology textbooks , written before even DF had been invented If you want an eye opener about what BF can do.

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Re: Viewing Bacteria With Bright-field Microscopy

#12 Post by dtsh » Thu Jun 10, 2021 4:53 am

apochronaut wrote:
Thu Jun 10, 2021 2:26 am
Have a look at turn of the century bacteriology textbooks , written before even DF had been invented If you want an eye opener about what BF can do.
Have any particular recommended titles?

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Re: Viewing Bacteria With Bright-field Microscopy

#13 Post by MicroBob » Thu Jun 10, 2021 9:34 am

I would be interested in this as well, Phil.
There really seem to be fashions of what scientfic methods to use and this leads to the use of sub-optimal methods in some cases. As much as I like DIC as a quick method to get a visually appealing image it doesn't really bring out more detail and adds artefacts.

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Re: Viewing Bacteria With Bright-field Microscopy

#14 Post by apochronaut » Thu Jun 10, 2021 11:14 am

In no specific order.

Henrici's Molds, Yeasts , Actinomyces 1930
Pathogenic Micro-Organisms including Bacteria and Protozoa William Hallock Park Anna W. Williams 1910
A Textbook of Bacteriology Hans Zinsser Frederick F. Russel 1922
Bergey's Manual of Determinative Bacteriology 1923
Bacteriology Richard Tanner Hewlett 1898
Verfahren zur Untersuching, zum Konservieren und Photographieren der Baktierien Robert Koch 1877

Although written about a century after Koch, Pasteur and Lister but still now, 40 years old. These two researchers offer much insight into the necessity of observing process and patience in the study of bacteria. Their work was revolutionary and modern bacteriology has still not caught up. We are generally, still stuck in the dark ages of morphology.
Cell Wall Defficient Bacteria : Stealth Pathogens Lida Mattman 1974
Cell Wall Deficient Bacteria : Basic Principles and Clinical Significance Gerald Domingue 1982
Last edited by apochronaut on Thu Jun 10, 2021 1:35 pm, edited 1 time in total.

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Re: Viewing Bacteria With Bright-field Microscopy

#15 Post by Hobbyst46 » Thu Jun 10, 2021 12:03 pm

apochronaut wrote:
Thu Jun 10, 2021 2:26 am
... When the electron microscope was invented, there was a bit of a dark age for microscopy. Everything became structure because the electron microscope only saw that. The poor old light microscope is powerless to see the stuctural detail of the electron microscope but it can reveal process and behaviour, well beyond the capability of the electron microscope....
Astonishingly, since the late 1980's, optical microscopy has been revolutionized, light microscopes feature super-resolution of live specimens and dominate several areas of research. For hobby use, however, it is just wonderful to relax and observe the micro structures; and even look at stained bacteria if one is so inclined...

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Re: Viewing Bacteria With Bright-field Microscopy

#16 Post by apochronaut » Thu Jun 10, 2021 2:02 pm

That's true but light microscopy still has severe limitations in viewing small structures but increasing capability in viewing process.

We still haven't broken the .2 micron barrier although there are rumours of various advances. Pre-magnifying with nano lenses, lots of digital modifications.

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Re: Viewing Bacteria With Bright-field Microscopy

#17 Post by viktor j nilsson » Thu Jun 10, 2021 2:57 pm

apochronaut wrote:
Thu Jun 10, 2021 2:02 pm
That's true but light microscopy still has severe limitations in viewing small structures but increasing capability in viewing process.

We still haven't broken the .2 micron barrier although there are rumours of various advances. Pre-magnifying with nano lenses, lots of digital modifications.
Can you elaborate what you mean? There are various super resolution techniques that can resolve things well below 0.2micron (200nm). Using structured illumination, many source images and image reconstruction algorithms of course. The recent paper linked below can resolve features as small as 40nm.

https://www.nature.com/articles/s41467-021-21835-8#

Or do you mean to reach below 200nm without using any image reconstruction techniques?

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Re: Viewing Bacteria With Bright-field Microscopy

#18 Post by apochronaut » Thu Jun 10, 2021 4:40 pm

of course. I was more talking about the possibilities for average microscopists with limited budgets.
Neatest thing I have seen so far is the concept , which has a prototype working at some level, of pre-magnification with nano lenses. They are claiming 25,000 diameters. Any image reconstruction system would be best useful in real time, otherwise it's use becomes limited . The point I was making that viewing process is what is lacking in high magnification microscopy.

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Re: Viewing Bacteria With Bright-field Microscopy

#19 Post by viktor j nilsson » Thu Jun 10, 2021 4:47 pm

I see. I have assembled most of the parts I need to build a structured illumination microscope using a 10lines/mm Ronchi grating as a mask, and some stepper motors and a raspberry pi to do the rotation and lateral shift. It should be able to achieve sub-diffraction resolution and good optical sectioning. It's not too complicated and definitely something that can be done at home:

https://nidhoggmicroscopy.wordpress.com ... vYEcRA3XGw

But yes, it's fairly slow, and not for viewing. And definitely not something for the average microscopist.

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Re: Viewing Bacteria With Bright-field Microscopy

#20 Post by MicroscopyLearning » Mon Jun 14, 2021 3:50 am

dtsh wrote:
Wed Jun 09, 2021 10:17 pm
Even with the 100x objective bacteria are still very small and it is difficult to see much detail.

Without staining or other techniques, there's usually not a lot to see aside from the basic shape with brightfield; even with phase there's not much to see, though it does make seeing the less visible ones much easier without the stain.
Would DIC be any more useful in seeing detail?

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Re: Viewing Bacteria With Bright-field Microscopy

#21 Post by dtsh » Mon Jun 14, 2021 4:19 am

MicroscopyLearning wrote:
Mon Jun 14, 2021 3:50 am
dtsh wrote:
Wed Jun 09, 2021 10:17 pm
Even with the 100x objective bacteria are still very small and it is difficult to see much detail.

Without staining or other techniques, there's usually not a lot to see aside from the basic shape with brightfield; even with phase there's not much to see, though it does make seeing the less visible ones much easier without the stain.
Would DIC be any more useful in seeing detail?
I don't know as I have zero experience with DIC, but given the typical size of bacteria and the limits of visible light microscopy, my guess would be no.

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Re: Viewing Bacteria With Bright-field Microscopy

#22 Post by apochronaut » Mon Jun 14, 2021 3:04 pm

I look at bacteria all the time. Yes, they are very small, so seeing structural details is a challenge. Staining provides aome assistance and in combination with a bunch of known details one can speciate them fairly accurately if that is the goal. If you just want to look at them and try to see their structure or features, it is impossible to get around the fact that high resolution microscopy requires high resolution optics. DIC is not a replacement for a high contrast 1.4 N.A. apochromat objective. D.I.C. and a 1.4 N.A. apochromat objective is better but so is phase contrast and a 1.4 N.A. apochromat objective, or DF and those or similar optics.

I use BF apochromats, oblique BF apochromats, DF apochromats, 4 different phase systems at the achromat level including a weird phase/DF hybrid, phase at the apochromat level. I can assure you that a change in contrast technology, while maintaining achromat objectives will only provide a slight improvement in what you can see. A change in the colour correction capability of the objectives will give you a base level of excellence , so that when you can apply an add on contrast enhancement system, it will provide a major difference.
Phase is a bit unique in that the objective type is usually coupled to the objective. Highly colour corrected phase objectives are usually expensive but the good thing is, you can start with achromats and move up as you can afford it , without having to change the condenser.

I have this anecdote, which I have mentioned in the past in some old thread. I will repeat it. I have a nice planachro dark phase infinity corrected system. It is a kind of default system because it is complete, has good working distance, provides excellent 20mm f.o.v. imaging for an achromat system and if something happens to one of the objectives, it is fairly easily repairable. I was provided with a milk sample from a situation where the milk was chronically clotting prior to it's b.b.d. In examining it under 100X 1.25 oil planachro dark phase, I could clearly see zillions of fat globules, the occasional bacterial cell and every now and then a large swelling movement of the sample, as though some leviathon in the Sargasso sea was on the move. Whatever was in there was big and motile. However, I could see nothing except the secondary impression of movement caused by the undulation of fat globules. Everywhere I moved the sample, it was the same. There were a lot of something in there that were invisible. With that system, I only have dark phase.
I switched to the planapo 100X 1.32 dark phase. Voila. There they were, all visible in their undulating glory. Bacillus Cereus. Very good planachros, even phase planachros couldn't see them.

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Re: Viewing Bacteria With Bright-field Microscopy

#23 Post by Hobbyst46 » Mon Jun 14, 2021 4:36 pm

apochronaut wrote:
Mon Jun 14, 2021 3:04 pm
I have this anecdote, which I have mentioned in the past in some old thread. I will repeat it. I have a nice planachro dark phase infinity corrected system. It is a kind of default system because it is complete, has good working distance, provides excellent 20mm f.o.v. imaging for an achromat system and if something happens to one of the objectives, it is fairly easily repairable. I was provided with a milk sample from a situation where the milk was chronically clotting prior to it's b.b.d. In examining it under 100X 1.25 oil planachro dark phase, I could clearly see zillions of fat globules, the occasional bacterial cell and every now and then a large swelling movement of the sample, as though some leviathon in the Sargasso sea was on the move. Whatever was in there was big and motile. However, I could see nothing except the secondary impression of movement caused by the undulation of fat globules. Everywhere I moved the sample, it was the same. There were a lot of something in there that were invisible. With that system, I only have dark phase.
I switched to the planapo 100X 1.32 dark phase. Voila. There they were, all visible in their undulating glory. Bacillus Cereus. Very good planachros, even phase planachros couldn't see them.
Successful use of the microscope to solve a practical problem is so rewarding !

A question: was there a green filter in the phase contrast illumination path ?

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Re: Viewing Bacteria With Bright-field Microscopy

#24 Post by apochronaut » Mon Jun 14, 2021 4:47 pm

No. I seldom use one. Occasionally, for photos. I guess the suggestion being that limiting extraneous colour artifacts might have allowed those lacto denizens to be visible with an achromat?

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Re: Viewing Bacteria With Bright-field Microscopy

#25 Post by Hobbyst46 » Mon Jun 14, 2021 4:54 pm

apochronaut wrote:
Mon Jun 14, 2021 4:47 pm
No. I seldom use one. Occasionally, for photos. I guess the suggestion being that limiting extraneous colour artifacts might have allowed those lacto denizens to be visible with an achromat?
Yes, that is what crossed my mind. The theoretical difference in resolution between NA 1.25 and 1.32 is small, and I read in Wikipedia that those bacilli are about 2 um long. Admittedly, I have never observed phase contrast images of bacteria.

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Re: Viewing Bacteria With Bright-field Microscopy

#26 Post by apochronaut » Mon Jun 14, 2021 8:52 pm

I actually have pictures but they are stuck on the hard drive of a computer that won't boot up.
Maybe had I used a green filter with the achromat the inevitable blurring due to refraction would have been filtered out but what I do recall was that the cell wall was indistinct with the planachro, complicated by a fair degree of chroma from the fat globules. You could sense movement but any detail was indistinct. It probably was not so much the difference in N.A., since the condenser is in fact a .90 but the difference in resolution masking caused by chroma. Achromats are much more prone to not achieving their theoretical resolution since different wavelenths spatially shift details.

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Re: Viewing Bacteria With Bright-field Microscopy

#27 Post by MicroscopyLearning » Thu Jul 01, 2021 5:00 am

apochronaut wrote:
Thu Jun 10, 2021 2:26 am
Even in the 19th century, bacteriologists were using BF to understand their chosen passion with a surprising degree of accuracy.They applied oblique illumination and with patience they observed behaviour and noted it. Early research microscopes were always equipped with sophisticated oblique condensers.
When the electron microscope was invented, there was a bit of a dark age for microscopy. Everything became structure because the electron microscope only saw that. The poor old light microscope is powerless to see the stuctural detail of the electron microscope but it can reveal process and behaviour, well beyond the capability of the electron microscope. The electron microscope is kind of like a tool for miniature bridge builders.
With better technique, lighting and optics in BF one can see details of bacteria that might not be obvious to the inexperienced . Patient observation will reveal details of behaviour that will complete the details.
Have a look at turn of the century bacteriology textbooks , written before even DF had been invented If you want an eye opener about what BF can do.
Hello. What are some methods they might have used to view bacteria. When I use brightfield at 1000x, I often only see a blurred image. Can this be resolved with adding oil to the objective?

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Re: Viewing Bacteria With Bright-field Microscopy

#28 Post by apochronaut » Thu Jul 01, 2021 12:37 pm

I have never encountered a 100X oil immersion objective that gave a good image without oil. Some are better than others but the image is of low contrast, lousy definition and sometimes impossible to focus at all. I have come across 2 lower magnification oil immersion objectives that did provide an acceptable image dry, although not as good as with immersion.

Almost all 100X objectives that would be fitted to a biological microscope will be oil immersion. If an objective has an N.A. of 1.0 or higher, it is an immersion objective. 99% of them will be oil. A few will be water or glycerin but the immersion medium is clearly marked.

It will also have a specification for coverslip correction. This will be either 0 or .17 on a 100X objective. If yours says .17 then you need a cover slip and likely immersion oil. If by some quirk it says 0 and it is still an oil immersion objective then it is to be used without a coverslip.

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Re: Viewing Bacteria With Bright-field Microscopy

#29 Post by MicroscopyLearning » Wed Dec 15, 2021 8:54 pm

DonSchaeffer wrote:
Wed Jun 09, 2021 8:49 pm
At least 40x objective...60X is better
Hello. I am interested in viewing bacteria with bright-microscopy. What would your general procedure for doing this be?

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Re: Viewing Bacteria With Bright-field Microscopy

#30 Post by DonSchaeffer » Wed Dec 15, 2021 9:05 pm

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.

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