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Observing bacteria under the light microscope

Can one see bacteria using a compound microscope? The answer is a careful “yes, but”.
Generally speaking, it is theoretically and practically possible to see living and unstained bacteria with compound light microscopes, including those microscopes which are used for educational purposes in schools. There are several issues to consider, however.

Why bacteria are difficult to see

Bacteria are difficult to see with a bright-field compound microscope for several reasons:

  • They are small: In order to see their shape, it is necessary to use a magnification of about 400x to 1000x. The optics must be good in order to resolve them properly at this magnification.
  • Difficult to focus: At a high magnification, the bacterial cells will float in and out of focus, especially if the layer of water between the cover glass and the slide is too thick.
  • They are transparent: Bacteria will show their color only if they are present in a colony. Individual cells present on the slide are clear. Regular bright-field optics will only show the bacteria if one closes the condenser iris diaphragm. This is due to the difference in the refractive index between the water and the bacterial cells.
  • Difficult to recognize: An untrained eye may have problems differentiating bacteria from small dust and dirt which is present on the slide. Some bacteria also form clumps and therefore it is difficult to see the individual cells.

Research organizations and advances amateurs use phase contrast optics to see bacteria. This system converts the differences of the refractive index of the bacteria into brightness. The transparent bacteria can then be seen dark on bright background. In bright-field, closing the condenser iris diaphragm will also make the bacteria appear darker, but at the same time one also introduces artifacts (“fringes”) around the individual cells. One possibility is to stain the bacteria, but in this case there fixing and staining process may introduce artifacts.

What is a safe source of bacteria? For recreational or educational purposes, one should never use spoiled food or (heaven forbid!) use bacteria obtained from the human body and grown on agar plates. The risks involved are simply not worth it, especially when working with students. Other sources, such as soil or humus have other disadvantages. The impurities make it difficult to keep bacteria from other particles apart, especially if one uses bright-field optics. Rather I recommend the use of yogurt. It should be possible to see small circular cells (cocci), which may also occur in pairs. It is also possible to scratch some bacterial cells off from certain kinds of cheese. Brevibacterium can be found on Limburger cheese, for example. One has to be aware that some cheeses use a combination of bacteria and fungi, however, and that the larger fungal cells may outweigh the bacteria.

In summary, there are easier (and maybe also more interesting) specimens to observe than bacteria. I you want to see individual cells, then I do recommend that you start out with yeast suspensions. These eukaryotic cells are much larger and can be more easily identified.

For pictures of bacteria in phase contrast read the following post: Bacteria in phase contrast

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32 Comments

  • Liz says:

    i have a microscope and i would like to see bacteria and i know about colonies. i know that my microscope is not that strong. i was wondering if i stained the slide if with my weak microscope would be able to see the colonies a little bit.

    • Oliver says:

      Hello, Do not observe bacteria which you do not know. Take bacteria from safe sources (yoghurt etc). You need to suspend some yoghurt in some water and then make a wet mount. Bacteria can be seen starting a magnification of 400x and more. athey are difficult to see, because they are quite small and transparent.
      Oliver.

  • John says:

    Dear Sir,
    I was wondering if you could help I am trying to find somebody to identify the different strains of bacteria found on the bottom of horses feet,as part of a paper I would like to do on a farrier article.or even guided in the right direction on where to start. there is a verry common problem that horses suffer with called “THRUSH” a seamingly colective name for a load of bad bacteria!! this is why i would like try to identify them,there doesnt see to be anybody that has this for many,many years.I am not a student or scholar but a full time working farrier try to improve the welfare of horses!
    Thank you
    John

  • Luther says:

    John,

    Usually thrush is a fungus. Ie Candida Albicans.

    Just an FYI,

    Luther

  • Molly says:

    I found this article very helpful. I have an inexpensive microscope (Vivitar) that is supposed to magnify to 1200. I wish to use it to view what is in pond water and was unsuccessful the first time. If anyone has suggestions they would be welcome.

    • Oliver says:

      Pond water itself contains only few observable organisms. The interesting things can be found in the material which can be found growing on rocks an decaying wood and leaves. Scratch off some of this material and observe this.

  • Justine says:

    I am trying to spot bacteria that is in meat under a microscope. I have a microscope that goes up to 1000x. I don’t quite know what to look for. Any sugestions?

    • Oliver says:

      sorry, but this is almost impossible. Unless the meat is totally spoiled (decomposing) the bacterial concentration is far too low. You would first have to isolate the bacteria and grow them before being able to analyze them. It is possible to do some staining to make the bacteria visible, but this is elaborate and the benefits are not clear. The question is, why you would like to do this. Is it for quality control? In this case microscopic analysis is not very helpful.
      Oliver

  • syed says:

    Hi Mr. Oliver,

    Thanks for such a knowledgeable article. I have a question,
    I scratched off a bit of a probiotic pill and put under the microscope , even at 1600x all i was able to see was black sponge like structures. I wonder what i saw was colonies or something else? The bottle says “active organisms” though, and they are lactobacillus acidophilus.

    Thanks again

    • Oliver says:

      Hello
      I think that you looked at some undissolved part of the probiotic pill. 400x-1000x magnification is enough to see bacteria. These pills are not pure bacteria but contain much filling material (sugar etc) so that it is possible to press them into a pill. The pill contains freeze dried bacteria, and these are not growing in colonies, but in compressed form. If you want to observe bacteria, then it is easier to observe some “yoghurt starter cultures” (search Amazon). These are bacteria in powdered form which can be dissolved in some water.
      Oliver.

  • Michelle says:

    Hello,
    I am the editor-in-chief for a school magazine. Yesterday, we had an idea that we would swab different surfaces in the school (e.g. stair railings, elevator buttons, library computers, cafeteria tables, and so on), observe the bacteria cultures, and compile them in an article because it seems interesting.

    After some researching and reading this article, though, I’m not really sure whether this is a plausible idea. Would you have any advice regarding this? Thank you!

    • Oliver says:

      Hello,

      The growth and cultivation of bacteria in a school environment does require a slightly more elaborate answer.

      First of all, it is not uncommon for teachers and students to cultivate bacteria in petri dishes (there are even instructions online on how to do this and it is not difficult), but I generally do not recommend this for several reasons. Even in our school teachers already considered doing these projects, but I generally advised against it. The reasons are:
      1. Legal aspects: The cultivation of unknown bacteria already belongs to Biohazard category 2 (out of 4), because the bacteria are unknown. Bacteria used for genetic engineering (such as E. coli) belong to the lower category 1, because they are known not to be dangerous. Depending on the legislation of the country, your school legally might have to have a license. Having said that, every rotting sandwich in the locker of a student is probably equally hazardous, but then again, these are not grown in a “controlled environment”.
      2. Safety issues: Bacteria grown on agar plates can reach incredibly high concentrations, densities which are rarely found elsewhere in nature. While there is no danger of the bacteria “escaping” from the plates (they stick to the colonies in which they grow), there is the possibility that students accidentally open the plates and touch the colonies. You then need to do elaborate disinfection. And the skin better be intact, otherwise there is the possibility of infection.
      3. When you collect samples from surfaces, then you will inevitably also collect spores of fungi. These have the unfortunate characteristics to grow fast and large, quickly covering the whole petri dish so that the bacterial colonies (much smaller) are overgrown and therefore not visible anymore.
      4. You need to know how to make sterile agar plates, otherwise there are contaminants on the plate, which form colonies. When you spread your sample from the surface, then you will also spread the contaminant over the plate, creating a so-called bacterial “lawn” and it will not be possible anymore to see the colonies of the surface. Alternatively, you must use very fresh plates, on which contaminants could not form colonies yet.
      5. Staphylococcus alert: Railings and other objects that came in contact with humans might contain Staphylococcus aureus, which can be (depending on the particular strain) quite pathogenic.
      6. microscopic observation of these colonies is therefore highly discouraged, because of safety reasons.

      In short, I would not do this, especially because other safer (and also interesting) alternatives are available.
      Oliver

  • Daniel says:

    Hello Sir,

    I found this article very helpful. I am doing a science project in which I will be attempting to find the amounts of bacteria on different types of vegetables. I plan to use swabs to collect samples and then observe them under a light microscope, and then count them. Will I be able to count the bacteria I collect? Also, is this the correct process to do this experiment?

    Thanks,
    Daniel

    • Oliver says:

      Hello Daniel,

      There’s a good and a bad news:
      1. Yes, it is possible to collect bacteria using swabs and yes, this is the way how bacteria are usually collected from surfaces.
      2. But (unfortunately) you are not going to obtain useful data this way. Direct bacterial count under the microscope is not a suitable method. And I even do hope (!) that you are NOT going to find many bacteria this way – let me explain below.

      To point 1: The general method is, theoretically, quite easy.
      a. You use a marker to draw a defined area (eg. 5 square cm) on the fruit/vegetable from which you want to obtain the bacteria
      b. You use a moist cotton swab ans streak over the area, rotating the swab, doing this a defined number of times
      c. You then streak a clean slide (a defined area, defined number of times)
      d. Allow to dry completely first and then heat-fix (Google it) by pulling the slide through a gas flame or briefly putting it on a hot plate or over a lighter. This bakes the bacteria to the slide and prevents them from being washed off later. The slide should be hot, but you should still be able to hold it in the open palm without burning yourself.
      e. Observe defined area (maybe 5suqre mm) using oil immersion and count the bacteria. Observe several squares and then average.

      To point 2: Here we have several problems.
      a. You will not be able to see many bacteria, because there are too few on the vegetables. If you do find several of them, then the vegetable should not have been sold, or it is already rotting away. This is why I said that I hope that you won’t find too many bacteria.
      b. You will have real problems identifying the bacteria among the dust and debris that can be found on the slide. Individual bacteria are difficult to identify. You really have to know what you are looking for. There will be many soil and dust particles on the slide and these will be much more prominent than the few individual bacteria.
      c. Due to the generally low number of bacteria (some will still be on the swab), the sample size is too low and the collected data will not be significant.
      d. You will not be able to see a difference in bacterial count on the different vegetables. Why should there be more/less bacteria on the different fresh vegetables? Bacteria that feed on the vegetables are in the process of decomposing it and in this case it would not make much difference in comparing bacterial count anyway.
      e. There are several sources for error: you will not be able to collect all of the bacteria from the surface; not all of the bacteria will be transferred to the slide; some bacteria will not be recognized as such; other structures will mistakenly be considered bacteria; if bacteria are present, they might be in clumps and they might therefore not be counted as separate. All of these factors contribute to a statistical uncertainty. There is the possibility that the error (the standard deviation of the sample) is so high that the data is not relevant.

      The folks who do quality control of food do not use microscopes to determine bacterial count. They use swabs to collect the bacteria and then streak out agar plates to do a so-called plate-count. They then count the number of colonies formed on the agar plates. Now this is something that I do not recommend to be done in schools, due to safety. You are actively multiplying bacteria and the bacterial concentrations are really high and there is danger of infection. After all, you do not know which bacteria you are growing (and therefore you are already in a higher Biohazard category, which may not even be legal in some countries).

      The problem of directly making bacteria visible using the microscope is not a new problem. During the 19th century Hans Christian Gram developed a staining method to make bacteria visible in infected lung tissue. Otherwise he could not keep apart the bacteria from the human lung cells under the microscope. These bacteria could not be grown in petri dishes to do a plate-count. So it is an old problem. http://en.wikipedia.org/wiki/Gram_staining

      Now don’t believe me, but simply try it out to collect some bacteria from vegetables/fruits. It’s only a few minutes worth of time, but I think that you will quickly see the difficulties.

      If you are looking for a science project topic, then there are other things I would consider: Is this for a science fair, where people are looking at posters that you made? In this case I would recommend a project that involves photography (such as pictures of pond life under the microscope, different plant cross sections, etc), as this is more impressive for the audience. If you need to collect data (such as for the International Baccalaureate Extended Essay or other related research), then you must make sure that the collected data is sufficiently statistically significant – this means you need a lot of numerical data to talk about. I kind of doubt that direct collection of bacteria from different surfaces is suitable, in this case.

      In case you are wondering, why I’m giving you such a long answer: A few years ago, I had the task to supervise one of my students for a science project and she came up with exactly the same idea. She wanted to collect bacteria from different fruits and then count them under the microscope. I told her that this is not a good project, because of the low statistical significance of the data. But then again, if the collection of data is not of main importance, then it might well be worth a try. It is not a lot of work, and not too complicated.

      Last, if you want to see a lot of (safe) bacteria under the microscope, then I would buy freeze dried “yoghurt starter culture” and dissolve some of this powder in water to be microscoped. This also gives you a reference on how bacteria look like under the microscope.

      Oliver.

      • Daniel says:

        Dear Sir,

        Thank you so much for the answer. I’m sorry for making you write such a long answer, but it helped me a lot. I greatly appreciate your reply.

        Thanks again,
        Daniel

  • Lea says:

    Dear Sir,

    What is the usual size of a bacteria cell obtained from yogurt? I did my calculations and got 7 micrometres- does this sound reasonable?

    Thanks.
    -Lea

    • Oliver says:

      Bacteria can vary in their size. Everything in the micrometer range is likely to be correct. bacteria have a size of about one micrometer (so 7 micrometers is in the correct range, but more towards the generous side), and eukaryotes have a diameter of about 10 micrometers. Filamentous bacteria can be even longer.

  • shakila says:

    hello sir…
    I have taken some pictures of microbes that i have isolated from distillery effluent sample sir.But my problem is that i could not do any other tests such as bio chemical tests etc to identify my microbes.the only test i did was gram staining and then captured the pictures using methylene blue stain…so will i be able to identify what microbes are actually present in my sample with the microscopic picture and gram stain results?

    • Oliver says:

      Hello, it depends much on the type of microorganism (but I assume that you refer to bacteria, having done the Gram test). Bacteria can generally not be identified using microscopy. Genetically very different species can look very similar, and genetically similar organisms can look very different. And: the shape of bacteria is not always consistent but depends on the stage of the growth. The information content of the shape is also not very high. It is like asking if it is possible to determine the brand of a car by looking at the color. Very different cars can have the same color and cars of the same brand can have different colors. So the problem is not microscopy per se, but rather that morphology is not a good criterion for identification. Microscopy is a good method to determine bacterial concentration of a sample and to determine if a certain sample has been contaminated. If you have a culture in which you grow round bacteria (cocci) and all of a sudden you see rods (long shape), then you know that the sample was contaminated.
      greetings, Oliver.

      • shakila says:

        thank you so much sir for responding to my question…i will surely do further characterizations to identify the microbes present in the sample that i am considering…thank you once again sir for explaining me and clarifying my doubt.

        • shakila says:

          i am really sorry to trouble you again sir…but my doubt now is that will the shapes and gram staining results alone be enough to identify the bacteria or is bio-chemical test necessary to find out the species?i know that my questions are too basic but i am running out of time and have to identify my bacterial species as soon as possible sir..so can you please help me out of it sir?

          • Oliver says:

            Hello,
            In short: it is impossible to identify bacteria down to the species level just by Gram staining and microscopy alone. There are probably millions of different bacterial species and only a small number of them have been described so far. Shape and staining reactions are simply not able to give you this information (you can not identify several million different bacterial shapes, and even if, the shape says nothing about the species). Can you identify the type of animal by counting the legs? Or by looking at the color of the fur? Or by looking at the size? These characteristics too, are not species specific. If I give you a bird (2 legs) and a cat (4 legs) and then ask you to identify which one is which by counting the number of legs, then this is possible, because I have already reduced the options for you so that an identification is possible. But if the options are not reduced, you have no possibility.

            If you need to identify the bacteria for a university course, then your teacher probably gave you some species which were already identified before, and which you now have to identify. For example, if you are given a Bacillus subtilis culture and an E. coli culture and then asked which one is which, then of course you can find this out by doing a Gram stain (knowing the B. subtilis stainas Gram positive and E. coli Gram negative). But if you have absolutely no idea what the samples are, because they were taken from the wild, then there is no possibility to find this out by microscopy alone. There are simply too many possibilities and bacterial shape is too unspecific.

            There are some rare exceptions. For example, if you see aerobic, Gram positive endospore forming bacteria, then these belong probably to the genus Bacillus. But you won’t be able to go down to the species level. Gram positive cocci in chains could be Streptococci, but they do not have to be. You can also reduce the possible options by growing them on selective growth media and by observing the shape of the colonies. There is no single method. If you need to identify completely unknown bacteria, then I suggest that you use 16s rDNA sequencing and then search the database for the nearest relative, but the chances are good that the isolated bacteria have not been classified before and therefore are not in the database. So far only several thousand bacterial species have been classified, and there are many more in nature. Biochemical tests are possible but unless you use a commercial kit, it is very time consuming and these tests are often optimized for medically relevant bacteria (so if you have a new species, then the test won’t work, because the chemical reactions of this new species are not yet known and therefore not in the database).

            Oliver.

  • Aneu Sood says:

    If i wish to examine saliva samples for bacteria and then show that on to a computer screen what would be the best microscope to buy?
    Thank you
    Also which microscope would you recommend to do this

  • Richard says:

    Sir, I am studying wet soil samples from my gardens. Some of the slides display white lines when they dry. What is the cause of this. I am using a light microscope at 400 power. The only thing I can think of is that the droplets are contracting causing division lines. They look like geomertric rice paddies as viewed from an airplane. Thank you! Richard

    • Oliver says:

      I have no idea what this could be. Can you post a picture of this? It could be that the drying water droplets start to accumulate salts (etc) forming these lines.
      Oliver.

  • Millet says:

    Hi Sir,

    Thanks for the helpful article.
    Im just interested in knowing why medtechs grow bacteria in a petridish and use the grown bacteria to put on the slide when they can just use the sample like saliva to see the bacteria?

    Thanks

    • Oliver says:

      Hello,
      Of course it is possible to observe bacteria directly in e.g. a sample of saliva, but there is little scientific meaning to this. We already know what to expect. You will see different bacteria of different shape and size. There is no “higher” scientific point to this. This knowledge is already there. It only becomes more interesting when a variety of chemical tests are done on the bacteria in order to identify them (microscopy alone is not enough to identify bacteria), eg for diagnosis of illnesses, etc. And for this you must grow them in pure culture. There are other reasons as well.

      1. Saliva contains a mixture of different bacteria, sometimes it is more useful to observe bacteria of one kind. To get this “pure culture” you need to grow them on petridishes and then observe one single bacterial colony (which contain bacteria of one kind).
      2. the concentration of bacteria in saliva is lower than in pure culture grown on petridishes
      3. The growth medium used for petridishes can promote the growth of certain bacteria but not others. This way it is possible to selectively grow only those bacteria that one is interested in.

      I can think of one thing where it would make sense to observe bacteria directly in saliva would be in order to directly count the bacterial density, if this is what is needed.

      All the best, Oliver

  • Abigail says:

    how much magnification do you need on a microscope to see cold of flu bacteria?

    • Oliver says:

      Cold flu is not a bacterium, it is a virus and viruses can not be seen with light microscopes. You need an electron microscope of a magnification of about 100000x or more. Light microscopes only go up to 1000x.
      Bacteria can be seen starting 400x – 1000x but you need phase contrast equipment (expensive) to see them well.

  • Eric says:

    Hello,

    I am interested in being able to show prepared slides of bacterial on a monitor through a digital microscope for museum education purposes. Is this possible if I use prepared and stained slides? Do I need optical zoom or will digital zoom work with stained slides?

    Thanks,

    Eric

    • Oliver says:

      Digital zoom alone is not enough to resolve bacteria. You need a microscope with a 40x or 100x objective to see them. Bacteria are generally difficult to see, due to their low contrast and small size. Regular bright field microscopes are not able to show bacteria well if they are not stained, so you need to get slides which are stained. Phase contrast microscopes (expensive) work best for bacteria (stained and unstained). If you just want to show cells (and if it does not matter if they are bacteria or not), then I would recommend that you use yeast (eukaryotic fungus), which are much larger and much easier to see.
      Oliver

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