There are millions of different species of bacteria, and they come in a diverse array of shapes, sizes, and other defining internal and external features. These physical characteristics are what helps us identify one type of bacteria from another, and to do that, we need a light microscope.
In this article, we’ll talk about how to visually inspect and identify bacteria under the microscope.
How to prepare microscope bacteria
Bacteria, or in scientific terms, prokaryotes, are single-celled organisms without a defined nucleus, nor specialized organelles. To view bacteria under the microscope, we first need to prepare the sample through slide preparation and staining.
Growing bacteria on culture media
To be able to see bacteria under a light microscope, growing the bacteria in a culture media that is rich in nutrients is often necessary. However, since different bacteria come with different nutrient requirements, it’s important to choose the right type of media based on the bacteria that is going to be cultured.
The most common types of bacteria culture media that are used in microbiology and microscopy include the following:
- Nutrient agar media – the most common kind of non-selective, nutrient-rich media is the nutrient agar, which allows for the growth of various species including microaerophilic, aerobic, and anaerobic microorganisms
- Selective agar media – meanwhile, the selective agar allows for selective bacterial growth, which means it only allows for one type of bacteria to grow while inhibiting another type of bacteria
- Differential culture media – another type of media is the differential culture, which is used for differentiating between different kinds of bacteria
After incubating and growing the bacteria on an agar plate, the next step is to perform a bacterial smear, which essentially means mounting a thin layer of the bacteria onto a glass specimen slide.
What you’ll need
- Bacteria sample
- Distilled water
- Glass slide
- Felt marker
- Inoculating loop
- Bunsen burner
How to prepare a slide
- Mark the glass slide with a felt marker in order to indicate where you intend to place the bacteria and make the smear.
- Use the inoculating loop to place a tiny drop of water on top of the marked spot.
- Prepare the smear by placing the inoculating loop in the blue flame part of a bunsen burner, allowing the loop to glow a bright red, then letting it cool.
- Use the heated and cooled loop to remove a decent sized bacteria colony from the broth or agar plate. To prevent sample contamination, remember to observe and maintain your aseptic technique.
- Mix the water on the slide with the bacteria on the inoculating loop. Afterwards, flame the loop one more time.
Leave the slide to air dry.
Staining is also an important part of preparing microscope slides of bacteria. This is because most bacteria can look transparent under the microscope without staining, but with staining, the different parts, cells, and structural components of the bacteria are more easily distinguishable against one another, including the membranes, cell wall, and cytoplasm.
There are various stains used for bacterial slides, with the most common ones being safranin, methylene blue, and crystal violet.
How to stain the slide
- On a staining rack or paper towel, place the prepared bacteria slides side by side.
- Cover each slide with your chosen stain. Let sit for 1 to 2 minutes.
- Run a gentle stream of water on the surface of the slide to remove any excess stain.
After you’re done staining the slide, mount the stained slide on the stage of the microscope, secure with clips, and view the specimen, starting at lower magnifications before gradually increasing until you reach the desired level of detail you want to see.
How to identify microscope bacteria
As we mentioned above, it is possible (and quite easy) to identify bacteria under a microscope based on its physical characteristics, most especially the shape and size of the bacteria. Now, bacteria come in various shapes, with the three main types being spiral, bacilli, and cocci.
Cocci – is the most common kind of bacteria, which typically appears in groups. The name “cocci” comes from the spherical shape of the bacteria. Some examples include diplococci (pairs), streptococci (chains), and staphylococci (clusters).
Bacilli – are similar to cocci in the sense that they can either come as in individual bacteria or in groups. This type of bacteria is rod-shaped. Examples of bacilli are diplobacilli (pairs) and streptobacilli (chains).
Spiral – any bacteria that appears spiral is called a spiral bacteria. For instance, the spirillum is a thick and durable spiral, while the spirochete is much more flexible and slender.
Gram positive vs gram negative bacteria
You can also identify a bacteria by determining whether it’s gram positive or gram negative, which can be done through the bacterial slide staining process. It’s a common and highly useful technique to identify bacteria, albeit not at a species level. Common stains you can use include iodine, crystal violet dye, and counterstain safranin.
Gram positive bacteria – are bacteria with a thick layer of peptidoglycan that an iodine- crystal violet complex stain is attracted to, which gives the bacteria a purple or bluish appearance under the microscope. Bacillus, Streptococcus, and Listeria are some of the many types of gram-positive bacteria.
Gram negative bacteria – are those that don’t have a thick peptidoglycan layer, so rather than using crystal violet-iodine complex, a safranin counterstain is used instead. This gives the bacteria a reddish appearance when viewed on a microscope. A couple of examples include cyanobacteria and proteobacteria.
Some real-life examples
Below are two of the most commonly known bacteria, including brief information about their appearance, physical characteristics, effects on humans, and so on:
Staphylococcus Aureus or S. Aureus is an extremely common round-shaped bacteria. It’s a gram-positive cocci bacteria that features a thick peptidoglycan layer which makes it ideal for gram staining techniques.
Significant amounts of S. Aureus can be found in the respiratory tract, in the nose, and on the skin. There are also various strains of the S. Aureus, with some of them being pathogenic, and these ones are responsible for respiratory inspections, abscesses, and skin infections.
Escherichia coli or E.coli is a gram-negative species of bacillus shaped bacteria that can be easily observed under a microscope, even for those with the untrained eye. This bacteria has a fast growth rate, doubling every 20 minutes, making them a common choice for bacterial related research purposes.
Contrary to popular belief, most strains of E.coli are actually harmless to humans. It’s just that there are a few pathogenic strains here and there that cause gastrointestinal infections, hence the inclusion of the term “E.coli” in our lexicon.
A few helpful tips
Before you begin on the wonderful journey of microbiology and microscopy, keep in mind these helpful tips for a better, safer, and more informative experience:
Prepare a good sample
Ensure that everything you’re working with is clean, from the water to the slide and the dropper. This will make things safer, and won’t skew with your results. Moreover, unless your bacteria sample is big enough to view without staining, don’t skip the staining procedure, and remember to use the right stains for your bacterial samples.
Experiment with your lens sizes
The reason for starting at lower magnifications is to make it easier for you to focus on what you want to see, which becomes progressively difficult as you reach higher magnifications. Skipping steps will only make it all the more difficult. That said, looking at bacteria at, say, 400X or 1000X magnification will offer stunning detail, including bacteria swimming, and different cell parts.
Be patient while viewing
The truth is, bacteria can be hard to recognize for beginners and expert microscope users alike, so you need to be patient when using the microscope. For instance, some bacteria may look like dirt at first glance, and sometimes, it’s hard to differentiate the ambient dust from the actual microorganisms, since some bacteria tend to clump together.
A good tip is to start with prepared slides and pre-made cultures with a single strain of bacteria to help you recognize different types under the microscope and train your eyes on how to look through a microscope lens, before experimenting with “real life” samples like your drinking water or your garden soil.
Prioritize your safety
Another benefit of prepared slides, especially for younger students, is that they are much safer to use at home. Other safety practices when handling microscopes and bacterial samples is to always wear gloves and to avoid using bodily fluids and spoiled food as your sample specimens so as not to risk contamination and infection.
Document your findings
After mastering the art of using microscopes and identifying bacteria with it, a great way to take your experiments one step further is to photograph your findings with the use of microscope cameras and other related accessories. With these, you can immortalize your samples, and even manipulate the images on your computer.
Bacteria are incredibly interesting samples to study under a microscope. However, viewing bacteria under the microscope can be a difficult task. Fortunately, there are many ways to make this process easier, from using the correct type of microscope, to preparing your samples correctly, staining them, and training your eye how to discern one bacteria from another.
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