Whether you’re simply passionate about observing microorganisms or you do that for a living, you need to use the right equipment and employ the proper techniques to get the best out of those specimens you want to observe under the microscope.
If you’re on the lookout for a quality lab microscope for sale, rest assured that the market is generous enough when it comes to such instruments yet if you’re new to this and you need to learn more about getting your sample ready and, especially, about staining, this post might be of help.
In order to observe microorganisms under the microscope, they need to undergo some processes. Growing the bacteria you want to observe is the first step to take and there are several ways and media used to do that.
Depending on the type of bacteria you’re interested in and your observation purposes, you can use one of the following: basal, selective, transport, or enriched media. These techniques will help you grow specific bacteria and/or prohibit the growth of others.
Once you’ve done that and made sure you took this step properly, you will have to get your microorganisms ready for observation and here is where the staining part comes into sight. Its main purpose is to highlight cells and their parts. Just as it happens with the growth techniques, staining can be done using various types of stain.
Bacteria and water have almost the same refractive index and they cannot be seen with the naked eye when under the microscope because they are almost invisible or opaque. Staining thus makes the cells and their components visible. The various substances used for staining adhere to the cell and give it color.
Without staining the bacteria, the microscope will be of little use. Therefore, the specimen to be examined must be fixed and stained properly in order to be visible. By staining them, their morphological features are highlighted for observation.
Depending on the type of stain and the bacteria used, you can observe cell walls and components in a way that will help you visualize metabolic processes, the number of cells within specific biomass, and so on. Not to mention that stains will also help you see dead cells and live cells.
As we’ve said before, the purpose of your examination will help you decide which type of stain to use. Since there are over 20 types of stains, it won’t be difficult to find one that will match your exact needs. You can thus find stains that will help you detect proteins and lipids or highlight spores. Of course, the purposes are various, hence the many stain types available.
However, not all stains can be used for living cells. The ones that can be used to observe living organisms, though, include toluene red, Bismarck brown, Nile red and Nile blue as well as fluorescent stains for DNA observation, just to name a few.
For example, Eosin Y is used by medical practitioners interested in conducting a PAP smear. When contacting cytoplasm, red blood cells, and cell membrane, this acid fluorescent stain becomes red. This stain type is also employed for testing blood marrow. What’s great about stains is that different types can be used for the same bacteria sample.
If you use eosin and hematoxylin, you will get a better contrast between the different parts of a cell. The eosin will make the cell turn red whereas the hematoxylin will stain the cell nuclei in blue. It will thus be easier for a medical practitioner to examine blood marrow samples and PAP smears when these two stain types are used together.
By opting for differential stains, that is, for two or more stains, the cells can be categorized into various types or groups. Even if both simple and differential stains allow for the observation of cell morphology, differential stains allow the observer to get more information about the cell wall.
A common type of stain used by hospital workers is Gram’s stain. This is employed to identify harmful microorganisms. The stain involves the use of different colorants that will trigger different effects on different types of microorganisms. There are three steps to take when using the Gram stain.
The first phase involves the use of Hucker’s crystal violet, a colorant that will make all the microorganisms in the sample turn violet. The second step requires the use of iodine to make the color adhere to all cells that are Gram-positive. These are primarily Streptococcus and Staphylococcus.
The stain will then be washed away and Safranin O will be added. This will enhance the contrast between the cells that are Gram-negative and the rest of the cells in the slide. Gram staining is of great importance in medicine as examining the bacteria using this staining method will help the observer know more about a bacterium’s susceptibility to certain antibiotics.
That’s why it is so commonly used. However, there are bacteria that cannot be stained using this standard laboratory procedure (Gram stain). Your observation purpose and the specimens to be examined will help you choose the right stain and method, though.
There are the so-called acid-fast bacteria that, because of the mycolic acid on their cell wall, are resistant to staining procedures. They are called acid-fast bacteria because of their resistance to decolorization with acid alcohol. For example, Mycobacterium tuberculosis is a Gram-resistant microorganism and thus it should be stained with an acid-fast stain.
This bacterium can be colored with the Ziehl-Neelsen staining which includes the use of red colored Carbol fuchsin to stain the microorganism and Malachite Green or Methylene blue as a counterstain to ensure a contrasting background.
Thanks to the phenol in the Carbol fuchsin used, the cell walls are solubilized. Heat should be used to help the stain penetrate the bacterium better, make it visible, and thus allow for proper observation.
Since there are different types of stains used for microscopy and different purposes for examining bacteria, the staining procedures employed can be different, too. The specimen that is being prepared for examination on the slide can be either dry-mounted or wet-mounted, smeared or sliced into a thin section.
If a stain is used, then the specimen should be wet-mounted. This procedure means that you should use a clean dropper to place a bit of water on the slide (just a drop), set your bacteria sample in the water, and then use a coverslip to cover it.
Use the dropper to apply the stain but place it at the corner of the slide in order for it to be drawn to the bacteria by capillary action. Use absorbent paper on the opposite side of the slide to remove excess water. Examine the specimen only when the stain has covered the entire slide.
Sometimes, when simple staining is required, the sample is immersed (before or after it has been fixed and mounted) in the dye solution, rinsed, and then observed. Of course, there are other staining procedures and techniques and your specific purposes and the specimens to be examined will help choose the appropriate one.
Regardless of the stain type and method employed, make sure you follow the required steps carefully. Not paying utmost attention can affect what you get to see when you place the sample under the microscope. A little mistake might render your sample useless.
However, when the sample is perfect for observation, the stain did its job and highlighted the cell and its parts, and you use a quality microscope, it is quite fascinating to see these tiny organisms and examine their morphology. One can almost never cease to be awed by these organisms and the forms life can take.