Mastering Simple Bacterium Examination Techniques Using Phase Contrast Microscopy
Experts universally agree that bacterium examination techniques using phase contrast microscopy yield the best results. They may not be perfect, but they definitely deliver better and more detailed images. Studying bacteria is considered vital because it allows scientists to learn more about the origins of diseases and consequently determine cures for them as well.
Bacterial Observation: Problems and Methods Bacteria have been in existence for billions of years already. Due to their size and near transparency, bacteria are impossible to observe with the naked eye. Even an ordinary microscope isn’t much help. Thankfully, various techniques have been discovered over the years to improve the study of bacteria.
One way of observing bacteria is by staining. This technique or Gram staining in particular has been used for more than one hundred and fifty years already. Invented by Hans Gram, it is still considered one of the best ways for identifying microbial species.
Optical contrast techniques may also be employed to make bacteria more visible. These are phase contrast and differential interference contrast. The latter is able to produce shadowy and three-dimensional relief images. Phase contrast is, however, considered the better technique. In phase contrast, specimens like bacteria are better seen by highlighting the difference in the specimen’s thickness with the use of phase differences of transmitted light. Microscopes that aren’t equipped with phase contrast can still use this technique by utilizing phase contrast lenses.
Bacterium Examination Techniques Using Phase Contrast Microscopy
The Antony Van Leeuwenhoeck Experiment – The first experiment of this type was done in the 17th century but it can still be applicable today. Do not brush your teeth for a day. Afterwards, use a toothpick to gently remove a bit of the plaque that has formed between your teeth.
Paste it on a glass slide. Pour a tiny amount of water on it before lowering the cover slip onto your slide. What you’ve just done is a wet mount – in contrast with a dry mount, which lets you observe your specimen without adding water.
Using a phase contrast microscope, you’ll be able to immediately see various types of microorganisms. It’s a bit discomfiting to accept that all of it came from your mouth but it’s true. While observing, try to distinguish the bacteria from eukaryotic cells or those accidentally scraped from your cheek. Size alone should let you know since cheek cells are quite larger than bacteria.
Observation of Living Bacteria – Make another wet mount but this time using Vaseline. Start observing the specimen at low power and using dark field lens; once you’ve focused on the sample properly, you can now slowly increase magnification. When you reach 100x, make sure to switch to phase contrast concurrently. Then go back to 40x and work on getting the right balance.
While observing, try answering the following questions: are there any other microorganisms outside the bacterial species included in the sample? What are the different species of bacteria present?
Observation of Stained Bacteria – Assuming that you know how to heat-fix a slide already, prepare one that’s already heat-fixed and stain them accordingly to see the difference between Gram positive and Gram negative microorganisms.
Start at low power once more. Use oil immersion lenses when you reach 400x. Move the fine focus of your microscope to ensure that the oil’s spread thoroughly on the lenses. Start observing. Consider its shapes and volume.
How to Set Up a Microscope for Phase Contrast If you’re using an ordinary microscope with optional phase contrast lenses, here’s how to set it up properly.
Check your equipment. You must have a phase contrast substage condenser and objective lenses with you.
Start by setting your microscope at bright field (BF). Start adjusting your coarse knob to focus properly on the sample. Move on to adjusting the condenser of your microscope. Look for optimal height before setting the condenser turret for phase contrast. Remove the specimen.
Remove one of the eyepiece lenses of your microscope. Afterwards, put the centering telescope in place. Use your microscope’s set screw to focus the telescope properly. If you try peeking, you’ll see a pair of rings. If they aren’t concentric or they don’t have the same center, you have to adjust the screws of your condenser once more. Continue adjusting until the rings are aligned with each other.
Lastly, remove the telescope and replace the eyepieces. Put your sample back and you’re now set to observe your specimen with phase contrast. If you’re going to change your objectives, remember to go through the whole process again!
Try mastering these simple bacterial examination techniques using phase contrast microscopy before moving on to advanced techniques.
