Bacteria in phase contrast
Categories: Microscopy Basics • Observations and pictures • Techniques | Tags: bacteria • phase contrast • prokaryotes
Cocci in packets
Cocci in pairs and in packets
Short rods
Rods, slightly curved
About phase contrast
Bacteria are transparent and therefore difficult to see using regular bright-field microscopy. The bacterial cells will appear just as bright as the surounding medium and there is no color contrast. Phase contrast optics provides a solution. Phase contrast optics convert the differences in optical density (i.e. the refractive index) of the bacterial cells into different shades of brightness. The optics achieves this by interference of the light which passes through the specimen (the bacteria) with the light that goes around the medium. Phase contrast optics therefore work only if the cells have a different refractive index compared to the medium.
How the bacteria were prepared
The bacteria were grown in pure culture in an appropriate microbiology laboratory. A colony was then suspended in saline (salt water) of right concentration and then microscoped with a 1000x magnification in oil immersion (using a 100x oil objective).
If one takes too much liquid, then the cells start to float in and out of focus and it is not easily possible to capture the shape of the individual cells. A similar problem can occur if the cells are much smaller than the film of liquid between the slide and cover slip. The evaporation of the liquid from the edges of the cover slip will cause a constant movement of the cells and make it difficult to take a steady picture. In this case it is necessary to heat-fix the bacteria. A colony was then suspended in saline and dried at room temperature. The slide was briefly pulled through the flame of a bunsen burner, with the bacteria on the opposite side of the the flame. This heating process fixed the bacteria to the glass slide. Immersion oil was then directly applied to the slide and the bacteria were observed without cover glass. One disadvantage of heat fixing is, that during the drying process the bacteria may aggregate (as the volume of liquid decreases) and it may become more difficult to see individual cells.
About the photographs
The pictures were taken on analog B/W film and then digitized with a camera and an adapter (see the following post for more info on the set-up: Digitizing photographic slides with a digital camera ). The negative was then inverted and the contrast levels adjusted. The soft, slightly blurry appearance of the pictures shows that we are already at the limits of the resolution. The images were not sharpened. Notice the bright halo around the bacterial cells. This is typical for phase contrast microscopy.
June 24th, 2010 at 23:03
Thanks for you enthusiastic web microscopy resource…sweet!!
I don’t know what level biology you teach, but your students must be lucky to engage your lecture/lab efforts.
Somewhere I once read that thanks to inter-net/online resources and technologies..’ a science of everything’ is really approaching educators/researchers/pupils/life-time learners. By contrast with my childhood nature/science/home hobbies/microscopy path to ‘life-time learning’…your interactive web site has everything but ‘the masses of real-time pupils and visitors talking to one another…your website is a self-contained first stop for a young student microscopist!
BRAVO…charlie guevar,fingerlakes/US
August 25th, 2010 at 20:27
Thanks for the feedback. I teach both middle and high school Biology.
May 15th, 2011 at 19:45
I want to find what kind of bacteria are present in my stools and in particular, observe the presence of C-diff bacteria but also study the demographics of the bacteria as much as possible. I am considering buying a 1000x-2000x compound microscope with phase contrast optics. I need some guidance about how to prepare the specimen and is there in biological danger in doing this. I am a retired electronics engineer.
I hope the bacteria do not have to be cultured because I do not understand the process and may not be practical for home use. Can anybody help?
May 20th, 2011 at 14:12
Sorry to disappoint you, but the type of investigations that you want to do are not possible with a microscope. The shape of bacteria can only be used in very rare cases for identification purposes. How do you want to identify Clostridium difficile in a mix of thousands of different kinds of bacteria? Concerning biological danger: yes. You are working with unknown bacteria of human origin, which are (potentially) pathogenic. This belongs to an elevated security level. Medical diagnostics is based on biochemical reactions or the analysis of DNA, but not microscopy. There are some cases when microscopes can be used, but this requires culturing the bacteria. Generally the bacteria do have to be cultured and then isolated. This takes time, experience and extensive laboratory equipment. The cultured bacteria are then chemically analyzed. If you take a stool sample and culture the bacteria on agar plates, then you will receive thousands of different kinds of bacterial colonies. Which one is C. difficile? You would have to analyze each one of them. There is also another problem: Clostridia are anaerobic, this means that they do not grow in presence of oxygen. You need a special incubation chamber, in which there is no air.
In short: I would not do any form of self-diagnosis, simply because it does not work. If you want to go into microscopy because of a hobby (and not for medical reasons), then I have to say that there are far more interesting things to observe than bacteria. Have a look at plain yogurt to observe bacteria. The maximum useful magnification of a light microscope is about 1000x, everything above this is empty magnification.
If you want to study the demographics ob bacteria (which bacteria occur where), then you would be better off doing a PCR study….. And as a matter of fact, this is indeed done. Simply because different bacteria have the same shape under the microscope does not mean that they are related, so the microscopic appearance is often pretty meaningless.
I know that this is kind of ironic. In the past, the microscope contributed greatly for understanding the microbial world, but nowadays the microscope only plays a minor role when doing bacteriological work.
Greetings, Oliver.