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Bacteria: Form and Function
Objective
In this lab, you will be working with living bacteria and should develop the following skills:
· To recognize the basic shapes and arrangements of bacteria.
· To examine the motility of bacteria.
· To use the oil immersion lens of the microscope efficiently to examine bacteria.
Bacteriology Safety Rules
The bacteria that we work with in this lab are considered to be nonpathogens (do not cause disease). However, almost any bacteria, given the opportunity, can cause disease. Therefore, when working with bacteria, we always work carefully, assuming that the bacteria is a pathogen. For your own safety and the safety of others, learn and follow the following safety rules.
1 Cover all cuts or open sores with bandages.
2 Remove all unnecessary objects from around your work area.
3 Wear a lab apron.
4 If you have long hair, tie it back.
5 If living bacteria are spilled, tell your teacher immediately — do not clean it up.
6 Be sure that all bacteria are killed before disposing of them.
7 Wash your hands well at the end of the lab period.
8 Do not bring food or drink into the lab during a bacteriology lab.
9 Always follow proper sterile techniques when handling bacteria.
10 Turn Bunsen burners off when not using them.
11 Assume that all bacteria are pathogens and treat them accordingly.
Materials
prepared slide: bacteria, three types
oil immersion microscope and immersion oil
concave slide and cover slip
vaseline
toothpicks
bean broth
lens paper
inoculating loops
Part 1: Observing Bacteria Forms
1 Obtain a prepared slide (one of each for coccus, bacillus, and spirillum) and a bottle of immersion oil. These slides are of different bacteria which have been stained to allow you to see them better. Each smear is one of the three forms of bacteria, coccus, bacillus, and spirillum.
2 Bacteria are extremely small and difficult to observe using the regular objective lenses on microscopes. To observe them, we need to use a special, high-powered objective lens called an oil immersion lens. On most microscopes, this lens has a magnification of 100x.
The immersion oil used with this lens has a refractive index identical to glass. We place a drop of immersion oil directly on top of the slide at the spot that we are observing and then dunk the oil immersion lens into the drop of oil. This increases the resolution that would be lost as light refracts between the glass of the slide and the air between the slide and the lens. As a result of the improved resolution, we are able to increase the magnification so we can see details in bacteria. Your teacher will demonstrate the correct procedure for using the oil immersion lens.
Examine each of the three samples of bacteria. On unlined paper, make a biological drawing of 10–20 representative examples of the bacteria that you see. Include magnification, title, and labels where appropriate. Each drawing should occupy about one-third to one-quarter of a page and be done in pencil.
Bacteria have a form (shape) and arrangement (how the individuals are growing together: solitary, diplo, strepto, staphylo, tetrads, etc.). Using the diameter of the field of view for your microscope estimate the size of individual bacteria. Fill in a data table with your observations of the bacteria on the prepared slides. The data table should have four columns: Bacteria Type (coccus, bacillus, and spirillum), Form, Arrangement, and True Size.
Part 2: Observing Motility in Bacteria
When observing living bacteria, two types of movement may be observed. One type is called Brownian motion and is due to collisions of the bacteria with the natural movement (vibrations) of the water molecules that suspend the bacteria. This type of movement involves no energy on the part of the bacteria and doesn't move them, necessarily, to a place that they want to be. The second type of motion is true movement or motility. Many bacteria have some mechanism for propelling themselves through the medium, using energy, to move somewhere that they wish. In bacteria, the most common movement structure is the flagellum.
To Make a Hanging Drop Suspension
To observe movement in bacteria, it is obvious that they must be alive! Observing living bacteria is difficult because of their extremely small size and the fact that most living bacteria are colorless and fairly transparent. We can't stain them because the stain kills them. We must also create an environment in which they can swim if we want to observe their ability to move.
Obtain and scrupulously clean a cover slip and a concave (depression) microscope slide. With a toothpick, add a tiny dab of vaseline to each corner of the cover slip. Place the cover slip, vaseline-side up, on a paper towel. With an inoculating loop, add a loopful of bean broth to the center of the cover slip. Now, pick up the concave slide and turn it upside down (so that the concavity is on the bottom of the slide). Place the slide over the cover slip such that the bean broth drop is below the center of the concavity. Place the slide down on top of the cover slip such that the Vaseline sticks the cover slip to the slide. Now lift up the slide with the cover slip attached and turn it right side up. The bean broth drop is now hanging from the underside of the cover slip in the concavity of the slide.
To observe the bacteria in the broth, place the slide under the microscope. Start with the 10x lens. Lower your light significantly by adjusting your diaphragm and condenser. At this magnification, you can just barely see bacteria. Locate some bacteria and get sharply focused. Rotate the nose piece to the high-dry 40x lens. Focus carefully to observe the bacteria. Adjust your light if necessary. Do not use the oil immersion lens. Make a drawing of a few motile bacteria.
Analysis Questions
1 What is the total magnification given by the oil immersion lens?
2 How many different types of bacteria can you identify? Describe each type.
3 Of the different bacteria observable, how many types show true motility?
4 What is the form of the bacteria that demonstrate true movement?
5 How common are the motile bacteria compared with the other bacteria present?
6 Do you see any colonies of bacteria? What type?
7 Can you see flagella in the motile bacteria?
Source: Nelson Biology 11, 2003
Adapted for Horton Biology April 2007
Mr. Fuller & Mr. Turner