Laboratory Notes for BIO 1003

© 30 August 1999, John H. Wahlert & Mary Jean Holland


DOMAIN BACTERIA

Prokaryotes represent the oldest kind of organisms on earth; their fossil record extends back over 3.5 billion years; all are members of the Domains Archaea and Bacteria. The term prokaryote means pre nucleus; they lack the membrane bound nucleus found in eukaryotes (members of the Domain Eukarya). In fact prokaryotes have no membranous cellular compartments called organelles in eukaryotes. There are about 2,700 known living species. In numbers prokaryotes are the most abundant kind of organisms. The examples you will see are all members of the Domain Bacteria. Most members of this group are heterotrophs and are important as decomposers. They play an extremely important role in nitrogen fixation, making atmospheric nitrogen (N2), available to eukaryotic organisms in the form of ammonia (NH3). Some bacteria are photosynthetic and have light absorbing pigments embedded in their plasma membranes. These bacteria appear green and are called Cyanobacteria.

Most bacteria are surrounded by a cell wall that is chemically different from those in fungi and plants. H. C. Gram discovered staining differences that are related to the wall structure. Gram-negative bacteria have a complex wall and are resistant to many antibiotics. Gram-positive bacteria, e.g., Staphylococcus, have a simpler wall and are more susceptible to antibiotics and to lysozyme, an enzyme in such secretions as tears. Some bacteria form a polysaccharide capsule around themselves and are thus very resistant to chemical attack, e.g., a certain form of Streptococcus pneumoniae.

Bacteria have three common shapes:

coccus (pl. cocci) = sphere
bacillus (pl. bacilli) = straight rods
spirillum (pl. spirilla) = spiral rods

Slides

Bacteria mixed shows bacteria of the three different shapes; some slides are Gram stained and bacteria may be dark purple (Gram positive) or light pink (Gram negative).
Escherichia coli is the laboratory bacterium commonly used in research. It occurs in the intestines of animals.
Typical spirillum gram stained

[mixed bacteria at 400 x][spirillum at 400 x]
mixed bacteria (left) and spirilla (right), both at 400 x

Examination of dental bacteria

Bacteria are present everywhere on the surface of your body and in your digestive tract. Your mouth has an especially rich flora. To examine your dental bacteria proceed as follows:

  1. Put a small drop of water on a microscope slide. Use a small drop because you must wait [step 4] until it evaporates.
  2. Take a sterile wooden stick and rub one end over your teeth close to the gum line.
  3. Swirl the bacteria laden end of the stick in the water on the slide, and spread it around over a one cm2 area.
  4. Wait for the slide to dry.
  5. Heat the slide with bacteria on the top over a flame; do not broil the bacteria, just get them good and warm so they stick to the slide.
  6. Put a drop of methylene blue stain on the bacteria. Let the stain sit for 1 minute.
  7. Rinse the slide with gently flowing tap water.
  8. Pat the slide dry with a piece of paper towel. Do not rub it.
  9. When the slide is totally dry, examine it with the microscope. The bacteria will be very tiny and stained blue. Examine areas where they are not clumped but are thinly spread out. Look for the three major shapes of bacteria: coccus, bacillus, spirillum. There may be a few cheek cells, and you can easily see how much smaller prokaryote cells are than those of eukaryotes.

Isolation of Bacteria from the Environment

Bacteria are everywhere. In this experiment you will collect bacteria from the environment and transfer them to a nutrient agar in a petri dish for incubation. Don't open the petri dish until step 2 below; bacteria from the air may settle on the agar and contaminate your experiment. Do note that the smaller half of the dish contains the gelatinous agar.

  1. Use a sterile cotton swab to collect organisms from the surface of your choice. These are sealed in paper; be careful not to touch the end you will use for collecting.
  2. Streak the swab or roll it gently across the surface of the agar in the petri dish.
  3. Close the dish promptly.
  4. Label the bottom of the dish with a wax pencil; note the environment sampled and your initials.
  5. As a control one petri dish should be inoculated with a sterile swab only.
  6. Your instructor will place all the dishes in the incubator at 30 degrees C; you will observe the growth of bacteria in your next laboratory class.

Cyanobacteria

Living Cyanobacteria are available for study. Prepare slides of Oscillatoria and Anabaena. Both are filamentous--chains of elongated cells. Occasional larger cells in the chain are heterocysts which have dark ends; these cells fix nitrogen. The green color in the cells is chlorophyll; note that the color is everywhere and is not localized in chloroplasts as you saw in the algae Elodea and Spirogyra, which are eukaryotes. If you look at free filaments at the edge of the Oscillatoria clump, you will observe their characteristic slow and smooth undulating (or oscillating) movement.

[Oscillatoria at 100 x][Anabaena at 400 x]
Oscillatoria (left) 100 x, Anabaena (right) 400 x

Return to index.

Last updated 22 Sept. 2007 (JHW & MJCH)