Laboratory Notes for BIO 1003, 2016, and 3001

© 24 August 2016, John H. Wahlert, Krista Dobi, & 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.

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

Isolation of Bacteria from the Environment

Are bacteria are everywhere? A growing area of research is interested in learning about all of the different types of microorganisms (bacteria and other small organisms such as unicellular eukaryotes and fungi) found in different places. This field of research studies the microbiome in places like your digestive tract and your skin.

With your group, design an experiment to test for the presence of bacteria on surfaces. You will be given sterile cotton swabs and petri dishes containing nutrient agar on which bacteria can grow. Feel free to swab any surface on your person, on your bag, in the laboratory, or outside of the classroom. What is your experiment (what surfaces would you like to test or compare)? Each bacterium that grows on the agar will multiply to produce a visible colony within a day or two; this is what you can observe.

An experiment needs controls to validate the results, and there are two kinds of controls, positive and negative. The controls will answer critical questions such as: How do you know the bacterial weren’t in the agar? How do you know that bacterial can grow on this kind of nutrient agar? A standard negative control would be an unopened petri dish; you would not expect any bacteria to grow if the agar is sterile. Another negative control would be to test something that would certainly not support bacterial life (what can you think of?) A positive control is a test of the agar; you would inoculate it with a known source of bacteria (did you see such a source already?) Due to limitations on materials, the controls must be agreed on and one set prepared for the entire class.

As a class, briefly discuss your planned experiments of what you will swab with your instructor before you start swabbing!

Don’t open the petri dish until step 2 below (Are there bacteria in the air that might settle on the agar and contaminate your experiment?). Note that the smaller half of the dish contains the gelatinous agar.

  1. Use a sterile cotton swab to collect a sample 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. Labeling the bottom is important because lids can be swapped, but the bottom stays with the sample on agar.
  5. Record your expected results and hypotheses; results might include numbers of bacteria and how many different kinds you expect to see; hypotheses are explanations for the results.
  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.
Sample (what)Location (where) Expected result & hypothesis
Negative control: unopened plate   
Negative control:
 
  
Positive control:
 
  
Experiment:
 
  

At the next class: Draw what you see on the plates. You may also take pictures. Fill out the table below with the data you observe.

Sample (what)Location (where) Observed results
Negative control: unopened plate   
Negative control:
 
  
Positive control:
 
  
Experiment:
 
  

Describe your experimental results—controls and surface sampled. How did you measure bacterial growth on the plates? How may different types of bacteria are growing: look at colony color, type of growth (circular, ragged), colony size, and surface appearance (glossy, matte). What is the abundance of each type of colony?

  • Do the controls support the validity of the experiment?
  • Do the observed results for the surface your sampled match your hypothesis? If your results are different from what you expected, how would you explain the difference (new hypothesis)?

Optional: Your instructor may ask you to draw a graph or chart to depict your data, or to prepare a laboratory report based on your data from this experiment.


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Last updated 24 Aug. 2016 (JHW & KD)