Chapter 1 - Microorganisms in the Environment

1 - 1 Microbes in the environment

Microorganisms exist in almost every environment on earth and maybe beyond this planet. They were most likely the first forms of life and will probably be the last. It is not too bold a statement to say that if water is in the liquid state and a source of carbon and energy is present, microorganisms can exist. Microbes can grow at temperatures from < 0°C (the snow alga, Chlamydomas nivalis) to 113°C (Pyrolobus fumarii). Think about that, a microbe that can grow in water over 100°C. Water boils at 100°C, how is that possible? The answer is that P. fumarii grows under the sea at hydrothermal vents where the water pressure is very high. Figure 1-1 shows a picture of a hydrothermal vent. Water therefore does not boil above 100°C. P. fumarii has very special proteins and membranes that help it deal with the intense heat and scientists are just beginning to understand the adaptations this microbe must make. While microbes that live at the extreme of temperatures are fascinating, most of the organisms we will examine will grow at 20-50°C. Microbes are also present in saturated salt lakes, in acid mine drainage that is below pH 1, in environments devoid of oxygen, in soil, and on you!

Whether you measure them by population or total biomass, microbes are the most common forms of life present in almost any environment. You contain more microbes in your digestive track (about 100 trillion) than you have cells in your body (about 10 trillion). The soil is also teeming with microbes. There is a rich nutrient source in the form of leaf litter, dead animals and plants, and organic waste.

Microbes have a large impact on the environments where they grow. In the most general terms, this impact is in the form of their metabolism where they consume available nutrients, use them to create energy and cell material, and then discard waste products. They use this bounty to grow to large numbers.

In nature, bacteria do not exist in isolation. Their biosphere is crowded with many different types of microorganisms competing for food and space.

1 - 2 Hay infusion - you try it

Producing a simulation of a pond environment in a cup is easy and fun to look at. The method used at UW-Madison is called a hay infusion and despite the concoctions being made for over 50 years, something novel pops up frequently. The below directions describe how to make a hay infusion.

1. Go to your nearest body of water and collect a water sample. Any natural water will work, but tap water will not. Tap water is chlorinated to remove microbes and the water out of your tap will have enough chlorine in it to kill or inhibit the growth of microbes.
2. Pour the water into a glass, or disposable cup, and add a handful of hay or grass to the pond water. A glass you do not care about should be used, as it is going to get pretty scummy.
3. Let the mixture incubate at room temperature or above for several days. If desired, the addition of a light source (either the sun or a lamp) will encourage the growth of photosynthetic microbes.
4. During the incubation, check the infusion and add more pond water as it evaporates.
5. In 5 to 10 days the broth should turn dark and turbid. Examination under a microscope will reveal a large number of microorganisms.

Some water from Lake Mendota in Madison, Wisconsin was placed in a plastic cup with hay and incubated at 30°C for 3 days. Because of the carbon source, a huge collection of microbes grew and and the video shows all the little critters swimming around in that cup.

1 - 3 Sampling various environments

Although microorganisms are present in or on nearly everything, it is usually not possible to demonstrate their presence by direct microscopic observation unless their density is high. However, if sterile culture media are exposed to air or inoculated with substances such as soil or lake water, a variety of microorganisms will multiply in the media and can be examined subsequently. To prove that microorganisms are in or on a substance, it is necessary that all media and equipment used be sterile and that aseptic technique be employed in performing inoculations and transfers.

The following procedures are meant to demonstrate colony formation by microbial cells inoculated onto a petri dish medium. Each cell which can utilize the medium as a source of nutrients and can tolerate the physical conditions present (temperature, pH, atmosphere, etc.) should multiply, resulting, during incubation, in a visible colony of like cells. Different-appearing colonies imply different species of microorganisms; colony appearance is often used in the characterization of bacterial species. When we observe colonies, we cannot assume each arose from just one cell originally planted on the medium, however. A pair, chain or cluster of cells or individual cells which "land" on the medium in close proximity to each other can multiply and produce a single colony. Thus, we use the term colony-forming unit when we consider the common origin for the cells of any colony.

Another term we will often use is culture which is simply a large population of living cells. Examples include a colony (above), a flask of organisms in a liquid medium, and a tube of slanted agar medium on which organisms are growing. A culture of cells, dividing every 20 minutes, can begin with one "new" cell and result in 16,777,216 (i.e., 2²⁴) cells after just 8 hours! A pure culture is composed of identical cells (except for occasional mutants), possibly having arisen from one cell. A mixed culture contains two or more different kinds of organisms.

We often refer to "young" and "old" cultures, depending on how long they have been incubating since inoculation. We do not, however, refer to "young" and "old" individual cells in the same way, as the cells of most of the bacterial species we work with undergo division every 15-30 minutes. Thus, an "old cell" - just about to divide into two "brand new" cells - may be less than a half-hour in age!

The three periods of this exercise are designed to coincide with Periods 1 through 3 of Experiment 2 during a regular semester when there are two or more days between periods.

Period 1

Materials

5 plates of Plate Count Agar (PCA)

4 sterile cotton swabs

1 tube of sterile saline (3-4 ml)

1. Remove the lid from one of the plates. Expose the surface of the medium to the air for 15-30 minutes and then replace the cover. Label the plate on the bottom lid. (This is standard procedure for labeling petri plates.)
2. For the remaining plates, various sites can be sampled with sterile cotton-tipped swabs moistened with the sterile saline. Each swab is then streaked across the entire surface of the medium in a petri plate and then discarded into disinfectant. (Who knows for sure if we're picking up any pathogenic organisms?) Examples of various items which can be sampled include your skin, the lab bench, a doorknob, an appliance, some other object in the vicinity, and one or more of the environmental samples provided for microscopic observation in Experiment 2. Discard the tube into the slanted tray on the discard cart.
3. Incubate the plates by placing them in an inverted (medium side up) position in the 30°C incubator for 2-5 days. Note: As a rule, we will always incubate our plates in an inverted position. Otherwise, moisture collecting on the top lid may drop down on the developing colonies, causing them to run together.

Period 2

Materials

Demonstrations of colonies of various species of bacteria and molds

Tube containing 1 ml of a soil suspension (a 1/10,000 dilution)

Tube containing 1 ml of lake water (a 1/100 dilution)

2 tubes of melted Plate Count Agar (PCA; 15-20 ml/tube) - in 50°C water bath

2 empty, sterile petri plates

1. Before observation of the plates prepared last period, another plating method will be performed:
2. For each of the two samples, dump the entire tube contents into an empty, sterile petri dish. Observe Figure 1-7 to see the correct method.
3. Obtain two tubes of melted PCA from the water bath. Wipe off the excess water with a paper towel, and pour the contents of each tube into a petri dish sitting upright on the table, opening the lid just enough to pour out the tube. Mix the sample and medium in each dish with a gentle, swirling motion and let the medium solidify.
4. Incubate the plates inverted at 30°C for 2-5 days.
5. Note the demonstration of colonies of various species of bacteria and molds. Keep the lids on the plates and observe the colonies through the top lid.
6. As time permits (i.e., with everything else having been done in Exps. 1 and 2 for today), the following can be done. Space for recording results is on page 3.
7. Observe the plates from Period 1, noting the various bacterial and mold colonies. At this point, do not open the plates, especially if molds are present. (Any fuzzy or hairy colonies are probably mold colonies. Their spores are very easily dispersed into the air, causing subsequent contamination problems and perhaps allergic reactions as well!) Note: As a rule, we always observe colonies through the top lids of the plates. Very little information about colony characteristics and differences can be obtained by looking through the medium. Note the various shapes, sizes and colors of the colonies.
8. From one or more of your plates which do not contain mold colonies, choose two or more different colonies and record their visible characteristics. Your observations can be recorded in the appropriate pages of the observation manual.
9. OPTIONAL: For each of your chosen colonies, prepare a smear (with a drop of water as described here) and stain by the gram-stain procedure. What is the gram reaction and morphology of the cells?

Period 3

1. Observe the plates prepared last period. Note and count the surface and sub-surface colonies. When counting the colonies, it is handy to draw a few lines with the wax pencil on the back of the plate to mark off a grid. The colonies can then be counted easily as you scan the sectors.
2. For the lake water sample, you can determine the density of colony-forming units (CFUs) that were in the original (undiluted) lake water if you know three things: the dilution of the sample (see the previous page), the amount inoculated into the plate (1 ml), and the colony count. For example, if you count 40 colonies on the plate, it follows that 40 CFUs had been in the 1 ml inoculum. As the inoculum was a 1/100 dilution of the lake water (i.e., "diluted 100 times"), there would have been 40 X 100 (i.e., 4000) CFUs per ml of the undiluted lake water. This result is expressed best in scientific notation: 4.0 X 10³ CFUs/ml. The same method applies to the soil sample, but the answer is expressed as CFUs per gram of the soil.
3. Time permitting (i.e., with everything else having been done in Exps. 1 and 2 for today), macro- and microscopic observation of colonies can be made as in the previous period.

As the instructor will explain, we treat milliliters and grams as equivalents, for convenience. Bacterial quantitation will be dealt with more fully in Experiment 4 (with Appendix C), and you will note that we are always interested in the concentration of CFUs (the number in one gram or ml) rather than the total number in the entire sample.