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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.

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