Bacteriology 102: Procedures for the First Day of Lab
Essential Material Reproduced from the Lab Manual
(Handout of equivalent material with space for results will be available in the lab.)
The three items under consideration in the first lab session are as follows.
- Microorganisms in the environment (Exercise 1; procedure starts here): For this we utilize petri dishes to sample the air and various items around the lab to get some idea that microorganisms are everywhere!
- Staining non-living preparations of bacteria for observation with our "regular light microscopes" (first part of Exercise 2; procedure starts here): Most of our microscopic observations in Bact. 102 are of bacterial smears which we have to stain in order for them to be visible with our microscopes. A handout will be provided which will outline how to adjust the illumination and focusing of these microscopes. More concise directions will be presented on the blackboard and have been added to the procedure below. Regarding focusing, note that we always start with the 10X objective and then, once the image is focused, switch over to the 100X objective with the use if immersion oil.
- Observing living preparations (wet mounts) of microorganisms with our phase contrast microscopes (second part of Exercise 2; procedure starts here): Occasionally we observe living organisms with the phase contrast microscopes which (as the instructor will point out sooner or later) are better suited for this purpose than our "regular light microscopes." All one has to do with the phase scopes is insert the slide and focus; all optical adjustments have been made by the instructor ahead of time.
EXERCISE 1. MICROORGANISMS IN THE ENVIRONMENT
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.
- 5 plates of Plate Count Agar (PCA)
- 4 sterile cotton swabs
- 1 tube of sterile saline (3-4 ml)
- 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.)
- 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.
- 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.
EXERCISE 2. MICROSCOPIC OBSERVATIONS OF BACTERIA
In the study and identification of bacteria, the microscope is indispensable! The series of microscopic observations in this exercise is designed to illustrate how bacteria may be viewed individually in their basic form, the cell. The second and third periods herein coincide with those of Experiment 1 where organisms isolated by the student are examined microscopically (and could be found to be more interesting than those provided in this exercise!).
For the observation of living microorganisms in Period 1, various samples including a "hay infusion" are available. The hay infusion consists of some hay or straw (usually the latter) immersed in pond water which has been incubating at 30°C for several days. Water leaches soluble materials from the plant material which many bacteria and fungi can utilize. In turn, protozoa ingest bacteria as their nutrient source. Certain other organisms such as nematodes, rotifers and algae may also be present. To study the microorganisms in the aqueous materials available, it is necessary to make "wet mounts." The procedure is relatively simple as seen below.
- Hay infusions and various other items from nature
- Slide with smears of two species of bacteria: Bacillus cereus and Staphylococcus epidermidis
Procedure for the Simple Stain
- You are provided with a microscope slide with two smears as indicated above. According to the instructor's explanation and demonstration, heat-fix the slide, making sure the slide goes through the flame smear-side up.
- Gloves are available for the staining procedure. Placing the slide on the staining rack in the sink, cover the slide with crystal violet for one minute. (Note the simple stain procedure below.)
- Carefully rinse off the dye with tap water and blot the slide dry with paper towel or blotting paper.
- With both hands, obtain the LIGHT MICROSCOPE from the cabinet (corresponding to your desk number). This is the type of microscope which we will always use to observe stained smears. Heed the instructor's comments on its handling and use. The special microscope handout can be consulted, but some more abbreviated and handy steps to achieve proper light and focusing are here.
- Place the slide on the stage such that it is oriented properly – i.e., with the check mark on the upper right. Make sure the clips on the stage hold the slide securely.
- Begin your observations with the Bacillus cereus smear. When observing this organism with the oil-immersion objective, you will notice that the cells are relatively large and rod-shaped ("bacilli") and are usually in chains. Record your observations.
- Repeat this procedure with the Staphylococcus epidermidis smear. Cells of this organism are spheres ("cocci") which are usually arranged in clusters ("staphylococci") and pairs.
- When you are through, be sure the microscope is put away properly (i.e., all oil wiped off, 10X objective lens in place, stage centered). We usually recommend that you keep the slide. (To remove immersion oil from smears, place a few pieces of lens paper on the slide to absorb the oil. Then, add several drops of xylol to the lens paper. Peel the paper, now soaked with xylol, off the slide. Xylol is flammable! Keep it away from flames!)
- Using a capillary pipette or inoculating loop, pick up some of the material from around the surfaces of grass and leaves and from the bottom of the sample. Place a drop of suspended material on a clean microscope slide.
- With a toothpick, spread a very thin layer of vaseline over a small part of the palm of your hand. Take a clean coverslip (always held by the edges) and gently scrape all four edges along your palm, picking up a thin line of vaseline along each edge.
- Place the coverslip directly onto the drop on the slide in such a manner that some air bubbles are trapped. Place a small, multilayered piece of paper towel over the coverslip and press down. Discard the piece of paper towel into the disinfectant.
- Examine the wet mount with a PHASE CONTRAST MICROSCOPE set up by the instructor at a special station in the back or side of the lab. Be sure to heed the instructor's precautions regarding the phase microscope. Until you become familiar with this type of microscope, use only the 40X objective lens which is already in position. Looking from the side, lower the lens until it almost touches the slide. Then turn the fine adjustment up while looking through the microscope. A piece of debris or the edge of an air bubble is a convenient object on which to focus.
- In addition to bacteria (the smallest life-forms evident), one may observe protozoa and algae in the hay infusion and other samples.
- Without removing the coverslip, discard the slide into the disinfectant container on the stage.
APPENDIX G.1 – THE SIMPLE STAIN
The smear is stained with a solution of a single dye which stains all cells the same color. Differentiation of cell types or structures is not the objective in the simple stain procedure. However, certain structures which are not stained by this method may be seen easily such as endospores and lipid inclusions.
- Prepare and heat-fix a smear of the organism to be studied.
- Cover the smear with the staining solution. If crystal violet is used, allow one minute for staining. The use of methylene blue requires 3-5 minutes to achieve good staining.
- Carefully wash off the dye with tap water and blot the slide dry with blotting paper, an absorbent paper pad or paper towel.