Introduction

Microbiologists grow and keep microorganism cultures on media. Media can be in either liquid (broth) or solid (agar) form. Agar is a complex carbohydrate isolated from seaweed. It has many industrial, food, and scientific uses. It is also a convenient solidifying agent for microbiology media as very few microorganisms can metabolize it. Agar liquefies at 90 - 100 °Celsius and solidifies at approximately 42 °Celsius.

Regardless of the type, all media must be prepared carefully to include the proper nutrients needed for microbial growth, and, to prevent unwanted growth before the addition of the microbe sample.

Sterile Procedures

Figure 1: The use of agar has greatly aided many advances in microbiology. It is also used in ice cream production.

Most media is prepared in a sterile procedure, and sterilized after production. Sterilization of media is commonly performed using an autoclave that uses pressure and steam to kill any unwanted organisms, or membrane filtration with positive pressure to filter out any particles above a specific size. Since you will not have access to these sterilization techniques, the solid media you use for the experiments in this lab manual will be prepared sterilely for you. However, you will be required to melt the media and pour it into Petri dishes in preparation for growing bacteria. This process must be performed in a manner that reduces the possibility of introducing unwanted organism into the media before being used in the experiment. Additionally, you will be required to inoculate (introduce a microorganism to) prepared media plates with samples containing bacteria that you have collected from your environment (or may be provided to you) and transfer bacteria from one media plate to another, or to other containers. These procedures must be accomplished without introducing contamination to the original plate, the new plate, the environment, or yourself. This method is called aseptic technique and is used for almost all procedures involving microbes.

Growth Media

Figure 2: Koch was the first scientist to isolate the causative agent of anthrax, Bacillus anthracis, using his potato slice method.

There are numerous types of growth media. Early microbiologists prepared their own media using available materials at the time, such as extracts of beef and vegetable slices. Robert Koch (1843-1910) was a German physician/scientist who pioneered many microbiology techniques that are still used today. Koch was one of the first microbiologists to use a solid form of media to cultivate microbes. He prepared slices of boiled potatoes and smeared them with samples from diseased animals. The potato served as a form of growth media for the bacteria and using this technique, Koch was able to isolate pure bacterial cultures that were derived from a single species.

Today, media are typically available in powdered form and only need to be rehydrated. All complete growth media need to provide the nutrient requirements of the diverse bacteria grown on them. These nutrients must include a carbon source (which can also supply energy) and a nitrogen source. Both of these nutrients can be supplied by plant or animal sources. Additional nutrients include phosphorus, sulfur, minerals, and water. Media made from these sources are called undefined or complex media since the exact makeup and amount of carbon and nitrogen are not known. Defined media is that in which each chemical component and its amount is precisely known and controlled to facilitate the growth of specific microbes. Various media are also used to enrich or select for particular types of bacterial growth; these are called selective media, which can be complex or defined.

Aseptic Technique

Figure 3: Different microorganisms colonize in different shapes and colors. The streaking (also referred to as plating) method may also influence a colony’s visual appearance.

Aseptic technique is used when adding bacterial samples to media plates and also when moving bacterial colonies from one plate to another. As mentioned above, aseptic techniques minimize the likelihood of contaminating the pure bacterial samples with unwanted microbes from the surrounding environment, or those that normally inhabit our bodies. Aseptic techniques also are important to reduce the spread of cultured bacteria (which may be potentially hazardous) to our surroundings, thereby limiting or preventing exposure to pathogenic agents. In a microbiology lab, scientists can sterilize instruments with heat (Bunsen burner), with an autoclave (high pressure and temperature), or by gas sterilization. Throughout the experiments in this lab kit, you will use sterile, disposable instruments or a candle (for sterilization) because you do not have access to lab-grade sterilization equipment. You will also need to use proper aseptic technique to ensure the purity of your cultures. Below is a list of some common aseptic techniques used in the microbiology lab:

• Reduce the potential to contaminate your surroundings by working in a clean, uncluttered space.
• Be organized and read through each experiment thoroughly before starting. Gather and prepare all necessary reagents and instruments before beginning the experiments.
• Immediately cover plates after pouring media into them. When opening a plate to either inoculate it or to transfer bacteria from it, use the lid as a shield to limit contamination from airborne sources.
• When using tubes containing media or other liquids, hold the tube at an angle and immediately recap to limit contamination from airborne sources.
• Always use a new, clean, sterile, disposable loop or transfer pipette when transferring multiple samples or colonies of bacteria, unless otherwise directed in the experiment instructions.
• Store prepared, solidified agar plates (not yet inoculated) upside-down so that any condensation that forms on them will not drop onto the media surface.

Figure 4: Chlorine bleach is an accepted liquid for sterilization.	Figure 5: Agar color can vary based on the ingredients.It is often clear-yellow or red.

• Disinfect all cultures at the end of every experiment (unless otherwise instructed) by adding a 10% bleach solution to the culture plates and letting them sit at room temperature for 20 minutes. Household bleach contains 5.25% sodium hypochlorite, and is corrosive so surfaces should be wiped with water after using bleach. A 10% solution of household bleach will kill many organisms immediately, but may take up to 20 minutes for full sterilization.
• After flooding the plates, pour the bleach down a drain, seal the plates with Parafilm™, and dispose of them in the trash.
• Be sure to clean and disinfect your work area with disinfecting wipes or 70% ethanol after each experiment.

Obtaining pure bacterial cultures is important for the microbiologist as this greatly facilitates identifying what types of bacteria occur in a mixed population. Frequently, a dilution series of the sample is prepared such that the initial number of bacteria is reduced so that when the sample is plated onto growth media, individual bacteria are separated from one another. These individually isolated bacteria cells then produce a colony of cells, all derived from the parent bacteria. Colonies arising from different types of bacteria often exhibit unique morphologies that can aid in identifying the original bacteria. In the experiments outlined in this manual, be sure to always note the appearance (morphology, color, edges, texture, pattern of growth) of the bacterial colonies that grow on your plates.