- Learn how to inoculate growth media using proper aseptic procedures
- Learn how to streak for single colonies
- Understand the uses of selective and differential growth media
- Determine the properties of some common bacterial types when grown on selective and differential growth media
To study bacteria and other microorganisms, it is necessary to grow them in controlled conditions in the laboratory. Growth media contain a variety of nutrients necessary to sustain the growth of microorganisms. There are two commonly used physical forms of growth media: liquid media and solid growth media. A liquid medium is called a broth. Solid growth media usually contains agar, which is a mixture of polysaccharides derived from red algae. It is used as a solidification agent because it (1) is not broken down by bacteria, (2) contains no nutrients that can be used by bacteria and (3) melts at high temperatures, and yet is solid at temperatures used for most bacterial growth. Solid growth media is used in the following forms: agar plates, agar slants, and agar deeps. To make agar deeps or agar slants, melted agar is poured into a test tube and then allowed to solidify vertically (agar deep), or at a slant (agar slant). Agar plates are made by pouring melted agar into a petri dish.
Figure 2.1.1: Solid growth media forms
Broths can be used to determine growth patterns in a liquid medium, and for certain types of inoculations and metabolic tests that you will be doing later in the semester. They are also the method of choice for growing large quantities of bacteria. Agar slants are commonly used to generate stocks of bacteria. Agar plates can be used to separate mixtures of bacteria and to observe colony characteristics of different species of bacteria (you will perform an experiment in this lab to illustrate this). Deeps are used for several different types of differential metabolic tests (e.g., the gelatinase test, which you will perform in Lab 5).
Growth media can be categorized based on their chemical constituents, or the purpose for which they are used.
- Complex growth media contain ingredients whose exact chemical composition is unknown (e.g. blood, yeast extract, etc.).
- Synthetic (also called chemically defined) growth media are formulated to an exactly defined chemical composition.
- A general purpose growth medium (e.g. tryptic soy agar (TSA) or Luria broth (LB) is used to grow a wide variety of non-fastidious bacteria. This type of medium is often a complex growth medium.
- A selective growth medium contains chemicals that allow some types of bacteria to grow, while inhibiting the growth of other types. An example of a purely selective growth medium is PEA, phenylethyl alcohol agar, which allows Gram positive bacteria to grow while inhibiting the growth of Gram negative bacteria.
- A differential growth medium is formulated such that different types of bacteria will grow with different characteristics (e.g. colony color). An example of a differential growth medium is blood agar, which differentiates among bacteria based on their ability to break down red blood cells and hemoglobin. Blood agar is also a complex growth medium because it contains blood.
A growth medium can be both selective and differential. For example, EMB (eosin methylene blue agar) inhibits the growth of Gram positive bacteria. Gram negative bacteria that grow on this medium are differentiated based on their ability to ferment the sugars lactose and sucrose. (Note: the Gram staining procedure divides bacteria into 2 main groups: Gram-positive bacteria and Gram-negative bacteria, based on their cell wall structure. You will be doing Gram staining in the next lab period.)
Characteristics of Bacterial Growth
Even on general purpose growth media, bacteria can exhibit characteristic growth patterns. On agar plates, bacteria grow in collections of cells called colonies. Each colony arises from a single bacterium or a few bacteria. Although individual cells are too small to be viewed, masses of cells can be observed. Colonies can have different forms, margins, elevations, and colors. Observing colony characteristics is one piece of information that microbiologists can use to identify unknown bacteria. Some examples of growth characteristics on different forms of growth media are shown at the end of the lab.
Aseptic Technique and Inoculation
Inoculation is the purposeful introduction of bacteria into a sterile growth medium. A material is sterile when it has no living organisms present; contamination is the presence of unwanted microorganisms. Aseptic techniques are practices that prevent the contamination of growth media.
When working in a microbiology laboratory, you must always remember that bacteria are present on all surfaces in the lab, as well as on your own hands and clothing. Aseptic techniques are designed to prevent the transfer of bacteria from the surrounding environment into a culture medium. These techniques require care and concentration. Pay attention to what you are doing at all times!
Aseptic techniques include the following practices:
1. Minimize the time that cultures and growth media are open to the environment.
2. Disinfect the work area before and after use.
3. Do not touch or breathe into the sterile culture media or the stock cultures.
4. Loops, needles, pipets, etc. should be sterilized before they are used.
5. When working with tubes, the tube caps should not be placed on the table top; they should be held in your hand while inoculating.
6. When removing the caps from test tubes, flame the lip of the test tube after the cap is removed. This heats the air inside the tube, so the air moves out of the tube, preventing contaminants from entering the tube.
7. Information about the use of the Bunsen burner can be found in the General Introduction in the Lab Manual.
General Procedure for inoculating media
See figure 2.1.2
1. Sterilize an inoculating loop or needle in the flame of a Bunsen burner. The portion of the loop or needle that will contact the stock culture or the growth medium must turn bright orange for effective sterilization. For the most rapid sterilization, place the loop at the top of the inner blue cone of flame—this is where the temperature of the Bunsen burner is the hottest. Remove the loop from the flame after it is properly heated- keeping the loops in the flame for too long will eventually cause them to crack.
2. If you are picking a colony from a plate, cool the inoculating loop on agar that does not contain any bacterial colonies.
3. Pick a small amount of bacteria (you do not need much). If you are inoculating a tube of broth or an agar slant, remove the cap of the tube (do not set the cap down on the table) and flame the lip of the tube. Throughout the procedure, hold the tube at an angle to reduce the probability of particles entering the opening. Insert the loop into the tube and transfer bacteria to the growth medium. Be careful that only the sterilized part of the loop touches the tube or enters the growth medium.
4. Flame the lip of the test tube before replacing the cap.
5. Sterilize the inoculating loop again.
Streaking for single colonies
In the real world outside the laboratory, bacteria grow in communities made of many bacterial species. If you need to identify the types of bacteria present in environmental or medical samples, you must have a way to separate out the different types and produce pure cultures. A pure culture contains a single bacterial species, whereas a mixed culture may contain many different types of bacteria. The process described in Procedure B (the streak plate method) describes the method that you will use to separate different types of bacteria in a mixture.
Inoculating a Plate from a Broth Culture
- Sterilize the inoculating loop.
- Remove the cap from tube. Do NOT put the cap of the tube down on the lab bench—hold it in your hand.
- Flame the lip of the tube.
- Place sterile portion of inoculating loop into broth, then remove.
- Flame the lip of the tube
- Replace the cap.
- Gently streak the surface of an agar plate with the inoculating loop.
- Sterilize the inoculating loop.
Figure 2.1.2: Procedure for inoculating a plate from broth culture
Notes about Labeling and Incubating Plates
1. Always label your plates/tubes BEFORE you do your inoculations. You can use Sharpies on the plates, but wax markers ONLY on tubes. When labeling tubes, label the tube itself—don’t label the cap!
2. Make sure you label the bottom of the plates (the part of the plate that holds the agar).
3. Place plates inverted (upside down) for incubation. This prevents condensation from falling on the surface of the agar and disrupting the streaking pattern.
Media Used in This Lab Exercise
See the Microbiology Review site for color pictures of media
Tryptic soy agar (TSA): General purpose complex growth medium.
Mannitol-salt agar (MSA): Differential and selective growth medium. This medium contains 7.5% NaCl, the carbohydrate mannitol and the pH indicator phenol red (yellow at pH 8.4). It is selective for staphylococci due to the high concentration of NaCl, and differentiates based on the ability to ferment mannitol. Staphylococci that ferment mannitol produce acidic byproducts that cause the phenol red to turn yellow. This produces a yellow halo in the medium around the bacterial growth.
|Growth||Growth Organism not inhibited by NaCl||E.g., Staphylococcus, Micrococcus|
|No Growth||Organism inhibited by NaCl||Not Staphylococcus|
|Yellow Halo||Organism ferments mannitol||Probable S. aureus|
|No Yellow Halo||Organism does not ferment mannitol||Staphylococcus species (other than S. aureus); Micrococcus (yellow colonies)|
Eosin-methylene blue agar (EMB): Differential and selective growth medium. This medium contains peptone, lactose, sucrose and the dyes eosin Y and methylene blue. Gram positive organisms are inhibited by the dyes, so this medium is selective for Gram negative bacteria. The medium differentiates based on the ability to ferment lactose (and/or sucrose.) Organisms that cannot ferment either of the sugars produce colorless colonies. Organisms that ferment the sugars with some acid production produce pink or purple colonies; organisms that ferment the sugars and produce large amounts of acid form colonies with a green metallic sheen. This medium is commonly used to detect the presence of fecal coliforms (like E. coli)—bacteria that grow in the intestines of warm-blooded animals. Fecal coliforms produce large amounts of acid when fermenting lactose and/or sucrose; non-fecal coliforms will produce less acid and appear as pink or purple colonies.
|No or poor growth||Organism inhibited by dyes||Organism is Gram-positive|
|Good growth||Organism not inhibited by dyes||Organism is Gram-negative|
|Colorless growth||Organism does not ferment sucrose or lactose||Non-coliform|
|Growth is pink and mucoid||Organism ferments lactose and/or sucrose with some acid production||Coliform bacteria|
|Growth is dark (purple to black with or without green metallic sheen)||Organism ferments lactose and/or sucrose, with large amounts of acid production||Possible fecal coliform (E. coli)|
agar, broth, general purpose medium, selective medium, differential medium, colony, aseptic techniques, inoculation, streak-plate technique, contamination, sterile, pure culture, mixed culture