Laboratory Investigations in Microbiology

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Chapter 8: Culture media 

Culture media

Many bacteria can be cultured, or grown, in the laboratory by making use of specific culture media. Culture media contain all the nutrients necessary to support the growth of the desired microbes. Culture media whose ingredients are known exactly are called simple, or defined, media. Culture media whose composition is not exactly known because they contain ground-up plant or animal tissue are called complex media. Complex media support a wide variety of microbes and are better suited as all-purpose media. We will most frequently use a complex medium known as Trypticase Soy (agar or broth) in this lab.

Culture media that are liquid are known as broths (C). With the addition of agar (a polysaccharide made by some algae), culture media can be made to solidify. When heated to 80°C, agar solutions are liquid, but will gel when cooled to below 40°C. Liquid agar media can be poured into Petri dishes or test tubes to harden there. In this way, one can produce agar plates (A), agar slants (D; agar hardens in a test tube at an angle), and agar deeps (B; the tube is mostly filled with solidified agar). Agar plates are usually used for isolating pure cultures of bacteria and for observing colony characteristics. However, the large surface area makes them more susceptible to contamination and to drying out. Agar slants are an excellent means for keeping a pure culture for several months. A smaller surface area and a much smaller opening keep moisture in and contaminants out. For longer-term cultures, agar deeps can be used. Deeps are stab-inoculated with an inoculating needle (see Ch. 3). Bacteria can last months to a year or more. Broths, in contrast, allow for very rapid growth of bacteria, but nutrients are quickly depleted and wastes accumulate. Broths are most often used to grow up large numbers of bacteria quickly. 

At times it is desirable to restrict the growth of some bacteria while allowing others to grow successfully. Culture media that accomplish this are termed selective media. Examples include Eosin Methylene Blue agar (EMB), which favors Gram-negative bacteria, and Mannitol Salt Agar (MSA), which favors Staphylococci. At other times, we want to distinguish between similar-looking bacteria based on biochemical abilities such as lactose fermentation. The use of a differential medium such as blood agar allows us to do this. EMB and MSA also happen to be differential. Bacteria which can ferment lactose have purple/green colonies on EMB, while those that cannot have pink/white colonies. On MSA, bacteria that can use mannitol change the agar to yellow; those that do not use mannitol leave the agar unchanged (or may even turn it red/magenta).

Table 1. Types of complex culture media used in Microbiology

Medium Selective Differential Enriched
Trypticase soy agar/broth NO NO NO
Nutrient agar/broth NO NO NO
citrate agar NO YES (citrate utilization) NO
SIM agar NO YES (H2S and indole production; motility) NO
Motility agar NO YES (motility) NO
Eosin Methylene blue agar YES (Gram negative) YES (lactose fermentation) NO
Mannitol salt agar YES (Gram positive) YES (mannitol fermentation) NO
Brilliant Green Lactose Bile broth YES (Gram negative) YES (lactose fermentation) NO
Phenol red (dextrose/lactose/sucrose) broth NO YES (glucose/lactose/sucrose fermentation) NO
MR-VP medium NO YES (butanediol or mixed acid fermentation) NO
Violet Red Bile agar YES (Gram negative) YES (lactose fermentation) NO
Lactose broth NO YES (lactose fermentation) NO
Blood agar NO YES (hemolysis) YES
Chocolate agar NO NO YES

Culture media may contain a wide range of ingredients, ranging from soy meal to blood. Often they contain a specific source of protein (tryptone, peptone, casein) which may be enzymatically treated (e.g. "trypticase") to make it smaller/more digestible. Media  may also include salts, dyes, pH indicators, and inhibitors such as bile.

Materials & Methods

A. Culture media preparation

Materials per group:
  1. Read the preparation instructions on the container. 
  2. Measure out 250 ml of distilled water using a graduated cylinder (back of the room). Add this to a 500 ml Erlenmeyer flask. 
  3. Weigh out the appropriate amount of culture medium using an electronic balance. 
  4. Gradually add the powder to the water while stirring:
    1. For agar media, use a stirring hot plate and heat the water while mixing
    2. For broth media use a stir plate (no heat).
    3. Add the powder a little bit at a time to avoid clumping
  5. When the powder has completely dissolved, take the flask off the stir/hot plate and cover with a square of aluminum foil. Label your flask.
    1. Broth media will dissolve quickly and appear clear
    2. Agar media will initially be cloudy. Agar does not completely dissolve until heated to ~ 80C. Maintain on the hot plate (medium heat) while stirring until agar clears.
  6. Complete the data sheet.

B. Culturing on selective and differential media

Materials per lab group
  1. Divide each plate in half (mark the bottom with a marker)
  2. Streak a loopful of each MIX on each EMB and MSA agar plate (Mix 1 on one side, Mix 2 on the other). 
  3. Label and turn in your plates for incubation

Next lab period

  1. Examine the agar plates for the presence and appearance of colonies. Complete the data sheet.
Data sheet & Review questions (printable)

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