Learning objectives
Learning Objectives
By the end of this section, you will be able to:
- Identify and describe culture media for the growth of bacteria, including examples of all-purpose media, enriched, selective, differential, defined, and enrichment media
The study of microorganisms is greatly facilitated if we are able to culture them, that is, to keep reproducing populations alive under laboratory conditions. Culturing many microorganisms is challenging because of highly specific nutritional and environmental requirements and the diversity of these requirements among different species.
Nutritional Requirements
The number of available media to grow bacteria is considerable. Some media are considered general all-purpose media and support growth of a large variety of organisms. A prime example of an all-purpose medium is tryptic soy broth (TSB). Specialized media are used in the identification of bacteria and are supplemented with dyes, pH indicators, or antibiotics. One type, enriched media, contains growth factors, vitamins, and other essential nutrients to promote the growth of fastidious organisms, organisms that cannot make certain nutrients and require them to be added to the medium. When the complete chemical composition of a medium is known, it is called a chemically defined medium. For example, in EZ medium, all individual chemical components are identified and the exact amounts of each is known. In complex media, which contain extracts and digests of yeasts, meat, or plants, the precise chemical composition of the medium is not known. Amounts of individual components are undetermined and variable. Nutrient broth, tryptic soy broth, and brain heart infusion, are all examples of complex media.
Media that inhibit the growth of unwanted microorganisms and support the growth of the organism of interest by supplying nutrients and reducing competition are called selective media. An example of a selective medium is MacConkey agar. It contains bile salts and crystal violet, which interfere with the growth of many gram-positive bacteria and favor the growth of gram-negative bacteria, particularly the Enterobacteriaceae. These species are commonly named enterics, reside in the intestine, and are adapted to the presence of bile salts. The enrichment cultures foster the preferential growth of a desired microorganism that represents a fraction of the organisms present in an inoculum. For example, if we want to isolate bacteria that break down crude oil, hydrocarbonoclastic bacteria, sequential subculturing in a medium that supplies carbon only in the form of crude oil will enrich the cultures with oil-eating bacteria. The differential media make it easy to distinguish colonies of different bacteria by a change in the color of the colonies or the color of the medium. Color changes are the result of end products created by interaction of bacterial enzymes with differential substrates in the medium or, in the case of hemolytic reactions, the lysis of red blood cells in the medium. In OSC_Microbio_09_05_MacMsa, the differential fermentation of lactose can be observed on MacConkey agar. The lactose fermenters produce acid, which turns the medium and the colonies of strong fermenters hot pink. The medium is supplemented with the pH indicator neutral red, which turns to hot pink at low pH. Selective and differential media can be combined and play an important role in the identification of bacteria by biochemical methods.
View original figure (external; image not reproduced in this text edition)
Original figure description: A light brown agar plate. Two streaks on the plate are bright pink and two streaks are beige.
Key Concepts and Summary
- Chemically defined media contain only chemically known components.
- Selective media favor the growth of some microorganisms while inhibiting others.
- Enriched media contain added essential nutrients a specific organism needs to grow
- Differential media help distinguish bacteria by the color of the colonies or the change in the medium.
Multiple Choice
EMB agar is a medium used in the identification and isolation of pathogenic bacteria. It contains digested meat proteins as a source of organic nutrients. Two indicator dyes, eosin and methylene blue, inhibit the growth of gram-positive bacteria and distinguish between lactose fermenting and nonlactose fermenting organisms. Lactose fermenters form metallic green or deep purple colonies, whereas the nonlactose fermenters form completely colorless colonies. EMB agar is an example of which of the following?
- a selective medium only
- a differential medium only
- a selective medium and a chemically defined medium
- a selective medium, a differential medium, and a complex medium
D
Haemophilus influenzae must be grown on chocolate agar, which is blood agar treated with heat to release growth factors in the medium. H. influenzae is described as ________.
- an acidophile
- a thermophile
- an obligate anaerobe
- fastidious
D
Fill in the Blank
Blood agar contains many unspecified nutrients, supports the growth of a large number of bacteria, and allows differentiation of bacteria according to hemolysis (breakdown of blood). The medium is ________ and ________.
complex, differential
Rogosa agar contains yeast extract. The pH is adjusted to 5.2 and discourages the growth of many microorganisms; however, all the colonies look similar. The medium is ________ and ________.
complex, selective
Short Answer
What is the major difference between an enrichment culture and a selective culture?
Critical Thinking
Haemophilus, influenzae grows best at 35–37 °C with ~5% CO2 (or in a candle-jar) and requires hemin (X factor) and nicotinamide-adenine-dinucleotide (NAD, also known as V factor) for growth. (Centers for Disease Control and Prevention, World Health Organization. “CDC Laboratory Methods for the Diagnosis of Meningitis Caused by Neisseria meningitidis, Streptococcus pneumoniae, and Haemophilus influenza. WHO Manual, 2nd edition.” 2011. http://www.cdc.gov/meningitis/lab-manual/full-manual.pdf) Using the vocabulary learned in this chapter, describe H. influenzae.