See also Differential Tests
Selective media, generally
Selective media can be used to inhibit unwanted bacterial and thereby differentiate bacterial types. Media for isolating intestinal pathogens (MacConkey agar, Hektoen enteric (HE) agar) contain bile salts as a selective agent. Other agents that have selective properties are dyes, such as methylene blue and crysal violet and antimicrobila drugs.
Selective media should be contrasted with differential media (see outline) which do not inhibit the growth of any particular microorganisms but are designed to display visible differences in how they grow. Differentiation shows up as variations in colony size or color (e.g., formations of gas bubbles and precipitates or media color changes). A single medium can be both selective and differential. MacConkey agar, for example is both slective and differentail due to its ability to suppress the growth of some organisms while producing a visual distinction among the ones that do grow.
Selective Media for Isolation of Gram-negative Rods
The following types of media are useful for isolation and differentiation of Gram-negative organisms such as members of the family Enterobacteriaceae (gut bacteria).
MacConkey Agar: contains lactose, bile salts, neutral red and crystal violet. Bile salts and crystal violet inhibit growth of Gram-positve bacteria. Neutral red dye is a pH indicator that is colorless above a pH of 6.8 and red at a pH less than 6.5. Acid accumulating from lactose fermentation turns the dye red. Lactose fermenters turn a shade of red on MacConkey Agar, whereas lactose nonfermenters retain their normal color or the color of the medium.
(1) Pour or no growth: organism is inhibited by crystal violet and/or bile so is Gram-positive.
(2) Good growth: organism is probably Gram-negative.
(3) Pink to red growth with or without bile precipitate: organism produces acid from lactose fermentation and is probably coliform
(4) Growth is colorless: organism does not ferment lactose is probably noncoliform.
Eosin Methylene Blue (EMB) Agar: is commonly used for the test for the presence of coliforms in environmental samples. It is a complex (chemically undefined), selective, and differential medium. It contains peptone, lactose, sucrose and the dyes eosin Y and methylene blue. The dyes inhibit the growth of Gram-positive organisms and they react with vigorous lactose fermenters and (in the acidic environment) turn the growth dark purple or black. This dark growth is typical of Escherichia coliand is usually accompanied by a green metallic sheen. Other less aggressive lactose fermenters such as Enterobacter or Klebsiella species produce colonies that can range from pink to dark purple on the medium.
(1) Poor growth or no growth: Organism is inhibited by eosin and methylene blue so is Gram positive.
(2) Good growth: organism is not inhibited by eosin and methylene blue os is gram-negative.
(3) Growth is pink and mucoid: organism ferments lactose with little acid production and is possible coliform.
(4) Dark purple to black, with or without green metallic sheen: organism ferments lactose and/or sucrose with acid production and is probable coliform.
(5) Growth is olorless (no pink, purple, or metallic sheen): organism does nto ferment lactose or sucrose and is probably noncoliform.
Oxygen Use: Thioglycollate
Oxygen is acted on by cellular enzymes. It is transformed into several toxic products. Single oxygen (O) is a very reactive molecule. In fact, it is one of the substances produced by phagocytes to kill invading bacteria. The buildup of singlet oxygen and the oxidation of membrane lipids and other moelcules can damage and destroy a cell. The very reactive superoxide ion (O2-), hydrogen peroxide (H2O2) and hydroxyl radicals (OH-) are other destructive metabolic by.produces of oxygen. To protect themselves against damage, most cells have developed enzymes that scavenge and neutralize these chemicals. The conversion of superoxide ion into harmless oxygen requires 2 steps using 2 diffferent enzymes, superoxide dismutase and catalse. Step 1: 2O2- + 2H+ —superoxide dismutase—H2O2 (hydrogen peroxide) + O2. Step 2 2 H2O2 —catalase–2H2O + O2. In this series of reactions (essential for aerobic organisms), the superoxide ion is first converted to hydrogen peroxide and oxygen by superoxide dismutase. Because hydrogen peroxide is also toxic (it is used as a disinfectant), it must be degraded by catalase into water and oxygen. If a microbe is not cabable of dealing with toxic oxygen by these or similar mechanisms, it is forced to live in habitats free of oxygen.
Thioglycollate allows one to differentiate the oxygen requirements of cultured microbes. Oxygen concetnration is highest at the top of the tub and lowest at the bottom.
Thioglycollate broth is designed to promote growth of a wide variety of fastidious microorganisms. Sodium thioglycollate in the media consumes oxygen and permits the growth of anaerobes. Oxygen removed during autoclaving will diffuse back into the medium as the tubes cool. This produces a gradient of concentration from full aerobic at the top to anaerobic at the bottom. Thus fresh media will appear clear to straw colored with a pink region at the top.
Aerobes: use gaseous oxygen in their metabolism and possess the enzymes needed to process toxic oxygen products. An organisms that cannot grow without oxygen is an obligate aerobe. Examples include most fungi, protozoa and many bacteria such as Bacillus species and Mycobacterium tuberculosis.
Microaerophiles: are harmed by normal atmospheric concentration of oxygen but require a small amount of it in metabolism. Examples include organisms that live in soil or water or in mammalian hosts, not directly expposed to atmosphere such as Helicobacter pylori and Borrelia burgdorferi.
Facutative anaerobes: do not require oxygen for metabolism but use it when it is present. They can also grow anaerobically. In a thioglycollate tube, the bacteria will thus grow throughout, but there is heavier growth in the aerobic portion (upper) because aerobic growth often goes more quickly in some facultative anaerobes. Examples include many gram negative intestinal bacteria such as staphylococci.
Facultative bacteria have developed elaborate mechanisms that permit the expression of the pathways only if required. In Escherichia coli, oxygen-regulated expression of the metabolic enzymes is effected by the transcriptional regulators ArcA (aerobic respiratory control) and FNR (fumarate and nitrate reductase regulation. FNR contains an oxygen-sensing domain at the N-terminus, which contains a Fe–S cluster. Reaction of the Fe– S cluster with oxygen converts FNR from the active to the inactive state. (See “The oxygen-responsive transcriptional regulator FNR of Escherichia coli : the search for signals and reactions” Molecular Microbiology (1997) 25(2), 205–210)
Anaerobes: lack the metabolic enzyme systems for using oxygen in respiration and also lack the enzymes for processing toxic oxygen and die in its presence. Examples include many oral bacteria and intestinal bacteria.
Aerotolerant anaerobes: no not use oxygen but can survive and grow to a limited extent in its presence. They are not harmed by oxygen minaly becasue they possess alternative mechanisms for breaking down peroxides and superoxide. Examples include certain lactobacilli and streptococci clostridial species.
Experiment: Label 4 tubes having thioglycollate broth medium as 1. E coli (this is a facultative anaerobe) , B. subtilis (strict aerobe so should see growth at top but nored because uses all the O2, 3. S. Aurues (facultative anaerobe (will see growth everywhere) and 4. control (expect to be just like the fresh medial with pink at top).
Protocol Strategies:
(1) look at streaked colonies.
(2) obtain 3 plaes, TP, EMB and MAC. Draw line down middle to separate the plate. Label one side “1” and the other “2”
(3) Using sterile techniques, touch one colony and single streak on side 1. Repeat for the other 2 plates. Then choose a different colony and repeat on side 2.