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Biochemical Test Media for Lab Unknown Identification—Part 2 Send Print

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Created: Thursday, 29 August 2002
Last update: Monday, 16 August 2010
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Mannitol salt agar (Enlarged view)
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Mannitol salt agar (Labeled view)
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Nitrate reduction test (Enlarged view)
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Nitrate reduction test (Labeled view)
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Phenol red broth (Enlarged view)
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Phenol red broth (Labeled view)
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Pour plate technique (Enlarged view)
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Pour plate (Enlarged view)
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Most bacteria of medical importance can be grown on artificial culture media. Culture media can be nonselective or selective. Nonselective media allow a wide variety of bacteria to grow (e.g., nutrient agar or blood agar). Selective media allow only certain organisms to grow because they have specific inhibitors added to the media (e.g., the bile salts in MacConkey agar). Additionally, culture media may also be differential, which allows groups of biochemically related bacteria to be distinguished from other groups of bacteria.

This series of images illustrates some common biochemical media reactions for identifying bacteria. Although some bacteria can be identified by visual observation using microscopy, definitive identification usually requires further tests, many of them biochemical. Diagnostic laboratories use various biochemical media to isolate and identify bacteria from clinical specimens.

These images can be used for practice questions on quizzes, lab practical reviews, or as guides for students as they are reading their own tests in lab.

Figure 6. Mannitol salt agar (MSA). MSA is both selective and differential and is used to differentiate Staphylococcus species from each other and from Micrococcus species. Because the medium contains 7.5% salt, it selects for organisms that can grow in a high salt content. Additionally, a pH indicator determines if an organism is able to ferment mannitol. Yellow indicates an acidic pH change, which is a positive indicator for mannitol fermentation. Positive growth (salt tolerance): Staphylococcus. Negative growth (salt intolerance): Micrococcus. Positive mannitol fermentation (yellow): S. aureus. Negative mannitol fermentation (no change in color): S. epidermidis, coagulase negative staphylococci.

Figure 7. Nitrate reduction test. This differential test determines the ability of an organism to reduce nitrate. Some organisms reduce nitrate to nitrite, while others reduce the produced nitrite even further to nitrogen gas. Following incubation in a nitrate broth, sulphanilic acid and a-naphthylamine are added. If nitrite is produced, the broth will turn red. If the broth remains clear, further testing is needed to determine if no reduction occurred or if the nitrite was further reduced. The addition of zinc dust will reduce any remaining nitrate, causing the broth to turn pink. This indicates a negative test for nitrate reduction. If the broth remains clear after the addition of the zinc dust, the organism reduced the nitrite all the way down to another nitrogenous compound. This is called a "positive complete." Positive for nitrite (red): Escherichia coli. Positive complete (full reduction—clear): Pseudomonas aeruginosa. Negative (pink): Acinetobacter calcoaceticus.

Figure 8. Phenol red broth. This differential test determines the ability of an organism to ferment sugars. A sugar (glucose, lactose, or sucrose) and phenol red (pH indicator) are added to a peptone medium with a small inverted tube to trap any produced gas. If an organism can metabolize the sugar, acid is produced and the indicator turns yellow. If gas by-products are produced a bubble will be present in the small, inverted tube.

Glucose fermentation
Positive (yellow), no gas: Staphylococcus aureus.
Positive (yellow), gas: Proteus vulgaris and Escherichia coli.
Negative (no change): Pseudomonas aeruginosa.

Lactose fermentation
Positive (yellow), no gas: Staphylococcus aureus.
Positive (yellow), gas: Escherichia coli.
Negative (no change): Proteus vulgaris and Pseudomonas aeruginosa.

Sucrose fermentation
Positive (yellow), no gas: Staphylococcus aureus.
Positive (yellow), gas: Proteus vulgaris.
Negative (no change): Escherichia coli and Pseudomonas aeruginosa.

Figure 9. Pour plate technique. This technique is used for bacterial enumeration and determines the bacterial count in a milliliter or gram of a specimen. After incubation, colonies appearing on the agar are counted. Each colony represents one colony forming unit (CFU). The CFU/ml or CFU/g is then calculated using a standard formula.

Figure 10. Close-up of pour plate.

See also:
Biochemical Test Media for Lab Unknown Identification—Part 1
Biochemical Test Media for Lab Unknown Identification—Part 3
Biochemical Test Media for Lab Unknown Identification—Part 4

Legend written by:
Kristen Catlin
American Society for Microbiology
Washington D.C. 20036

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