Biochemical Test Media for Lab Unknown Identification

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Biochemical Test Media for Lab Unknown Identification—Part 1

Resource Type: Visual: ImagePublication Date: 8/29/2002

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Authors Jackie Reynolds Math and Science Richland College Dallas, Texas 75243USAEmail:
jackiesr@dcccd.edu

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 1. Simmon citrate. This differential test determines the ability of an organism to use citrate as its sole
carbon source and is used to differentiate the Enterobacteriaceae. The uninoculated medium is green. The
bromothymol blue pH indicator changes to blue when an organism is able to metabolize the citrate and
produce alkaline by-products. Positive: Klebsiella pneumoniae and Enterobacter cloacae. Negative:
Escherichia coli.

Figure 2. Phenylalanine deaminase. This differential media identifies bacteria which possess the enzyme
phenylalanine deaminase. One of the primary uses is to differentiate Proteus and Providencia from the rest
of the Enterobacteriaceae. The medium contains phenylanine which, in the presence of the enzyme
phenylalanine deaminase, is reduced to phenylpyruvic acid. This by-product can be detected by the addition
of an oxidizing agent, which will turn the media green if the acid is present. Yellow indicates a negative
result.

Figure 3. Amino acid decarboxylase. This differential test determines an organism's ability to decarboxylate
an amino acid. An amino acid (either lysine or ornithine) is added to the broth with a pH indicator. After
inoculation, the broth is sealed with mineral oil to promote fermentation. Fermentation causes an
accumulation of acid products, which in turn induces the decarboxylase enzyme (if present).
Decarboxylation produces alkaline end products, indicated by a purple color. Yellow indicates a negative
test.
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Figure 4. Litmus milk—lactose fermentation. This differential test distinguishes organisms that can ferment
lactose. Lactose fermentation causes a lowering of the pH, which in turn causes the litmus to change from
purple to pink.

Figure 5. Litmus milk—casein precipitation. This differential test distinguishes organisms that can
precipitate casein. Sometimes the accumulation of acid following lactose fermentation can lead to the
precipitation of casein, forming an acid clot.

Biochemical Test Media for Lab Unknown Identification—Part 2

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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 α-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.
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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.

Biochemical Test Media for Lab Unknown Identification—Part 3

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Figure 11. Spirit blue lipase test. This differential test determines if an organism produces the secreted
enzyme lipase. The bacterial sample is streaked onto an agar plate containing tributyn, a triglyceride
hydrolyzed by the enzyme lipase. If the bacteria secretes lipase, there will be a zone of clearing surrounding
the sample. If the bacteria does not produce and secrete lipase, the agar will remain opaque. Positive:
Serratia marcescens. Negative: Escherichia coli.

Figure 12. Starch agar. This differential test determines an organism's ability to produce amylase. The
bacterial sample is incubated on an agar plate containing starch and iodine (as an indicator). If the organism
produces amylase, a zone of clearing will surround the inoculation. Positive: Bacillus subtilis. Negative:
Escherichia coli.

Figure 13. Unified-Oxidation-Fermentation (Uni-OF) glucose test (a variation of the Oxidation-

Fermentation Hugh-Leifson Base). This single tube test determines if an organism is oxidative or
fermentative. Carbohydrates may be metabolized by one of two processes: oxidation (aerobic) or
fermentation (anaerobic). Uni-OF glucose media is supplemented with glucose as the carbohydrate source
and a pH indicator. The lower half of the tube allows for anaerobic conditions, while the upper half contains
aerobic conditions. If an organism is able to metabolize the glucose in the condition present, acid is
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produced and the media turns yellow. A color of blue or green indicates that metabolism did not occur. An
organism with oxidative metabolism will display yellow in the upper half of the tube and green in the lower
half. An organism with fermentative metabolism will display yellow in both halves of the tube. Blue or
green in both halves indicates that the organism cannot metabolize glucose. Fermentative metabolism
(yellow in both halves): Vibrio cholerae. Oxidative metabolism: (yellow in upper half, green in lower half):
Bacillus subtilis. Negative (green in both halves): Pseudomonas.

Biochemical Test Media for Lab Unknown Identification—Part 4

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Figures 14–16. Oxidation-fermentation Hugh-Leifson base. This test determines if an organism is oxidative
or fermentative. Carbohydrates may be metabolized by one of two processes: oxidation (aerobic) or
fermentation (anaerobic). Hugh-Leifson glucose medium is supplemented with glucose as the carbohydrate
source and a pH indicator. One tube is covered with vaspar wax for anaerobic conditions, while the other
tube is incubated under aerobic conditions. If an organism is able to metabolize the glucose in the condition
present, acid is produced and the medium turns yellow. A color of blue or green indicates that metabolism
did not occur.

Figure 14. Represents uninoculated control or a negative reaction (no change). Blue or green in both tubes
indicates that the organism cannot metabolize glucose. Example: Pseudomonas.

Figure 15. Oxidative metabolism indicated by yellow in the aerobic tube and green in the anaerobic tube.
Example: Bacillus subtilis.

Figure 16. Fermentative metabolism indicated by yellow in both tubes. Example: Vibrio cholerae.

Figure 17. Methylene blue tributyrin lipase test. This differential test determines if an organism produces the
secreted enzyme lipase. The bacterial sample is streaked onto an agar plate containing tributyrin, a
triglyceride hydrolyzed by the enzyme lipase. If the bacteria secretes lipase, there will be a zone of clearing
surrounding the sample. If the bacteria does not produce and secrete lipase, the agar will remain opaque.
Positive: Serratia marcescens. Negative: Escherichia coli.
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Legend written by:

Kristen Catlin
American Society for Microbiology
Washington, D.C. 20036
kcatlin@asmusa.org

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