Indole test

The indole test is a biochemical test performed on bacterial species to determine the
ability of the organism to split indole from the amino acid tryptophan. This division is
performed by a chain of a number of different intracellular enzymes, a system
generally referred to as "tryptophanase."

Biochemistry
Indole is generated by reductive deamination from tryptophan via the intermediate
molecule indolepyruvic acid. Tryptophanase catalyzes the deamination reaction,
during which the amine (-NH2) group of the tryptophan molecule is removed. Final
products of the reaction are indole, pyruvic acid, ammonia (NH3) and energy.
Pyridoxal phosphate is required as a coenzyme.

Performing a Test
Like many biochemical tests on bacteria, results of an indole test are indicated by a
change in color following a reaction with an added reagent.

Pure bacterial culture must be grown in sterile tryptophan or peptone broth for 24-48
hours before performing the test. Following incubation, add 5 drops of Kovac's
reagent (isoamyl alcohol, p-Dimethylaminobenzaldehyde, concentrated hydrochloric
acid) to the culture broth.

A variation on this test using Ehrlich's reagent (using ethyl alcohol in place of isoamyl
alcohol, developed by Paul Ehrlich) is used when performing the test on
nonfermenters and anaerobes.

A positive result is shown by the presence of a red or red-violet color in the surface
alcohol layer of the broth. A negative result appears yellow. A variable result can also
occur, showing an orange color as a result. This is due to the presence of skatole, also
known as methyl indole or methylated indole, another possible product of tryptophan
degradation.

Indole-Positive Bacteria

Bacteria that test positive for cleaving indole from tryptophan include: Aeromonas
hydrophilia, Aeromonas punctata, Bacillus alvei, most Citrobacter sp., Edwardsiella
sp., Escherichia coli, Flavobacterium sp., Haemophilus influenzae, Klebsiella
oxytoca, Proteus sp. (not P. mirabilis), Plesiomonas shigelloides, Pasturella
multocida, Pasturella pneumotropica, Streptococcus faecalis, and Vibrio sp.

Indole-Negative Bacteria

Bacteria which give negative results for the indole test include: Actinobacillus spp.,
Aeromonas salmonicida, Alcaligenes sp., most Bacillus sp., Bordtella sp.,
Enterobacter sp., Lactobasillus spp., most Haemophilus sp., most Klebsiella sp.,
Neisseria sp., Pasturella haemolytica, Pasturella ureae, Proteus mirabilis,
Pseudomonas sp., Salmonella sp., Serratia sp., Yersinia sp.

References
• MacFaddin, Jean F. "Biochemical Tests for Identification of Medical
Bacteria." Williams & Wilkins, 1980, pp 173 - 183.
• Example of typical indole reactions

Retrieved from "http://en.wikipedia.org/wiki/Indole_test"

Voges–Proskauer test
Voges–Proskauer (English pronunciation: /ˈfoʊɡəs ˈprɒskaʊ.ər/) or VP is a test used to
detect acetoin in a bacterial broth culture. It is designed to distinguish E. coli from
Enterobacter aerogenes.[1] By adding a concentrated solution of sodium hydroxide a
red color appears.[2] [3] The test depends on the digestion of glucose to
actylmethylcarbinol. If Glucose is being broken down, it will react with alpha-
naphthol (VP reagent #1) and potassium hydroxide (VP reagent #2) to form a red
color. Alpha-naphthol and potassium hydroxide are chemicals that detect acetoin.

Procedure: First, add the alpha-naphthol; then, add the potassium hydroxide. A
reversal in the order of the reagents being added may result in a weak-positive or
false-negative reaction. Red color means a positive result. If the color does not
change, the test is negative.

VP is one of the four tests of the IMViC series, which tests for evidence of an enteric
bacterium. The other three tests include: the indole test [I], the methyl red test [M],
and the citrate test [C]. [4]

History
The reaction was developed by Daniel Wilhelm Otto Voges and Bernhard Proskauer
— German bacteriologists in 1898 at the Institute for Infectious Diseases.

References
 1. ^ Bachoon, Dave S., and Wendy A. Dustman. Microbiology Laboratory
Manual. Ed. Michael Stranz. Mason, OH: Cengage Learning, 2008. Exercise
15, "Normal Flora of the Intestinal Tract" Print.
2. ^ Manual of Clinical Microbiology, Fifth Edition, Chapter 120, "Quality
Control" and Chapter 122, "Reagents and Stains"
3. ^ Bailey and Scott's Diagnostic Microbiology, Seventh Edition, Chapter 27,
"Enterobacteraceae"
4. ^ Bachoon, Dave S., and Wendy A. Dustman. Microbiology Laboratory
Manual. Ed. Michael Stranz. Mason, OH: Cengage Learning, 2008. Exercise
15, "Normal Flora of the Intestinal Tract" Print.

External links
• Voges–Proskauer Tests at Key Scientific
• Voges–Proskauer reaction at Merriam–Webster Online

Oxidase test
The oxidase test is a test used in microbiology to determine if a bacterium produces
certain cytochrome c oxidases.[1] It uses disks impregnated with a reagent such as
N,N,N′,N′-tetramethyl-p-phenylenediamine (TMPD) or N,N-Dimethyl-p-
phenylenediamine (DMPD), which is also a redox indicator. The reagent is a dark
blue to maroon color when oxidized, and colorless when reduced.

Classification
Strains may either be oxidase positive (OX+) or negative (OX-).

OX+

OX+ normally means that the bacterium contains cytochrome c oxidase and can
therefore utilize oxygen for energy production with an electron transfer chain.

Typically the Pseudomonadaceae are OX+[citation needed]

Another example is the preliminary identification of Neisseria and Moraxella genera,

which are both oxidase positive, Gram-negative diplococci.

Many Gram-negative spiral curved rods are also oxidase positive, which includes
Helicobacter pylori, Vibrio cholera, and Campylobacter jejuni.

Also Legionella pneumophila is oxidase positive. A trick to remember the most

medical relevant bacteria is: "VIce President CHeNEy MOstly LEads" (Vibrio,
Pseudomonas, Campylobacter, Helicobacter, Neisseria, Moraxella, and Legionella,
respectively).
OX-

OX- normally means that the bacterium does not contain cytochrome c oxidase and
therefore cannot utilize oxygen for energy production with an electron transfer chain.

Typically Enterobacteriaceae are OX-.[2]

Procedures
1. Wet each disk with about 4 inoculating loops of de-ionized water.
2. Use a loop to aseptically transfer a large mass of pure bacteria to the disk.
3. Observe the disk for up to 3 minutes. If the area of inoculation turns dark blue
to maroon to almost black, then the result is positive. If a color change does
not occur within three minutes, the result is negative.

Alternatively, live bacteria cultivated on trypticase soy agar plates may be prepared
using sterile technique with a single-line streak inoculation. The inoculated plates are
incubated at 37°C for 24–48 hours to establish colonies. Fresh bacterial preparations
should be used. After colonies have grown on the media, two-to-three drops of the
reagent DMPD is added to the surface of each organism to be tested.

• A positive test (OX+) will result in a color change to pink, through maroon
and into black, within 10–30 seconds.
• A negative test (OX-) will result in a light pink coloration or absence of
coloration.

References
1. ^ "Oxidase Test and Modified Oxidase Test".
http://web.mst.edu/~microbio/Lab_Supplement/Oxidase.html. Retrieved 2008-
11-07.
2. ^ Farmer JJ, Fanning GR, Huntley-Carter GP, et al. (May 1981). "Kluyvera, a
new (redefined) genus in the family Enterobacteriaceae: identification of
Kluyvera ascorbata sp. nov. and Kluyvera cryocrescens sp. nov. in clinical
specimens". J. Clin. Microbiol. 13 (5): 919–33. PMID 7240403. PMC 273917.
http://jcm.asm.org/cgi/pmidlookup?view=long&pmid=7240403.

External links
• Oxidase test video
• Oxidase Test Procedure