The Voges-Proskauer (VP) test is a biochemical assay used in microbiology to detect whether a bacterium produces acetoin (also called acetyl methyl carbinol) during glucose fermentation. Developed in the late 1800s by Arthur Voges and Bernhard Proskauer, the test remains a workhorse in diagnostic labs for sorting out which bacteria are which, particularly among the large family of gram-negative rods known as Enterobacteriaceae. The chemistry is straightforward, the procedure takes less than a day, and the result is a simple color change, but knowing what the test actually measures and how to interpret it correctly matters more than its apparent simplicity might suggest.
What the Test Actually Detects
At its core, the VP test identifies bacteria that ferment glucose through the butanediol pathway rather than the mixed-acid pathway. When certain bacteria metabolize glucose, they first break it down to pyruvate, the same step that occurs in virtually all sugar-fermenting organisms. What happens next is where the paths diverge. Some bacteria send pyruvate through a series of reactions that produce a mixture of organic acids. Others shunt pyruvate toward a neutral end product called 2,3-butanediol, with acetoin as a key intermediate along the way.
The enzymes responsible for this detour have been well characterized. Acetolactate synthase converts pyruvate into acetolactate, and acetolactate decarboxylase then converts acetolactate into acetoin.1PubMed. Identification of acetoin reductases involved in 2,3-butanediol pathway in Klebsiella oxytoca Acetoin can be further reduced to 2,3-butanediol by a reductase enzyme. The VP test catches the process at the acetoin stage. If acetoin is present in the culture medium, the reagents added during the test will oxidize it to diacetyl, which then reacts to produce a visible red or pink color.
This distinction between the butanediol pathway and the mixed-acid pathway is not just an academic curiosity. It directly reflects how a bacterium handles its metabolic waste. Mixed-acid fermenters acidify their environment heavily, while butanediol fermenters produce a more neutral pH. That difference in acid production is precisely what the companion test, the methyl red (MR) test, picks up, which is why the two are almost always performed together.
How the Test Is Performed
The VP test starts with inoculating a tube of MR-VP broth, also called Clark-Lubs medium, with the bacterial isolate you want to identify. This broth contains glucose and peptone in a buffered solution, giving the organism a sugar source to ferment. The tube is incubated aerobically at roughly 35°C for 18 to 24 hours. If no color develops after adding reagents at that point, the remaining broth can be re-incubated for an additional 24 hours and tested again, since some slower-growing organisms need the extra time.
After incubation, a small aliquot of the broth is transferred to a clean tube. The standard protocol, known as Barritt’s method, calls for adding alpha-naphthol solution (the 5% concentration is typical) first, followed by potassium hydroxide solution (usually 40% KOH). The order matters: alpha-naphthol goes in first, and you shake or swirl the tube after each addition. The KOH serves two purposes. It raises the pH of the medium and provides the alkaline environment needed for acetoin to be oxidized to diacetyl. The alpha-naphthol acts as a catalyst and color intensifier, reacting with the diacetyl to produce the characteristic red or copper-pink color.
One practical detail that trips people up: after adding both reagents, the tube should be left with the cap loosened or at a slant so the surface of the medium is exposed to air. Oxygen is needed for the oxidation step, and sealing the tube tightly can delay or prevent color development. Results are typically read within 15 to 60 minutes, though some protocols allow up to four hours for weakly positive reactions to appear.
Reading and Interpreting Results
A positive VP test shows a red to pink color developing at the surface of the broth and gradually spreading downward. The color should be clearly distinguishable from the yellowish or straw-colored broth of an uninoculated control. A strong positive can appear within 15 minutes; a weak positive may take closer to an hour. If the broth stays yellow or shows only a faint copper tinge that does not deepen, the result is negative.
A few things can go wrong with interpretation. Adding too much KOH relative to alpha-naphthol sometimes produces a copper color that looks ambiguous. Reversing the order of reagents (KOH before alpha-naphthol) tends to reduce sensitivity and can give false negatives. Over-incubation of the broth beyond 48 hours can also cause problems, because some organisms that initially produce acetoin will eventually metabolize it further, leaving nothing for the reagents to detect. Under-incubation is equally problematic for slow growers, which is why the re-incubation step exists.
False positives are less common but can occur if the medium is contaminated or if the alpha-naphthol solution has degraded. Freshness of reagents matters more than most lab manuals emphasize. Old alpha-naphthol solutions can darken and produce misleading background color.
The MR-VP Pairing and Why They Are Run Together
In most microbiology labs, you will almost never see a VP test ordered in isolation. It is nearly always paired with the methyl red (MR) test, and the two are run from the same MR-VP broth, which is why the medium carries both names. After incubation, the broth is split: one aliquot goes for the VP reagents, the other gets methyl red indicator dropped in.
The logic behind pairing them is that the two results are generally inversely correlated. An organism that ferments glucose through the mixed-acid pathway produces enough acid to keep the broth pH low, giving a positive MR result (the methyl red stays red below about pH 4.4). That same organism typically does not produce significant acetoin, so its VP result is negative. Conversely, an organism using the butanediol pathway produces acetoin (VP positive) and does not acidify the broth as heavily (MR negative).
This inverse pattern holds well enough to be diagnostically useful, but it is not absolute. Some organisms give positive results for both tests or negative for both, depending on incubation time, growth phase, and the specific strain. Treating MR and VP as a perfect either/or can lead to misidentification if you do not consider the full panel of biochemical results.
Which Organisms Are VP Positive and Which Are Not
The VP test is most frequently used to sort members of the Enterobacteriaceae family and related gram-negative rods. Classic VP-positive organisms include species of Klebsiella, Enterobacter, Serratia, and Hafnia. Among gram-positive bacteria, many species of Bacillus (including B. subtilis) and certain streptococci and enterococci are VP positive as well.
On the VP-negative side, Escherichia coli is the textbook example. Studies characterizing E. coli isolates from diverse biological and environmental sources have consistently found them to be VP negative while testing positive for catalase, methyl red, and indole.2Bangladesh Journals Online (BanglaJOL). Characterization of Escherichia coli isolated from samples of different biological and environmental sources Other reliably VP-negative genera include Salmonella, Shigella, Proteus, and Citrobacter.
This split is one of the main reasons the VP test exists in routine identification workflows. When you have a gram-negative rod growing on a plate and you need to narrow down whether it is something like Klebsiella pneumoniae or E. coli, the VP result (combined with MR, indole, and citrate utilization) gets you most of the way there. That four-test combination, sometimes called IMViC (Indole, Methyl red, Voges-Proskauer, Citrate), has been a cornerstone of enterobacterial identification for decades.
Applications Beyond Enterobacteriaceae
While the VP test is most associated with sorting gram-negative rods, it plays an important role in identifying gram-positive cocci as well. Distinguishing aerococci from enterococci and streptococci, for instance, relies on a panel of physiological tests that includes VP. Research comparing rapid identification systems with conventional methods found that the VP test was a satisfactory substitute for more time-consuming conventional approaches when classifying these gram-positive groups.3PubMed Central. Comparison of physiologic tests used to identify non-beta-hemolytic aerococci, enterococci, and streptococci
The test also shows up in food microbiology and agricultural research. Screening probiotic bacteria from animal sources, for example, uses MR-VP alongside oxidase, catalase, and sugar fermentation profiles to characterize isolates across genera like Lactobacillus, Bacillus, Streptococcus, Enterococcus, and Bifidobacterium.4Asian Journal of Medical and Biological Research. Isolation, identification and molecular detection of selected probiotic bacteria from broiler chickens and their related environment In the fermented-food industry, identifying Bacillus strains in products like fermented sausages similarly relies on biochemical panels where the VP test is a standard component.5Meat Science. Evaluation of different systems for the identification of Bacillus strains isolated from Spanish fermented sausages
In water quality testing, the IMViC panel that includes the VP test has long been used to distinguish fecal coliforms from non-fecal coliforms. A water sample growing E. coli (indole positive, MR positive, VP negative, citrate negative) tells a different public health story than one growing Enterobacter (VP positive, citrate positive). That distinction can determine whether a water supply is considered contaminated with fecal matter or harboring environmental organisms that are less concerning from a sanitation standpoint.
Reproducibility and Automated Systems
One practical advantage of the VP test is its reproducibility. When researchers evaluated the API 20E system, a widely used commercial kit for identifying gram-negative bacteria, they found the VP test results to be among the most reproducible of all the biochemical reactions in the panel.5Meat Science. Evaluation of different systems for the identification of Bacillus strains isolated from Spanish fermented sausages This consistency matters because some biochemical tests are notoriously finicky, giving different results depending on slight variations in incubation temperature or inoculum density. The VP test, by comparison, tends to give clean results when the reagents are fresh and the protocol is followed.
That said, the broader landscape of bacterial identification has shifted dramatically. Automated and semi-automated systems like Vitek, Phoenix, and MicroScan have incorporated the VP reaction into panels that run dozens of biochemical tests simultaneously, reducing hands-on time and standardizing the process. More significantly, technologies like MALDI-TOF mass spectrometry have begun replacing biochemical testing altogether for routine bacterial identification. One reference laboratory’s ten-year study documented how MALDI-TOF MS was validated in 2014 as a replacement for both 16S rRNA sequencing and traditional biochemical testing.6Open Forum Infectious Diseases. P-1700. A Ten-year study on Bacterial Identification using MALDI-TOF Mass Spectrometry in a Reference Laboratory
MALDI-TOF identifies bacteria by their protein fingerprint rather than by what they do metabolically, which means a result can come back in minutes rather than overnight. For well-resourced clinical labs, this has made conventional tube-based biochemical tests less central to daily workflows. But the VP test has not disappeared. Smaller labs, field settings, and teaching institutions still rely heavily on manual biochemical panels. And when automated systems return ambiguous identifications, going back to basics with tests like VP, MR, indole, and citrate remains a common troubleshooting strategy.
Common Mistakes and Misconceptions
A few recurring errors are worth flagging for anyone performing or learning the VP test. The first is reagent order. Barritt’s method specifies alpha-naphthol before KOH. Flipping them does not just change the intensity of the color; it can genuinely produce a false negative, because the chemistry depends on the alpha-naphthol being present when the oxidized diacetyl forms. Some older protocols (notably the O’Meara modification, which uses creatine instead of alpha-naphthol) do not follow the same order, and mixing up which protocol you are using leads to unreliable results.
The second common mistake is reading results too early or too late. Checking the tube five minutes after adding reagents and calling it negative is premature; weakly positive organisms need time. On the other hand, leaving the tube on the bench for several hours and then reading a faint pinkish tinge as positive is overcalling it. The reliable reading window is roughly 15 to 60 minutes after reagent addition, and the color should be unambiguous.
A subtler misconception involves treating the MR and VP results as a perfectly binary toggle. Students often learn that an organism is either MR positive/VP negative or MR negative/VP positive, with no overlap. In practice, some organisms produce enough acid to give a positive MR result while also generating detectable acetoin for a positive VP. This tends to happen with prolonged incubation or with organisms that use both fermentation pathways to some degree. Relying on the MR-VP pair as the sole basis for identification, without corroborating with other tests, can lead you astray.
The Butanediol Pathway in Industrial Microbiology
The same biochemistry that makes the VP test work has attracted interest well outside the diagnostic lab. 2,3-butanediol and its precursor acetoin are industrially valuable chemicals. Butanediol is used as a precursor for synthetic rubber, plasticizers, and fuel additives, and acetoin itself serves as a flavoring agent in food production, contributing buttery notes to dairy products and baked goods.
Because certain bacteria naturally produce these compounds in large quantities, there has been substantial research into engineering microbial strains for industrial-scale butanediol production. Klebsiella oxytoca and Klebsiella pneumoniae are among the most studied organisms for this purpose, precisely because their butanediol pathways are robust and well characterized.1PubMed. Identification of acetoin reductases involved in 2,3-butanediol pathway in Klebsiella oxytoca Researchers have cloned and expressed the key pathway enzymes in E. coli and other production hosts to optimize yields, effectively taking the metabolic machinery that makes certain bacteria VP positive and transplanting it into organisms better suited for fermentation at scale.
This industrial angle highlights something worth appreciating about the VP test. It is not just a diagnostic curiosity or a relic of pre-molecular microbiology. The metabolic pathway it detects sits at the intersection of clinical diagnostics, food science, and industrial biotechnology. The test itself may eventually fade from routine clinical use as mass spectrometry and genomic methods take over, but the underlying biology it reveals continues to drive research in fields that the test’s nineteenth-century inventors could never have imagined.