Microbiologists have differentiated bacterial based on their biochemical diversity using various biochemical tests. Such tests often include differential medial which are artificial mixtures of chemicals and organic substrates intended to exploit different microbial abilities to perform in ways that might never actually be expressed in nautre. For example, even though an organisms may never greatly lower the pH of its natural environgment, the microbiologist can manipulate conditions in a lab such as by cultivating a microbe with medium spiked with a specific sugar or other substrate.

Oxidation-Fermentation (O-F) Test: is designed to differentiate bacteria on the basis of fermentative or oxidative metabolism of carbohydrates.

In oxidation pathways a carbohydrate is direclty oxidized to pyruvate and is further converted to CO2 and energy by way of the krebs cycle and the electron transport chain (ETC). An inorganic molecule such as oxygen is required to act as the final electron acceptor.

Fermentation also converts carbohydrates to pyruvate but uses it to produce one or more acids (as well as other compounds). As a consequence, fermenters identified by this test acidify O-F medium to a greater exent that do oxidizers.

The O-F test is used to differentiate bacteria based on their ability to oxidize or ferment specific sugars.

Triple Sugar Iron Agar (TSIA) / Kligler Iron Agar: TSIA is a rich medium designed to differentiate bacteria on the basis of glucose fermentation, lactose fermentation, sucrose fermentation and sulfur reduction. In addition to the three carbohydrates, it includes animal proteins as sources of carbon and nitrogen, and both ferrous sulfate and sodium theiosulfate as sources of oxidized sulfure. Phenol red is the pH indicator, and the iron in the ferrous sulfate is the hydrogen sulfide indicator.

The medium is prepared as a shallow agar slant with a deep butt, thereby providing both aerobic and anaerobic growth envirnoments. It is inoculated by a stab in the agar butt followed by a fishtail streak of the slant. When TSIA is inoculated with a glucose only fermenter, acid products lower the pH and turn the entire medium yellow within a few hours. As the glucose depletes, the organisms located in the aerobic region (slant) will begin to break down available amino acids, producing NH3 and raising the pH (18-24 hours). This only occurs in the slant due to the anaerobic conditions in the butt. Thus, a TSIA with a red slant and yellow butt after 24 hour incubation indicates that the bacteria ferments glucose but not lactose.

Organisms that are able to ferment glucose and lactose and/or sucrose also turn the medium yellow throughout. However, because the lactose and sucrose concentrations are 10 times higher than that of glucose, resulting in greater acid production, both slant and but will remain yellow after 24 hours.

Catalase test: The electron transport chains of aerobic and facultatively anaerobic bacterial are composed of molecules capable of accepting and donating electrons as conditions require. One carier molcule in the ETC called falvoprotein can bypass the next carrier in the chain and transfer electrons direclty to oxygen. This alternative pathway produces hydrogen peroxide (H2O2) and superoxide radical (O2-). Organisms that produce these toxins also produce enzymes capable of breaking them down. Superoxide dismutasecatalyzes conversion of superoxide radicals to hydrogen peroxide. Catalase converts hydrogen peroxide into water and gaseous oxygen.

Bacterial that produce catalase can be detected easily using hydrogen peroxide. When hydrogen peroxide is added to a catalase-postive culture, oxygen gas bubbles form. The test is commonly used to differentiate members of the catalase-positive Micrococcaceae from the catalase negative Streptococcaceae.

Oxidase Test: When glucose enters a cell, it is first split (oxidzied) in glycolysis where it is converted to two molecules of pyruvate and reduces two NAD (coenzyme) molecules to NADH (+H+). Then each of the pyruvate molecules is oxidized and converted to a two carbon molecule called acetyl-CoA and one molecule of CO2, which reduces another NAD to NADH. Then the Krebs cycle finishes the oxidation by producing two more molecules of CO2 (per acetyl-CoA) and reduces three more NADs and one FAD to FADH2. The cell is thus beocming full of reduced coenzymes. In order to continue oxidizing glucose, these coenzymes must be converted back to the oxidized state. This i the job of the electron transport chain.

Many aerobes, microarophiles, facultative anaerobes, and even some anaerobes have ETCs. The functions of the ETC are to transport electrons down a chain of molecules with increasingly positive reduction potentials to the terminal electron acceptor (1/2O2, NO32-, SO43-) and generate a protein motive force by pumping H+ out of the cell thus creating an ionic imbalance that will drive the production of ATP by way of membrane ATPases. The protons pumped out of the cell come from the hydrogen atoms whose electrons are being transferred down the chain. Some organisms use more than one type of ETC depending on the availability of oxygen or toher preferred terminal electron acceptor. E coli, for example, has two pathways for respiring aerobically and at least one for respiring anaerobically. Many bacteria have ETCs resembling mitochondria ETCs in eukaryotes. These chains contain a series of foru large enzymes broadly named Complexes I, II, III, and Iv, wach of which contains several molecules jointly able to transfer electrons and use the free energy released in the reactions. The last enzyme in the chain, Complex IV, is called cytochrome c oxidasebecause it makes the final electron transfer of the chain from cytochrome c, residing in the periplasm to oxygen inside the cell.

The oxidase test is designed to identify the presence of cytochrome c oxidase which has the ability to not only oxidize cytochrome c, but to catalyze the reduction of cyctochrome c by a chromogenic reducing agent called tetramethyl-p-phenylenediamine. Chromogenic reducing agents are chemicals that develop color as they become oxidized.

Tests Detecting Hydrolytic Enzymes: Reactions that use water to split complex molecules are called hydrolysis (or hydrolytic) reactions. The enzymes required for these reactions are called hydrolytic enzymes.

(i) Starch Hydrolysis: Starch is too large to pass through bacterail cell membranes and thus must be split into smaller fragments or individual glucose molecules. Organisms that produce and secrete the extracellular enzymes alpha-amylase and oligo-1,6-glucosidase are able to hydrloyze starch by breaking the glycoside linkages between sugar subunits. Starch agar is a medium of beef extract, soluble starch and agar. When organisms produce alpha-amylase and oligo-1,6-glucosidase they hydrolyze the starch in the area surrounding their growth. Because both starch and its sugar subunits are nearly invisible in the medium, iodine is added to detect the presence or absence of starch in the vicinity around the bacterail growth. Iodine reacts with starch and produces a blue or dark brown color.

(ii) Casein Hydrolysis Test: Casease is an enzyme that some bacteria produce to hydrolyze the milk protein casein. The presence of casease can be detected with milk agar. Casease positive organisms will secrete casease which will diffuse into the medium around the colonies and create a zone of clearing where the casein has been hydrolyzed.

Nitrate Reduction Test: Anaerobic respiration involves the reduction of (i.e., transfer of electrons to) an inorganic molecule other than oxygen. Nitrate reduction is one such example. Many Gram-negative bacteria (including most Enterobacteriaceae) contain the enzyme nitrate reductase and perform a single step reduction of nitrate to nitrite (NO3- to NO2).

Nitrate broth is an undefined medium of beef extract, peptone, and ptoassium nitrate (KNO3). An inverted Durham tube is placed in each broth to trap a portion of any gase produced. In contrast to many differential media, no color indicators are included. The color reactions obtained in nitrate broth take place as a result of reactions between metabolic products and reagents added after incubation.

Differential Tests for Enterobacteriaceae   See right hand panel

Motility Tests: See outline