Differential Tests for Enterobacteriaceae See right hand panel
Motility Tests: See outline
Introduction:
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 nature. For example, even though an organisms may never greatly lower the pH of its natural environment, the microbiologist can manipulate conditions in a lab such as by cultivating a microbe with medium spiked with a specific sugar or other substrate.
Differential Tests:
Gran Stain: is a differential stain that differentiates by color two majory groups of bacteria: gram-positive and gram-negative bacteria. The response (positive or negative) of cells for this staining technique is based on the composition of the cell wall. The cell wall of gram-positive cells is made up of a very thick layer of peptidoglycan. Peptidoglycan is a long, fibrous network of NAM (N-acetyl muramic acid and NAG (N-acetyl glucosamine) cross-linked by short peptide chains. The cell wall of gram negative cells consists of a very thin layer of peptidoglycan surrounded by an outer membrane countaining lipopolysaccharide (LPS).
Acid fast staining: is a differential staining technique that differentiates between acid fast and non-acid fast bacteria. Acid fast genera include Mycobacterium and ome members of Nocardia. Pathogenic mycobacterial species include the causative agents of tuberculosis (TB) and leprosy.
Acid fast cells ahve a waxy cell wall. Mycolic acid in the cell walls of acid fast bacteria makes these organism resistant to desiccation (drying) and disnfectants. The waxy cell wall also amkes it difficult to stain these organisms with traditional stains that are water soluble. Water soluble stains are repelled by the cell wall in the same way that water beads up on a paraffin candle.
There are two different means commonly used to stain acid fast bacteria: (1) the Ziehl Neelsen method uses a steaming water bath to facilitate staining of the cell and (2) the Kinyoun method uses more concentrated ragents and does not require steam (cold method).
(1) Ziehl-Neelsen technique for acid fast staining entails steaming heat fixed slides over boiling water with the primary stain, carbol fuchsin which melts or increases the fluidity of the waxy cell wall (mycolic acid) of acid fast cells and permits the peentration of the primary stain. After staining,t he slide is cooled. As the slid cools, the waxy cell wall firms up and loses its fluidity, trapping the stain int he cell. Smears are delolorized with acid alchol. Cells that retain the primary stain after declolorization are called acid fast, that is the bright red fuchsia color reamins fast int he cells. The slide is then counterstained with a secondary stain, methylene blue, to dye the non acid fast cells which will appear blue.
(2) Kinyound acid fast staining is nearly the same as the Ziehl Neelsen method but uses carbol funchsin, acid alcohol, and methylene blue and does nto requrie the slide to be steamed. The primary stain contains higher concentrations of phenol and basic fuchsin than in the Ziehl-Neelsen reagent. Phenol is a lipd solvent. Incdreasing the concentraiton of phenol increases the solubility of the cell wall or dissolves the waxy cell wall and allows the basic fuchsin to peentrate the cells.
Endospore Stain (Schaeffer-Fulton Method): When exposed to conditions such as nutrient limitation or waste accumulation, vegetative cells (actively metabolizing) of the genera Bacillus (aerobe) and Clostridium (anaerobe) begin the process of sporulation and eventually form endospores. The term “endospore” is used to describe location, referring to a spore that is contained in the cytoplasm of a bacterium. When released or the bacterium is lysed, the endospore is then re-termed an “exospore” or “spore”.
The endospore stain is a differential stain. Heat fixed smears are steamed over boiling water with the primary stain, malachite green. Heating the endospores with the primary stain allows the stain to penetrate and dye the pore caot. The slide is then cooled. As the spore cools, the stain is trapped inside. Smears are declorized adn then counter stained with a secondary stain (safranin) to dye the vegetative cells.
Oxidation-Fermentation:
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 directly 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.
Sugar Fermentation:
Because sugar fermentation capabilities vary widely among bacteria, carbohydrate testing is often among the first tests done in the identification of an unknown organism. Sugar fermentation studies are especially useful for differentiating gram-negative enteric bacteria from each other and from other gram-negative organisms. In the bacterial cell, sugars are metabolized in the glycolytic pathway, resulting in pyruvate and ATP. Under oxygen-limiting conditions, pyruvate is further metabolized to a variety of end products, including various alcohols, gases, and acids. It is these acidic or alkaline end products that are detectable in the testing.
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 environments. 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.
Phenol red broth (PRB) is commonly used to determine the test organism’s ability to utilize a specific sugar. PRB includes the pH indicator phenol red, a sugar of interest, buffers, salts, and small peptide fragments. In practice, an organism is inoculated into a series of broth tubes, each containing a single sugar. The three most commonly tested sugars are glucose, lactose, and sucrose.
Metabolism of the selected sugar generates acidic end products, lowering the pH of the tube. The pH indicator, phenol red, changes color from red to yellow if acidic products are present, indicating a positive result for sugar fermentation. Some organisms may not ferment the sugar in question. In this case, the bacteria may catabolize the peptide amino acids into ammonia. This breakdown increases the pH of the medium to alkaline and causes the phenol red to appear bright red, magenta, or fuschia in color, signifying a negative result for usage of the sugar.
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 carrier 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.
Other Types of Differential Media
HE Agar: differentiates Salmonella and Shigella from each other and form other enterics based on their ability to overcome the inhibitory effects of bile, reduce sulfur to H2S and ferment lactose, sucrose or salicin.
The test is based on the ability to ferment lactose, sucrose, or salicin and to reduce sulfur to hydrogen sulfide gas (H2S). Sodium thiosulfate is included as the source of oxidized sulfur. Ferric ammonium citrate is included as a source of oxidized iron to react with any sulfur that becomes reduced (H2S) to form the black precipitate ferrous sulfide (FeS). Bile salts are included to prevent or inhibit growth of Gram positive organisms. The bile salts also have a moderate inhibitory effect on enterics, so relatively high concentrations of animal tissue and yeast extract are included to offset this situation. Bromthymol blue and acid fuchsin dyes are added to indicate pH changes. Differentation is possible as a reslt of the various colors produced in the colonies and in the agar.
Enterics that produce acid from fermentaiton will produce yellow to salmmon pink colonies. Neither Salmonella nor Shigella species ferment any of the sugars but do break down the animal tissue which raises the pH and gives the colonies a blue-green color. Salmonella species also reduce sulfur to H2S, so the solonies formed also contain FeS which makes them partially or completely black.
XLD Agar: favors growth of Salmonella, Shigella or Providencia based on its ability to overcome the inhibitory effects of desoxycholate and differentiates them based on their ability to reduce sulfur to H2S, decarboxylate the amino acid lysine and ferment xylose or lactose.
Blood agar is an enriched differential medium that contains 5–10% sheep red blood cells (RBCs). Many bacteria can grow on this medium. The iron and other extra nutrients provided by the RBCs are needed by fastidious bacteria in order to grow.
Some bacteria simply grow on the agar, whereas others partially or completely break down the RBCs embedded in the agar, using toxins called hemolysins. The ability to break down RBCs (or not) makes this medium differential. Depending on the hemolysin produced by the bacteria, it can cause several outcomes.
Hemolysis will appear as a “halo” or partial clearing around the bacterial colonies. Partial lysis of RBCs leads to green-colored discoloration of the blood agar around the colony called alpha-hemolysis. Hemolysins that cause complete lysis of the RBCs lead to yellow or colorless clearing of the blood agar around the colony called beta-hemolysis. If an organism causes no hemolysis, it is reported as gamma-hemolysis.
Many different species of Streptococcus have been characterized for their hemolytic abilities, which helps in diagnosing one species from another. Throat swabs can be used to test for possible strep throat. Those samples were likely grown on blood agar to look for beta-hemolytic Streptococcus pyogenes, a causative agent for strep throat.