Microbiology Lab 2-6 Aerotolerance, 2-8 temperature, 2-12 effects of UV, Disinfectant and antiseptic lab, page 227 selective vs differential, 4-3 mannitol salt agar, 4-4

application of uv lab

Ultraviolet light is commonly used to disinfect laboratory and health-care environment work surfaces and surrounding air.

mesophiles

15-45 C

MR-VP

MR-VP broth is a combination medium used for both methyl red (MR) and Voges-Proskauer (VP) tests. It is a simple solution containing only peptone, glucose, and a phosphate buffer. The peptone and glucose provide protein (with its nitrogen) and fermentable carbohydrate, respectivel民 and the potassium phosphate resists pH changes in the medium. The MR test is designed to detect organisms capable of performing a mixed acid fermentation, which overcomes the phosphate buffer in the medium and lowers the pH (Figs. 5.6, 5.7, and A.6). Succinate is produced from the addition of C02 to phosphoenolpyruvate, whereas the other end products are derived from the reduction of pyruvate to lactate or its oxidation to acetyl-CoA and formate. Conversion of acetyl-CoA to acetate results in the formation of one ATP or it can be reduced to ethanol. Formate can be further broken down into H2 and C02 gas. The acids produced by these organisms tend to be stable, whereas acids produced by other organisms may be converted to more neutral products or their fermentation shifts to produce more neutral products in response to the lowered pH. Mixed acid fermentation is verified by the addition of methyl red indicator dye following incubation. Methyl red is red at pH 4.4 and yellow at pH 6.2. Between these two pH values, it is various shades of orange. Red color is the only true indication of a positive result. Orange is negative or inconclusive. Yellow is negative (Fig. 5.8). The Voges-Proskauer test was designed to identify organisms that are able to ferment glucose, with the production of acetoin and 2,3-butanediol, which have a neutral pH (Figs. 5.7, 5.9, and A.6). VP-positive organisms can produce other fermentation end products in addition to 2,3-butanediol. They can reduce pyruvate to lactate (which lowers the pH), and they can make formate (which is broken down into H2 and C02 gases) and acetyl-CoA (which is reduced to ethanol). The acetyl-CoA path results in no pH change, but formate (at least until its conversion to H2 and C02) and lactate production lower pH. VP-positive organisms compensate for the lowered pH by shifting to 2,3-butanediol production. Adding VP reagents to the medium oxidizes the acetoin to diacetyl, which in turn reacts with guanidine nuclei from peptone to produce a red color (Fig. 5.10). A positive VP result, therefore, is red. No color change NAD+ F ~ ／＼.、 (or development of copper color) after the addition of reagents is negative. The copper color is a result of interactions between the reagents and should not be confused with the true red color of a positive result (Fig. 5 .11). Use of positive and negative controls for comparison is usually recommended. To avoid conflicting results of the two tests, two 1 mL volumes are transferred from the MR-VP broth to separate test tubes after incubation. Methyl red indicator reagent is added to one tube, and VP reagents are added to the other. Color changes then are observed and documented. Refer to the procedural diagram in Figure

application MacConkey agar

MacConkey agar= isolate and differentiate members of the Enterobacteriaceae based on the ability to ferment lactose. Variations on the standard medium include MacConkey agar w/o CV (without crystal violet) to allow growth of Gram-positive cocci, or MacConkey agar CS to control swarming bacteria that interfere with interpretation of results and isolation of other species.

mannitol salt agar

Mannitol=substrate for fermentation and makes the medium differential. used for isolation and differentiation of Staphylococcus aureus from other Staphylococcus species. Sodium chloride=selective because its concentration is high enough to dehydrate and kill most bacteria. Phenol red=used for fermentation Indicator

reduction potential

e tendency of a molecule to donate electrons, measured in volts. Without the input of energy, electron transfers will always go from a molecule with a higher reduction potential to a molecule with a lower reduction potential

optimum temperature

grow its fastest and most growth rate

Obligate Aerobes

require oxygen

microaerophiles

require oxygen concentration lower than air

selective media

suppress growth of unwanted bacteria and encourage growth of desired microbes

bacteriostatic agents

temporary inhibition of growth

electromagnetic energy

ultraviolet radiation

Eosin Methylene Blue Agar

Eosin methylene blue=, selective, and differential medium. It contains digest of gelatin, lactose, and the dyes eosin Y and methylene blue. gelatin=nitrogen and organic carbon. Lactose is fermented to acid end-products by coliforms such as Escherichia coli and Enterobacter aerogenes, whereas it is not by pathogens such as Proteus, Shig·ella, and Salmonella species. The purpose of the dyes is twofold: ( 1) they inhibit the growth of most Gram-positive organisms (some Enterococcus and Staphylococcus species are exceptions), and (2) they react with vigorous lactose fermenters whose acid end-products turn the growth dark purple or black. This dark growth is typical of Escherichia coli and is usually accompanied by a green metallic sheen (Fig. 4.7). Other, less-aggressive lactose fermenters, such as Enterobacter or Klebsiella species, produce colonies that can range from pink to dark purple on the medium. Lactose non-fermenters typically retain their normal color or take on the coloration of the medium. 4

differential test

, bacteria have been differentiated and characterized based on their enormous biochemical and physiological diversity

redox

, metabolic free energy changes are associated with 。xidation-reducti。n, or"red。x吁eactions. Reduction occurs when a molecule (called an 。1xidant) gains one or more electrons, which requires oxidation of another molecule (called a reductant) to provide those electrons. Because electrons possess potential energy, a redox reaction results in an energy transfer.

psychrotrophs

0-30 C

conjugate pair

An acid/base pair that differ only by one H+. Acids turn into conjugate bases, bases turn into conjugate acids.

fluid thyoglycollate broth (aerotolerance)

As such, this medium can support growth of a broad variety of aerobic and anaerobic, fastidious and nonfastidious organisms. yeast and pancreatic digest of caesin=nutrients sodium thioglycollate and L-cystine=reduce O2 and H2O resazurin=O2 indicator (pink oxidized, colorless when reduced) aneorobic and microaerophillic=primary use

Oxidase

Consider the fate of a glucose molecule in a r臼piring cell 1. If it is destined to be used for ATP production, it will become oxidized to 6 C02 with the concurrent reduction of the coenzymes 10 NAD+ to 10 NADH + H + and 2 FAD to 2 FADH2. In addition, 4 ATPs are made by substrate phosphorylation. These occur in reactions throughout glycolysis (Fig. A.1 ), the transition step, and the citric acid cycle (Fig. A.4). As you can see, reduced coenzymes have the potential to accumulate rapidly. Therefore, in order to continue oxidizing glucose, these coenzymes must be converted back to their oxidized state. This is the job of the electron transport chain (Fig. 5.18). Many aerobes, microaerophiles, facultative anaerobes, and even some anaerobes have ETCs. One function of the ETC is to pass electrons down a chain of membranebound molecules with increasingly positive reduction potentials (Fig. 5 .1) to the terminal electron acceptor (e.g., 1h0 2, N03 - , 5042-). A second function of some carriers in the ETC is to act as proton pumps, using the energy lost by the electrons to create a proton gradient across the cytoplasmic membrane that is higher on the outside. Because of their charge, protons are incapable of diffusing back into the cell through the phospholipid bilayer of the membrane. However, the cytoplasmic membrane has channels through which protons can diffuse, and the kinetic energy of their diffusion is coupled to oxidative phosphorylation-ATP synthesis-catalyzed by membrane-bound ATPases. So, the energy possessed by electrons stripped away from glucose ultimately are used in ATP synthesis. There are many different types of electron transport chains, but all share the characteristics listed above. Some organisms use more than one type of ETC, depending on the availability of oxygen or other 且nal electron acceptor(s). Escherichia coli, for example, has two ETCs for respiring aerobically and at least one for respiring anaerobically (using nitrate as the FEA). Many bacteria have ETCs resembling mitochondrial ETCs in eukaryotes. These chains contain a series of four large enzymes broadly named Complexes I, II，田， and IV, each of which contains several molecules jointly able to transfer electrons 1 As you have seen in previous labs, glucose is not con1pletely oxidized to 6 C02 in fermenting cells. and use the free energy released in the reactions. The last enzyme in the chain, Complex IV, is called cytochrome c oxidase because it makes the final electron transfer of the chain from cytochrome c, residing in the periplasm or attached to the periplasmic side of the cytoplasmic membrane of Gram-negative cells, to oxygen inside the cell. In Gram-positive cells, it is membrane-bound. The oxidase test is designed to identify the presence of cytochrome c oxidase. It is able to do this because cytochrome c oxidase has the unique ability to not only oxidize cytochrome c, but to catalyze the reduction of cytochrome c by a chromogenic reducing agent called tetramethyl-p-phenylenediamine. Chromogenic reducing agents are chemicals that develop color as they become oxidized (Fig. 5 .19). In the oxidase test, the reducing reagent is added directly to bacterial growth on solid media (Fig. 5.20A), or more conveniently, a bacterial colony is transferred to paper and reagent is added (Fig. 5.20B), or even more conveniently, bacteria are added to paper already containing the reagent (5.20C). A color change occurs within seconds if the reducing agent becomes oxidized, thus indicating that cytochrome c oxidase is present. Lack of color change within the allotted time means that cytochrome c oxidase is not present and signifies a negative result

Urea

Decarboxylation of certain amino acids produces urea, which is the primary nitrogenous waste in the urine of many land animals (including mammals). It can be hydrolyzed to ammonia and carbon dioxide by bacteria possessing the enzyme urease (Fig. 5 .49). Urea hydrolysis provides nitrogen in a usable form (ammonia) and it also acts as a virulence factor for some pathogens ( e.g., Helicobacter pyloη） by counteracting acid in the environment. Streptococcus salivarius, an oral commensal, also produces urease. Many enteric bacteria possess the ability to metabolize urea, and the urease genes are activated when a usable nitrogen source is absent or urea is present. The original urease test used urea aga鸟 which was formulated to differentiate enteric organisms capable of rapid urea hydrolysis ("rapid urease-positive bacteria," such as Proteus, Morganella morganii, and some Providencia stuartii strains) from slower urease-positive and ureasenegative bacteria. A pH indicator was used to track the rising pH due to accumulation of ammonia. Rapid urease-positive organisms produce a positive result within a day; slower urease-positive organisms may take several days to produce a readable result. In this exercise you will be using urea broth, which differs from urea agar in two important ways. First, its only nutrient source other than urea is a trace (0.0001 o/o) of yeast extract. Second, it contains buffers strong enough to inhibit alkalinization of the medium by all but the rapid urease-positive organisms mentioned previousl予 Phenol red, which is yellow or orange below pH 8.4 and red or pink above, is included to expose any increase in pH. Pink color in the medium in less than 24 hours indicates a rapid urease-positive organism. Orange or yellow is negative (Fig. 5.50 and Table 5-17).

application

EMB agar (Levine) is used for the isolation of fecal coliforms. It can be streaked for isolation or used in the Membrane Filter Technique

Selective Media for Isolation of Gram-Negative Rods pg 247

Enterobacteriaceae- the enteric gut"bacteria Depending on the circumstances, some organisms in a mixed sample are contaminants and relatively benign, whereas others are potentially harmful and must be isolated and identi币ed.The media in this part of Section 4 are designed to isolate and differentiate these organisms from each other and to discourage growth of other organisms that are not currently of interest to the microbiologist

Gelatin

Gelatin is a protein derived from collagen-a component of vertebrate connective tissue. Gelatinases comprise a family of extracellular enzymes produced and secreted by some microorganisms to hydrolyze gelatin. Subsequent!民 the cell can absorb individual amino acids and use them for metabolic purposes. Bacterial hydrolysis of gelatin occurs in two sequential reactions, as shown in Figure 5 .46. The presence of gelatinases can be detected using nutrient gelatin, a simple test medium composed of gelatin, peptone, and beef extract. Nutrient gelatin differs from most other solid media in that the solidifying agent (gelatin) is also the substrate for enzymatic activity. Consequently, when a tube of nutrient gelatin is stab inoculated with a gelatinase-positive organism, secreted gelatinase (or gelatinases) will liquefy the medium. Gelatinase-negative organisms do not secrete the enzyme and do not liquefy the medium (Figs. 5.47 and 5.48). A 7-day incubation period is usually sufficient to see liquefaction of the medium. However, gelatinase activity is very slow in some organisms. All tubes still negative after 7 days should be incubated an additional 7 days. A slight disadvantage of nutrient gelatin is that it melts at 28。C (82。F). Therefore, inoculated stabs are typically incubated at 25。C along with an uninoculated control to verify that any liquefaction is not temperature related.

pg 269

Heterotrophic bacteria obtain their energy by performing either respirati。n or fermentati。n using ingested organic molecules. Both catabolic systems oxidize the organic molecules and transfer their chemical energy into high-energy bonds in aden。sine triph。sphate (ATP). They di忏er principally in what而nal electr。n accept。r (FEA), which acts as an oxidant, is used; the presence or absence of an electron transport chain (ETC); and in their ATP yield. Fermentations are characterized by the absence of an ETC and use an organic compound as the FEA, whereas respirations use an inorganic compound and possess an ETC. Oxygen serves as the FEA in aerobic respirati。n, whereas another inorganic compound (e.g., N03 or 504) acts in that role in anaerobic respiration. Fermentation pathways are many and varied, but all use the process of substrate-level ph。sphorylati。n to make ATP, in which the energy for adding the phosphate comes directly from breaking a bond in a biochemical. Respiration accomplishes ATP

5. Indicators

Indicators make a desired or expected reaction visible. An indicator is virtually always included in differential media because bacterial growth, by itself, is usually not enough to reliably differentiate between microbial groups. The medium must change fairly dramatically for us to be able to see it. Subtle changes can lead to false readings. The indicator frequently is a dye that changes color as pH changes. All pH dyes have a speci币c functional range and are chosen based on the starting pH of the medium, which in turn is based on the optimum pH of the organisms being tested. Indicators can also be chemicals that react with products of a reaction and produce a color change. For example, if the organism being tested reduces the medium substrate, an indicator that becomes oxidized can produce a pretty spectacular change. Oxidized iron is a common reactant used to form a visible precipitate upon its reduction.

3. Inhibitors

Inhibitors make the medium selective. They are designed to exploit weaknesses in spec的c groups of organisms and thus prevent or inhibit their growth, while allowing other organisms to grow. Some inhibitors function by interrupting DNA synthesis or expression of a gene. Other inhibitors function at the enzymatic level, interfering with a critical reaction. Still other inhibitors interfere with membrane permeability, thus upsetting homeostasis and starting a cascade of catastrophic changes inside the cell. Sodium chloride can be added to inhibit organisms that can't tolerate high osmotic pressures. The precise "menu"of nutritional components can enhance selectivity by excluding certain ingredients required by undesired organisms in the sample (In the absence of an inhibitor, this strategy would make it an enrichment medium.)

Capnophiles

Microbes that grow better at high CO2 concentrations

2. Nutritional Components

Nutritional components are selected to obtain the optimum growth of the organisms being tested for or suspected of being in the sample. Although many ingredients are suitable for many different types of microorganisms, typically slight variations are made to tailor the medium for a specified group. For instance, yeast extract can be added for fastidious organisms requiring specific vitamins.

application

PR broth is used to differentiate members of Enterobacteriaceae and to distinguish them from other Gram-negative rods. It also is used to distinguish between Gram-positive fermenters, such as Streptococcus and Lactobacillus species. •

Anaerobic

Process that does not require oxygen

carboxylic acid

R-COOH

Sim

SIM medium is used for determination of three bacterial activities：主ulfur reduction, indole production from tryptophan, and 旦otility (from which the acronym " SIM" is derived). The semisolid medium includes casein and animal tissue as sources of amino acids, an ironcontaining compound, and sulfur in the form of sodium thiosulfate. Sulfur reduction to H2S can be accomplished by bacteria in two different ways, depending on the enzymes present. 1. The enzyme cysteine desulfurase catalyzes the hydrolysis of the sulfur-containing amino acid cysteine to pyruvate and H2S during putrefaction (Fig. 5 .5 6). 2. In a totally unrelated way, the enzyme thiosulfate reductase catalyzes the reduction of sulfur (in the form of sulfate) to H2S at the end of an anaerobic respiratory electron transport chain (Fig. 5 .57). Both systems produce hydrogen sulfide (H2S) gas. When either reaction occurs in SIM medium, the H2S produced combines with iron, in the form of ferrous ammonium sulfate, to form ferric sulfide (FeS ), a black precipitate (Fig. 5 .58). Any blackening of the medium is an indication of sulfur reduction and a positive test. No blackening of the medium indicates no sulfur reduction and a negative reaction (Fig. 5.59). Indole production in the medium is made possible by the presence of the amino acid tryptophan ( contained in casein and animal protein). Bacteria possessing the enzyme tryptophanase can hydrolyze tryptophan to pyru飞rate, ammonia (by deamination), and indole (Fig. 5.60). Tryptophan hydrolysis in SIM medium can be detected by the addition of Kovac's reagent after the incubation period. Kovac's reagent contains p-dimethylaminobenzaldehyde (D MABA) and HCl dissolved in amyl alcohol. When a few drops of Kovac's reagent are added to the tube, it forms a layer over the agar. DMABA reacts with any indole present and produces a quinoidal compound that turns the reagent layer red (Figs. 5.61 and 5.62). The formation of red color in the reagent layer indicates a positive reaction and the presence of tryptophanase. No red color is indole negative. 5 Determination of motility in SIM medium is made possible by the reduced agar concentration (0.35o/o vs. 1.5 % in nutrient agar) and the method of inoculation. The medium is inoculated with a single stab from an inoculating needle. Motile organisms are able to move about in the semisolid medium and can be detected by the radiating growth extending outward in all directions from the central stab line. Growth that radiates in all directions and appears slightly fuzzy is an indication of motility (Fig. 5 .63 ). This should not be confused with the (seemingly) spreading growth produced by lateral movement of the inoculating needle when stabbing. 5Determination of motility in SIM medium is made possible by the reduced agar concentration (0.35o/o vs. 1.5 % in nutrient agar) and the method of inoculation. The medium is inoculated with a single stab from an inoculating needle. Motile organisms are able to move about in the semisolid medium and can be detected by the radiating growth extending outward in all directions from the central stab line. Growth that radiates in all directions and appears slightly fuzzy is an indication of motility (Fig. 5 .63 ). This should not be confused with the (seemingly) spreading growth produced by lateral movement of the inoculating needle when stabbing. 5

application

SIM medium is used to identify bacteria that are capable of producing indole using the enzyme tryptophanase. The indole test is one component of the IMViC battery of tests (Jndole, Methyl red, Voges-Proskauer, and Citrate) used to differentiate the Enterobacteriaceae, especially E. coli-positive, from Enterobacteγ， Klebsiella, Hafn饵， and Serratia-negative. SIM medium also is used to differentiate sulfur-reducing members of Enterobacteriaceae, especially members of the genera Salmonella, Shigella, and Proteus, from the negative Morganella morganii and Providencia rettgeri. In addition to the first two functions of SIM, motility is an important differential characteristic of Eηteγobacteγiaceae.

Staphylococci on MSA

Staphylococci thrive on the medium, largely because of their adaptation to salty habitats such as human skin. Phenol red indicates whether fermentation with an acid end-product has taken place by changing color as the pH changes. Most staphylococci are able to grow on MSA, but do not ferment mannitol, so their growth appears pink or red and the medium remains unchanged. Staphylococcus aureus ferments mannitol, which produces acids and lowers the pH of the medium (Fig. 4.3). The result is formation of bright yellow colonies usually surrounded by a yellow halo

4. Substrates

Substrates are almost always what make the medium differential. Differentiation and identi币cation of organisms frequently relies on their differing abilities to perform a speci币c chemical reaction or set of reactions and to do them in a way that can be observed. This can all be staged in an art的cial environment (differential medium) by providing the organisms all the required components for growth and by including at least one substrate that only one organism or group of organisms can utilize. Once the reaction has taken place, presumabl弘 organisms that before the test were indistinguishable are now (with the help of indicators-see #5) easily differentiated. For example, if a fecal sample is being tested for the presence of a pathogen known not to ferment lactose, then lactose is an important substrate needed to identify and isolate f. coli and other coliforms, all of which ferment lactose and all of which are likely to be found in the sample. Colonies of lactose non-fermenters will be visibly different and would be grown in pure culture for further testing.

application

The citrate utilization test is used to determine the ability of an organism to use citrate as its sole source of carbon. Citrate utilization is one part of a test series referred to as the IMViC (Jndole, Methyl Red, Voges-Proskauer and Citrate tests) that distinguishes between members of the family Enterobacteriaceae and also from other Gramnegative rods. •

citrate

The citrate utilization test was designed to differentiate members of the Enterobacteriaceae, all of which are facultative anaerobes. That is, they have the ability to ferment carbohydrates and they also have the ability to aerobically respire, which means they have a functional citric acid cycle. However, the citrate utilization test does not tell us about the citric acid cycle. Instead, it tells about the ability of organisms to use citrate as their sole carbon source and perform citrate fermentation. Simmons citrate agar is a defined medium. That is, the amount and source of all ingredients are carefully controlled. Because sodium citrate is the only carbon source in the medium1 it will not support a complex, high-energy yielding respiratory process like the citric acid cycle. It does, however, provide the means for bacterial species that possess the enzyme citrate permease (an oxaloacetate decarboxylase Na+ pump) to transport citrate ( actua酌， oxaloacetate-see below) into the cell and perform citrate fermentation. These organisms must also be able to survive with ammonium (in the form of ammonium phosphate) as the sole nitrogen source. Bacteria that do not possess citrate permease will not grow on this medium. Citrate-positive bacteria hydrolyze citrate into oxaloacetate and acetate using the enzyme citrate lyase (Fig. 5 .25). From there, oxaloacetate is decarboxylated to pyruvate, simultaneously using the energy released to pump oxaloacetate/pyru飞rate into the cell. A variety of products can be formed from pyruvate depending on the cell's pH. Here is the secret: some bacterial fermentation pathways can make ATP and reducing power (NADH or NADPH). For instance, Klebsiella and Enterobacter, both of which are citrate positive, have the ability to make acetyl-CoA from pyruvate (nothing new) but then have the ability to convert it to acetyl phosphate, which can then phosphorylate ADP to AT卫 In addition, the same bacteria are capable of using H2 as a source of electrons to reduce NAD and/or NADP, the latter of which is used in synthesis reactions. Both of these are shown in Figure 5.25. Bacteria that survive in the medium and utilize the citrate also convert the ammonium phosphate to ammonia (NH3) and ammonium hydroxide (NH40H), both of which tend to alkalinize the agar. Bromothymol blue dye, which is green at pH 6.9 and blue at pH 7.6, is an indicator, and as the pH goes up the medium changes from green to blue (Fig. 5.26). Thus, conversion of the medium to blue is a positive citrate test result (Table 5-9). Occasionally a citrate-positive organism will grow on a Simmons citrate slant without producing a change in color. In most cases, this is because of incomplete incubation. In the absence of color change, growth on the slant indicates that citrate is being utilized and is evidence of a positive reaction. To avoid confusion between actual growth and a heavy inoculum, which may be misinterpreted as growth, citrate slants typically are inoculated lightly with an inoculating needle rather than a loop.

Catalase test

The electron transport chains (ETC) of aerobic and facultatively anaerobic bacteria are composed of molecules capable of accepting and donating electrons as conditions dictate. As such, these molecules alternate between their oxidized and reduced forms, passing electrons down the chain to the 且nal electron accepto鸟 02. Energy lost by electrons in this sequential transfer is used to perform oxidative phosphorylation (i.e., phosphorylate ADP to ATP). In most cases, electrons in the aerobic ETC follow the step飞;vise path to oxygen, but other paths can be followed and these result in production of toxic forms of reduced oxygen 1. For instance, one ETC carrier molecule called flavoprotein can bypass the next carrier in the chain and transfer electrons directly to oxygen (Fig. 5.13), which produces hydrogen peroxide (H20 2), a highly potent cellular toxin. Reduced flavin adenine dinucleotide (FADH2) is capable of the same reaction Even if the electrons follow the complete ETC another toxin, the superoxide radical (02-), can be produced in the 且nal step because electrons reduce oxygen one at a time and sometimes it is released before it is completely reduced to H20 (Fig. 5.13). Hydrogen peroxide and the superoxide radical are toxic because they oxidize biochemicals and make them nonfunctional. However, organisms that produce them also produce enzymes capable of breaking them down. Superoxide dismutase catalyzes conversion of superoxide radicals (the more lethal of the two compounds) to hydrogen peroxide (Fig. 5 .13). Catalase converts hydrogen peroxide into water and gaseous oxygen (Fig. 5.14). In large part (though exceptions exist), the ability to synthesize these protective enzymes accounts for an organism's ability to live in the presence of oxygen (Table 5-5). Bacteria that produce catalase can be detected easily using typical store-grade hydrogen peroxide. When hydrogen peroxide is added to a catalase-positive culture, oxygen gas bubbles form immediately. If no bubbles appea乌 the organism is catalase-negative. This test can be performed on a microscope slide (Fig. 5 .15) or by adding hydrogen peroxide directly to the bacterial growth

6. Positive or Negative?

The last item covered is a description of the positive and negative reactions. We tell you what to look for

application

The methyl red and Voges-Proskauer tests are components of the IMViC battery of tests (Jndole, Methyl red, VogesProskauer, and Citrate) used to distinguish between members of the family Enterobacteriaceae and differentiate them from other Gram-negative rods. Species in the genera Escherichia, Shigella, and Salmonella are MR positive, whereas Enterobacter, Serratia, and Erwinia species are VP positive. For more information about these fermentations, refer to Figure A.6 on page 597

free energy

The potential energy of the bonds within a biochemical

application

This test is used to identify bacteria containing the respiratory enzyme cytochrome c oxidase. Among its many uses is the presumptive identification of the oxidasepositive Neisseria and Moraxella. It also can be useful in differentiating the oxidase-negative Enterobacteriaceae from the oxidase-positive Pseudomonadaceae. SECTION

application gelatin

This gelatin hydrolysis test is used to determine the ability of a microbe to produce gelatinases. Staphylococcus aureus, which is gelatinase-positive, can be differentiated from S. epidermidis. Serratia and Proteus species are gelatinase-positive members of Enterobacteriaceae, whereas most others in the family are negative. Bacillus anthracis, B. cereus, and several other Bacillus species are gelatinase-positive, as are Clostridium tetani and C. per斤ingens.

application

This test is used to differentiate organisms based on their ability to hydrolyze urea with the enzyme urease. Urinary tract pathogens from the genus Proteus may be distinguished from other enteric bacteria by their rapid urease activity. It is also used in identifying H. pylori, which is associated with gastric and duodenal ulcers, as well as stomach cancer. •

Catalyse test

This test is used to identify organisms that produce the enzyme catalase. It is frequently used to differentiate catalase-positive Micrococcus and Staphylococcus from the catalase-negative Streptococcus, Enterococcus, and Lactococcus. Variations on this test also may be used in identification of Mycobacterium species. •

catabolic

a decrease in the reactant molecule's complexity

Aerotolerance

ability or inability to live in the presence of oxygen

thermophiles

above 40C

PH indicator

acidophiles: organisms adapted to grow well in environments below about pH 5 .5 2. neutrophiles: organisms that prefer pH levels between 5 .5 and 8 .5 3. alkaliphiles: organisms that live above pH 8 .5.

psychrophiles

below 20C

anabolic

building up

falcultative anaerobes

can grow with or without oxygen

disinfectants

chemical agents used on inanimate objects to lower the level of microbes on their surfaces

anitmicrobial agents

chemicals that destroy or suppress the growth of infectious organisms

antiseptics

chemicals used on living tissue to decrease the number of microbes

Phenol Red Fermentation Broth

differential fermentation medium composed of standard ingredients (the " base broth") to which a single carbohydrate is added. After inoculation and incubation, an organism's ability to ferment that particular carbohydrate can be determined, as can the end products of its fermentation. Because virtually any carbohydrate can be added to the base, PR broth is a versatile medium. Figure 5.4 shows fermentation pathways for glucose, lactose, and sucrose. Included in the base medium are peptone and the pH indicator phenol red. Phenol red is yellow below pH 6.8, pink to magenta above pH 7.4, and red in between. During preparation the pH is adjusted to approximately 7.3 so it appears red. Finally, an inverted Durham tube is added to each tube as an indicator of gas production. Acid production from fermentation of the carbohydrate lowers the pH below the neutral range of the indicator and turns the medium yellow (Fig. 5.5). Gas production, also from fermentation, is indicated by a bubble, or pocket, in the Durham tube where the broth has been displaced. Deamination of amino acids supplied by casein (milk protein) results in an alkaline reaction from the ammonia (NH3) that is produced. Ammonia raises the pH and turns the broth pink if no acid is produced or if acid production has ceased due to consumption of the carbohydrate

UVC rays

is related to time of exposure, lamp intensity, and distance to the target UV-C as a germicidal agent has its limited applications. UV-C, wavelengths ranging from 100 nm to 280 nm. (These wavelengths-more specifically, 240 nm-280 nm-are most detrimental to bacteria. Bacterial exposure to UV-C for more than a few minutes usually results in irreparable DNA damage and death of the organism.

extreme thermophiles

live in very hot environments 65-110C

differential media

many types of selective media contain indicators to expose differences between organisms,

entropy

measure of disorder

obligate anaerobes

organisms that cannot live where molecular oxygen is present

Aerobic

requires oxygen

bacterialcidal agents

result in bacterial death

MacConkey Agar

selective and differential medium containing lactose, bile salts, neutral red, and crystal violet. Bile salts and crystal violet= inhibit growth of Gram-positive bacteria. Neutral red dye= pH indicator that is colorless above a pH of 6. 8 and red at a pH less than 6.8. Organisms that ferment lactose to acid end-products lower the pH (Fig. 4.5) and their colonies turn a pink to red color. On occasion, a pink to red bile precipitate will also form in the agar due to the lowered pH. Lactose nonfermenters retain their normal color or the color of the medium Formulations without crystal violet allow growth of Enterococcus and some species of Staphylococcus, which ferment the lactose and appear pink on the medium.

1. Selective, Differential, Defined, or Undefined-Which Is It?

selective, it encourages growth of some organisms and discourages (inhibits) growth of others differential, it allows us to distinguish between different microbes defined, or"chemically defined，each of its ingredients is known and in exactly what amounts undefined, or "complex;' it contains one or more ingredient(s) of unknown composition and/or amount, such as yeast extract, beef extract, digest of gelatin, etc.

American official analytical chemists use-dilution test

standard method for measuring the effectiveness of a chemical agent

Deamination

the removal of an amino group from an organism, particularly from an amino acid

facultative thermophiles

thermophiles that will grow below 40 degrees

aerotolerant anaerobes

tolerate but cannot use oxygen

coliform bacteria

used to find in selective and differential Coliforms= Enterobacteriaceae (Escherichia coli being the most prominent member) that produce acid and gas from lactose fermentation. coliforms=inhabitants of the human intestinal tract fecal contamination.

diagnostic test

used to identify unknown bacteria in a patient or environmental sample by matching the unknown's test results to the characterization of " known" bacteria that fits bes

obligate thermophiles

will not grow below 40 degrees C

metabolism

ζells are chemical entities and the chemical reactions they perform