Unit Nine Biochemical Tests PDF

UNIT NINE BIOCHEMICAL biochemicalTEST test BIOCHEMICAL TEST Ammonia production results in an In the microbiology laboratory, alkal...

UNIT NINE BIOCHEMICAL biochemicalTEST test BIOCHEMICAL TEST Ammonia production results in an In the microbiology laboratory, alkaline pH, causing the medium to biochemical and identification testing change color from green to royal blue. plays an important role in accurately Result identifying bacteria. Positive: BLUE COLOR Biochemical tests are based on the Negative: No color change metabolic processes that occur in microorganisms along with enzymatic Quality Control activities that allow for rapid Positive: Pseudomonas aeruginosa identification of microorganisms. Negative: Escherichia coli Each biochemical test is viewed directly when adding one or more reagents to the microorganism. Acetate Utilization Individual bacterial species have a Purpose unique set of biochemical reactions Differentiate that distinguish the microorganism from organisms based on other species. ability to use acetate Sometimes even a single result from as the sole source of one test can identify a bacterial genus carbon. Generally and species. used to differentiate Shigella sp. from Biochemical tests are comprised of three Escherichia coli. methods: spot, rapid, and conventional procedures. Principle Spot procedures provide results within This test is used to differentiate an seconds to minutes. organism capable of using acetate as Rapid test procedures take longer than the sole source of carbon. Organisms spot tests, but not as long as capable of using sodium acetate grow conventional tests. Results can be on the medium, resulting in an alkaline observed within a few minutes to a few pH, turning the indicator from green to hours. blue. Conventional methods are incubated at least 18 to 24 hours before results can Result be interpreted but can take up to 72 Positive: Medium becomes alkalinized = BLUE hours. Negative: No growth or no indicator change to blue Acetamide Utilization Purpose Quality Control Differentiate Positive: Escherichia coli microorganisms based Negative: Shigella sonnei on the ability to use acetamide as the sole Bacitracin Susceptibility /TAXO A source of carbon. Purpose Principle This test is used Bacteria capable of growth on this for presumptive medium produce the enzyme identification acylamidase, which deaminates and acetamide to release ammonia. differentiation of beta-hemolytic  UNIT NINE BIOCHEMICAL biochemicalTEST test group A streptococci (Streptococcus inhibited by the bile salts in this pyogenes–susceptible) from other beta- medium. hemolytic streptococci. It is also used Organisms capable of growth in the to distinguish staphylococci species presence of 4% bile and able to (resistant) from micrococci hydrolyze esculin to esculetin. (susceptible). Esculetin reacts with Fe3+ and forms a dark brown to black precipitate. Principle Result The antibiotic bacitracin inhibits the Positive: Growth and blackening of the agar synthesis of bacterial cell walls. slant A disk (TaxoA) impregnated with a small Negative: Growth and no blackening of amount of bacitracin (0.04 units) is medium placed on an agar plate, allowing the antibiotic to diffuse into the medium Quality Control and inhibit the growth of susceptible Positive: Enterococcus faecalis organisms. Negative: Escherichia coli After incubation, the inoculated plates are examined for zones of inhibition Bile Solubility Test surrounding the disks. Purpose This test Result differentiates Positive: zone of inhibition >10 mm Streptococcus Negative: No zone of inhibition pneumoniae (positive– Quality Control soluble) from alpha-hemolytic Positive: Streptococcus pyogenes, streptococci (negative– insoluble). Micrococcus luteus Negative: Streptococcus agalactiae, Principle Staphylococcus aureus Bile or a solution of a bile salt (e.g., sodium desoxycholate) rapidly lyses Bile Esculin Test pneumococcal colonies. Purpose Lysis depends on the presence of an This test is used for intracellular autolytic enzyme, amidase. the presumptive Bile salts lower the surface tension identification of between the bacterial cell membrane enterococci and and the medium, thus accelerating the organisms in the organism’s natural autolytic process. Streptococcus bovis group. Result The test differentiates enterococci and Positive: Colony disintegrate group D streptococci from non–group D Negative: Intact colonies viridans streptococci. Quality Control Principle Positive: Streptococcus pneumonia The bile esculin test identifies if a Negative: Enterococcus faecalis microorganism can hydrolyze esculin in the presence of bile. Gram-positive bacteria other than some streptococci and enterococci are  UNIT NINE BIOCHEMICAL biochemicalTEST test Butyrate Disk extracellular hemolytic protein (CAMP Purpose factor) that acts synergistically with the This is a rapid beta-lysin of Staphylococcus aureus to test to detect cause enhanced lysis of red blood cells. the enzyme The group B streptococci are streaked butyrate perpendicular to a streak of S. aureus esterase, to aid identification of on sheep blood agar. Moraxella (Branhamella) catarrhalis. A positive reaction appears as an arrowhead zone of hemolysis adjacent Principle to the place where the two streak lines Organisms capable of producing come into proximity. butyrate esterase hydrolyze bromochlorindolyl butyrate. Result Hydrolysis of the substrate in the Positive: Arrowhead-shaped zone of beta- presence of butyrate esterase releases hemolysis indoxyl, which in the presence of oxygen Negative: No enhancement of hemolysis spontaneously forms indigo, a blue to blue-violet color. Quality Control Result Positive: Streptococcus agalactiae Positive: Development of a BLUE COLOR (5- Negative: Streptococcus pyogenes minute incubation Negative: No color change CAMP Positive Organisms: 1. Streptococcus Quality Control agalactiae Positive: Moraxella catarrhalis 2. Listeria Negative: Neisseria gonorrhoeae monocytogenes 3. Proprionibacterium Christie, Atkins, and Munch-Peterson (CAMP) acnes Reverse CAMP Positive Organisms: Test 1. Clostridium Purpose perfringens The Christie, 2. Corynebacterium Atkins, and pseudotuberculosis Munch-Peterson 3. Corynebacterium (CAMP) test is ulcerans used to 4. Corynebacterium urealyticum differentiate group B streptococci (Streptococcus agalactiae–positive) from other streptococcal species. Catalase Test Listeria monocytogenes also produces Purpose a positive CAMP reaction. This test differentiates Principle catalase- The CAMP factor test detects a positive micrococcal and diffusible protein that acts staphylococcal species from catalase- synergistically with beta-lysin produced negative streptococcal species. by Staphylococcus aureus to produce The catalase test detects the presence an increased zone of hemolysis. of catalase enzymes, which hydrolyzes Certain organisms (including group B hydrogen peroxide into water and streptococci) produce a diffusible gaseous oxygen (bubbles form).  UNIT NINE BIOCHEMICAL biochemicalTEST test Principle Result Aerobic and facultative anaerobic Positive: Growth, variation in color of colonies organisms produce two toxins during Negative: No growth normal metabolism, hydrogen peroxide (H2O2) and superoxide radical (O2−). Quality Control These bacteria have two enzymes that Positive: Pseudomonas aeruginosa detoxify the products of normal Negative: Escherichia coli metabolism. One of these enzymes, catalase, is Citrate Utilization capable of converting hydrogen Purpose peroxide to water and oxygen. The purpose of this The presence of the enzyme in a test is to identify bacterial isolate is evidenced when a organisms small inoculum introduced into capable of using hydrogen peroxide (30% for the slide sodium citrate as test) causes rapid elaboration of oxygen the sole carbon bubbles. source and The lack of catalase is evident by a lack inorganic ammonium salts as the sole of or weak bubble production. nitrogen source. The test is part of a series referred to as Result IMViC (indole, methyl red, Voges- Positive: Copious bubbles are produced Proskauer, and citrate), which is used to Negative: No or few bubbles are produced differentiate Enterobacteriaceae from other gram-negative rods. Quality Control Positive: Staphylococcus aureus Principle Negative: Streptococcus pyogenes Bacteria that can grow on this medium produce an enzyme, citrate-permease, Cetrinamide Utilization capable of converting citrate to Purpose pyruvate. This test is Pyruvate can then enter the organism’s primarily used to metabolic cycle for the production of isolate and energy. B purify acteria capable of growth in this Pseudomonas medium use the citrate and convert aeruginosa from contaminated ammonium phosphate to ammonia and specimens. ammonium hydroxide, creating an alkaline pH. Principle The pH change turns the bromthymol The test is used to determine the ability blue indicator from green to blue. of an organism to grow in the presence of cetrimide, a toxic substance that Result inhibits the growth of many bacteria by Positive: Growth on the medium = BLUE causing the release of nitrogen and Negative: Absence of growth phosphorous, which slows or kills the organism. P. aeruginosa is resistant to Quality Control cetrimide. Positive: Enterobacter aerogenes Negative: Escherichia coli  UNIT NINE BIOCHEMICAL biochemicalTEST test Coagulase Test Decarboxylase Tests (Moeller’s Method) Purpose Purpose The test is used to differentiate This test is used Staphylococcus aureus (positive) from to differentiate coagulase-negative staphylococci decarboxylase (negative). producing The coagulase test is performed to Enterobacteriaceae from other gram- identify if a microorganism produces negative rods. coagulase. Principle Principle This test measures the enzymatic ability S. aureus produces two forms of (decarboxylase) of an organism to coagulase, bound and free. Bound decarboxylate (or hydrolyze) an amino coagulase, or “clumping factor,” is acid to form an amine. bound to the bacterial cell wall and Decarboxylation, or hydrolysis, of the reacts directly with fibrinogen. amino acid results in an alkaline pH and This results in the precipitation of a color change from orange to purple. fibrinogen on the staphylococcal cell, pH indicator: BROMCRESOL PURPLE causing the cells to clump when a bacterial suspension is mixed with Result plasma. Positive: Alkaline (PURPLE) color change The presence of bound coagulase Negative: No color change or acid (yellow) correlates with free coagulase, an extracellular protein enzyme that Quality Control causes the formation of a clot when S. Positive: aureus colonies are incubated with Lysine— Klebsiella pneumoniae plasma. Ornithine—Enterobacter aerogenes The clotting mechanism involves the Arginine—Enterobacter cloacae activation of a plasma coagulase- Negative: reacting factor (CRF), which is a Lysine—Enterobacter cloacae modified or derived thrombin molecule, Ornithine—Klebsiella pneumoniae to form a coagulase-CRF complex. Arginine—Klebsiella pneumoniae This complex in turn reacts with fibrinogen to produce the fibrin clot DNA Hydrolysis (DNase Test Agar) Purpose Result This test is used to 1.Slide Test differentiate Positive: Clumping in 10 organisms based seconds or less on the production Negative: No clumping of deoxyribonuclease. 2.Tube Test It is used to distinguish Serratia sp. Positive: Clot of any size (positive) from Enterobacter sp., Negative: No clot Staphylococcus aureus (positive) from other species, and Moraxella catarrhalis Quality Control (positive) from Neisseria sp. Positive: Staphylococcus aureus Negative: Staphylococcus epidermidis  UNIT NINE BIOCHEMICAL biochemicalTEST test Principle Andrade’s formula is used to The test is used to determine the ability differentiate enteric bacteria from of an organism to hydrolyze DNA. coryneforms, and bromocresol purple is The medium is pale green because of used to distinguish enterococci from the DNA–methyl green complex. streptococci. If the organism growing on the medium hydrolyses DNA, the green color fades Principle and the colony is surrounded by a Carbohydrate fermentation is the colorless zone. process microorganisms use to produce energy. Result: Most microorganisms convert glucose Positive: COLORLESS around the test organism to pyruvate during glycolysis; however, Negative: Medium remains green some organisms use alternate pathways. Quality Control A fermentation medium consists of a Positive: Staphylococcus aureus basal medium containing a single Negative: Escherichia coli carbohydrate (glucose, lactose, or sucrose) for fermentation. Esculin Hydrolysis However, the medium may contain Purpose various color indicators, such as This test is used for the Andrade’s indicator, bromocresol, or presumptive others. identification and In addition to a color indicator to detect differentiation of the production of acid from Enterobacteriaceae. fermentation, a Durham tube is placed in each tube to capture gas produced by Principle metabolism. This test is used to determine whether an organism can hydrolyze the ANDRADE’S FORMULA glycoside esculin. differentiate enteric bacteria from Esculin is hydrolyzed to esculetin, coryneforms which reacts with Fe3+ and forms a dark Result brown to black precipitate. Positive: Indicator change to PINK Result Negative: Growth, but no Positive: BLACKENED MEDIUM change in color Negative: No blackening Quality Control Quality Control Positive, with gas: Escherichia coli Positive: Enterococcus faecalis Positive, no gas: Shigella flexneri Negative: Escherichia coli BROMOCRESOL PURPLE Fermentation Media distinguish enterococci Purpose from streptococci Fermentation media are used to Result differentiate organisms based on their Positive: Indicator change to ability to ferment carbohydrates YELLOW incorporated into the basal medium. Negative: Growth, but no change in color  UNIT NINE BIOCHEMICAL biochemicalTEST test Quality Control This test is used to determine the ability Positive, with gas: Escherichia coli of an organism to produce extracellular Negative, no gas: Moraxella osloensis proteolytic enzymes (gelatinases) that liquefy gelatin, a component of Flagella Stain (Wet Mount Technique) vertebrate connective tissue. Purpose Nutrient gelatin medium differs from This technique is traditional microbiology media in that used to visualize the solidifying agent (agar) is replaced the presence and with gelatin. arrangement of When an organism produces gelatinase, flagella for the the enzyme liquefies the growth presumptive identification of motile medium. bacterial species. Result Principle Positive: Partial or total liquefaction at 4°C Flagella are too thin to be visualized within 14 days using a bright field microscope with Negative: Complete solidification ordinary stains, such as the Gram stain, or a simple stain. Quality Control A wet mount technique is used for Positive: Bacillus subtilis staining bacterial flagella, and it is Negative: Escherichia coli simple and useful when the number and arrangement of flagella are critical to Growth at 42°C the identification of species of motile Purpose bacteria. This test is used The staining procedures require the use to differentiate a of a mordant so that the stain adheres in pyocyanogenic layers to the flagella, allowing pseudomonads visualization. from other Pseudomonas Quality Control sp. Peritrichous: Escherichia coli Polar: Pseudomonas aeruginosa Principle Negative: Klebsiella pneumonia The test is used to determine the ability of an organism to grow at 42°C. Several Gelatin Hydrolysis Pseudomonas species have been Purpose isolated in the clinical laboratory that The production are capable of growth at elevated of gelatinases temperatures. capable of hydrolyzing Result gelatin is used as Positive: Good growth at both 35°and 42°C a presumptive Negative: No growth at 42°C but good growth at test for the identification of various 35°C organisms, including Staphylococcus sp., Enterobacteriaceae, and some Quality Control gram-positive bacilli. Positive: Pseudomonas aeruginosa Negative: Pseudomonas fluorescens Principle  UNIT NINE BIOCHEMICAL biochemicalTEST test The test is used to determine an Hippurate Hydrolysis organism’s ability to hydrolyze Purpose tryptophan to form the compound Production of the indole. enzyme hippuricase Tryptophan is present in casein and is used for the animal protein. Bacteria with presumptive tryptophanase are capable of identification of a hydrolyzing tryptophan to pyruvate, variety of ammonia, and indole. microorganisms. Kovac’s reagent (dimethylamine- benzaldehyde and hydrochloride), when Principle added to the broth culture, reacts with The end products of hydrolysis of the indole, producing a red color. hippuric acid by hippuricase include An alternative method uses Ehrlich’s glycine and benzoic acid. reagent. Ehrlich’s reagent has the same Glycine is deaminated by the oxidizing chemicals as the Kovac preparation, agent ninhydrin, which is reduced but it also contains absolute ethyl during the process. alcohol, making it flammable. Ehrlich’s The end products of the ninhydrin reagent is more sensitive for detecting oxidation react to form a purple-colored small amounts of indole. product. The test medium must contain only Result hippurate because ninhydrin might Positive: PINK- TO WINE-COLORED RING react with any free amino acids present Negative: No color change in growth media or other broths. Quality Control Result A. KOVAC’S METHOD Positive: Deep purple color Positive: Escherichia coli Negative: Colorless or slightly yellow pink color Negative: Klebsiella pneumoniae Quality Control Positive: Streptococcus agalactiae B. EHRLICH’S METHOD Negative: Streptococcus pyogenes Positive: Haemophilus influenzae Indole Production Negative: Haemophilus parainfluenzae Purpose This test is used to identify organisms C.EHRLICH’S METHOD (ANAEROBIC) that produce the enzyme Positive: Porphyromonas asaccharolytica tryptophanase. Negative: Bacteroides fragilis The indole test can help distinguish Escherichia coli from Enterobacter and Leucine Aminopeptidase (LAP) Test Klebsiella. Purpose The LAP test is Principle used for the The indole test detects tryptophanase presumptive production and determines the ability of identification of a microorganism to produce indole from catalase- negative the degradation of the amino acid gram-positive cocci. tryptophan.  UNIT NINE BIOCHEMICAL biochemicalTEST test Principle Some bacteria hydrolyze casein, The LAP disk is a rapid test for the causing the milk to become straw detection of the enzyme leucine colored and resemble turbid serum. aminopeptidase. Additionally, some organisms reduce Leucinebeta- naphthylamide– litmus, in which case the medium impregnated disks serve as a substrate becomes colorless in the bottom of the for the detection of leucine tube. aminopeptidase. Quality Control After hydrolysis of the substrate by the Fermentation: Clostridium perfringens enzyme, the resulting beta- Acid: Lactobacillus acidophilus naphthylamine produces a red color Peptonization: Pseudomonas aeruginosa upon addition of cinnamaldehyde reagent. Lysine Iron Agar (LIA) Purpose Result This test is used Positive: Development of a red color within 1 to differentiate minute gram-negative Negative: No color change or development of a bacilli based on slight yellow color decarboxylation or deamination of lysine and the Quality Control formation of hydrogen sulfide (H2S). Positive: Enterococcus faecalis Negative: Aerococcus viridans Principle Lysine iron agar contains lysine, Litmus Milk Medium peptones, a small amount of glucose, Purpose ferric ammonium citrate, and sodium This test thiosulfate. differentiates The medium has an aerobic slant and microorganisms an anaerobic butt. When glucose is based on various fermented, the butt of the medium metabolic reactions in litmus milk, becomes acidic (yellow). including fermentation, reduction, clot If the organism produces lysine formation, digestion, and the formation decarboxylase, cadaverine is formed. of gas. Cadaverine neutralizes the organic Litmus milk is also used to grow lactic acids formed by glucose fermentation, acid bacteria. and the butt of the medium reverts to the alkaline state (purple). Principle If the decarboxylase is not produced, This test is used to determine an the butt remains acidic (yellow). organism’s ability to metabolize litmus If oxidative deamination of lysine milk. occurs, a compound is formed that, in Fermentation of lactose is the presence of ferric ammonium demonstrated when the litmus turns citrate and a coenzyme, flavin pink as a result of acid production. If mononucleotide, forms a burgundy sufficient acid is produced, casein in color on the slant. the milk is coagulated, solidifying the If delamination does not occur, the LIA milk. slant remains purple. Bromocresol With some organisms, the curd shrinks, purple, the pH indicator, is yellow at or and whey is formed at the surface.  UNIT NINE BIOCHEMICAL biochemicalTEST test below pH 5.2 and purple at or above pH Principle 6.8. This test is used to determine the ability Result of an organism to produce and maintain Alkaline slant/alkaline butt stable acid end products from glucose (K/K)—lysine decarboxylation fermentation, to overcome the buffering and no fermentation of capacity of the system, and to glucose determine the ability of some organisms Alkaline slant/acid butt to produce neutral end products (e.g., (K/A)—glucose fermentation 2,3-butanediol or acetoin) from glucose Red slant/acid butt (R/A)—lysine deamination fermentation. and glucose The methyl red detects mixed acid fermentation that lowers the pH of the Quality Control broth. The MR indicator is added after Alkaline slant and butt: H2S positive: incubation. Methyl red is red at pH 4.4 Citrobacter freundii and yellow at pH 6.2. A clear red is a Alkaline slant and butt: Escherichia coli positive result; yellow is a negative Alkaline slant and butt: H2S positive: result; and various shades of orange are Salmonella typhimurium negative or inconclusive. Red slant, acid butt: Proteus mirabilis The VP detects the organism’s ability to convert the acid products to acetoin LIA Interpretation Bacteria and 2,3-butanediol. reaction Organisms capable of using the VP pathway produce a smaller amount of K/K (-) Lysine Salmonella acid during glucose fermentation and Deamination therefore do not produce a color change and when the methyl red indicator is added. (+) Lysine A secondary reagent is added, alpha- Decayboxylati naphthol, followed by potassium on hydroxide (KOH); a positive test result is K/A (-) Lysine Shigella indicated by a red color complex Deamination Citrobacter and(-) Lysine Result Decayboxylati Positive: Red color, indicative of acetoin on production R/A (+) Lysine Proteus, Negative: Yellow color Deamination Providencia and(-) Lysine Morganella Quality Control Decayboxylati (PPM) MR positive/VP negative: Escherichia coli on MR negative:/VP positive: Enterobacter aerogenes Methyl Red/Voges-Proskauer (Mrvp) Tests Microdase Test (Modified Oxidase) Purpose Purpose The combination test methyl red (MR) This test is used and Voges-Proskauer (VP) differentiates to differentiate members of the Enterobacteriaceae gram-positive, family. catalase-  UNIT NINE BIOCHEMICAL biochemicalTEST test positive cocci (micrococci from MRS Broth staphylococci Purpose Principle This test is used to The microdase test is a rapid method to determine whether an differentiate Staphylococcus from organism forms gas Micrococcus spp. by detection of the during glucose enzyme oxidase. In the presence of fermentation. Some atmospheric oxygen, the oxidase Lactobacillus spp. and enzyme reacts with the oxidase reagent Leuconostoc sp. produce gas. and cytochrome C to form the colored compound, indophenol. Principle The MRS broth contains sources of Result carbon, nitrogen, and vitamins to Positive: Development of BLUE TO PURPLE- support the growth of lactobacilli and BLUE COLOR other organisms. Negative: No color change It is a selective medium that uses sodium acetate and ammonium citrate Quality Control to prevent overgrowth by contaminating Positive: Micrococcus luteus organisms. Negative: Staphylococcus aureus Growth is considered a positive result. A Durham tube may be added to Motility Testing Oxidase differentiate Lactobacillus spp. from Purpose Leuconostoc sp. These tests are used to determine Result whether an enteric Positive: Leuconostoc sp. - Growth, gas organism is motile. production indicated by a bubble in the An organism must Durham tube have flagella to be motile. Positive: Lactobacillus spp. - Growth, no gas production Principle Negative: No growth The inoculum is stabbed into the center of a semisolid agar deep. Bacterial Quality Control motility is evident by a diffuse zone of Positive: Lactobacillus lactis growth extending out from the line of Negative: Escherichia coli inoculation. Some organisms grow throughout the 4-Methylumbelliferyl-β-D- Glucuronide entire medium, whereas others show (MUG) Test small areas or nodules that grow out Purpose from the line of inoculation. This test is used to Result presumptively Positive: Spread out from the site of identify various inoculation genera of Negative: Remain at the site of inoculation Enterobacteriaceae and verotoxin- producing Escherichia coli. Quality Control Positive: Escherichia coli Negative: Staphylococcus aureus  UNIT NINE BIOCHEMICAL biochemicalTEST test Principle naphthylamine. The sulfanilic acid and The MUG (4-Methylumbelliferyl-β-D- nitrite react to form a diazonium salt. Glucuronide) test detects the presence The diazonium salt then couples with of the β-glucuronidase enzyme. E. coli the alphanaphthylamine to produce a and other Enterobacteriaceae produce red, water-soluble azo dye. the enzyme β-d-glucuronidase, which If no color change occurs, the organism hydrolyzes β-d-glucopyranosid-uronic did not reduce nitrate or reduced it derivatives to aglycons and d- further to NH3, NO, or N2O2. Zinc is glucuronic acid. added at this point; if nitrate remains, The substrate 4-methylumbelliferyl-β- the zinc will reduce the compound to d-glucuronide is impregnated into the nitrite and the reaction will turn positive, disk and is hydrolyzed by the enzyme to indicating a negative test result for yield the 4-methylumbelliferyl moiety, nitrate reduction by the organism. If no which fluoresces blue under long color change occurs after the addition wavelength ultraviolet light. However, of zinc, this indicates that the organism verotoxin producing strains of E. coli do reduced nitrate to one of the other not produce MUG, and a negative test nitrogen compounds previously result may indicate the presence of a described. clinically important strain. A Durham tube is placed in the broth for two reasons: (1) to detect deterioration Result of the broth before inoculation, as Positive: Electric blue fluorescence evidenced by gas formation in the tube; Negative: Lack of fluorescence and (2) to identify denitrification by organisms that produce gas by alternate Quality Control pathways; if gas is formed in the tube Positive: Escherichia coli before the addition of the color Negative: Klebsiella pneumoniae indicator, the test result is negative for nitrate reduction by this method. Nitrate Reduction Purpose Result This test is used to determine the ability Positive: RED of an organism to reduce nitrate to Negative: No color change nitrite. All members of the Enterobacteriaceae family reduce Quality Control nitrate, but some members further Positive: NO3+, no gas: Escherichia coli metabolize nitrite to other compounds. Positive: NO3+, gas: Pseudomonas aeruginosa Negative: Acinetobacter baumannii Principle Anaerobic metabolism requires an electron acceptor other than atmospheric oxygen (O2). Many gram- negative bacteria use nitrate as the final electron acceptor. The organisms produce nitrate reductase, which converts the nitrate (NO3) to nitrite (NO2). The reduction of nitrate to nitrite is determined by adding sulfanilic acid and alpha-  UNIT NINE BIOCHEMICAL biochemicalTEST test Nitrite Reduction Organisms unable to produce β- Purpose galactosidase may become genetically This test is used to altered through a variety of mechanisms determine whether and be identified as late-lactose an organism can fermenters. reduce nitrites to ONPG enters the cells of organisms that gaseous nitrogen do not produce the permease but are or to other compounds containing capable of hydrolyzing the ONPG to nitrogen. galactose and a yellow compound, o nitrophenol, indicating the presence of Principle β-galactosidase. Microorganisms capable of reducing nitrite to nitrogen do not turn color and Result do produce gas in the nitrate reduction Positive: YELLOW (presence of β- test. The test does not require the galactosidase) addition of zinc dust. Negative: Colorless Result Quality Control Positive: No color change to red 2 minutes; gas Positive: Shigella sonnei production in Durham tube Negative: Salmonella typhimurium Negative: Broth becomes red ; no gas production is observed Optochin (P disk) Susceptibility Test / TAXO P Purpose Quality Control This test is used Positive: Proteus mirabilis to determine Negative: Acinetobacter baumannii the effect of Optochin (ethyl o-Nitrophenyl-β-D-Galactopyranoside hydrocupreine (ONPG) Test hydrochloride) on an organism. Purpose Optochin lyses pneumococci (positive This test is used to test), but alpha-streptococci are determine the resistant (negative test). ability of an The Optochin test is used to organism to differentiate Streptococcus produce β pneumoniae from other alpha- galactosidase, an enzyme that hemolytic streptococci. hydrolyzes the substrate ONPG to form a visible (yellow) product, Principle orthonitrophenol. Optochin is an antibiotic that interferes The test distinguishes late lactose with the ATPase and production of fermenters from non–lactose adenosine triphosphate (ATP) in fermenters of Enterobacteriaceae. microorganisms. The Optochin impregnated disk (TaxoP) Principle is placed on a lawn of organism on a Lactose fermenters must be able to sheep blood agar plate, allowing the transport the carbohydrate (β- antibiotic to diffuse into the medium. galactoside permease) and hydrolyze The antibiotic inhibits the growth of a (β-galactosidase) the lactose to glucose susceptible organism, creating a and galactose. clearing, or zone of inhibition, around  UNIT NINE BIOCHEMICAL biochemicalTEST test the disk. A zone of 14 to 16 mm is Negative: Absence of color considered susceptible and presumptive identification for Quality Control Streptococcus pneumoniae. Positive: Pseudomonas aeruginosa Negative: Escherichia coli Result Positive: ZOI ≥ 14 mm in diameter, with 6-mm Oxidation/Fermentation (of) Medium disk (CDCMethod) Negative: No zone of inhibition Purpose This test is used Quality Control to differentiate Positive: Streptococcus pneumoniae microorganisms Negative: Streptococcus pyogenes based on the ability to oxidize Oxidase Test (Kovac’s Method) or ferment Purpose specific The oxidase test carbohydrates. identifies Hugh and Leifson’s formula low microorganisms peptone-to-carbohydrate ratio and a that produce the enzyme cytochrome limiting amount of carbohydrate. oxidase. reduced peptone limits the formation of This test determines the presence of alkaline amines that may mask acid cytochrome oxidase activity in production resulting from oxidative microorganisms for the identification of metabolism. oxidase negative Enterobacteriaceae, Carbohydrate Fermentation Medium differentiating them from other gram- negative bacilli. A. Base Medium-sugar-free It is used to differentiate oxidase a. Peptone water positive microorganisms such as b. Trypticase Aeromonas spp., Pseudomonas spp., c. Tryptone Broth and Haemophilus spp. from the oxidase negative Enterobacteriaceae. B. pH Indicators a. Bromcresol Purple ---Purple (alk) to Principle Yellow(acid) at pH 6.3 To determine the presence of bacterial b. Andrade’s Acid Fuchsin Pale Yellow (alk) to cytochrome oxidase using the oxidation Reddish Pink (acid) at pH 5.5 of the substrate tetramethyl-p- c. Phenol Red Red (alk) to Yellow at pH 7.9 phenylenediamine dihydrochloride to indophenol, a dark purple colored end Other Methods product. 1.Rusell’s Double Sugar (RDS) A positive test (presence of oxidase) is contains glucose and lactose with indicated by the development of a dark Andrade’ Acid Fuchsin purple color. No color development 2.Smith Fermentation Tube indicates a negative test and the absence of the enzyme. Result Positive: Development of a dark purple color within 10 seconds  UNIT NINE BIOCHEMICAL biochemicalTEST test Result L-Pyrrolidonyl Arylamidase (PYR) Test Positive: Acid production (A) indicated by the Purpose color indicator changing to This test is used for the presumptive yellow identification of group A streptococci Weak-positive (Aw): (Streptococcus pyogenes) and Weak acid formation enterococci by the presence of the Negative: enzyme L-pyrrolidonyl arylamidase. Red or alkaline (K) color in the deep Principle No change (NC) or neutral (N) The Pyrrolidonyl Arylamidase (PYR) test is a colorimetric test that detects Quality Control pyrrolidonyl peptidase by the hydrolysis Fermenter (Glucose): Escherichia coli of the substrate, L-pyroglutamic acid Oxidizer (Glucose): Pseudomonas aeruginosa beta-naphthylamide. The enzyme L- pyrrolidonyl arylamidase hydrolyzes the Phenylalanine Deaminase Agar L-pyrrolidonyl- β-naphthylamide Purpose substrate to produce a β- This test is used to naphthylamine. determine the The β-naphthylamine can be detected in ability of an the presence of N,N- organism to methylaminocinnamaldehyde reagent deaminate by the production of a bright red phenylalanine to precipitate. phenylpyruvic acid oxidatively. The genera Morganella, Proteus, and Result Providencia can be differentiated from Positive: Bright red color other members of the within 5 minutes Enterobacteriaceae family. Negative: No color change or an orange color Principle Microorganisms that produce Quality Control phenylalanine deaminase remove the Positive: Enterococcus faecalis, amine (NH2) from phenylalanine. The Streptococcus pyogenes reaction results in the production of Negative: Streptococcus agalactiae ammonia (NH3) and phenylpyruvic acid. The phenylpyruvic acid is detected by Pyruvate Broth adding a few drops of 10% ferric Purpose chloride; a green colored complex is This test is used to formed between these two compounds. determine the ability of an Result organism to utilize Positive: GREEN COLOR on slant pyruvate. This Negative: Slant remains original color aids in the differentiation between Enterococcus faecalis (positive) and Quality Control Enterococcus faecium (negative). Positive: Proteus mirabilis Negative: Escherichia coli Principle MEDIUM: PHENYLALANINE AGAR Pyruvate broth is a carbohydrate-free, nutrient limited medium. Pyruvic acid is  UNIT NINE BIOCHEMICAL biochemicalTEST test added to the broth to determine Spot Indole Test whether the microorganism is able to Purpose use pyruvate, resulting in the formation This test is used to of metabolic acids. Bromthymol blue determine the indicator changes from blue to yellow in presence of the the presence of acid as a result of the enzyme tryptophanase. decrease in pH. Principle Result Tryptophanase breaks down tryptophan Positive: Indicator changes from green to to release indole, which is detected by yellow its ability to combine with certain Negative: No color change aldehydes to form a colored compound. For indole-positive bacteria, the blue- Quality Control green compound formed by the reaction Positive: Enterococcus faecalis of indole with cinnamaldehyde is easily Negative: Streptococcus bovis visualized. The absence of enzyme results in no color production (indole Salt Tolerance Test negative). Purpose This test is used to Result determine the ability Positive: Development of a blue color within 20 of an organism to seconds grow in high Negative: No color development or slightly pink concentrations of salt. color It is used to differentiate enterococci (positive) from nonenterococci Quality Control (negative). Positive: Escherichia coli Negative: Klebsiella pneumoniae Principle The salt tolerance test is a selective and Triple Sugar Iron Agar (TSI) differential medium. Enterococci are Purpose resistant to high salt concentration. TSI is used to determine whether a A heart infusion broth containing 6.5% gram-negative rod ferments glucose NaCl is used as the test medium. This and lactose or sucrose and forms broth also contains a small amount of hydrogen sulfide (H2S). The test is used glucose and bromcresol purple as the primarily to differentiate members of indicator for acid production. the Enterobacteriaceae family from other gram-negative rods. Result Triple sugar iron agar contains three Positive: Visible turbidity in the broth, with or sugars (lactose, sucrose, and glucose), without a color change from purple to yellow ferrous sulfate, and a pH indicator. Negative: No turbidity and no color change Composition: 10 parts Lactose, 10 parts sucrose, 1 part glucose and peptone Quality Control Phenol Red pH: indicator Positive: Enterococcus faecalis Ferrous Ammonium Sulfate: H2S Negative: Streptococcus bovis Indicator CO2 and hydrogen gas (H2): presence of bubbles or cracks or by separation of the agar  UNIT NINE BIOCHEMICAL biochemicalTEST test Hydrogen sulfide production (H2S) = black Reactions include: color or precipitate Acid reaction (A) = yellow color Gas production (G) = bubbles, cracks, or Alkaline reaction (K) = red color media displacement  UNIT NINE BIOCHEMICAL biochemicalTEST test Result detected by the color change of phenol Alkaline slant/ Alkaline butt (K/K) red from light orange at pH 6.8 to glucose, lactose, and sucrose magenta (pink) at pH 8.1. nonutilizer; Rapid urease- positive organisms turn Alkaline slant/Acid butt (K/A) glucose the entire medium pink within 24 hours. fermentation only Weakly positive organisms may take Acid slant/Acid butt (A/A) glucose, several days, and negative organisms sucrose, and/or lactose fermenter produce no color change or yellow as a Black Precipitate production of ferrous result of acid production. sulfide and H2S gas (H2S+) Medium: CHRISTENSEN’S UREA AGAR Bubbles or cracks gas production or BROTH Quality Control Result A/A gas production: Escherichia coli Positive: Change in color of slant from light K/A, +/− gas production, H2S+: orange to magenta Salmonella typhimurium Negative: No color change K/K: Pseudomonas aeruginosa K/A, H2S+: Proteus mirabilis Quality Control K/A: Shigella Flexner Positive: Proteus vulgaris Weak positive: Klebsiella pneumonia Triple Sugar Iron Agar (TSI) Negative: Escherichia coli Result A/A GAS(+) H2S(-) E.coli, Klebsiella X and V Factor Test Pneumonaie, Enterobacteriaceae Purpose K/A GAS(+) H2S (+) Salmonella typhi, The X and V factor test is used to Proteus spp., Salmonella Arizonae, differentiation Haemophilus species. Citrobacter, Edwardsiella. Members of the genus Haemophilus K/A GAS (-)H2S (-) Shigella sonnei require accessory growth factors in K/K GAS (-) H2s(-) Pseudomonas vitro. Aeroginosa Some Haemophilus spp. require X factor (hemin) alone, V factor Urease Test (Christensen’s Method) (nicotinamide adenine dinucleotide Purpose [NAD]) alone, or a combination of the This test is used to two. determine an organism’s ability to Principle produce the enzyme A lawn of the test organism is streaked urease, which onto heart infusion agar, tryptic soy hydrolyzes urea. agar, Haemophilus agar, or nutrient Proteus sp. may be agar. presumptively identified by the ability to The impregnated disks or strips (X, V, or rapidly hydrolyze urea. XV) are placed directly on the confluent inoculation, allowing diffusion of the Principle accessory growth factor into the Urea is the product of decarboxylation medium. of amino acids. Hydrolysis of urea The organisms will grow only around the produces ammonia and CO2. disk that provides the appropriate factor The formation of ammonia alkalinizes for the growth of the organism. the medium, and the pH shift is  UNIT NINE BIOCHEMICAL biochemicalTEST test Result Positive: Growth around the XV disk : requirement for both factors Growth around the V disk, no growth around the X disk, and light growth around the XV disk shows a V factor requirement Negative: Growth over the entire surface of the agar indicates no requirement for either X or V factor Quality Control Haemophilus influenza- halo of growth around the XV disk, no growth on the rest of the agar surface Haemophilus parainfluenzae- halo of growth around the XV and V disks Haemophilus ducreyi -halo of growth around the XV and X disks