Bacte LEC - Module 1 (Biochemical Test) PDF

Biochemical Test- Part 2 Supplemental Lecture Reminders: 1. Fill-up the Student information Sheet thru your Section Leader. 2. Study the assigned Modules before virtual meeting. 3. Check schedule of the quiz. 4. Don’t be Absent o...

Biochemical Test- Part 2 Supplemental Lecture Reminders: 1. Fill-up the Student information Sheet thru your Section Leader. 2. Study the assigned Modules before virtual meeting. 3. Check schedule of the quiz. 4. Don’t be Absent or Late during the our virtual meeting 5. Avoid Cyberbullying, all concerns and suggestions must be address to the person professionally. 6. Note down all your inquiries before our Virtual Class. Prepared by: RSCRUZ 10/7/2020 6:40 AM 1 Centro Escolar University-Manila  Principle of Biochemical Reaction based on enzymatic reactions Synthesis of enzymes produce by the organisms or utilization of substrate Available substrate in the culture medium Formation of the End-product after enzyme- substrate reaction Addition of the indicator/s The color change, initial to the final color reaction Prepared by: RSCRUZ 10/7/2020 6:40 AM 2 Centro Escolar University-Manila  Carbohydrate Utilization Supplemental readings: Chapter 13. pp 216- 247. BAILEY & SCOTT’S DIAGNOSTIC MICROBIOLOGY. 12 Ed. Forbes,BA, Sahm, DF, Weissfeld, AS. Prepared by: RSCRUZ 10/7/2020 6:40 AM 3 Centro Escolar University-Manila  Bacteria has a greatly diversity in the ability to use Carbohydrates LACTOSE utilization is the most important carbohydrate determination test A disaccharide consisting of glucose and LACTOSE galactose connected by a galactoside bond. LACTOSE DEGREDATION is used to differentiate bacterial species Lactose Fermenters non-lactose fermenters (LF) (NLF) which able to which unable to ferment lactose ferment lactose Prepared by: RSCRUZ 10/7/2020 6:40 AM 4 Centro Escolar University-Manila  enzymes necessary for bacterium to take up lactose: 1. β-galactoside permease - serves as a transport enzyme that facilitates entry of lactose molecule across plasma membrane. 2. β-galactosidase - it hydrolyzes lactose into glucose and galactose  After hydrolyzed, glucose is available for metabolism through the pathways: 1. Embden-Meyerhof pathway (glycolysis) or 2. Entner-Doudoroff pathway.  LFs posses both enzyme unlike the NLFs which do not posses either.  LATE/DELAYED LACTOSE FERMENTERS (dLFs) - bacteria that have β-galactoside permease but possess β- galactosidase Prepared by: RSCRUZ 10/7/2020 6:40 AM 5 Centro Escolar University-Manila  BIOCHEMICAL TEST: 1.Triple Sugar Iron agar (TSI) -contains glucose, lactose, sucrose containing ammonium sulfate - red colored medium alkaline) - yellow colored medium acid ) TSI Reaction Grp I Ac slant & butt with gas,no H2S Grp II Ac slant & butt with gas & H2S Grp III Alk slant/ac butt with gas Grp IV Alk slant/ac butt with out gas Grp V Alk slant & butt with out gas * red colored medium (alkaline) change to yellow— acid *red colored medium— no change (red) — alkaline Initial pH Indicator used — phenol red 7.3-7.6 Prepared by: RSCRUZ 10/7/2020 6:40 AM 6 Centro Escolar University-Manila Triple Sugar Iron Agar Triple Sugar Iron Agar (TSI) and Kligler Iron Agar (KIA) Useful in the presumptive identification of gram-negative bacteria pH indicator: Phenol Red ferrous sulfate and sodium thiosulfate is added to detect production of H₂S Useful in detecting the ability of the microorganism to ferment carbohydrate, glucose, and lactose To produce gas from fermentation of sugars To detect the production of H₂S Both put on slant(aerobic) or butt (anaerobic) Reactions must be read within 18-24 hours otherwise it will be erroneous Prepared by: RSCRUZ 10/7/2020 6:40 AM 7 Centro Escolar University-Manila Reactions on TSI Agar or KIA 1. No Fermentation ALK/ALK or K/K – alkaline slant/alkaline butt ALK/no change or K/NC – alkaline slant/no change Unable to ferment either lactose or glucose Organism can degrade the peptones present in the medium aerobically or anaerobically Resulting in production of alkaline byproducts in slant/deep Indicator: deep color red 2. Glucose fermentation only, no lactose fermentation K/A - alkaline slant/acid butt TSI agar and KIA contains glucose conc. 0.1%. Yellow indicator: because of acid produced in concentration of glucose deep color red indicator: because of after 12 hours, glucose will be consumed and bacteria utilized the peptones producing an alkaline reaction Prepared by: RSCRUZ 10/7/2020 6:40 AM 8 Centro Escolar University-Manila 3. Lactose fermentation A/A – Acid/acid Glucose fermenters attacked the glucose then the lactose Acid production from the fermentation of the additional sugar is suffering to keep both the slant and the butt acid (yellow). 4. Hydrogen Sulfide Production K/A, H₂S – alkaline slant/acid butt, H₂S in butt A/A, H₂S – acid slant/acid butt, H₂S in butt 2 indicators (to detect H₂S): Sodium thiosulfate and Ferrous sulfate 2 step process: a. Bacterium (acid environment) + sodium thiosulfate H₂S gas b. H₂S + Ferric ion ferrous sulfide (black precipitate) 5. Gas production (aerogenic) or no gas production (nonaerogenic) Formation of bubbles poor splitting of the media in the butt or complete displacement 10/7/2020 6:40 AM Prepared by: RSCRUZ 9 Centro Escolar University-Manila  Triple Sugar Iron Agar K/A- glucose is fermented A/A- two or more sugars fermented K/K – no sugar fermented Space or crack – gas formation Blackening – H2S production Prepared by: RSCRUZ 10/7/2020 6:40 AM 10 Centro Escolar University-Manila  Understanding the Concept: Read the following TSI reactions Prepared by: RSCRUZ 10/7/2020 6:40 AM 11 Centro Escolar University-Manila  Understanding the Concept: Interpret the TSI Reaction Acid/Acid Akaline/Acid Prepared by: RSCRUZ 10/7/2020 6:40 AM 12 Centro Escolar University-Manila Sugar Fermentation Test 2. Krigler iron agar(KIA) - glucose and lactose with ferrous ammonium sulfate ( for hydrogen sulfide production) pH indicator -phenol red 3. Russell Double sugar agar(RDS) - contains glucose, lactose, no ferrous ammonium sulfate pH incator use: Andrades indicator Dissolve the fuchsin in distilled water and add the sodium hydroxide. If the fuchsin is not sufficiently decolorized after several hours, an additional 1 or 2 ml sodium hydroxide is added (pH = 7.2- colorless). - pale yellow or colorless (alkaline) - pink or red ( acid) * All are distributed or dispense at butt/slant * Ferrous ammonium sulfate - H2S indicator black - (+) reaction Prepared by: RSCRUZ 10/7/2020 6:40 AM 13 Centro Escolar University-Manila  Glucose Metabolism and Its Metabolic Products Prepared by: RSCRUZ 10/7/2020 6:40 AM 14 Centro Escolar University-Manila  PROCESS OF METABOLISM Methyl Red (MR) and Voges Proskauer (VP) test detect the end products of glucose fermentation part of the IMViC Reactions: indole, methylred, voges- proskauer, citrate Bacteria can be positive for either of two but not for both, Bacteria can be negative for both  Prepared by: RSCRUZ 10/7/2020 6:40 AM 15 Centro Escolar University-Manila  OTHER CARBOHYDRATES: ▪ Monosaccharides, Disaccharides, Polysaccharides ▪ Examples: maltose, rhamnose, sucrose, raffinose, arabnose ▪ Sugars – collective polyhydric alcohols - ex: adonitol, dulcitol, mannitol, sorbitol  Bacteria can utilize carbohydrates by: 1. OXIDATION – aerobically 2. FERMENTATIVELY – anaerobically 3. Or both - Facultative anaerobes  Asaccharolytic – they do not use any carbohydrate instead they use other organic molecules for energy and carbon sources. Prepared by: RSCRUZ 10/7/2020 6:40 AM 16 Centro Escolar University-Manila OXIDATION-FERMENTATION TESTS (O/F) -Important test in the identification of bacteria CARBOHYDRATE FERMENTATION TEST determine the ability of a microorganism to ferment a specific carbohydrate incorporated into a basal medium. glucose enters the glycolysis pathway formation of pyruvic acid end product is an acid or acid with gas Acid formation – detected with pH indicators added to medium Homolactic acid fermenters – bacteria that produce primarily a single acid. Example;: streptococci Mixed acid fermenters – bacteria that produce several acids like lactic acid, propionic acid, and succinic acid. Prepared by: RSCRUZ 10/7/2020 6:40 AM 17 Centro Escolar University-Manila  OXIDATION TEST glucose enter the glycolysis pathway pyruvic formed oxidized to CO₂ requires: oxygen (aerobic respiration) inorganic molecule (anaerobic respiration) - eg. Nitrate (NO₃) as a terminal electron acceptor Higher acidity is form during fermentation than in oxidation The same medium is for oxidation and fermentation test Oxidizers and fastidious fermenters often produce either weak or small amounts of acids from carbohydrates Prepared by: RSCRUZ 10/7/2020 6:40 AM 18 Centro Escolar University-Manila  Fermentation/Oxidation Reaction ▪ Energy production by breakdown of sugars through degradation process coupled with oxidation-reduction reaction. ▪ Substrate is oxidized as it donates electron to e’-acceptor molecules, w/c then reduced ▪ Mediated by carrier molecules such as NAD and NADP ▪ Which involved fermentative metabolism ( anaerobic) producing alcohols, acids, co2 and H2 ▪ Oxidative phosphorylation involved e’ transport system in w/c O2 is the final e’ acceptor ( aerobic respiration) Hugh-Leiffson O-F Medium ▪ Anaerobic respiration- O2 is not the final e’ acceptor 10/7/2020 6:40 AM Prepared by: RSCRUZ 19 Centro Escolar University-Manila Indol Production Tryptone broth is rich in amino acid tryptophan which can be used by some bacteria as a source of carbon and nitrogen. The organism hydrolyzed tryptophan producing indol, pyruvic acid (alanine) and ammonia. plus ether or xylene Ehrlich and Kovack’s reagent react to methylindol producing red colored reaction Prepared by: RSCRUZ 10/7/2020 6:40 AM 20 Centro Escolar University-Manila Indole Production Indole – one of the degradation products of the amino acid tryptophan organism that possess the enzyme tryptophan are capable of deaminating tryptophan, with formation of degradation products of indole, pyruvic acid, and ammonia Ehrlich indole test ❑ indole is extracted from the broth culture by addition of xylene and Ehrlich reagent(with PDAB) ❑ Positive for indole: red color ❑ More sensitive than Kovac reagent Kovac reagent test ❑ Kovac reagent use as an alternative because it contains PDAB(Para-dimethylaminobenzaldehyde) ❑ Xylene is not used in this method ❑ Positive for indole: red color Prepared by: RSCRUZ 10/7/2020 6:40 AM 21 Centro Escolar University-Manila  METHYL RED - pH indicator Clark-Lub’s broth or Glucose broth,MR-VP contains peptone, dextrose or glucose and dipotassium phosphate. Dextrose is utilized by the organism producing acidty and can be observed upon the addtion of methyl red (indicator). If the pH of the media reaches 4.5 or less the positive reation appears with a red color, but if the pH is less acidic (6.2), it displays a negative reaction with a yellow color (4 days incubation at 37C). Indicator: Methyl red (4.4-6.4,acid) red to yellow * Positive result— red (acid) *Negative result— yelllow (alkaline) 10/7/2020 6:40 AM Prepared by: RSCRUZ 22 Centro Escolar University-Manila Methyl Red (MR) test bacteria are incubated in a broth medium containing glucose (3-5 days) glucose is metabolized by the mixed acid fermentation pathway, stable acid, end products are produced, low pH MR (+) develops red color upon addition of indicator methyl red MR (-) remain yellow Prepared by: RSCRUZ 10/7/2020 6:40 AM 23 Centro Escolar University-Manila  Voges-Proskauer test Production of acetyl methyl carbinol from glucose after 48hrs. by adding 15 drops of 5% alphanapthol in absolute ethanol (Barritt’s rgts A) and 1O drops of.3% creatine in 4O% KOH (Barritt rgt B) producing positive rxn upon the apperance of pink red color in 15-3O mins. Media: Clark Lub’s broth , glucose broth, MR-VP broth Positive result - red from oxidation of acethylmethylcarbinol in the presence of potassium hydroxide alkaline & atmospheric air Prepared by: RSCRUZ 10/7/2020 Negative6:40 AMresult - colorless Centro Escolar University-Manila 24 Voges Proskauer (VP) test Acids formed during fermentation metabolized to 2,3 butanediol through he acetoin α-napthol is added as a catalyst 40% KOH or NaOH is added VP (+); presence of potassium forms red complex, pH is neutral Prepared by: RSCRUZ 10/7/2020 6:40 AM 25 Centro Escolar University-Manila  Citrate utilization Utilization citrate as the only source of carbon in a medium containing inoganic nitrogen giving a prussian blue color or blue which is a positive result in 24-48 hrs. incubation(using Simmon Citrate Agar). 1.Simmon citrate agar Bromthymol blue (pH 6.9) * Positive result - blue color medium, * Negativeresult -green color med 2. KOSER CITRATE BROTH Result - presence of growth of organism (turbidity) is the positive result disadvantage: turbidity of the medium when large inoculum is used. Prepared by: RSCRUZ 10/7/2020 6:40 AM 26 Centro Escolar University-Manila Citrate Utilization determines whether an organism can see sodium citrate as a sole carbon source simmons citrate (medium) contains ammonium salt as the sole nitrogen source thereby releasing ammonia changes the pH indicator (bromcresol blue) from green to blue light inoculum is important to use because dead organisms can be a source of carbon, producing a false positive reaction. Christensen citrate (medium) incorporates phenol red (pH indicator) and organic nitrogen at alkaline pH, indicator turns from yellow to pink Prepared by: RSCRUZ 10/7/2020 6:40 AM 27 Centro Escolar University-Manila  CATALASE TEST Decomposition of H2O2 into free O2 and H2O by enzyme catalase from the organism resulting to a rapid evolution of oxygen gas which is the positive reaction. * Positive result Micrococci, Staphylococci, Bacillus species are all Gram (+) organisms * Negative result Streptococci and Pneumococci - no evolution of gas * Reagent : 3% H2O2 Organism enzyme (Catalase) + H2O2 --H2O + O2 gas * False (+) result obtain due to contamination of RBC. * Do not use blood agar:Use tryptic soy agar(TSA); blood has catalase and will give a false positive test. Prepared by: RSCRUZ 10/7/2020 6:40 AM Centro Escolar University-Manila 28  OXIDASE TEST- Colonies producing enzymes indophenol oxidase become pink changing to red and finally black or dark purple upon addtion of oxidase reagen(1% tetramethylparaphenylene-diamine-dihydrochloride or paraamino- dimethyl-aniline-monohydrochloride). * MEDIA USE: Chocolate Agar for Neisserria Nutrient Agar for Pseudomonas, Moraxella and Aeromonas, Campylobacter fetus POSITIVE RESULT: Black or dark purple Neisserria, Pseudomonas, Moraxella, Aeromonas, Campylobacter,Vibrio NEGATIVE RESULT : no change in color colorless * Not satisfactory for colonies growing on selective media or media containing glucose. * Fermentation inhibits enzyme oxidase activity and lead to a false negative result for Indophenol oxidase * Use platinum loop or wooden applicator, iron in the nichrome loop can give false positive reaction Prepared by: RSCRUZ 10/7/2020 6:40 AM 29 Centro Escolar University-Manila Oxidase Test Determines the presence of the cytochrome oxidase system that oxidizes cytochrome with molecular oxygen. Oxidase negative: enterobacteriaceae Oxidase positive: Pseudomonas, Neisseria, Campylobacter, Vibrio Other oxidase test Modified Oxidase test - To distinguish Staphylococcus from Micrococcus Kovac Oxidase test - Lavender color within 10 to 15seconds is a positive reaction Prepared by: RSCRUZ 10/7/2020 6:40 AM 30 Centro Escolar University-Manila  UREASE TEST Some organisms can hydrolyze urea producing urease and ammonia,thus making the medium alkaline and change of color from salmon to cerise or cherry color. * Proteus hydrolyzed urea rapidly 2-4 hours Christensen’s Urea Agar Phenol red-indicator UREA SERVE AS SUBTRATE: * Positive result: pink- red color - Proteus Negative result: salmon color, no change Prepared by: RSCRUZ 10/7/2020 6:40 AM 31 Centro Escolar University-Manila  Urease Test determines whether a microorganism can hydrolyze urea Urease hydrolyze urea to form ammonia, water and CO₂ Christensen urea broth or agar Phenol red is the pH indicator Alkaline pH is bright pink color Prepared by: RSCRUZ 10/7/2020 6:40 AM 32 Centro Escolar University-Manila  Decarboxylation/ Dihydrolase Test Determines whether the bacterial species possess enzyme capable of decarboxylating (removing the carboxyl group) the specific amino acids. DEGRADATION OF AMINO ACIDS AND THEIR SPECIFIC END PRODUCTS: o Lysine (amino acid) Lysine decarboxylase cadaverine(amine) + CO₂ o Ornithine ornithine decarboxylase putrescine o Arginine arginine dihydrolase citrulline ornithine putrescine Prepared by: RSCRUZ 10/7/2020 6:40 AM 33 Centro Escolar University-Manila  Moeller Decarboxylase base medium o test to detect decarboxylation o A broth containing glucose; peptones o2 indicators: bromcresol purple and cresol red oSpecific amino acid concentration; 0.1% oinitial pH of the medium 6.0 ( purple) Prepared by: RSCRUZ 10/7/2020 6:40 AM 34 Centro Escolar University-Manila  Amino Acid Utilization Bacteria use amino acids as energy and carbon sources Prepared by: RSCRUZ 10/7/2020 6:40 AM 35 Centro Escolar University-Manila  RESULTS: First few hours of incubation, the organism attack glucose first, changing pH to ACID (yellow, negative result) If organism produce decarboxylase and hydrolyzes the amino acid in the medium, the enzyme attacks the amino acid and release amine product – changing pH to ALKALINE (purple = + result) Prepared by: RSCRUZ 10/7/2020 6:40 AM 36 Centro Escolar University-Manila  LYSINE DECARBOXYLASE Some organisms can decarboxylate lysine converting it to cadavering,producing a purple color as a positive result. Some organisms deaminate lysine producing red slant and yellow butt due to fermentation of glucose. * For differentiation of member of Enterobacteriaceae MEDIA USED: LYSINE-IRON AGAR positive reaction yellow color will be produced its due to alkalization of medium which is the sign of initial fermentation of glucose. * Yellow color will be turned into violet color due to decarboxy lation of the amino acid. * Yellow color after several days of incubation is (-) or absence of enzymes decaboxylase or dihydrolase. * All positive tests should be compared with the control tube , which remains yellow. Prepared by: RSCRUZ 10/7/2020 6:40 AM 37 Centro Escolar University-Manila Lysine Iron Agar Slant ( LIA) It is the most useful in conjunction with TSI in screening tool specimens for the presence of pathogens (agar slant) Contains the amino acid lysine, glucose, ferric ammonium citrate, and sodium thiosulfate. pH indicator: bromcresol purple it is primarily to determine bacteria decarboxylate or deamine lysine H₂S production is detected + result for lysine decarboxylation: dark purple or black precipitate(due to the H₂S) + result for lysine deamination: light purple color to plum or reddish- purple color Ex: salmonella (lysine+) and Citrobacter (lysine-) Prepared by: RSCRUZ 10/7/2020 6:40 AM 38 Centro Escolar University-Manila ○ Motility-Indole-Ornithine Agar A semisolid agar used to detect motility and indole and ornithine decarboxylase production. ○ Useful in differentiating Klebsiella spp. From Enterobacter and Serratia spp. ○ Motility is shown by a clouding of the medium or spreading growth from the line of inoculation ○ Ornithine decarboxylation is indicated by a purple color through out the medium ○ It does not overlayed with mineral oil to provide anaerobic condition. ○ It is detected by adding Kovac’s reagent ○ Positive result: pink to red color ○ Motility is read before addition of Kovac reagent Prepared by: RSCRUZ 10/7/2020 6:40 AM 39 Centro Escolar University-Manila  PHENYL ALANINE DEAMINASE TEST Some organisms can deaminate phenylalanine converting it to phenyl acid by phenylalanine deaminase produced by bacteria. Then later reacts with ferric ions to produce a green color. MEDIA: Phenylalanine Agar (+) result - rxn appear after addittion of ferric chloride * Positive result: dark green - Proteus, Klebsiella, and Yersinia * Negative result: yellow - other Enterobacteriaceae Prepared by: RSCRUZ 10/7/2020 6:40 AM 40 Centro Escolar University-Manila  Deaminase Test Removed an amine group Phenylalanine deaminase (PAD) test Determines whether an organism possess the enzyme that deaminates phenylalanine to phenylpyruvid acid. Medium: agar slant (with 0.2% conc. Of phenylalanine) Deamination of Phenyalanine Phenylalanine Phenylalanine deaminase Phenylpyruvic acid + (FeCl ₃) green Prepared by: RSCRUZ 10/7/2020 6:40 AM 41 Centro Escolar University-Manila Ortho-Nitrophenyl-β-D-galactopyranoside Test ONPG and PNPG determines whether the organism is a dLF or NLF ONPG is similar to lactose but easily transported through the bacterial plasma membrane and does not require β-galactoside permease Test can be performed by making a heavy suspension of bacteria in sterile saline and adding commercially prepared ONPG disks or tablets. The suspension is incubated at 37◦C and positive results can be seen within 6 hours …ONPG - o-nitrophenyl-β-d-galactopyranoside test …PNPG – p-nitrophenyl-β-d-galactopyranoside test …NLF - non - lactose fermenters …DLFs - late/delayed lactose fermenters Prepared by: RSCRUZ 10/7/2020 6:40 AM 42 Centro Escolar University-Manila  DNase DNA – a polynucleotide composed of repeating purine and pyrimidine mononucleotide monomeric units. Bacterial DNases – are undonuclease cleaving internal phosphodiester bonds resulting in smaller subunits. DNase test medium usually contains 0.2% DNA inoculum of bacteria is streak in a straight line plate is incubated at 35 C̊ for 18-24 hours 1N HCl is added (unhydrolyzed DNA is insoluble in HCl and form precipitate) Olignucleotide formed and dissolved in acid forming clear zone around inoculum Prepared by: RSCRUZ 10/7/2020 6:40 AM 43 Centro Escolar University-Manila DNase Test ▪ Hydrolysis/ depolymerization of DNA by enzyme DNAse w/c produce a clear zone in a DNA-methyl green medium. ▪ Positive: clear zone ▪ Negative: pale green 1% toluidine 10/7/2020 6:40 AM Prepared by: RSCRUZ 44 Centro Escolar University-Manila Gelatin Liquefaction Gelatin – a protein derived from animal collagen Gelatinases- Proteolytic enzymes that break down gelatin into amino acids produced by bacteria Gelatinase activity - detected by loss of gelling (liquefaction) of gelatin affected by some factors including size of inoculum and incubation at room temperature. Prepared by: RSCRUZ 10/7/2020 6:40 AM 45 Centro Escolar University-Manila Gelatinase Test ▪ Hydrolysis of gelatine by enzyme gelatinase that causing liquefaction. ▪ Positive: liquid medium ▪ Negative: solid medium after refrigeration 10/7/2020 6:40 AM Prepared by: RSCRUZ 46 Centro Escolar University-Manila Malonate Utilization Malonate test determines whether the organism is capable of using sodium malonate as its sole carbon source. Malonate broth contains bromthymol blue as a pH indicator Positive test: increase alkalinity, changing indicator from green to blue Prepared by: RSCRUZ 10/7/2020 6:40 AM 47 Centro Escolar University-Manila  Motility test media Have agar concentration of Motility 0.4% (to allow for the free spread microorganism) A single stab into the center of medium is made (best result if it is made straight) After incubation: movement away from the stub or hazy appearance indicates a motile organism Incubation temperature is important for some bacteria are motile only at room temperature. Prepared by: RSCRUZ 10/7/2020 6:40 AM 48 Centro Escolar University-Manila  Nitrate Test ▪ Reduction of nitrate to nitrite which can be determined after the addition of sulfanilic acid and alpha- naphthylamine, nitrite react with sulfanilic acid to form diazonium salt w/c couples with alpha- naphthylamine to form red, water soluble azo dye. ▪ Positive: red or gas ▪ Negative: yellow no gas 10/7/2020 6:40 AM Prepared by: RSCRUZ 49 Centro Escolar University-Manila Nitrate Reduction Determines the ability of the organism to reduce nitrate to nitrite and further reduce nitrite to nitrogen gas. N,N dimethyl-α-naphthylamine and sulfanilic acid is added (indicator). Positive for presence of nitrite: red No color develops indicate that nitrate has not been reduced or nitrate is reduced to N₂, nitric oxide, nitrous oxide Zinc dust - help to detect the result if it is a true negative result or due to the reduction beyond nitrite. Prepared by: RSCRUZ 10/7/2020 6:40 AM 50 Centro Escolar University-Manila  Sulfide-indole-motility agar A semisolid agar helpful in differentiating gram-negative bacteria in the family Enterobacteriaceae Positive for motility: cloudiness spreading from the inoculation Production of H₂S: formation of black precipitate Positive result: colorless to red color after addition of Kovac’s reagent or Erlich’s reagent + chloroform or ether Prepared by: RSCRUZ 10/7/2020 6:40 AM 51 Centro Escolar University-Manila Hippurate Hydrolysis ▪ Hydrolysis of hippuric acid by enzyme hippuricase that produce glycine. Glycine is detected by oxidation with ninhydrin reagent resulting the production of purple color. ▪ Negative-yellow or pink 10/7/2020 6:40 AM Prepared by: RSCRUZ 52 Centro Escolar University-Manila  PYR test ▪ L-pyrroglutamyl aminopeptidase hydrolyzes the substrate L- pyrrolidonyl-B-naphthylamide (PYR) to produce a B- naphthylamine that combines with cinnamaldehyde reagent producing bright red color ▪ Negative: orange (other streptococci) ▪ Positive for S.pyogenes, Enterococci 10/7/2020 6:40 AM Prepared by: RSCRUZ 53 Centro Escolar University-Manila Acetamide Utilization ▪ Acetamide as the source of carbon, which deaminate acatamide to form ammonia resulting to increase pH that change the medium from green to royal blue 10/7/2020 6:40 AM Prepared by: RSCRUZ 54 Centro Escolar University-Manila Bile Esculin Agar Bile inhibition of organisms other that streptococci and enterococci which grow on 40% bile and hydrolyze esculin forming esculetin which turn the indicator ( ferric ammoium citrate) a dark brown color due to combination of esculetin with ferric ions to form a phenolic iron complex. 10/7/2020 6:40 AM Prepared by: RSCRUZ 55 Centro Escolar University-Manila  Bile Solubility Test ▪ Differentiates S. pneumoniae from alpha hemolytic streptococci (negative). Sodium desoxycholate lyses the cell due to the presence of intracellular autolytic enzyme. Bile salt lower the surface tension between the cell membrane and the medium. 10/7/2020 6:40 AM Prepared by: RSCRUZ 56 Centro Escolar University-Manila CAMP test Group B streptococci produce a diffusible extracellular protein ( CAMP factor) that acts synergistically with the beta-lysin of S. aureus to cause enhanced lysis of red blood cells forming an arrowhead zone of beta hemolysis e.g. S.agalactiae. 10/7/2020 6:40 AM Prepared by: RSCRUZ 57 Centro Escolar University-Manila Cetrimide Test Determine the ability of the organism to grow on cetrimide which is toxic to many organism. (+) growth/green 10/7/2020 6:40 AM Prepared by: RSCRUZ 58 Centro Escolar University-Manila  LAP ( Leucine Aminopeptidase) Test Used to idenify catalase negative gram positive cocci. Leucine B- naphthylamide impregnated on a disk is hydrolyze by lap forming a red B- naphthylamine upon the addition of cinnnamaldehyde reagent. 10/7/2020 6:40 AM Prepared by: RSCRUZ 59 Centro Escolar University-Manila  Rapid and Automated Identification Systems Prepared by: RSCRUZ 10/7/2020 6:40 AM 60 Centro Escolar University-Manila Rapid Methods ▪ Conventional methods ▪ Miniaturized biochemical test using small amount of substrates and inoculum. 10/7/2020 6:40 AM Prepared by: RSCRUZ 61 Centro Escolar University-Manila  Principles of Identification Categories: pH-based reactions enzyme-based reactions utilization of carbon sources visual detection of bacterial growth detection of volatile or non-volatile fatty acids by gas chromatography Numeric code - generated based on the metabolic profiles of each organism Metabolic reaction translated into 2 responses: Plus (+) for positive Minus(-) for negative Binary codes- are computer converted into code profile numbers that represent the identifying phenotype of specific organism Prepared by: RSCRUZ 10/7/2020 6:40 AM 62 Centro Escolar University-Manila  Clinical outcome of rapid and accurate reporting of results should directly affect the patient care in 2 ways: 1. Early diagnosis 2. Subsequent selection of appropriate antimicrobial therapy Rapid method – encompasses variety of procedure and techniques and has been loosely applied to any procedure affording results faster than conventional method. ex: common stains and fluorescent antibody to detect specific organism Rapid test for detection of end products resulting from carbohydrate metabolism or enzymatic tests using chromogenic substrate produce reaction endpoints in minutes to hours. A positive reaction is measured by enzymatic activity and color change Prepared by: RSCRUZ 10/7/2020 6:40 AM 63 Centro Escolar University-Manila Rapid Biochemical Test Performed On Isolated Colonies Test Bacterial enzyme Mode of action applications ONPG Β-galactoside An ester linkage of ortho-nitrophenyl Determine lactose moieties to various carbohydrates fermentation in slow Hydrolysis results in release of yellow lactose fermenters ortho- nitrophenol Oxidase Cytochrome C A blue compound is produce when Differentiation of non- oxidase tetramethyl-phenlenediamine reacts fermenters; aids in with Cytochrome C identification Neisseria sp., Aeromonas sp. LAP Leucine LAP hydrolyzes the substrate to leucine Presumptive aminopeptidase and α-naphthylamine which reacts identification of catalase with DMAC to form a red color negative, gram-poiistive cocci Catalase Catalase Breakdown of hydrogen peroxide into Differentiation of oxygen and water resulting in rapid staphylococci from production of bubbles streptococci Prepared by: RSCRUZ 10/7/2020 6:40 AM 64 Centro Escolar University-Manila Rapid Methods Microtiter tray for MIC detection (2 hours) Minitek system – biochem in filter paper disks impregnated with substate (4 hours) Vitex 2 system Analytical Profile Index (API) with data recording form 10/7/2020 6:40 AM Prepared by: RSCRUZ 65 Centro Escolar University-Manila Rapid Methods ▪ Enterotube ID system- one shot inoculation Uni-N/F Tek plate ▪ Other rapid methods include: ▪ Pie-shapedwedges in a circular ▪ Micro –ID plastic tray ▪ Rap ID ▪ Enteric –Tek ▪ Quantum II- multipurpose instrumental system using plastic cartridge 10/7/2020 6:40 AM Prepared by: RSCRUZ 66 Centro Escolar University-Manila  Automated Identification Systems These systems uses turbidity, colorimetric, fluorescent assay principles Lyophilized reagents are provided in micro titer trays Advantages: 1. Interface to laboratory information systems leading to decreased turn around times for of results 2. Statistical prediction of correct identification 3. Increase data acquisition and epidemiologic analysis 4. Automated standardization of identification profiles that can reduce analytical errors Prepared by: RSCRUZ 10/7/2020 6:40 AM 67 Centro Escolar University-Manila  Micro Scan System ❑ Consist of plastic standard-sized 96-well micro titer trays in which as many as 32 reagent substrates are included for the identification of bacteria and yeasts 3 systems: ❖ Touch scan - Reagents are added to some wells, and the panels are interpreted visually. Biochemical results are recorded with a computer ❖ Auto scan - Can be used to detect bacterial growth or color changes by differences in light transmission. ❖ Walk away - Fully automated with capabilities to incubate more than one panel; (add reagent, interpret panel results, and print results), all without operator intervention Walk away Prepared by: RSCRUZ 10/7/2020 6:40 AM 68 Centro Escolar University-Manila Sensititre System ❑Offers two automated systems: ❑Sensititre Autoreader ❑FullAutomated Sensitire ❑It use the same gram-negative intracellular diplococcic (GNID) for determination for gram negative bacteria ❑It use fluorescent technology to detect bacterial growth and enzyme activity ❑Comprises 32 biochemical test Prepared by: RSCRUZ 10/7/2020 6:40 AM 69 Centro Escolar University-Manila Vitek System First introduced in 1980’s EBC provide automatic identification of Enterobacteriaceae within 8 hours of incubation. The computer software collates the reading and matches them to the automated database for final identification. Prepared by: RSCRUZ 10/7/2020 6:40 AM 70 Centro Escolar University-Manila BD Phoenix 100 system Released in 2003, it is the latest automated system Panels are read every 20 minutes for up to 16 hours. Results are generally available in 8 to 12 hours Prepared by: RSCRUZ 10/7/2020 6:40 AM 71 Centro Escolar University-Manila Sherlock Microbial Identification System Takes a totally different approach; identification is based on the 9 to 20 carbon fatty acid composition of microorganisms Fatty acids are located in the plasma membrane of bacteria and are modified by the bacteria depending on the environmental condition. This system compares the fatty acid profile of the unknown with the database and determines a similarity index. Prepared by: RSCRUZ 10/7/2020 6:40 AM 72 Centro Escolar University-Manila  Sherlock Microbial Identification System Evaluation of Identification Systems To provide results with greater speed and precision than traditional method Highly accurate and can provide results in as soon as 2 to 4 hours New system for accuracy Cost effectiveness Effect on work flow Provide decrease sensitivity Prepared by: RSCRUZ 10/7/2020 6:40 AM 73 Centro Escolar University-Manila Molecular Method ▪ Nucleic Acid-Based Methods a. Hybridization b. Amplification & Sequencing c. Enzymatic Digestion of Nucleic Acid NonNucleic Acid- based Analytic Methods a. Chromatography b. High-Performance Liquid Chromatography c. Electrophoretic Protein Analysis 10/7/2020 6:40 AM Prepared by: RSCRUZ 74 Centro Escolar University-Manila Molecular ID 10/7/2020 6:40 AM Prepared by: RSCRUZ 75 Centro Escolar University-Manila Electrophoretic analysis ▪ Which of the bacterial isolates are of the same species? ▪ Which of the isolates are related to one another? ▪ Which of the isolates is not totally related to other? 10/7/2020 6:40 AM Prepared by: RSCRUZ 76 Centro Escolar University-Manila Immunochemical ID ▪ Production of Antibodies ▪ Precipitin test ▪ Particle Agglutination ▪ Immunofluorescent ▪ Enzyme Immuno Assays 10/7/2020 6:40 AM Prepared by: RSCRUZ 77 Centro Escolar University-Manila Serological ID 10/7/2020 6:40 AM Prepared by: RSCRUZ 78 Centro Escolar University-Manila  Good day, God bless. 10/7/2020 6:40 AM Prepared by: RSCRUZ 79 Centro Escolar University-Manila