Biochemical Identification of Bacteria

Biochemical Identification of BacteriaBacterial Identification Techniques Classification Based on Genotype Based on Serotype Based on Phenotype Methods Nucleic acid amplificatio n tests Serological tests {Lancefield Classificatio n Scheme, Widal, WeilFelix Test) Staining, bacterial and colonial morphology, hemolytic pattern, biochemica l tests Genotypic test • NAAT equinus C D Group C Strep Nonenterococcus Enterococcus Pneumococcus Viridans Strep Enterococcus faecalis.β/NONE Streptococcus agalactiae S. E. E. equi subsp. D faecium. Mitis group. equisimilis. bovis.Serotypic Test LANCEFIELD CLASSIFICATION SCHEME SPECIES Streptococcus pyogenes LANCEFIELD HEMOLYSIS GROUP TYPE ANTIGEN A B COMMON TERMS Group A Strep Group B Strep β β β α/NONE α. Pneumoniae Mutans group. S. S. durans S. Anginosus group - α α/NONE . zooepidemicus S. Phenotypic tests • Bacterial Hemolysis (Using BAP) TYPES OF HEMOLYSIS HEMOLYSIS ALPHA (α) BETA (β) NONHEMOLYTIC (γ) ALPHA-PRIME (α’) OR WIDE ZONE DESCRIPTION Partial lysis of RBC around colony Complete lysis of RBC around colony No lysis of RBC Small area of intact RBC around colony surrounded by a wider zone of complete hemolysis . Hemolytic Patterns . Lipids. CHON. urease.Phenotypic tests Basis of Biochemical tests • Bacteria are able to release enzymes (catalase. coagulase. NA) • Metabolic pathway (Methyl Red Test. VogesProskauer test) . and other hydrolysis tests) • Metabolize different substrates (CHO. Phenotypic tests Basis of Biochemical tests • Susceptible or resistant to certain AMA (Bacitracin. Optochin. Novobiocin disc) • Able to tolerate extreme environment (SaltTolerance test) • Able to tolerate or utilize poisons (Cetrimide test) . Biochemical Tests Gram Negative Gram Positive . Biochemical Tests Catalase test CAMP test Bile Esculin Optochin disk SaltTolerance Novobiocin Disk Gram Positive Gram Negative Coagulase Bacitracin disk PYR test Hippurate Hydrolysis . .Catalase test Principle: Bubble formation/effervescence Reagents: 3% H2O2 Positive Control: Staphylococcus sp. Negative Control: Streptococcus sp. Coagulase Test Clouding and solidification of plasma Principle: Coagulase is an enzyme that clots plasma similar to the coagulation cascade/process. Reagents: Rabbit’s plasma (Citrate/EDTA) Positive Control: Staphylococcus aureus Negative Control: other species of Staph. it is produced by bacteria to protect itself from the host’s defenses. . Susceptibility to Bacitracin presumptively identifies Streptococcus pyogenes.Bacitracin Susceptibility Any Zone of Inhibition is interpreted as SUSCEPTIBLE Principle: Group A Strep. Reagents: 5% BAP Bacitracin disk (0. Are susceptible to low levels of Bacitracin. whereas other Groups are resistant.04 units) Positive Control: Streptococcus pyogenes Negative Control: Other Streptococci . PYR Hydrolysis Test RED Principle: PYR-impregnated disks serve as the substrate to produce α-naphthylamine. which is detected in the presence of D-dimethylaminocinnamaldehyde by prodcution of a red color Reagents: L-pyrrolidonyl-α-naphthylamide (PYR) in disk Positive Control: Streptococcus pyogenes and Enterococcus faecalis Negative Control: Other Streptococci . Used to differentiate S.Hippurate Hydrolysis Test Purple-colored complex Principle: Hippuricase hydrolyzes hippurate/ic acid to form sodium benzoate and glycine. Reagents: Sodium hippurate (substrate) Ninhydrin (indicator) Positive Control: Streptococcus agalactiae Negative Control: Other beta-hemolytic Streptococci . agalactiae from other β-hemolytic streptococci. Subsequent addition of Ninhydrin yields a purple-colored product. agalactiae produces CAMP Factor that enhances the lysis of sheep RBC by staphylococcal β-lysin.CAMP Test Arrowhead-shaped area of enhanced hemolysis where the two streaks (staphylococcal and streptococcal) approach each other. aureus Or disk impregnated with β-lysin Positive Control: Streptococcus agalactiae Negative Control: Other beta-hemolytic Streptococci . Requirement: Isolates of S. Principle: S. agalactiae Isolates of β-lysin producing S. . Bile Esculin Test Blackening of the agar slant (Esculetin combines with Ferric Citrate forming black complex. Esculetin diffuses intothe agar and combines with ferric citrate in the medium to give a black complex Requirement: Bile Esculin agar Positive Control: Grp D Enterococcus Negative Control: Other gram positive cocci .) Principle: Group D strep and Enterococcus grow in the presence of bile and also hydrolyzes esculin to esculetin and glucose. pneumoniae.Optochin Susceptibility Susceptble if: ZOI= >14mm (6mm disk) ZOI=> 16mm (10mm disk) Principle: Ethylhydrocuprein hydrochloride (optochin) inhibits the growth of S. Requirement: Disk impregnated with Optochin (ethylhydrocuprein hydrochloride) CO2 incubator Positive Control: Streptococcus pneumoniae Negative Control: Other alpha-hemolytic streptococci . the organism’s cell wall lyses during cell division. Requirement: Sodium deoxycholate/detergent Positive Control: Streptococcus pneumoniae Negative Control: Other alpha-hemolytic streptococci . pneumoniae in a solution of sodium deoxycholate lyses and the solution becomes CLEAR. Other alpha-hemolytic strep do not lyse and the solution remains cloudy. A suspension of S.Bile Solubility Test Clear solution (dissolved colonies) Principle: Under the influence of a bile salt (sodium deoxycholate) or detergent. . Negative Control: Other gram positivec streptococci . Requirement: 6. Aerococcus. and some species of Pediococcus and Leuconostoc can withstand a higher salt concentration than other gram positive cocci.Salt-Tolerance Test Turbidity (presence of growth) Principle: Enterococcus.5% NaCl Nutrient broth Positive Control: Enterococcus sp. Novobiocin susceptibility Susceptible=presence of ZOI Resistant=absence of ZOI Principle: Presumptive identification of Staphylococcus saprophyicus is accomplished by testing for Novobiocin Susceptibility using 5µg Novobiocin disk. S.saprophyticus is RESISTANT while other Coagulase Negative Staph are Susceptible. Requirement: 5µg Novobiocin disk Resistant: Staphylococcus saprophyticus Susceptible Other Coagulase Negative Staph Biochemical Tests Gram Positive Gram Negative Amino Acid Utilization CARBOHYDRATE UTILIZATION TRIPLE SUGAR IRON (TSI) Decarboxylase test Deaminase test NA and others Lipids and Others Gelatin Liquefaction O-F Test ONPG test IMViC LIA Urease test Nitrate and Nitrite Oxidase Dnase test SIM Malonate test Lipid Hydrolysis TSI A/A, ±gas, ±H2S K/A, ±gas, ±H2S K/K distinguish the members of Enterobacteriaceae from other enteric bacteria by their ability to metabolize glucose, lactose or sucrose and to liberate hydrogen sulfide (H2S) gas. Principle: Acid production when glucose, lactose or sucrose is catabolized. H2S production when thiosulfate is reduced by bacteria. Positive Organisms: Lactose Fermenters and Late Lactose Fermenters 1%) Lactose (1%) Sucrose (1%) 2% Phenol red ( yellow at pH<6. ±H2S K/K Triple Sugar Iron Agar Glucose (0. ±H2S K/A. ±gas. presence of acid) Acid /alkaline slant Acid butt Alkaline slant Alkaline butt Ferrous sulfate . ±gas.8.Composition of TSI Medium Carbohydrates (concentration) Peptone Indicator for acid production Fermenter Nonfermenter Indicator for H2S production A/A. ±H2S K/A. ±gas.Interpretation of TSI results A/A. ±H2S K/K Phenol Red in TSI turns: Yellow(A)=if there is acid production Purple(K)=if no acid produced or if acid is neutralized by peptone products . ±gas. ±H2S K/K GAS is formed= splitting of the TSI agar H2S gas is formed= blackening of agar . ±gas. ±gas.Interpretation of TSI results A/A. ±H2S K/A. ±gas. ±H2S K/K A/A. ±gas.Interpretation of TSI results A/A. ±gas. ±gas = Lactose Fermenters K/A. ±H2S K/A. ± H2S = Non-Lactose Fermenters K/K= Nonfermenters . ±H2S K/K K/K . ±H2S K/A.Interpretation of TSI results A/A. ±gas. ±gas. ±H2S K/A. ±gas.Interpretation of TSI results A/A±gas A/A. ±H2S K/K attacks all sugars or lactose & sucrose only K/A ± gas±H2S LACTOSE FERMENTER Only glucose is fermented NON-LACTOSE FERMENTER . ±gas. ±H2S K/K ? . ±gas. ±H2S K/A. ±gas.Interpretation of TSI results A/A. 2% Open tube: acid Sealed tube: acid Open tube: acid Sealed tube: no acid .Triple Sugar Iron Agar Carbohydrates Glucose (0.1%) (concentration) Lactose (1%) Sucrose (1%) Kliger’s Iron Agar Glucose (0.1%) Lactose (1%) Hugh-Leifson OxidationFermentation Basal Medium (OFBM) Glucose or other carbohydrate being tested (1%) Peptone Fermenter Nonfermenter 2% Acid /alkaline slant Acid butt Alkaline slant Alkaline butt 2% Acid /alkaline slant Acid butt Alkaline slant Alkaline butt 0. Hugh-Leifson OxidationFermentation Basal Medium (OFBM) Fermenter: Open tube: acid Sealed tube: acid Nonfermenter: Open tube: acid Sealed tube: no acid Determines the ability of microorganism to ferment/oxidize specific type of sugars Makes use of: Basal medium without seal Basal medium with seal (mineral oil)-oxidation tube-fermentation tube . Hugh-Leifson OxidationFermentation Basal Medium (OFBM) Fermenter: Open tube: acid Sealed tube: acid Nonfermenter: Open tube: acid Sealed tube: no acid Both Oxidizer Non-oxidizer. and Oxidizer only= Determines the ability of microorganismnonto Fermenter= Obligate fermenter= Facultative ferment/oxidize specific Aerobes type of sugars asaccharolytic Anaerobes Makes use of: Basal medium without seal Basal medium with seal (mineral oil)-oxidation tube-fermentation tube Open tube Acid Sealed tube Acid Open tube Acid Sealed tube Acid Open tube Acid Sealed tube Acid . β-galactoside permease and β-galactosidase Rapid Lactose Fermenters= possess both enzymes Late Lactose Fermenters= possess only β-galactosidase Positive Organisms: Late Lactose Fermenters .ONPG Test Yellow Two enzymes are required to effectively ferment lactose. ONPG Test Yellow Positive Organisms: Late Lactose Fermenters . Decarboxylase Test Purple (indicates decarboxylation) Moeller Decarboxylase base medium Bromcresol and cresol red as pH indicator Medium has to be acidified first (add glucose) Positive Organisms: Klebsiella pneumoniae . Phenylalanine Deaminase Test (PAD) Green (indicates deamination of F) Positive Organisms: differentiates Tribe of Proteae from the rest of Enterobacteriaceae . yellow butt: R/A Positive Organisms: differentiates Tribe of Proteae from the rest of Enterobacteriaceae .Lysine Iron Agar + decarboxylation=K/K±H2S + deamination=R/A LIA is a tubed agar butt/slant (lysine.ferric ammonium citrate and sodium thiosulfate) To determine whether bacteria decarboxylate or deaminate LYSINE Lysine decarboxylation=purple slant and butt: K/K±H2S Lysine deamination=red slant.glucose. with H2S K/K. with H2S K/K R/A DECARBOXYLATION DEAMINATION + - - + + + .K/A. IMViC Test INDOLE TEST METHYL RED AND VOGES PROSKAUER TEST CITRATE TEST Positive Organisms: . ammonia.Indole Test Red Organisms that possess the enzyme tryptophanase are capable of deaminating Windole.pyruvic acid Tryptophan broth is incubated for 48 hrs Xylene and Ehrlich’s reagent (PDAB)is used to detect indole Kovac’s rgt is also used (less sensitive) Positive Organisms: Proteus vulgaris. Providencia rettgeri. Providencia stuartii . Providencia alkalifaciens. Ehrlic’s reagent (PDAB) + Xylene (more sensitive) . Methyl Red Test Red Organisms that possess the enzyme tryptophanase are capable of deaminating Windole. ammonia.pyruvic acid Positive Organisms: Escherichia coli . Voges-ProskauerTest Cherry Red Positive Organisms: Enterobacter aerogenes . . Providencia sp.Citrate Utilization Blue Simmons Citrate Medium(bromthymol blue)green to blue Christensen’s citrate medium(phenol red)—yellow to pink Positive Organisms: Klebsiella pneumoniae. NFO . Enterobacter cloacae. Salmonella typhimurium. Enterobacter aerogenes. Late lactose fermenters.Proteus sp. Enterobacter cloacae.. Salmonella typhimurium. Late lactose fermenters. Providencia sp.Citrate Utilization Blue Positive Organisms: Klebsiella pneumoniae.Proteus sp. NFO . Enterobacter aerogenes. Yersinia enterocolitica.Urease Test Deep Pink pink Positive Organisms: Tribe of Proteeae. Serratia marcescens . . Phenylalanine Deaminase Test (PAD) Green (indicates deamination of F) Positive Organisms: differentiates Tribe of Proteae from the rest of Enterobacteriaceae . Oxidase Test Purple/Lavender Tetramethyl-p-phenylenediamine dihydrochloride (homolog of cytochrome c) Positive Organisms: Pseudomonads (diff. Enterobacteriaceae-negative) . . Rapid Multitest System • API (Analytical Profile Index) • API 20E System – Standardized. of Enterobacteriaceae and other Gram Negative Bacteria .D. miniaturized version of conventional biochemical tests used in the I. . AST . Reasons and Indications for Performing AST • If the isolate is determined to be the probable cause of infection • Susceptibility of the isolate to the AMA is not reliable predicted . Factors to Consider When Determining Whether AST is Warranted • The body site from which the organism was isolated • The presence of other bacteria and the quality of the specimen from which the organism was grown • The host’s status . 5 Turbidity Standard • Inoculum standardization • Barium Sulfate • Turbidity comparable to that of a bacterial susp.=1.5 x 108CFU/mL .McFarland 0. more organism is added and reincubated • Once standardized the inoculum should be used within 15 minutes .• If the bacterial suspension is too dense than the McFarland=add more broth/sterile saline • If the suspension is too light. Types of AST • Broth Dilution – Different concentrations of one AMA against one bacterial isolate – MIC and MBC can be determined • Agar Dilution – One concentration of AMA againts several bacterial isolates (32 in 100mm Petri dish) – MIC only • Disk Diffusion – Kirby Bauer test – Several AMA with standardized concentrations against one isolate . 1-2mL of AMA • MH Broth is used • Standardized Suspension is added to each tube until 1.Broth Dilution • Two-fold serial dilution series.5 x 105 CFU/mL is obtained • Incubated overnight at 35oC • MIC and MBC can be determined . Broth Dilution • The MIC (minimum inhibitory concentration) is determined visually as the lowest concentration that inhibits growth. as demonstrated by absence of turbidity. . . MBC • To get the Minimum Bactericidal Concentration: – Subculture all tubes with no growth into broth/plates – MBC is read as the: “Minimum concentration of AMA with no growth (clear/no visible colonies)” . Agar Dilution Test • Specific volumes of AMA is dispensed into premeasured molten and cooled agar • MHA-aerobic bacteria • MHA + 5% Sheep’s RBC-fastidious bacteria • 1.0 x 104CFU/mL • Drawback: – Shelf life of agar dilution plates is only one week . Agar Dilution Test • MIC is read as the lowest concentration of AMA that inhibits the visible growth of the bacterium (1 or 2 colonies are ignored) . Disk Diffusion Testing • AMA are impregnated onto paper disks 1. Incubated for 16-18 hours @ 35oC 3. I. Measurement is interpreted as S. R. The diameter of the zone of inhibition is measured (mm) 4. . AMA disks are placed on MHA seeded with standardized inoculum 2. 5mg/L Mg++ Minimal or Absent 7.5 x 108CFU/mL MHA 25mg/L Ca++ 12.Standardization VARIABLE Inoculum Medium Ca++ and Mg++content STANDARD 1.2-7.4 3-5 mm Humidified ambient air Thymidine content pH Agar depth Atmosphere . and Enterococci with Vancotransmitted light) and the plate is held against black background Zones of Inhibition is read from back of plate .Standardization VARIABLE Temperature Length of incubation Placement on agar Endpoint measurement STANDARD 350C 16-18 hrs (16-20hrs for broth dil) 12 or fewer disks/150mm plate Reflected light (except for Staph with Oxa and Vanco. • Utilizes a rectangular strip that has been impregnated with the drug to be studiedAfter 24 hours of incubation. E-Test . an elliptical zone of inhibition is produced and the point at which the ellipse meets the strip gives a reading for the minimum inhibitory concentration (MIC) of the drug. 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