Biochemical Identification of Bacteria
Bacterial Identification Techniques
Classification
Methods
Based on Genotype
Nucleic acid amplificatio n tests
Based on Serotype
Serological tests {Lancefield Classificatio n Scheme, Widal, WeilFelix Test)
Based on Phenotype
Staining, bacterial and colonial morphology, hemolytic pattern,
biochemica l tests
Genotypic test • NAAT
Serotypic Test LANCEFIELD CLASSIFICATION SCHEME SPECIES
LANCEFIELD HEMOLYSIS GROUP TYPE ANTIGEN
Streptococcus pyogenes
A
Streptococcus agalactiae
B
S. equisimilis, S. equi subsp. zooepidemicus
C
S. bovis, S. equinus
D
β β β α/NONE
Enterococcus faecalis, E. D faecium, E. durans
α,β/NONE
S. Pneumoniae
-
α
Mutans group, Mitis group, Anginosus group
-
α/NONE
COMMON TERMS Group A Strep Group B Strep
Group C Strep Nonenterococcus Enterococcus Pneumococcus Viridans Strep
Phenotypic tests • Bacterial Hemolysis (Using BAP) TYPES OF HEMOLYSIS
HEMOLYSIS
DESCRIPTION
ALPHA (α)
Partial lysis of RBC around colony
BETA (β)
Complete lysis of RBC around colony
NONHEMOLYTIC (γ)
No lysis of RBC
ALPHA-PRIME (α’) OR WIDE ZONE
Small area of intact RBC around colony surrounded by a wider zone of complete hemolysis
Hemolytic Patterns
Phenotypic tests Basis of Biochemical tests • Bacteria are able to release enzymes (catalase, coagulase, urease, and other hydrolysis tests) • Metabolize different substrates (CHO, CHON, Lipids, NA) • Metabolic pathway (Methyl Red Test, VogesProskauer test)
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
Coagulase
Bile Esculin
Bacitracin disk
Optochin disk
PYR test
SaltTolerance
Hippurate Hydrolysis
Novobiocin Disk
Gram Negative
Gram Positive
Catalase test
Bubble formation/effervescence
Principle:
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, it is produced by bacteria to protect itself from the host’s defenses.
Reagents: Rabbit’s plasma (Citrate/EDTA)
Positive Control: Staphylococcus aureus
Negative Control: other species of Staph.
Bacitracin Susceptibility
Any Zone of Inhibition is interpreted as SUSCEPTIBLE
Principle: Group A Strep. Are susceptible to low levels of Bacitracin, whereas other Groups are resistant. Susceptibility to Bacitracin presumptively identifies Streptococcus pyogenes.
Reagents: 5% BAP Bacitracin disk (0.04 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
Hippurate Hydrolysis Test
Purple-colored complex
Principle: Hippuricase hydrolyzes hippurate/ic acid to form sodium benzoate and glycine. Subsequent addition of Ninhydrin yields a purple-colored product. Used to differentiate S. agalactiae from other β-hemolytic streptococci.
Reagents: Sodium hippurate (substrate) Ninhydrin (indicator)
Positive Control: Streptococcus agalactiae
Negative Control: Other beta-hemolytic Streptococci
CAMP Test
Arrowhead-shaped area of enhanced hemolysis where the two streaks (staphylococcal and streptococcal) approach each other.
Principle: S.agalactiae produces CAMP Factor that enhances the lysis of sheep RBC by staphylococcal β-lysin.
Requirement: Isolates of S. agalactiae Isolates of β-lysin producing S. aureus Or disk impregnated with β-lysin
Positive Control: Streptococcus agalactiae
Negative Control: Other beta-hemolytic Streptococci
Bile Esculin Test
Blackening of the agar slant (Esculetin combines with Ferric Citrate forming black complex.)
Principle: Group D strep and Enterococcus grow in the presence of bile and also hydrolyzes esculin to esculetin and glucose. 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
Optochin Susceptibility
Susceptble if: ZOI= >14mm (6mm disk) ZOI=> 16mm (10mm disk)
Principle: Ethylhydrocuprein hydrochloride (optochin) inhibits the growth of S. pneumoniae.
Requirement: Disk impregnated with Optochin (ethylhydrocuprein hydrochloride) CO2 incubator
Positive Control: Streptococcus pneumoniae
Negative Control: Other alpha-hemolytic streptococci
Bile Solubility Test
Clear solution (dissolved colonies)
Principle: Under the influence of a bile salt (sodium deoxycholate) or detergent, the organism’s cell wall lyses during cell division. A suspension of S. 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.
Requirement: Sodium deoxycholate/detergent
Positive Control: Streptococcus pneumoniae
Negative Control: Other alpha-hemolytic streptococci
Salt-Tolerance Test
Turbidity (presence of growth)
Principle: Enterococcus, Aerococcus, and some species of Pediococcus and Leuconostoc can withstand a higher salt concentration than other gram positive cocci.
Requirement: 6.5% NaCl Nutrient broth
Positive Control: Enterococcus sp.
Negative Control: Other gram positivec streptococci
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 Negative
Gram Positive
Amino Acid Utilization
CARBOHYDRATE UTILIZATION
TRIPLE SUGAR IRON (TSI)
Decarboxylase test
O-F Test
Deaminase test
NA and others
ONPG test
Lipids and Others
IMViC
Gelatin Liquefaction
Urease test
Nitrate and Nitrite
Oxidase
SIM
Dnase test
Malonate test
LIA
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
Composition of TSI Medium
A/A, ±gas, ±H2S K/A, ±gas, ±H2S K/K
Triple Sugar Iron Agar Carbohydrates (concentration)
Glucose (0.1%) Lactose (1%) Sucrose (1%)
Peptone
2%
Indicator for acid production
Phenol red ( yellow at pH<6.8, presence of acid)
Fermenter
Acid /alkaline slant Acid butt
Nonfermenter
Alkaline slant Alkaline butt
Indicator for H2S production
Ferrous sulfate
Interpretation of TSI results
A/A, ±gas, ±H2S K/A, ±gas, ±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
Interpretation of TSI results
A/A, ±gas, ±H2S K/A, ±gas, ±H2S K/K
GAS is formed= splitting of the TSI agar H2S gas is formed= blackening of agar
Interpretation of TSI results
A/A, ±gas, ±H2S K/A, ±gas, ±H2S K/K
A/A, ±gas = Lactose Fermenters K/A, ±gas, ± H2S =
Non-Lactose Fermenters
K/K= Nonfermenters
Interpretation of TSI results
K/K
A/A, ±gas, ±H2S K/A, ±gas, ±H2S K/K
Interpretation of TSI results
A/A, ±gas, ±H2S K/A, ±gas, ±H2S K/K
A/A±gas
attacks all sugars or lactose & sucrose only K/A ± gas±H2S
LACTOSE FERMENTER
Only glucose is fermented
NON-LACTOSE FERMENTER
Interpretation of TSI results
A/A, ±gas, ±H2S K/A, ±gas, ±H2S K/K
?
Triple Sugar Iron Agar
Kliger’s Iron Agar
Hugh-Leifson OxidationFermentation Basal Medium (OFBM)
Carbohydrates Glucose (0.1%) (concentration) Lactose (1%) Sucrose (1%)
Glucose (0.1%) Lactose (1%)
Glucose or other carbohydrate being tested (1%)
Peptone
2%
2%
0.2%
Fermenter
Acid /alkaline slant Acid butt
Acid /alkaline slant Acid butt
Open tube: acid Sealed tube: acid
Nonfermenter
Alkaline slant Alkaline butt
Alkaline slant Alkaline butt
Open tube: acid Sealed tube: no acid
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
Sealed tube
Open tube
Sealed tube
Open tube
Sealed tube
Acid
Acid
Acid
Acid
Acid
Acid
ONPG Test
Yellow
Two enzymes are required to effectively ferment lactose; 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
Positive Organisms:
Late Lactose Fermenters
Yellow
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
Lysine Iron Agar
+ decarboxylation=K/K±H2S + deamination=R/A
LIA is a tubed agar butt/slant (lysine,glucose,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, yellow butt: R/A
Positive Organisms: differentiates Tribe of Proteae from the rest of Enterobacteriaceae
K/A, with H2S
K/K, with H2S
K/K R/A
DECARBOXYLATION
DEAMINATION
+ -
-
+ +
+
IMViC Test
INDOLE TEST METHYL RED AND VOGES PROSKAUER TEST
CITRATE TEST Positive Organisms:
Indole Test
Red
Organisms that possess the enzyme tryptophanase are capable of deaminating Windole, ammonia,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 alkalifaciens, Providencia stuartii
Ehrlic’s reagent (PDAB) + Xylene (more sensitive)
Methyl Red Test
Red
Organisms that possess the enzyme tryptophanase are capable of deaminating Windole, ammonia,pyruvic acid
Positive Organisms: Escherichia coli
Voges-ProskauerTest
Positive Organisms: Enterobacter aerogenes
Cherry Red
Citrate Utilization
Blue
Simmons Citrate Medium(bromthymol blue)green to blue
Christensen’s citrate medium(phenol red)—yellow to pink
Positive Organisms: Klebsiella pneumoniae, Enterobacter aerogenes, Enterobacter cloacae, Late lactose fermenters,Proteus sp, Providencia sp., Salmonella typhimurium, NFO
Citrate Utilization
Blue
Positive Organisms: Klebsiella pneumoniae, Enterobacter aerogenes, Enterobacter cloacae, Late lactose fermenters,Proteus sp, Providencia sp., Salmonella typhimurium, NFO
Urease Test
Deep Pink
pink
Positive Organisms: Tribe of Proteeae, Yersinia enterocolitica, 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, miniaturized version of conventional biochemical tests used in the I.D. of Enterobacteriaceae and other Gram Negative Bacteria
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
McFarland 0.5 Turbidity Standard • Inoculum standardization • Barium Sulfate • Turbidity comparable to that of a bacterial susp.=1.5 x 108CFU/mL
• If the bacterial suspension is too dense than the McFarland=add more broth/sterile saline • If the suspension is too light, more organism is added and reincubated
• Once standardized the inoculum should be used within 15 minutes
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
Broth Dilution • Two-fold serial dilution series, 1-2mL of AMA • MH Broth is used • Standardized Suspension is added to each tube until 1.5 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. AMA disks are placed on MHA seeded with standardized inoculum 2. Incubated for 16-18 hours @ 35oC 3. The diameter of the zone of inhibition is measured (mm) 4. Measurement is interpreted as S, I, R.
Standardization VARIABLE
STANDARD
Inoculum Medium Ca++ and Mg++content
1.5 x 108CFU/mL MHA 25mg/L Ca++ 12.5mg/L Mg++ Minimal or Absent 7.2-7.4 3-5 mm Humidified ambient air
Thymidine content pH Agar depth Atmosphere
Standardization VARIABLE
STANDARD
Temperature Length of incubation Placement on agar
350C
Endpoint measurement
Reflected light (except for Staph with Oxa and Vanco, and Enterococci with Vancotransmitted light) and the plate is held against black background
16-18 hrs (16-20hrs for broth dil)
12 or fewer disks/150mm plate
Zones of Inhibition is read from back of plate
E-Test
• Utilizes a rectangular strip that has been impregnated with the drug to be studiedAfter 24 hours of incubation, 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.