Biochemical Identification of Bacteria

Methods

Classification

Bacterial Identification

Techniques

Based on Phenotype

Staining, bacterial and

colonial morphology,

hemolytic pattern,

biochemical tests

Based on Serotype

Serological tests

{Lancefield Classification Scheme,

Widal, Weil-Felix Test)

Based on Genotype

Nucleic acid

amplification tests

Genotypic test

• NAAT

Serotypic TestLANCEFIELD CLASSIFICATION SCHEME

SPECIESLANCEFIELD

GROUP ANTIGEN

HEMOLYSIS TYPE

COMMON TERMS

Streptococcus pyogenes Aβ

Group A Strep

Streptococcus agalactiae

Bβ

Group B Strep

S. equisimilis, S. equisubsp. zooepidemicus

C β Group C Strep

S. bovis, S. equinus Dα/NONE

Nonenterococcus

Enterococcus faecalis, E. faecium, E. durans

Dα,β/NONE

Enterococcus

S. Pneumoniae - α Pneumococcus

Mutans group, Mitisgroup, Anginosus group

-α/NONE

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, Voges-Proskauer test)

Phenotypic tests

Basis of Biochemical tests

• Susceptible or resistant to certain AMA (Bacitracin, Optochin, Novobiocin disc)

• Able to tolerate extreme environment (Salt-Tolerance test)

• Able to tolerate or utilize poisons (Cetrimide test)

Biochemical Tests

Gram Negative

Gram Positive

Biochemical Tests

Gram Negative

Gram Positive

Catalasetest

Coagulase

Bacitracindisk

PYR test

HippurateHydrolysis

CAMP test

Bile Esculin

Optochindisk

Salt-Tolerance

NovobiocinDisk

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.

BacitracinSusceptibility

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% BAPBacitracin disk (0.04 units)

Positive Control: Streptococcus pyogenesNegative 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 pyogenesand 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 TestArrowhead-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. agalactiaeIsolates of β-lysin producing S. aureusOr disk impregnated with β-lysin

Positive Control: Streptococcus agalactiae

Negative Control:Other beta-hemolytic Streptococci

Bile Esculin TestBlackening 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 intotheagar 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 SusceptibilitySusceptble 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 TestClear solution (dissolved colonies)

Requirement:Sodium deoxycholate/detergent

Positive Control: Streptococcus pneumoniae

Negative Control: Other alpha-hemolytic streptococci

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.

Salt-Tolerance TestTurbidity (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 susceptibilitySusceptible=presence of ZOIResistant=absence of ZOI

Principle: Presumptive identification of Staphylococcus saprophyicus is accomplished by testing for Novobiocin Susceptibility using 5µg Novobiocindisk. S.saprophyticus is RESISTANT while other Coagulase Negative Staph are Susceptible.

Requirement:5µg Novobiocin disk

Resistant:Staphylococcus saprophyticus

SusceptibleOther Coagulase Negative Staph

Biochemical TestsGram

Negative

CARBOHYDRATE UTILIZATION

TRIPLE SUGAR IRON (TSI)

O-F Test

ONPG test

Amino Acid Utilization

Decarboxylasetest

Deaminasetest

LIA

NA and

others

IMViC

Ureasetest

Oxidase

Dnase test

Lipids and Others

Gelatin Liquefaction

Nitrate and Nitrite

SIM

Malonatetest

Lipid Hydrolysis

Gram Positive

distinguish the members of Enterobacteriaceaefrom 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.

TSIA/A, ±gas, ±H2S K/A, ±gas, ±H2SK/K

Positive Organisms:

Lactose Fermenters and Late Lactose Fermenters

Composition of TSI Medium

A/A, ±gas, ±H2SK/A, ±gas, ±H2SK/K

Triple Sugar Iron AgarCarbohydrates (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 slantAlkaline butt

Indicator for H2S production

Ferrous sulfate

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 resultsA/A, ±gas, ±H2SK/A, ±gas, ±H2SK/K

GAS is formed= splitting of the TSI agar

H2S gas is formed= blackening of agar

Interpretation of TSI resultsA/A, ±gas, ±H2SK/A, ±gas, ±H2SK/K

A/A, ±gas = Lactose Fermenters

K/A, ±gas, ± H2S =

Non-Lactose Fermenters

K/K= Nonfermenters

Interpretation of TSI resultsA/A, ±gas, ±H2SK/A, ±gas, ±H2SK/K

K/K

Interpretation of TSI resultsA/A, ±gas, ±H2SK/A, ±gas, ±H2SK/K

Interpretation of TSI resultsA/A, ±gas, ±H2SK/A, ±gas, ±H2SK/K

A/A±gas

attacks all sugars or lactose & sucrose only

LACTOSE FERMENTERK/A ± gas±H2S

Only glucose is fermented

NON-LACTOSE FERMENTER

Interpretation of TSI resultsA/A, ±gas, ±H2SK/A, ±gas, ±H2SK/K

?

Triple Sugar Iron Agar

Kliger’s Iron Agar

Hugh-LeifsonOxidation-Fermentation Basal Medium (OFBM)

Carbohydrates (concentration)

Glucose (0.1%)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: acidSealed tube: acid

Nonfermenter Alkaline slantAlkaline butt

Alkaline slantAlkaline butt

Open tube: acidSealed 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 Oxidation-Fermentation Basal Medium (OFBM)

Fermenter: Open tube: acidSealed tube: acid

Nonfermenter: Open tube: acidSealed 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 Oxidation-Fermentation Basal Medium (OFBM)

Fermenter: Open tube: acidSealed tube: acid

Nonfermenter: Open tube: acidSealed tube: no acid

Open tube Sealed tube

Acid Acid

Open tube Sealed tube

Acid Acid

Open tube Sealed tube

Acid Acid

Both Oxidizer and

Fermenter=Facultative Anaerobes

Oxidizer only=Obligate Aerobes

Non-oxidizer, non-

fermenter= asaccharolytic

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

ONPG Test Yellow

Positive Organisms:

Late Lactose Fermenters

ONPG Test Yellow

Positive Organisms:

Late Lactose Fermenters

Decarboxylase Test Purple (indicates decarboxylation)

Positive Organisms: Klebsiella pneumoniae

Moeller Decarboxylase base mediumBromcresol and cresol red as pH indicator

Medium has to be acidified first (add glucose)

Phenylalanine DeaminaseTest (PAD)

Green(indicates deamination of F)

Positive Organisms: differentiates Tribe of Proteae from the rest of Enterobacteriaceae

LIA is a tubed agar butt/slant (lysine,glucose,ferricammonium 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

Lysine Iron Agar + decarboxylation=K/K±H2S+ deamination=R/A

Positive Organisms: differentiates Tribe of Proteae from the rest of Enterobacteriaceae

K/K

K/A, with H2S K/K, with H2S

R/A

DECARBOXYLATION + - + -

DEAMINATION - - + +

INDOLE TESTMETHYL RED AND

VOGES PROSKAUER TEST

CITRATE TEST

IMViC Test

Positive Organisms:

Organisms that possess the enzyme tryptophanase are capable of deaminating Windole, ammonia,pyruvicacid

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)

Indole Test Red

Positive Organisms: Proteus vulgaris, Providencia rettgeri, Providencia alkalifaciens, Providencia stuartii

Ehrlic’s reagent (PDAB)+ Xylene(more sensitive)

Organisms that possess the enzyme tryptophanase are capable of deaminating Windole, ammonia,pyruvicacid

Methyl Red Test Red

Positive Organisms: Escherichia coli

Voges-ProskauerTest Cherry Red

Positive Organisms: Enterobacter aerogenes

Simmons Citrate Medium(bromthymol blue)green to blue

Christensen’s citrate medium(phenol red)—yellow to pink

Citrate Utilization Blue

Positive Organisms: Klebsiella pneumoniae, Enterobacter aerogenes, Enterobactercloacae, Late lactose fermenters,Proteus sp, Providencia sp., Salmonella typhimurium, NFO

Citrate Utilization Blue

Positive Organisms: Klebsiella pneumoniae, Enterobacter aerogenes, Enterobactercloacae, Late lactose fermenters,Proteus sp, Providencia sp., Salmonella typhimurium, NFO

Urease Test Deep Pink

Positive Organisms: Tribe of Proteeae, Yersinia enterocolitica, Serratia marcescens

pink

Phenylalanine DeaminaseTest (PAD)

Green(indicates deamination of F)

Positive Organisms: differentiates Tribe of Proteae from the rest of Enterobacteriaceae

Oxidase Test Purple/Lavender

Positive Organisms: Pseudomonads (diff. Enterobacteriaceae-negative)

Tetramethyl-p-phenylenediamine dihydrochloride (homolog of cytochrome c)

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 1.5 x 108CFU/mL

Medium MHA

Ca++ and Mg++content 25mg/L Ca++

12.5mg/L Mg++

Thymidine content Minimal or Absent

pH 7.2-7.4

Agar depth 3-5 mm

Atmosphere Humidified ambient air

Standardization

VARIABLE STANDARD

Temperature 350C

Length of incubation 16-18 hrs (16-20hrs for broth dil)

Placement on agar 12 or fewer disks/150mm plate

Endpoint measurement Reflected light (except for Staph with Oxaand Vanco, and Enterococci with Vanco-transmitted light) and the plate is heldagainst black background

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.