Biochemical Identification of Bacteria

Bacterial 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

Serotypic Test
LANCEFIELD CLASSIFICATION SCHEME
SPECIES
Streptococcus pyogenes LANCEFIELD HEMOLYSIS GROUP TYPE ANTIGEN A B

COMMON TERMS
Group A Strep Group B Strep

/NONE
,/NONE

Streptococcus agalactiae S. equisimilis, S. equi subsp. zooepidemicus S. bovis, S. equinus

C D

Group C Strep Nonenterococcus Enterococcus Pneumococcus Viridans Strep

Enterococcus faecalis, E. D faecium, E. durans

S. Pneumoniae Mutans group, Mitis group, Anginosus group

/NONE

Phenotypic tests
Bacterial Hemolysis (Using BAP)
TYPES OF HEMOLYSIS

HEMOLYSIS ALPHA ()
BETA () NONHEMOLYTIC () ALPHA-PRIME () 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

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
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, it is produced by bacteria to protect itself from the hosts defenses.

Reagents: Rabbits 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 -naphthylamine, which is detected in the presence of D-dimethylaminocinnamaldehyde by prodcution of a red color

Reagents: L-pyrrolidonyl--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 -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 -lysin.

Requirement: Isolates of S. agalactiae Isolates of -lysin producing S. aureus Or disk impregnated with -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 organisms 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 5g Novobiocin disk. S.saprophyticus is RESISTANT while other Coagulase Negative Staph are Susceptible.

Requirement: 5g 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

Composition of TSI Medium

Carbohydrates (concentration) Peptone Indicator for acid production Fermenter Nonfermenter Indicator for H2S production

A/A, gas, H2S K/A, gas, H2S K/K

Triple Sugar Iron Agar

Glucose (0.1%) Lactose (1%) Sucrose (1%) 2% Phenol red ( yellow at pH<6.8, presence of acid) Acid /alkaline slant Acid butt Alkaline slant Alkaline butt 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

A/A, gas, H2S K/A, gas, H2S K/K

K/K

Interpretation of TSI results

A/Agas

A/A, gas, H2S K/A, gas, H2S K/K

attacks all sugars or lactose & sucrose only K/A gasH2S

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

Carbohydrates Glucose (0.1%) (concentration) Lactose (1%) Sucrose (1%)

Kligers 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

0.2%
Open tube: acid Sealed tube: acid 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
Acid

Sealed tube
Acid

Open tube Acid

Sealed tube Acid

Open tube
Acid

Sealed tube
Acid

ONPG Test

Yellow

Two enzymes are required to effectively ferment lactose; -galactoside permease and -galactosidase Rapid Lactose Fermenters= possess both enzymes Late Lactose Fermenters= possess only -galactosidase

Positive Organisms:

Late Lactose Fermenters

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

Lysine Iron Agar

+ decarboxylation=K/KH2S + 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/KH2S
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 Ehrlichs reagent (PDAB)is used to detect indole

Kovacs rgt is also used (less sensitive)
Positive Organisms: Proteus vulgaris, Providencia rettgeri, Providencia alkalifaciens, Providencia stuartii

Ehrlics 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

Cherry Red

Positive Organisms: Enterobacter aerogenes

Citrate Utilization

Blue

Simmons Citrate Medium(bromthymol blue)green to blue

Christensens 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 hosts 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% Sheeps 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
Inoculum Medium Ca++ and Mg++content

STANDARD
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
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, and Enterococci with Vancotransmitted light) and the plate is held against black background

Zones of Inhibition is read from back of plate

 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.

E-Test