biochemical tests (2nd part)

PURPOSE

To determine the deaminase activity using the amino acids phenylalanine or tryptophan.

Only Proteus, Providencia and Morganella species possess the deaminase enzyme.

PRINCIPLE

Deamination of the amino acid results in a colored compound with the addition of 10% ferric chloride

Phenylalanine ----------------PPA + 10% FeCl3 Phenylalanine deaminase green

Tryptophan-------------indole-pyruvic acid +10% FeCl3 Tryptophan deaminase brown

INTERPRETATION

Positive deamination for phenylalanine – intense green color Positive deamination for tryptophan – brown color Negative – slant retains its original color after the addition of ferric chloride

A. Negative – Escherichia coliB. Positive – Proteus vulgaris

A B

Phenylalanine deamination test

PURPOSE:

To determine the ability of the organism to deaminate lysine, decarboxylate lysine and produce H2S.

To identify Salmonella, Proteus, Providencia and Morganella.

COMPOSITION

1. Proteins2. Sugar- Glucose3. Amino acid - Lysine4. Sulfur5. indicators a. ferric ammonium citrate – H2S production b. bromcresol purple – carbohydrate fermentation

PRINCIPLE:

As glucose fermentation occurs, deep of the tube turns yellow.Lysine decarboxylation produces alkaline cadaverine and leads to reversion of the deep from yellow to purple.Lysine deaminatiion occurs in the presence of oxygen (on the slant) and results in the production of a red color.H2S production is noted by a black precipitate in the deep as H2S reacts with ferric ammonium citrate.

INTERPRETATION

Lysine decarboxylation - butt Positive – purple Negative – yellow

Lysine deamination - slant Positive – red Negative –purple

K/K alkaline slant/alkaline butt H2S(-) purple/ purple

Negative deaminationPositive decarboxylationNo blackening of the butt

Escherichia coli

Lysine iron agar

K/K alkaline slant/alkaline butt H2S + purple/purple

Negative deamination Positive decarboxylationWith black precipitate in the butt

Salmonella typhimurium

Lysine iron agar

K/A alkaline slant/acid butt H2S(-) (purple/yellow)

Negative deaminationNegative decarboxylationNo black precipitate in the butt

Shigella flexneri

Lysine iron agar

R/A red slant/acid butt H2S(-) red/yellow

Positive deaminationNegative decarboxylationNo black precipitate in the butt

Proteus vulgaris

Lysine iron agar

PURPOSE:

To determine if a member of the Enterobacteriaceae is capable of utilizing citrate as the sole source of carbon.

Useful in the identification of the lactose fermenting Enterobacteriaceae: Escherichia coli is citrate negative; Enterobacter and Klebsiella are positive

PRINCIPLE

Sodium citrate is the only carbon source in Simmons citrate agar.If the organism can utilize citrate, the sodium citrate is converted to ammonia, which is then converted to ammonium hydroxide.The alkalinity of the compound formed raises the pH of the medium, and the bromthymol blue indicator takes on its alkaline color which is blue.

INTERPRETATION

Positive – growth with an intense blue color on the slant or solely the presence of growth

Negative – absence of growth and no color change in the medium (remains green)

Citrate Utilization test

A. Positive - Klebsiella pneumoniaeB. Negative - Escherichia coli

A B

PURPOSE

To determine the ability of an organism to use acetate as the sole source of carbon.

PRINCIPLE Breakdown of the sodium acetate causes the pH of the medium to shift toward the alkaline range, turning the indicator from green to blue.

INTERPRETATION

Positive – Medium becomes alkalinized(blue) because of the growth of the organism

Negative – no growth or growth with no indicator change to blue

A B

Acetate utilization test

A. Positive - Klebsiella pneumoniaeB. Negative – Escherichia coli

PURPOSE

To determine the ability of an organism to use acetamide as the sole source of carbon.

PRINCIPLE Bacteria that can grow on this medium deaminate acetamide to release ammonia. The production of ammonia results in a pH-driven color change of the medium from green to royal blue.

INTERPRETATION

Positive – deamination of the acetamide resulting in a blue color

Negative – no color change

Acetamide utilization test

A. Positive – Klebsiella pneumoniaeB. Negative – Escherichia coli

PURPOSE:

To differentiate Micrococccus and Stomatococcus from Staphylococcus when combined with other procedures such as the modified oxidase test.

For presumptive identification of Group A streptococcus

Bacitracin(0.04 units) inhibits the growth of Micrococcus and Stomatococcus and Group A streptococcus while having no effect on Staphylo- coccus which is resistant.

PRINCIPLE:

INTERPRETATION

susceptible – zones of inhibition greater than or equal to 10 mm resistant – zones of inhibition less than or equal to 9 mm.

A. Susceptible – Micrococcus and StomatococcusB. Resistant – Staphylococcus epidermidis

A B

Bacitracin susceptibility test

PURPOSE:

To identify the different species of Streptococcus especially Group A and Group B beta hemolytic streptococci.

PRINCIPLE

Group A beta hemolytic streptococci (Streptococcus pyogenes) are susceptible to 0.04 units bacitracin but resistant to 1.25 ug sulfamethoxazole-trimethoprim (SXT)Group B beta hemolytic streptococci – resistant to both bacitracin and SXT

INTERPRETATION

Susceptible: any zone of inhibition around either diskResistant: growth up to the disk(no zone of inhibition

Organism Bacitracin SXT

Group A susceptible resistant Group B resistant resistant Group C,F,G resistant susceptible

PURPOSE

To differentiate the different species of coagulase negative staphylococci.

PRINCIPLE

After incubation with 5 ug of novobiocin, Staphylococcus saprophyticus is not inhibited by the antibiotic whereas Staphylococcus epidermidis are susceptible to novobiocin.

INTERPRETATION:

susceptible – zone greater than 16 mm

resistant – zone diameter less than or equal to 16 mm

A B

Novobiocin susceptibility test

A. Susceptible - Staphylococcus epidermidisB. Resistant - Staphylococcus saprophyticus

PURPOSE

To differentiate Pediococcus from other alpha hemolytic streptococcus.

PRINCIPLE

After incubation with 5 ug of vancomycin, Pediococcus is not inhibited by the antibiotic whereas Viridans streptococcus is susceptible to vancomycin.

INTERPRETATION

Susceptible – zone of inhibition

Resistant – no zone of inhibition

Vancomycin susceptibility test

A. Susceptible - Viridans streptococcus B. Resistant - Pediococcus

A B

PURPOSE

To determine an anaerobe’s inhibition that can be used for presumptive identification based on its characteristic susceptibility pattern to colistin (10 ug), vancomycin(5 ug) and kanamycin(1 mg).

PRINCIPLE Most anaerobes have a characteristic susceptibility pattern to colistin(10 ug), vancomycin( 5 ug), and kanamycin(1 mg) disks.

kanamycin – inhibits facultative gram-negative bacilli vancomycin- inhibits facultative and obligate gram-positive bacteria colistin- inhibits facultative gram-negative bacilli

K Va

Co

INTERPRETATION

Susceptible – zone greater than 10 mmResistant – zone of 10 mm. or less

Antibiotic Disks for the Presumptive Identification of Anaerobes

PURPOSE

To classify bacteria based on their ability to grow in the presence of 6.5% NaCl, a characteristic of certain species of gram positive and gram negative bacilli.

To differentiate the Group D(salt tolerant) from the nonenterococci(intolerant).

PRINCIPLE

Nutrient broth or 6.5%NaClTrypticase broth-salt free medium

Positive equal equal

Negative good very weak

INTERPRETATION

Positive – if growth is equivalent to both media – tolerant of salt

Negative- growth on the salt containing medium is very weak or absent growth in the salt free medium is good - intolerant of salt

Indicator: bromcresol purple Positive: medium turns yellow from purple or the appearance of growth

SALT TOLERANCE TEST

A. Positive - Enterococcus faecalis ( salt tolerant)B. Negative - Streptococcus bovis(salt intolerant)

To distinguish Group D streptococci and Enterococcus species from other Lancefield group of streptococci

based on the organisms ability to grow in 40% bile and to hydrolyze esculin to produce esculitin Esculin reacts with ferric citrate to form a brown black precipitate.

PURPOSE

PRINCIPLE

INTERPRETATION

Positive growth indicates tolerance to 40% bile(40% oxygall) blackening indicates hydrolysis of esculin

Negative lack of growth indicates inability to grow in 40% bile lack of color change indicates inability to hydrolyze esculin

A. Positive - Enterococcus faecalisB. Negative - Streptococcus viridans

Bile esculin agar

A B

PURPOSE

To differentiate Streptococcus pneumoniae from other alpha hemolytic streptococci

PRINCIPLE In the presence of optochin, colonies of Strepto- coccus pneumoniae are selectively lysed indicated by a zone of inhibition after incubation under increased CO2. Other alpha hemolytic streptococci are resistant to optochin.

Positive – zone of inhibition at least 14 mm. in diameter using a 10 ug P disk and at least 10 mm. using a 6 mg P disk

Negative – growth up to the disk or a zone of inhibition less than 14 mm with a 10 ug P disk or less than 10 mm. with a 6 ug P disk

INTERPRETATION

Optochin susceptibility test

A. Positive – Streptococcus pneumoniaeB. Negative – Viridans streptococci

A B

PURPOSE

To differentiate Streptococcus pneumoniae(positive) from other alpha hemolytic streptococci.

PRINCIPLE

Pneumococcal colonies are rapidly lysed by bile or a solution of a bile salt such as sodium deoxycholate. Lysis depends on the presence of an intracellular autolytic enzyme. Bile salts lower the surface tension between the bacterial cell membrane and the medium thus accelerating the organism’s natural autolytic process.

INTERPRETATION

Positive – colony disintegrates; an imprint of the lysed colony may remain within the zone

Negative – intact colonies

BA

Bile solubility test

A. Positive – Streptococcus pneumoniaeB. Negative – Viridans Streptococci

PURPOSE

to demonstrate the phenomena of synergistic hemolysis between group B streptococcus and beta hemolytic Staphylococcus aureus

PRINCIPLE

a characteristic “arrowhead” hemolytic pattern results when the organism is streaked perpen- dicular to beta hemolytic Staphylococcus aureus

INTERPRETATION

Positive – a zone of enhanced hemolysis given by an arrowhead appearance at the junction of the Staphylococcus and Streptococcus – indicative of Group B streptococcus

Negative – no zone of enhanced hemolysis- not indicative of Group B streptococcus

CAMP REACTION

A. Positive - Streptococcus agalactiae B. Negative - Streptococcus bovis

A B