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