(Laboratory) Identification of Streptococcus and Enterococcus Flashcards
List down the step-by-step procedure in the Catalase Test of Streptococcus/Enterococcus:
- Place 1 drop of hydrogen peroxide reagent in a glass slide
- Emulsify a colony from the plate using applicator stick
- Observe
List down the step-by-step procedure in the Gram Staining of the Streptococcus/Enterococcus:
- Using a sterile loop, place a loopful of saline into a labelled slide
- Emulsify a colony from Plate into the saline
- Air dry and heat fix
- Stain with gram stain
- View under microscope
List down the step-by-step procedure in the Bacitracin disc test (Taxo A) of Streptococcus/Enterococcus:
- Make a bacterial suspension (pure inoculum) by placing 3-5 colonies from plate into sterile distilled water. Match the turbidity with the MacFarland standard
- Using lawn culture method, streak the pure inoculum into a blood agar plate
- After drying for 10 minutes, place the 0.04 units bacitracin disc into the blood agar plate using sterile forceps
- Incubate
List down the step-by-step procedure in the CAMP Test for Streptococcus/Enterococcus:
- Inoculate beta-toxin producing S. aureus straight on the center of BAP
- Using bacteria from plate, inoculate a straight line at right angle to Staphylococcus streak. Make sure the two streak lines do not touch.
- Incubate at 35 C for 18 hours in a candle jar
- Observe
List down the step-by-step procedure in the Colonial Morphology of Streptococcus/Enterococcus:
- Using multiple interrupted streak, inoculate a BAP with bacteria from plate.
- Incubate for 18-24 hours
List down the step-by-step procedure in the Hippurate Hydrolysis:
- Inoculate bacteria on sodium Hippurate substrate to produce a milky suspension
- Incubate at 35 C for 2 hours
- Add 0.2 mL ninhydrin reagent and further incubate for 15 minutes
List down the step-by-step procedure in the Bile Esculin Test:
- Using multiple interrupted streak, inoculate a BEA plate with bacteria from plate.
- Incubate for 18-24 hours
List down the step-by-step procedure in the Growth on 6.5% NaCl:
- Inoculate loopful of bacteria to 6.5% NaCl broth
- Incubate at 35 C for 18 hours
Gram Stain Morphology of Streptococcus/Enterococcus:
Gram-positive cocci in pairs or chains
What is the Catalase Test Result of Enterobacteriaceae:
Catalase Negative
No Effeverscence
A test that differentiates Group A Streptococci from other Streptococci
Bacitracin Susceptibility Test (Taxo A)
What is the positive result in Bacitracin Susceptibility Test (Taxo A)?
Zone of Inhibition
Group A Strep
What is the negative result in Bacitracin Susceptibility Test (Taxo A)?
Negative Zone of Inhibition
Other Beta-hemolytic Streptococci spp.
Test for Group B Streptococci except for Hippurate Test:
CAMP Test
Positive Result in CAMP Test:
Arrowhead hemolysis [Group B Strep] or bowtie appearance [S. agalactiae]
CAMP means:
Christie, Atkins, Munch and Petersen
A diffusible extracellular hemolytic heat stable protein
CAMP Factor
Test for Group A and Group D Streptococci
PYR Hydrolysis Test
Positive Result in the PYR Hydrolysis Test:
Cherry Red/Pink
Substrate of the PYR Hydrolysis Test:
L-pyrrolidonyl-beta-napthylamide
Reagent of the PYR Hydrolysis Test:
p-dimethyl-aminocinnamaldehyde
Enzyme of PYR Hydrolysis Test:
Pyrroglutamylamino peptidase or Pyrrolidomylaryl amidase
The only species of streptococcus that is PYR positive
Streptococcus pyogenes
A test used to detect the ability of bacteria to hydrolyze substrate hippurate into glycine and benzoic acid by the action of the hippuricase enzyme present in the bacteria.
Hippurate Hydrolysis Test
A constitutive enzyme that hydrolyzes the substrate hippurate to produce the amino acid glycine
Hippuricase
Medium of the Hippurate Test:
Broth with Hippurate
Reagent Indicator in the Hippurate Test
Ninhydrin
Substrate of the Hippurate Test
Sodium Hippurate
What is the positive result of the Hippurate Hydrolysis Test?
Purple Color
Streptococcus agalactiae
_____ is detected by the oxidation with Ninhydrin which results in the production of a deep purple.
Glycine
A test that differentiate Group D from Beta-hemolytic
Bile Esculin Test
Enzyme of the Bile Esculin Test
Esculinase
Media used for the Bile Esculin Test
Bile Esculin Agar
What is the color of the Bile Esculin Agar?
Slightly Brown in color
Substrate used in the Bile Esculin Test
Esculin
What is the positive result of the Bile Esculin Test?
Blackening of Medium
Group D
Further test from Bile Esculin Test
Salt Tolerance Test (6.5% NaCl)
What is the negative result of the Salt Tolerance Test (6.5% NaCl)?
Clear/Transparent
Non-enterococci
What is the positive result of the Salt Tolerance Test (6.5% NaCl)?
Turbid
Enterococci
Media used in the Salt Tolerance Test (6.5% NaCl)
6.5% NaCl Broth
Substrate in the Leucine Aminopeptidase Test
Leucine-β-naphthylamide → β-naphthylamine
Reagent in Leucine Aminopeptidase Test
Paradimethylaminocinnamaldehyde reagent
Positive result in the Leucine Aminopeptidase Test
Red Color
Viridans Streptococci
A susceptibility test in conjunction with bacitracin is used for the presumptive identification of beta hemolytic Streptococci on blood agar.
SXT Susceptibility Test
What specie/s is resistant to SXT Susceptibility Test?
Group A & B Strep
What specie/s is susceptible in the SXT Susceptibility Test?
Other Strep spp.
A test that detect carbohydrate component of the cell wall of Streptococci
Serological Test
Principle of Serological Tests
Antigen (from colonies) - Antibody (from Reagent) Reaction
Positive result in the Serological Tests
Agglutination/clumping [Group A Strep]
Negative result in the Serological Tests
No agglutination [S. agalactiae]
A rapid latex test system for use in the qualitative detection and identification of the lancefield group of Streptococci.
Serological Tests
Incubation period of CAMP Test
35 C for 18 hours
How many units of Bacitracin Disc is placed into plate?
0.04 units
Incubation period of CAMP Test
35 C for 18 hours
How many units of Bacitracin Disc is placed into plate?
0.04 units
Incubation period of Hipurate Hydrolysis Test
35 C for 2 hours and then after adding 0.2 mL ninhydrin reagent, further incubate for 15 minutes
How many ml of ninhydrin reagent is added into the tube for Hippurate Hydrolysis Test?
0.2 mL of ninhydrin reagent
Incubation period for the Bile Esculin Test
18-24 hours
Incubation period for the 6.5% NaCl growth
35 C for 18 hours