ID: Staphylococcus and Micrococcus Flashcards
What are the basic characteristics for staphylococcus
(6)
No haemolysis
No odour
Catalase positive*
Oxidase negative
KoH negative
What Preliminary ID test results indicate Staphylococci
Cocci
Gram positive
Catalase positive
What other genus might by cocci, gram positive, catalase positive other staphylococci?
Micrococci
What are the three clinically significant staphylococci?
Aureus
Epidermidis
Saprophyticus
What five tests would you do to speciate staphylococci?
DN’ase test
Mannitol salt agar
SAIDE agar -> Chromid S.aureus ELITE agar
Novobiocin susceptibility test
Coagulase test -> Staphaurex P test -> protein A test
How is DN’ase used to speciate staphylococci?
What is the principle behind the DN’ase test?
(4)
S. aureus produces DN’ase
DN’ase agar contains 0.2% DNA
After growth, plate is flooded with 1M hydrochloric acid which precipitates DNA and turns the medium cloudy
S. aureus colonies will show a distinct clear zone around inoculum as they have broken down the DNA and is therefore no longer available for precipitation by HCL
How do you carry out a DN’ase test?
(8)
Ask for DN’ase agar containing 0.2% DNA
Split plate down the middle
Split one half into halves again for the positive and negative control
Spot inoculate test strain and controls
Incubate at 37 degrees Celsius
After incubation put int fume cupboard and flood plate with 1M HCL
Allow HCL to permeate for 10 minutes then carefully pour off excess
Observe plate for distinct clear zones around colonies (S. aureus) or cloudy DNA precipitates (non S. Aureus)
What is a positive DN’ase test
Zone of clearance around colonies
What does a positive DN’ase test indicate?
S. aureus
What is a negative DN’ase tes
Cloudy cultures
What does a negative DN’ase test indicate?
S. epidermidis
S. saprophyticus
What are your controls for the DN’ase tes
+ S. aureus
- S. epidermidis or S. saprophyticus
What is the principle behind mannitol salt agar?
(3)
Selective and differential for Staphylococci
High salt concentration of salt (7.5%) selects for staphylococci
Mannitol and pH indicator phenol red is differential and will turn yellow if organism can ferment mannitol
What is a positive result for mannitol salt agar
Yellow coloured colonies
What is a negative result for mannitol salt agar
Pink coloured colonies
What does a yellow coloured colony on mannitol salt indicate?
Pathogenic Staphylococci such as S. aureus
What does a pink coloured colony on mannitol salt indicate?
Non pathogenic staphylococci such as S. epidermidis (most likely) or S. saprophyticus
What is the principle behind SAIDE (Chromid S. aureus Elite agar)?
(2)
SAIDE agar is a chromogenic medium designed for the selective culture of S. aureus
Chromogenic substrates in the agar target specific enzymes in S. aureus to enable the differential growth of S. aureus colonies as pink colonies
What does pink colonies on SAIDE agar indicate?
S. aureus
What is the principle behind the Staphaurex Plus test?
(4)
Protein A is found on about 95% of Staph aureus strains
Protein A has the ability to bind to the Fc portions of IgG
Staphaurex Plus uses latex particles coated with porcine fibrinogen and rabbit IgG including specific polyclonal antibodies raised against capsular polysaccharides of S. aureus
When the reagent is mixed on a card with colonies of S, aureus, rapid agglutination occurs through the reaction between fibrinogen and clumping factor, Fc portion of IgG and Protein A, specific IgG and capsular polysaccharide
What is a positive Staphaurex Plus result
Agglutination
What is a negative Staphaurex Plus result
No agglutination
What does a positive Staphaurex result indicate
S. aureus
What does a negative Staphaurex result indicate
Coagulase negative staph
Non S. aureus staph
S. epidermidis or S. saprophyticus
Why should Staphaurex Plus be used instead of a coagulase test?
(3)
Some strains of methicillin-resistant strains of S. aureus may express undetectable levels of clumping facto and protein A
These MRSA strains possess capsular polysaccharide
The capsule can mask both Protein A and the clumping factor thereby preventing agglutination
How do you carry out a Staphaurex Plus test
(6)
Ask for latex reagent, reaction card, control latex (some sticks to mix)
Shake the latex reagent to mix
Dispense one drop of test latex onto one of the circles and one drop of control reagent onto another circle
Using a loop (wooden end of swab/stick) pick up and smear 2-3mm of Staph growth onto a circle and mix this in the control latex reagent
Using a clean loop/stick proceed in the same way with the Test Latex
Pick up and rock the card for 20 seconds
How do you differentiate S. epidermidis from S. saprophyticus
Test for novobiocin resistance
(Saprophyticus is resistant to novobiocin while epidermidis is not)
How do you test for novobiocin resistance?
Ask for blood agar and novobiocin disc and (bijou bottle? containing nutrient broth - don’t necessarily need this you can just streak out a plate and add disk)
Lawn inoculum
What is the principle of the tube coagulase test?
How do you carry it out in theory?
(5)
Designed to detect both free and bound coagulase
Done by emulsifying 1 colony of test organism in 1ml rabbit plasma and incubating at 37 degrees
Check for clot production at 4 hours and again after incubation
Positive = visible clot formation or lose web of fibrin
Negative = no clot formation, plasma flows freely
