Tests for G+ Flashcards
What does the catalase test try to determine and how?
This test separates staphylococci form streptococci. When an organism possesses the catalase enzyme is combined with hydrogen peroxide (typ. 3%) the result is release of water and oxygen (bubble formation).
Staphylococci –> bubble formation –> POSITIVE
Streptococci –> no bubbles –> Negative
What does the slide and tube coagulase test determine?
Determines if a Staphylococcus ssp. (e.g. confirmed from the Catalase Test) is a S. aureus or not. S. aureus produces an enzyme called Coagulase found in bound and free coagulase forms.
Slide Test –> tests for bound coagulase (clumping factor) on the surface of the bacterial cell wall
Tube Test –> tests for free coagulase
What does the clumping factor (bound coagulase) react with from the rabbit plasma?
The clumping factor will react directly with fibrinogen in plasma resulting in rapid cell agglutination.
Why is the slide test not as accurate as the tube coagulase test?
The slide test is a great, quick and easy test but not as accurate as the tube coagulase because some strains might produce free coagulase and NOT bound coagulase. But if limitations are considered regarding the morphology of the colonies, it is a reliable test for the routine Micro lab.
What does DNAse determine in the lab?
The test determines the presence of the enzyme deoxyribonuclease which splits DNA into nucleotides which helps confirm identification of S. aureus (but also other organisms in other groups).
A test plate with either
a) Methyl green indicator–>white zone around inoculum if POS.
b) Toluidine blue indicator–>pink zone around inoculum if POS
are used.
No colour change in either plate –> NEG.
Why is Novobiocin susceptibility test performed on a coagulase negative specimen?
Used to differentiate between Staphylococcus saprophyticus and the rest of the most common Staphylococcus. Its use is specially useful in urines for pathogen ID because S. sapro is a frequent pathogen compared to other CoNS which tend to be contaminants.
Staph. saprophyticus is Novobiocin Res
Recall:
CoNS = Coagulase negative Staphylococci (M2 Slide 9)
Why is Bacitracin (antimicrobial) susceptibility test performed?
Used to differentiate between Staphylococcus (Res) and Micrococcus (Sens).
(Although colony differences may be sufficient for ID, as micrococcus spp. are typically a strong yellow colour, and it also shows tetrads on gram stains.)
What does the bile solubility test determine? What type of organism is it performed on?
Differentiates between Strep. pneumoniae from other alpha Strep (e.g. Strep viridans Group).
Used only for alpha-hemolytic streptococci.
Principle: Bile Salts (sodium deoxycholate) lyse Streptococcus pneumoniae when added to actively growing cells.
How does the bile solubility test work?
The bile salts in 10% sodium desoxycholate are capable of lowering the surface tension between the bacteria and the BA surface. This accelerates the organism natural autolytic activity.
If the colonies are lysed (colonies disappear) then the interpretation is positive (for streptococcus pneumoniae); intact colonies are interpreted as negative.
What does the PYR test determine?
The PYR test determines if it is positive –> Streptococcus pyogenes and Enterococcus spp. But will be negative for other streptococci.
What is the bile esculin test?
The bile esculin test determines the presence of Streptococcus Group D (Enterococcus + Strep bovis) as all other growth of Streptococcus species are inhibited.
Esculin is a carbohydrate that Group D Strep will hydrolyze to esculetin. This reacts with the iron salts from the ferric citrate in the medium to turn the medium from brown to black.
Describe the Elek Assay for Diphtheria Toxin.
- A paper strip or disk is saturated with diptheria antitoxin is placed in molten agar at 55degC and allowed to sink to the bottom of the plate.
- Agar allowed to solidify at room temp.
- Streaks of unknown test organisms are placed at a right angle to the strip.
- Development of an agar precipitin line at a 45deg within 1-2 days of the incubation at 35degC is a positive result for diptheria toxin. The precipitin line forms between the strip and test organism.
Describe what the Urease Test does.
Test organism is inoculated into this media which contains urea and a pH indicator (phenol red). If the organism possesses urease then it will be able to convert the media from a yellow color to pink, due to production of ammonia (alkaline product).
Procedure:
Pick a colony of the test organism and streak it over the surface of the agar slant using a straight wire
Loosen the cap.
Incubate at 35°C for 24 hours.
Interpretation: Positive: pink to red colour Negative: no colour change Quality control: Positive: Proteus mirabilis ATCC 12453 Negative: Escherichia coli ATCC 25922
Describe the wet mount test.
Wet Mount From broth culture: Place a drop of the suspension on a slide Place coverslip on top of suspension Examine under 40x objective
Describe how tube media is used to help identify listeria.
Tube media
Inoculate tube
Observe for turbidity
Listeria produces unique umbrella morphology
