Module 8 Flashcards
Catalase test
Staphylococcus and streptococcus Genus test
detects the enzyme catalase which causes breakdown of H2O2 into water and oxygen
Bubbles of oxygen = positive test
Nichrome wire or wooden sticks used
3 groups of bacteria that Catalase test is useful for
Differentiating Staphylococci from Streptococci
Species identification of Mycobacterium
Identification of anaerobes
Reagent used in catalase test
3% hydrogen peroxide
What causes H2O2 to break down
exposure to light and warm temps
Do QC daily or per shift
What happens if decomposed H2O2 is used for catalase test
Tests would be falsely negative as the reagent is actually just water
3 methods of the Catalase test
Rapid tube method
Rapid slide test
Overnight culture method
Catalase test: Rapid tube method
Control: Dip wire/stick into H2O2 and observe for bubbles
Should show no bubbles = used as negative control
Pick up portion of colony, immerse in small test tube with H2O2
Positive test = bubbles form
Negative test = no bubbles
Catalase test: Rapid Slide Test method
Control: sterile stick for negative control
Place drop of H2O2 on glass slide
Pick up small portion of colony on stick and immerse in drop
Pos test = bubbles
neg test = no bubbles
Catalase test: Overnight culture method
Pour 1mL of H2O2 reagent over 24hr growth on a nutrient agar slant or plate Observe for bubbles Bubbles = pos test no bubbles = neg test May also be done with 24hr broth culture
Use of platinum wires in catalase test
DO NOT USE
PLAT = BAD
platinum may cause breakdown of H2O2 with formation of bubbles (false positive result)
Why to be careful when taking colonies off blood agar plates for catalase test
blood contains the enzymes catalase
If you accidentally scrape the plate when picking up a colony, the H2O2 may react with the agar to produce a FALSE POSITIVE
This can be avoided by using a chocolate agar plate
How old should cultures be when doing a catalase test
incubated for 18-24hrs
Older cultures may lose catalase activity resulting in FALSE NEGATIVE
Controls for Catalase test
Positive: Any staphylococcus species
Negative: Any streptococcus species from blood agar
Nitrate Reduction test
determines ability of an organism to reduce nitrate
Many different forms of reduced nitrogen, therefore the medium is tested for UNREDUCED nitrate
If unreduced nitrate is found in the medium, nitrate reduction has NOT taken place and the test result is NEGATIVE
4 groups that the Nitrate Reduction test is useful for
Enterobacteriaceae
non fermentative gram negative bacilli
Separation of streptococci and staphylococci
anaerobes
Durham tube
“upside-down barbie test tube”
Nitrogen gas collects in tube in Nitrate reduction test
2 reagents required for Nitrate Reduction test
Reagent A: sulfanilic acid in acetic acid
Reagent B: alpha naphthylamine in acetic acid (stored in brown bottle/fridge)
End products for Nitrate reduction test
test medium inoculated/incubated for 18-24hrs
Checked for growth (cloudy) and for gas in tube
Gas in tube is assumed to be nitrogen gas and test is POS
No gas in tube? equal amounts of reagent A and B are added
Red color = presence of nitrite in the medium (POS RESULT FOR STAPH)
No red color after adding A and B?
Add zinc dust
Red color with zinc dust indicated unreduced nitrate remains in the medium = NEG RESULT (zinc reduces the unreduced nitrate present to nitrite which then reacts with reagents A and B that were previously added)
No color after zinc? Nitrate has been reduced beyond nitrite to one of the other end products = POS RESULTS
Quality control for Nitrate reduction test
Pos = Staphylococcus aureus (nitrite endproduct) Neg= Streptococcus species
Coagulase Test
Tests for bacterial enzyme, coagulase
2 forms of coagulase
Bound coagulase or clumping factor
Free coagulase
Bound coagulase
AKA clumping factor Attached to bacterial cell wall Not found in extracellular fluid Acts of fibrinogen in plasma to form fibrin Detected in the SLIDE coagulase test
clumping = pos test
Free coagulase
extracellular enzyme found in fluids and culture filtrates
Produces a thrombin-like substance from plasma which then converts into a fibrin clot
Detected in the TUBE coagulase test
Reagents used in coagulase test
Human or rabbit plasma Rabbit does not need to be screened for hep/HIV Lyophilized form Refrigerated NOT BROKEN DOWN BY LIGHT
Anticoagulant used in coagulase test
heparin
EDTA
Not sodium citrate (contaminating bacteria may use it as a nutrient causing false clotting in tube = false positive)
Slide coagulase test procedure
for bound coagulase
Place drop of saline on slide
Emulsify several colonies of Staphylococci to give THICK emulsion
Stir in loop of plasma and examine for macroscopic clumping or agglutination
Autoagglutination in coagulase test
when bacteria clump with sterile saline before plasma is added
If this happens, slide coagulase cannot be performed and a tube coagulase is required
Most common error in performing slide coagulase
making too weak of a bacterial suspension
Will give false negative result
Tube coagulase test procedure
0.5ml plasma in small tube (may be diluted 1:4 for test)
Emulsify a portion of a colony in the plasma
Incubate at 35C for 4 hours
If negative, incubate at RT for 24hrs and examine
Formation of a partial or complete clot = positive
Incubation temp for tube coagulase
incubate at 35C
Temps over 35C may prevent clot formation in some strains of S. aureus = false negative tube coagulase
Excess agitation in coagulase incubation =
prevent clot formation
False negative
Over incubation in coagulase test
Full 24hrs required for clot to form
BUT Incubation should not exceed this time
Staphylokinase
produced by S. aureus that may cause clot dissociation on prolonged incubating
Causes false negative
Use of citrated plasma in coagulase test
DO NOT use
bacteria use citrate was nutrient which may use up the anticoagulant causing the plasma to clot and a false positive test result
* staphylococci do not use citrate but other bacteria may
Use of serum in coagulase test
DO NOT use
serum is plasma minus fibrinogen
use will cause false neg/no clot formation
QC for coagulase test
Positive = Staphylococcus aureus Negative = Staphylococcus epidermidis
DNase test
Determines if bacteria produce enzymes that will depolymerize (unwind) DNA
*virulence factor for bacteria
DNase test clinical applications
Differentiate S. aureus from other Staphylococci
Differentiate Moraxella catarrhalis from Neisseria species
Differentiate Serrate species from other Enterbacteriaceae
Differentiate Xanthomonas maltophilia from other non-fermentative gram neg rods
Oligonucleotides
final product after DNase from bacteria has unwound DNA in the host cell
3 ways to test for DNase
Hydrochloric acid
Methyl Green
Toluidine Blue
Hydrochloric acid for DNase test
Inoculate a plate containing DNA with he bacterium in question
Incubate overnight, then flood plate with HCl
Whole/intact DNA is precipitated by HCl = cloudy appearance = Negative DNase
Oligonucleotides are insoluble in HCl = clearing around bacterial growth = positive DNase
Methyl Green for DNase test
Inoculate Methyl Green Dnase agar with bacterium
Incubate and observe for clear areas around colonies
Green = whole/intact DNA = Negative DNase Colorless = oligonucleotides present = positive DNase
Store plates in dark
Toluidine Blue for DNase test
Inoculate DNase agar with bacterium and incubate overnight
Flood plate with 0.1% Toluidine blue dye after growth
Blue= whole/intact DNA = Negative DNase
Pink = oligonucleotides = Positive DNase
QC for DNase test
Pos = S. aureus, Serratia marcescens Neg = S. epidermidis, E. coli
Why is toluidine blue NOT incorporated into medium
it may inhibit growth of some bacteria
Beta-Lactamase Test
detect presence of beta-lactamase enzymes that inactivate certain beta-lactam antibiotics
*considered virulence factor
2 groups of antibiotics with beta-lactam rings
penicillins and cephalosporins
*Only those with EXPOSED B-lactam rings are susceptible to b-lactamase
These include: Pen G (benzylpenicillin), Ampicillin, Carbenicillin and a few cephalosporins
Induced B-bactamase
Enzymes in bacteria can be induced by growing up organism in the presence of b-lactam antibiotics (eg. oxacillin disc)
Bacterial growth taken from edge of disc and tested with B-lactamase reagent
3 methods of testing for B-lactamase
Acidimetric method
Idometric method
Chromogenic Cephalosporin test
Acidimetric Method for B-lactamase
Rub bacterial growth on a moistened strip (combined strip with benzylpenicillin and a pH indicator) and observe for color change indicating acid
Can take up to 30 min
Hydrolysis of b-lactam ring by b-lactamase yields an acid pH
Iodometric Method for B-lactamase
Uses combo of penicillin, starch and iodine
When penicillin is hydrolyzed to penicilloic acid by B-bactamase, the acid converts iodine to iodide with a subsequent loss of purple color
Purple + Bacterical B-lactamase enzyme = no purple (pos result)
Performed by preparing filter paper strips soaked in benzylpenicillin and starch
Iodine added to give deep purple color
Test colonies rubbed on strip and clear area indicated B-lactamase
Up to 30 min for reaction
Chromogenic Cephalosporin Test for B-lactamase
NITROCEFIN - cephalosporin with b-lactam ring that is hydrolyzed by b-lactamse
Yellow in color but RED when hydrolyzed
Commercial strip is moistened and colonies are rubbed on the surface RED = POS YELLOW = NEG Up to 15 min Nitrocefin kept away from light Store in drawer while waiting the 15 min
Most sensitive b-lactamase test method
Chromogenic cephalosporin test
uses Nitrocefin
