Tests Flashcards
Principle of Catalase
get rid of the peroxide in a cell
Positive results of catalase
immediate appearance of bubbles
Negative result of catalase
no or few bubbles after 20 sec
Errors with catalase
Picking up blood agar with colony. RBC produce catalase and may give a false positive
Catalase procedure
- Remove colony from blood or chocolate plate with wooden stick.
- Apply to dry glass slide
- Add 1 drop of 3% hydrogen peroxide (H2)2) to drop
What does catalase test differentiate?
Differentiates between Staph (positive) from Strep (negative). Enterococcus can produce a pseudocatalase result
Principle of coagulase
identifies whether an organism produces the exoenzyme coagulase, which causes the fibrin of blood plasma to clot
Positive result of coagulase
clot of any size
Negative result of coagulase
no clot
Slide Coagulase Procedure
- Place a drop of coagulase on one side of slide with a drop of saline/water on the opposite
- Remove a colony from plate using a loop or stick and mix in saline/water
- Remove a separate colony from plate and mix with coagulase serum
- Rock slide for 5-10 seconds.
- Within 10 seconds clumps will appear if positive.
Tube Coagulase Procedure
- Add several colonies from plate with loop to coagulase serum in test tube
- Incubate at 35°C for up to 4 hours
- Check for clotting during incubation
- If no clots incubate for 24 hours and recheck
Principle of staph latex agglutination
detects both clumping factor and protein A, which are used to distinguish S. aureus from other staphylococcus species
Positive results of staph latex agglutination
large black clumps of agglutination with loss of black background
Negative results of staph latex agglutination
little or no agglutination (without loss of black background)
staph latex agglutination Procedure
- Pretest colonies with gram stain and catalase.
- Warm reagents to room temperatiure
- Mix latex reagent by mixing
- Use dropper to dispense one drop of reagent onto the test slide
- Select 2-5 isolated colonies from an 18-24 hour culture to dry area inside circle
- Slowly blend the staphylococcus into the reagent using the stick
- Spread mixure over the entire area of the circle
- Circulate the slide for up to 60 seconds
Microdase principle
differentiates staphylococcus from micrococcus
Positive Microdase
blue or purple-blue color development within 2 minutes
Negative Microdase
no color change within 2 minutes
Microdase procedure
- Test should be performed on aerobic, catalase +, gram pos cocci. Colonies should be from a blood agar <24 hours.
- Place disk on clean glass slide using forceps
- Use wooden stick to inoculate the disk with several well isolated colonies.
- Examine for up to 2 minutes for blue color development
Optochin Principle
determines the effect of optochin on an organism. Pneumococci will be lysed but other alpha streptococci will not.
Optochin Positive Result
Zone is ≥ 14mm
Optochin Negative Result
No zone
Optochin Equivocal
Zone is < 14mm. Questionable, perform bile solubility
Optochin Procedure
- Streak colonies onto SBA
- Place optochin disk in upper third of streaked area
- Incubate at 35° in 5% CO2
- Measure zones of inhibition
Bile Solubility Principle
differentiates S. pneumonia from alpha hemolytic streptococci
Bile Solubility Positive
colony disintegrates; an imprint of the lysed colony may remain in the zone
Bile Solubility Negative
intact colony
Bile Solubility Procedure
- After 12-24 hours incubation on SPA, place 1-2 drops of 10% sodium desoxycholate on a well isoled colony
- Gently wash liquid over the colony without dislodging the colony from the agar
- Incubate the plate at 35°-37°C in ambient air for 30 minutes
- Examine for lysis of colony
Bile Solubility Errors
Because of nutritional requirements, some organisms may grow poorly or not at all on this media
Salt Tolerance Test Principle
determines ability of an organism to tolerate high salt concentrations. Differentiates enterococci from non enterococci
Salt Tolerance Test Positive
Visible turbidity in broth with or without a color change
Salt Tolerance Test Negative
No turbidity or color change
Salt Tolerance Procedure
- Inoculate 1-2 colonies from 18-24 hr culture into 6.5% salt tube
- Incubate for 24-48 hours at 35°C
- Check daily for growth
CAMP test Principle
differentiate group B streptococci from other streptococcal species
CAMP test Positive
Enhanced hemolysis in the shape of an arrowhead
CAMP test Negative
No enhanced hemolysis
CAMP test Procedure
- Streak a beta hemolytic S. aureus down the center of a SBA plate
- Streak test organisms perpendicular to the S. aureus streak
- Incubate overnight
CAMP test errors
Small percentage of group A streptococci may have a positive CAMP. Limit test to colonies with characteristic group B strep morphology
Motility (Listeria) Principle
determines if an enteric organism is motile. Organism must have flagella to be motile
Motility Positive
organisms will spread out into the medium from the site of inoculation
Motility Negative
organisms will remain at the site of inoculation
Motility Procedure
- Touch a straight needle to a colony of a young culture on agar medium
- Stab once to a depth of 1/3 to ½ inch in the middle of the tube
- Incubate and examine daily for up to 7 days
Motility Errors:
some organisms will not display sufficient growth in the medium to make an accurate determination. Followup test may be required
PYR Principle
presumptive ID of group A streptococci
PYR Positive
Bright red color within 5 minutes
PYR Negative
No color change or an orange color
PYR Procedure
- Moisten disk without flooding
- Use a wooden stick to put colonies on the PYR disk
- Incubate at room temp for 2 minutes
- Add a drop of detector and observe for a red color within 1 minute
Lancefield Grouping Principle
identify beta hemolytic streptococci into a lancefield group. Specific antibodies on latex particles react with and agglutinates
Lancefield Grouping Positive
2+ or greater reaction observed within 60 seconds
Lancefield Grouping Negative
A uniform or milky appearance with no agglutination
Lancefield Grouping Procedure
- Pick 3-4 colonies from SBA plate
- Rub the colonies on the slide in oval
- Add a drop of latex A to oval and mix with stick
- Repeat for latex B, C, F, or G
- Rock slides back and forth for up to a minute. Observe for agglutination
