Tests

Question 1

Principle of Catalase

Answer 1

get rid of the peroxide in a cell

Question 2

Positive results of catalase

Answer 2

immediate appearance of bubbles

Question 3

Negative result of catalase

Answer 3

no or few bubbles after 20 sec

Question 4

Errors with catalase

Answer 4

Picking up blood agar with colony. RBC produce catalase and may give a false positive

Question 5

Catalase procedure

Answer 5

1. Remove colony from blood or chocolate plate with wooden stick.
2. Apply to dry glass slide
3. Add 1 drop of 3% hydrogen peroxide (H2)2) to drop

Question 6

What does catalase test differentiate?

Answer 6

Differentiates between Staph (positive) from Strep (negative). Enterococcus can produce a pseudocatalase result

Question 7

Principle of coagulase

Answer 7

identifies whether an organism produces the exoenzyme coagulase, which causes the fibrin of blood plasma to clot

Question 8

Positive result of coagulase

Answer 8

clot of any size

Question 9

Negative result of coagulase

Answer 9

no clot

Question 10

Slide Coagulase Procedure

Answer 10

1. Place a drop of coagulase on one side of slide with a drop of saline/water on the opposite
2. Remove a colony from plate using a loop or stick and mix in saline/water
3. Remove a separate colony from plate and mix with coagulase serum
4. Rock slide for 5-10 seconds.
5. Within 10 seconds clumps will appear if positive.

Question 11

Tube Coagulase Procedure

Answer 11

1. Add several colonies from plate with loop to coagulase serum in test tube
2. Incubate at 35°C for up to 4 hours
3. Check for clotting during incubation
4. If no clots incubate for 24 hours and recheck

Question 12

Principle of staph latex agglutination

Answer 12

detects both clumping factor and protein A, which are used to distinguish S. aureus from other staphylococcus species

Question 13

Positive results of staph latex agglutination

Answer 13

large black clumps of agglutination with loss of black background

Question 14

Negative results of staph latex agglutination

Answer 14

little or no agglutination (without loss of black background)

Question 15

staph latex agglutination Procedure

Answer 15

1. Pretest colonies with gram stain and catalase.
2. Warm reagents to room temperatiure
3. Mix latex reagent by mixing
4. Use dropper to dispense one drop of reagent onto the test slide
5. Select 2-5 isolated colonies from an 18-24 hour culture to dry area inside circle
6. Slowly blend the staphylococcus into the reagent using the stick
7. Spread mixure over the entire area of the circle
8. Circulate the slide for up to 60 seconds

Question 16

Microdase principle

Answer 16

differentiates staphylococcus from micrococcus

Question 17

Positive Microdase

Answer 17

blue or purple-blue color development within 2 minutes

Question 18

Negative Microdase

Answer 18

no color change within 2 minutes

Question 19

Microdase procedure

Answer 19

1. Test should be performed on aerobic, catalase +, gram pos cocci. Colonies should be from a blood agar <24 hours.
2. Place disk on clean glass slide using forceps
3. Use wooden stick to inoculate the disk with several well isolated colonies.
4. Examine for up to 2 minutes for blue color development

Question 20

Optochin Principle

Answer 20

determines the effect of optochin on an organism. Pneumococci will be lysed but other alpha streptococci will not.

Question 21

Optochin Positive Result

Answer 21

Zone is ≥ 14mm

Question 22

Optochin Negative Result

Answer 22

No zone

Question 23

Optochin Equivocal

Answer 23

Zone is < 14mm. Questionable, perform bile solubility

Question 24

Optochin Procedure

Answer 24

1. Streak colonies onto SBA
2. Place optochin disk in upper third of streaked area
3. Incubate at 35° in 5% CO2
4. Measure zones of inhibition

Question 25

Bile Solubility Principle

Answer 25

differentiates S. pneumonia from alpha hemolytic streptococci

Question 26

Bile Solubility Positive

Answer 26

colony disintegrates; an imprint of the lysed colony may remain in the zone

Question 27

Bile Solubility Negative

Answer 27

intact colony

Question 28

Bile Solubility Procedure

Answer 28

1. After 12-24 hours incubation on SPA, place 1-2 drops of 10% sodium desoxycholate on a well isoled colony 2. Gently wash liquid over the colony without dislodging the colony from the agar 3. Incubate the plate at 35°-37°C in ambient air for 30 minutes 4. Examine for lysis of colony

Question 29

Bile Solubility Errors

Answer 29

Because of nutritional requirements, some organisms may grow poorly or not at all on this media

Question 30

Salt Tolerance Test Principle

Answer 30

determines ability of an organism to tolerate high salt concentrations. Differentiates enterococci from non enterococci

Question 31

Salt Tolerance Test Positive

Answer 31

Visible turbidity in broth with or without a color change

Question 32

Salt Tolerance Test Negative

Answer 32

No turbidity or color change

Question 33

Salt Tolerance Procedure

Answer 33

1. Inoculate 1-2 colonies from 18-24 hr culture into 6.5% salt tube 2. Incubate for 24-48 hours at 35°C 3. Check daily for growth

Question 34

CAMP test Principle

Answer 34

differentiate group B streptococci from other streptococcal species

Question 35

CAMP test Positive

Answer 35

Enhanced hemolysis in the shape of an arrowhead

Question 36

CAMP test Negative

Answer 36

No enhanced hemolysis

Question 37

CAMP test Procedure

Answer 37

1. Streak a beta hemolytic S. aureus down the center of a SBA plate 2. Streak test organisms perpendicular to the S. aureus streak 3. Incubate overnight

Question 38

CAMP test errors

Answer 38

Small percentage of group A streptococci may have a positive CAMP. Limit test to colonies with characteristic group B strep morphology

Question 39

Motility (Listeria) Principle

Answer 39

determines if an enteric organism is motile. Organism must have flagella to be motile

Question 40

Motility Positive

Answer 40

organisms will spread out into the medium from the site of inoculation

Question 41

Motility Negative

Answer 41

organisms will remain at the site of inoculation

Question 42

Motility Procedure

Answer 42

1. Touch a straight needle to a colony of a young culture on agar medium 2. Stab once to a depth of 1/3 to ½ inch in the middle of the tube 3. Incubate and examine daily for up to 7 days

Question 43

Motility Errors:

Answer 43

some organisms will not display sufficient growth in the medium to make an accurate determination. Followup test may be required

Question 44

PYR Principle

Answer 44

presumptive ID of group A streptococci

Question 45

PYR Positive

Answer 45

Bright red color within 5 minutes

Question 46

PYR Negative

Answer 46

No color change or an orange color

Question 47

PYR Procedure

Answer 47

1. Moisten disk without flooding 2. Use a wooden stick to put colonies on the PYR disk 3. Incubate at room temp for 2 minutes 4. Add a drop of detector and observe for a red color within 1 minute

Question 48

Lancefield Grouping Principle

Answer 48

identify beta hemolytic streptococci into a lancefield group. Specific antibodies on latex particles react with and agglutinates

Question 49

Lancefield Grouping Positive

Answer 49

2+ or greater reaction observed within 60 seconds

Question 50

Lancefield Grouping Negative

Answer 50

A uniform or milky appearance with no agglutination

Question 51

Lancefield Grouping Procedure

Answer 51

1. Pick 3-4 colonies from SBA plate 2. Rub the colonies on the slide in oval 3. Add a drop of latex A to oval and mix with stick 4. Repeat for latex B, C, F, or G 5. Rock slides back and forth for up to a minute. Observe for agglutination