Gram positive cocci

Question 1

staphylococcus

Answer 1

-are gram-positive cocci that form into clusters and are catalase positive
-have to perform a certain test to differentiate between staph species
ex: staph aureus: coagulase-positive, often pathogenic
ex: coagulase-negative staphylococcus: often a part of normal flora, may be pathogenic

Question 2

Staphylococcus Aureus

Answer 2

-more pathogenic organism
-it is distinguished by hemolysis of red cells in blood agar plate resulting in clear zones around opaque creamy white colonies

Question 3

Coagulase Negative staph

Answer 3

-generally considered to be normal flora unless isolated from sterile body sites
-it is distinguished by the absence of red cell hemolysis on the blood agar plate and white opaque colonies

Question 4

Catalase test: staph identification

Answer 4

-a small amount of growth from an agar plate is placed on a glass slide
-a drop of 3% H2O2 is placed on the growth
-the presence of strong bubbles is a positive test indicating staph species

Question 5

Slide coagulase: staph identification

Answer 5

-detects bound coagulase (clumping factor) which is attached to the cell wall
-converts fibrinogen to fibrin clots directly
-fibrin strands are formed between bacterial cells causing clumping
-a negative result may need to be confirmed with a tube coagulase test

Question 6

Steps to a slide coagulase test

Answer 6

1. place a drop of coagulase rabbit plasma and a drop of saline on a glass slide
2. a colony of staph is mixed with each drop
3. the presence of clumping in the plasma along with the absence of clumping in the saline is positive for staph aureus
   -the absence of clumping in both drops is a negative result

Question 7

Tube coagulase: staph identification

Answer 7

-a tube of coagulase rabbit plasma is inoculated with a colony of staph species
-the tube is incubated overnight (24 hours)
-staph aureus will clot the plasma- a positive result
-the coagulase plasma will remain unchanged if inoculated with coag neg staph

Question 8

Novobiocin: staph identification

Answer 8

-novobiocin disks are used to distinguish staph saprophyticus from another coag-negative staph in urine cultures, where staph saprophyticus may be pathogenic in females
-S. saprophyticus has a similar colonial appearance but is usually brighter white
-a novo disk is placed on broth inoculated Mueller-Hinton plate
-after overnight incubation, the zone size is measured at 17mm or less (resistant) is a positive result for S. saprophyticus

Question 9

Micrococcaciae

Answer 9

-Gram-positive cocci
-no longer classified with the staph spp.
-rarely cause infection except in immunocompromised patients and endocarditis
-they are distinguished by tetrads- on the gram stain a yellow colony color
-furazolidone (FX) susceptibility test can be used to distinguish micrococcus from staph species

Question 10

Furazolidone (FX)

Answer 10

susceptibility test can be used to distinguish micrococcus from staph species
-micrococcus is resistant
-staph species is sensitive

Question 11

Streptococcus

Answer 11

-are gram-positive cocci found in pairs/and or chains, the colonies are translucent and are catalase negative

Question 12

Bacitracin disk: beta-hemolytic streptococcus

Answer 12

-this test or A disc determines the presence or absence of Group A beta-hemolytic strep (strep pyogenes)
-a blood plate is inoculated with isolated beta-hemolytic strep
-An A disc is placed on the primary quadrant and incubated overnight
-a zone of inhibition around the disc is positive for group A beta-hemolytic

Question 13

CAMP Test: beta-hemolytic strep identification

Answer 13

-group B beta-hemolytic strep (strep agalactiae) is most pathogenic in newborns
- a CAMP test can be performed from isolated colonies of beta strep
-CAMP factor acts synergistically with beta hemolysins of some staph aureus strains
-a streak of the organism is placed perpendicular to a line of staph aureus on a BAP
-after overnight incubation the presence of a clear hemolytic arrow at the inner end of the strep streak is positive for group B beta strep

Question 14

PYR- Strep Identification

Answer 14

-group A strep and enterococci contain an enzyme called pyrrolidinyl peptidase which hydrolyzes L- pyrrolidinyl-beta-naphthylamide (PYR) with the addition of cinnamaldehyde reagent to produce a deep red color

Question 15

Bile Esculin/Salt- Gamma strep Identification

Answer 15

The -a plate is inoculated on both the bile esculin and salt sides and incubated overnight
-growth and the development of black color from esculin hydrolysis is a positive bile esculin
-on the salt side, growth in the presence of a high concentration of NaCl is positive
-enterococcus species are positive on both sides

Question 16

optochin- P disc: Alpha strep identification

Answer 16

-optochin reagent causes the lysis of strep pneumoniae
-place P disc on the primary quadrant of a blood plate that has been inoculated with strep pneumoniae and incubates overnight
-examine the plate for a zone of inhibition of 14mm or greater around the P disc, this is positive for strep pneumoniae
-a zone between 7 mm and 13 mm needs confirmation with DOC
- a zone size of 6mm (resistant) would indicate alpha strep, not strep pneumo

Question 17

DOC: Bile solubility: Alpha strep Identification

Answer 17

-DOC (sodium desoxycholate) is a bile salt that weakens the cell wall of strep pneumonia by enhancing its autolytic enzymes
-this causes lysis of the strep pneumoniae colonies
-a DOC reagent is placed on isolated colonies and incubated at 35 degrees for 30 minutes
-if the colonies dissolve completely the test is positive for strep pneumoniae