VPM 201: Veterinary Bacteriology & Mycology, 2012
Laboratory 3a & b. Gram-positive cocci: Staphylococci, Streptococci & Enterococci
A. Detailed flowcharts summarizing major Genera & species.
1. Catalase-positive cocci.
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2. Catalase-negative cocci.
G-positive
cocci
Catalase
Test
Positive
Negative
Staphylococci
&
Micrococci
Lancefield
Grouping
Group D
Streptococci
Enterococci
Group B & C
16s rDNA
Bovine Streps
S. agalactiae CAMP+ve
S. dysgalactiae
*S. uberis Esc
Porcine Streps
*S. suis
*S. porcinus
Group C
E. faecalis
E. faecium
Equine Streps S. equi (- / -)
S. zooepidemicus (- / +)
*S. equisimilis (+ / -)
Group G
"-" or "+" indicate ability to ferment
Trehalose/Sorbitol respectively
People Streps
S. pyogenes Group A
*S. pneumoniae
Canine Strep(s)
S. canis
* =Variable or no Lancefield Group
beta hemolysis
= alpha hemolysis (zone-of-greening)
nonhemolytic
Esc = Positive Esculin hydrolysis
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Esc
Esc
B. Outline of primary culture (colony) assessment steps. [modified from L.S. Garcia (editor-inchief), Clinical Microbiology Procedures Handbook, 3rd edition. ASM press, 2007, pp. 3.3.2.1-3.3.2.5.]
1. Examine plates closely for growth and distinct colony types. A magnifying lens may be
required to see very small colonies.
2. Record information on a “Diagnostic Worksheet” and use simple “tracking” terminology such
as Col # 1, Col # 2 for distinct colony types. It is not unusual for a single Streptococcal isolate to
display two distinct colony types on the same plate.
3. Indicate relative colony numbers (growth).
No growth (NG):
Scant growth (SG): 1 – 5 colonies (are these colonies along streak lines or contaminants?)
Few: Growth primarily in first quadrant.
Moderate growth (MG): First & second quadrants.
Heavy growth (HG): Third or fourth quadrant growth.
4. Use gross colonial morphology terminology introduced in Lab 2.
5. Assess colonies for evidence of hemolysis.
- 𝛼 (alpha)-hemolysis – “zone of greening” resulting from conversion of hemoglobin to
methemoglobin adjacent to, or beneath, bacterial colonies. Good examples include Strep.
pneumoniae & Strep. dysgalactiae.
- β (beta)-hemolysis – generally a wide, clear, single-zone of hemolysis around colonies.
Good examples include the “equine” streps: Strep. equi subsp. equi and zooepidemicus.
Note: There are β-type “variants” that are typical for a given species. A good example is
Streptococcus agalactiae.
- Double-zone (or “target-hemolysis”) is often seen with Staph. aureus & Staph.
intermedius Group colonies.
C. Things in your Laboratory Handbook that you may find helpful include:
1. Gram-positive flowchart (cocci section) page 5.
2. CAMP, Catalase, Coagulase and Carbohydrate fermentation test descriptions in
Appendix C.
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General Worksheet (modified from AVC Diagnostic Bacteriology 27-8-12)
Exercise 1: Gram-stain (Direct Smear) : _not available _______________________________________
1
Media
BA
2
Colony Characteristics
Moderate size (1.0 mm),
circular, convex, off-white,
opaque
Double-zone (target) hemolysis
NG
MAC
3
Gram-stain
Gram-positive cocci
Clumps, pairs,
occasional short (3-4)
chains
Additional Tests
Catalase (CAT) +ve
Coagulase +ve
Tentative i.d.
Staph. intermedius Group
Or
Staph. pseudintermedius
Complex
Or
Staph. pseudintermedius
Generally not done
from MAC colonies.
1. see Laboratory Handout manual for media descriptions. 2. Relative amount of colony type(s): Hg/Mg/Sg/Ng – Heavy, Moderate, Few/Scant
and No growth, respectively. 3. Examples include; TSI, SIM, Urea, Citrate, Indole, CO, CAT, Coagulase (see Lab Handout for test descriptions).
Exercise 2: Gram-stain (Direct Smear): _not available _______________________________________
1
Media
BA
2
Colony Characteristics
Gram-stain
Pinpoint to small (<0.5 mm),
circular, translucent
“subtle” β-type hemolysis
Gram-positive cocci,
smaller than Exercise 1
Clumps, short chains
Small (<0.5 mm), circular,
translucent
α-type hemolysis
Gram-positive cocci,
smaller than Exercise 1
Clumps, short chains
NG
MAC
3
Additional Tests
Tentative i.d.
CAT –ve
Mastitis Strep. spp
CAMP test set up
CAT –ve
Mastitis Strep. spp
CAMP test set up
Generally not done from
MAC colonies.
Exercise 3: Gram-stain (Direct Smear): _not available _______________________________________
1
Media
BA
2
Colony Characteristics
Moderate size (1.0 mm),
circular, convex, off-white,
opaque
No hemolysis
NG
MAC
Gram-stain
Gram-positive cocci
Clumps, pairs, occasional
short (3-4) chains
Generally not done from
MAC colonies.
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3
Additional Tests
Tentative i.d.
CAT +ve
Coagulase +ve
Staph. hyicus
D. Lab 3a: Student Exercises
For each of your exercises use culture, Gram-stain and test results, as well as colony and
microscopic observations, in the context of the animal(s) involved and clinical presentation, to
tentatively identify pathogens. The Gram-positive cocci flow charts in Part A of this lab can
help.
Exercise 1. A dog presents with pyoderma of the face and limbs. Material from pustules was
plated on Blood (BA) and MacConkey (MAC) agars and incubated overnight at 37 ºC. BA plates
are provided - there was no growth on MAC.
a. Use the guidelines in Part B to assess any colonies that might be present on your plates. Use the
worksheet on the opposite page to record information.
b. Is there hemolysis? Is there an obvious type? Recall that “double-zone” hemolysis (target-hemolysis)
is generally limited to several coagulase-positive Staphylococci (ex. S. aureus, S. intermedius Group) and
Clostridium perfringens (gram-positive bacilli, spore-forming, obligate anaerobe).
c. Do a Gram-stain on distinct colony types that might be present. Examine and record your results on
the worksheet.
d. Perform a catalase test. Is it positive or negative? Positive
e. Is your colony coagulase positive or negative? (see Demo 2 – Exercise 1). Positive
Exercise 2. The colonies on BA are from a Holstein with acute mastitis. The organism did not
grow on MAC. Please perform the following tests.
a. Use the same process from Exercise 1 to assess your plate.
b. Set up a CAMP reaction on a BA plate provided – an instructor will provide you with a procedure. Each
student should make an attempt.
Exercise 3. Colonies growing on BA are from an acute exudative dermatitis in a recently weaned
pig. The plates were incubated at 37 ºC for 24 hrs. There was no growth on MAC.
a. Use the same process from Exercise 1 to assess your plate. .
b. Is this pathogen coagulase positive? (see Demo 2- Exercise 3). This strain is positive
E. Demonstration Materials
1. Bacterial cultures on BA plates: S. aureus, S. intermedius Group & S. hyicus
2. Coagulase tests
a. Pathogens from Exercise 1 & 3.
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Exercise 1: Gram-stain (Direct Smear) : _not available _______________________________________
1
Media
2
Colony Characteristics
Gram-stain
3
Additional Tests
CAT –ve
BA
Small (<0.5 mm), circular,
translucent, shiny/mucoid
“dew-drop”
β-type hemolysis
NG
MAC
Gram-positive cocci,
smaller than Exercise 1
Clumps, short chains
Note: Sugar
fermentation will
distinguish – trehalosi,
sorbitol
Tentative i.d.
“Equine Strep”
- S. equi subsp. equi
- S. equi subsp.
zooepidemicus
- S. dysgalactiae
subsp. equisimilis
Generally not done from
MAC colonies.
1. see Laboratory Handout manual for media descriptions. 2. Relative amount of colony type(s): Hg/Mg/Lg/Ng – Heavy, Moderate, Low and No
growth, respectively. 3. Examples include; TSI, SIM, Urea, Citrate, Indole, CO, CAT, Coagulase (see Core lab manual for test descriptions).
Exercise 2: Gram-stain (Direct Smear): _not available _______________________________________
1
Media
2
3
Colony Characteristics
Gram-stain
Additional Tests
see Exercise 2, Lab 3A
(subtle – β-type hemolysis)
see Exercise 2, Lab 3A
CAMP +ve
“arrowhead zone”
see Exercise 2, Lab 3A
(α-type hemolysis)
see Exercise 2, Lab 3A
CAMP –ve
NG
Generally not done from
MAC colonies.
Tentative i.d.
Strep. agalactiae
BA
MAC
Strep. dysgalactiae
Exercise 3: Gram-stain (Direct Smear): _not available _______________________________________
1
Media
2
Colony Characteristics
Pinpoint, circular, shiny,
Faint α-type hemolysis
developing.
BA
Gram-positive cocci,
Pairs with an elongated
(lancet) morphology
Note: Colony variation is a
feature of a number of Strep.
spp.
NG
MAC
Gram-stain
Generally not done from
MAC colonies.
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3
Additional Tests
CAT –ve
Tentative i.d.
Strep. suis
Lab 3b: Gram-positive cocci
A. Student Exercises.
Use the same guidelines and support information utilized for exercises yesterday to work though these
cases and tentatively identify the most likely pathogen.
Exercise 1: This organism was isolated from a 2-year old horse with a nasal discharge and fever.
The horse had difficulty breathing and swallowing. The organism did not grow on MAC.
a. Use the worksheets on the opposite page to record your results.
b. What is your tentative identification and why? animal, clinical problem, culture, gram-stain & tests
consistent with i.d. (see sheet)
Exercise 2: Examine your CAMP test results that you set up from Exercise 2 yesterday.
a. Is your organism CAMP-positive, Camp-negative or CAMP-inhibitory? see worksheet
b. Compare your results to an adjacent group.
c. What is happening when an organism is CAMP positive? Synergistic hemolysis between test-bug & S.
aureus (in this case) outer-zone hemolysin.
d. See the CAMP tests (Demo 2).
e. Using your CAMP results and information gathered from your primary plate yesterday you should be
able to identify this pathogen to the species level.
What is it and why? animal, clinical problem, culture, gram-stain & tests consistent with i.d. (see sheet)
Exercise 3. Joint fluids from a few slaughter hogs showing signs of arthritis and/or valvular
endocarditis were collected using sterile swabs and submitted for standard aerobic culture.
BA and MAC plates were inoculated and gown overnight at 37 oC. There was no growth on
MacConkey’s.
a. Use the worksheets on the opposite page to record your results.
b. What is your tentative identification and why? animal, clinical problem, culture, gram-stain & tests
consistent with i.d. (see sheet)
B. Demonstration materials
1. Cultures: S. agalactiae, S. dysgalactiae, S. equi, S. suis
2. CAMP test reactions: S. agalactiae, S. dysgalactiae and S. uberis
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C. Questions
1. What simple test will distinguish Staphylococci & Streptococci taken from pure colonies?
Answer - Catalase test
2. Is Gram-staining and microscopic examination a reliable technique for distinguishing
Staphylococci & Streptococci?
From colony material? (Y/N) – generally no
From clinical samples? (Y/N) – liquid samples ( exudate, pus, milk) are more reliable
3. Historically the “coagulase test” involved a “rapid” slide test and a “slower” tube test when
assessing Staphylococci? We now know that one test measures a “true” secreted-coagulase while
the other measures a cell-associated Clumping Factor (ClfA). Indicate which does what below.
Rapid Slide test: ClfA – agglutination occurs by binding fibrinogen
Tube Test: Coagulase – a clot forms by polymerization of fibrinogen to fibrin
4. In what disease conditions are staphylococci commonly associated with in:
Dogs: SSTI’s (Skin + Soft Tissue Infections), otitis externa, UTI, reproductive etc..
Currently, S. pseudintermedius (of the S. intermedius group) and to a lesser extent S.
schleiferi subsp..
Cows: S. aureus - mastitis, pustular dermatitis on udder (impetigo), CoNS – mastitis.
Pigs: S. hyicus - young pigs (dermatitis, septicemia, arthritis); S. aureus – mastitis,
udder impetigo, endometritis in sows.
Poultry: S. aureus – Bumblefoot, septicemia & arthritis in turkeys
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5. Name the major disease conditions caused by the species of Streptococci dealt with today?
Streptococcus agalactiae – mastitis in ruminants
Streptococcus dysgalactiae subsp. dysgalactiae (Strep. dysgalactiae) – mastitis in ruminants
Streptococcus equi subsp. equi – Strangles (submandibular, retropharyngeal lymphadenitis
with abscessation) and/or Bastard Strangles (metastatic abscessation to: mediastinal lymph
nodes, lungs, liver, spleen, brain, guttural pouch – from retropharyngeal L.N.).
Streptococcus suis – septicemia, bronchopneumonia, endocarditis, arthritis, meningitis in
pigs.
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