Staphylococcus aureus and Streptococcus pyogenes are common pathogens found in wound infections. Swabs are used to collect samples for culture and Gram stain. Growth on blood agar allows identification by colony morphology and hemolytic patterns. Biochemical tests and antibiotic susceptibility testing can further identify and characterize the bacteria. Both are gram-positive cocci that differ in arrangements, virulence factors, and typical infection sites on the human body.

1. Wound Swab
Ashely Hamilton
Angela Thetford
MLT-2010
Professor Tiffany Gill
CSM
June 15, 2015

2. Collection

3. Wound Swab

4. Wound Swab
Swabs tipped with Dacron or Rayon polyester are optimum for
recovery of infective bacteria.

5. Primary Set Up

6. Primary Set Up
Nutritional Media
for most bacterial
growth
Enriched media for
fastidious bacteria
Selective Media for
Gram Negative
bacteria
2 Bacterial Smears
for Gram Stain

7. Primary Set Up
• BAP- Staphylococcus aureus and Streptococcus
pyogenes will both grow on this plate.
• CHOC- Media to enhance growth of fastidious
organisms such as; Haemophilus influenzae,
Neisseria gonorrhoeae and Neisseria meningitidis
• MAC- Media selective for Gram Negative bacteria
and differential for lactose fermenting and non
lactose fermenting bacteria such as; E. coli and
Enterobacter aerogenes. (Enteric bacteria)

8. Media and Slide
Inoculation

9. Media/slide inoculation
CDC,Atlanta

10. Gram Stain

11. Gram Stain
• Perform a Gram Stain on your mixed colony smear.
o Gram Stain reaction
• Positive or Negative
o Bacterial morphology
• Cocci or Bacillus
o Bacterial arrangement
• Pairs, clusters, chains
• Preliminary ID of bacteria

12. Morphology and
Arrangement

13. Morphology and
Arrangement
GPC clusters
S. aureus
GPC chains
S. pyogenes
GNR single
and pairs
GNR pairs and
chains

14. Pathogens

15. Pathogens
• The 2 pathogens that can be seen in a
wound infection are:
• Gram Positive Streptococcus
pyogenes
• Gram Positive Staphylococcus
aureus and/or MRSA (Methicillin
Resistant Staphylococcus aureus)

16. Media to detect
pathogens
• Blood agar (BAP) is a nutrient/differential growth
medium and contains 5% sheep blood. Certain
bacteria produce exotoxins called hemolysins,
which act on the red blood cells to lyse, or break
them down. This hemolysis pattern can help identify
or narrow down a species of bacteria.
•

17. Morphology
S. aureus
and
S. pyogenes

18. Distinguishing Features
Morphology
Staphylococcus aureus Streptococcus pyogenes
Gram positive
Cocci
Grape like clusters
Gram positive
Cocci
Chains

19. Colonial Growth
S. aureus
and
S. pyogenes

20. Distinguishing Features
Colonial Growth
Staphylococcus aureus Streptococcus pyogenes
Golden yellow
entire, smooth,
convex colonies
on BAP
Double zone
Beta hemolysis
on BAP
Opaque,
pearly grey,
smooth, flat
colonies on
BAP
Wide zone of
Beta
hemolysis on
BAP
Facultative anaerobes will grow in CO2 incubator
Growth period 18 to 24 hours @ 35 to 37C

21. Normal Flora or
Pathogen

22. Normal Flora or
Pathogen?
Staphylococcus aureus and
Normal Flora
Streptococcus pyogenes and
Normal Flora
Contaminated
Streptococcus
pneumoniae plate
with P disc

23. Virulence Factors
S. aureus

24. Virulence Factors
Staphylococcus aureus
• Protein A-major component of
cell walls, prevents opsonization
from occurring
• Extotoxins
o Exfoliatin-cause scaled skin
syndrome
o Panton-Valentine-destroys
leukocytes
o TSST-1-Toxic Shock Syndrome
• Exoenzymes
o Hyaluronidase-destroys
tissues
o Hemolysins-destroy RBC
o Coagulase-clot formation
o Staphylokinase-dissolves clots

25. Virulence Factors
S. pyogenes

26. Virulence Factors
Streptococcus pyogenes
• Fibrils on the surface that are
composed of lipoteichoic acid
and M protein: enables S.
pyogenes to adhere to surfaces
and functions as an
antiphagocytic
• Exotoxins
o Streptolysin S and O: Beta
hemolytic component
o Streptococcal pyrogenic
exotoxin: toxin that causes
Toxic shock-like syndrome
o Hyaluronidase: spreading
factor
o Streptokinase: digests fibrin

27. Differential/Selective
Media

28. Differential/Selective
Media
• MSA-Mannitol salt agar
o Selective-bacteria that can tolerate high salt
concentrations
o Differential-differentiates Staphylococcus
aureus from Staphylococcus epidermidis

29. Biochemical Testing
S. aureus
and
S. pyogenes

30. Biochemical Testing
Staphylococcus aureus Streptococcus pyogenes
• Catalase positive
• Coagulase positive
• Bacitracin Resistant
• Catalase negative
• PYR positive
• CAMP negative
• Bacitracin Sensitive

31. Susceptibility Testing
S. aureus
and
MRSA

32. Susceptibility Testing
Results
Staphylococcus aureus
• Sensitive
o Ampicillin/Sulbactam
o Amoxicillin/ Clavulante
o Oxacillin
o Naficillin
o Cefazolin
o Clindamycin
• Resistant
o Bacitracin
o Penicillin
MRSA
• Sensitive
o Vancomycin
o Moxifloxacin
o Doxycycline
o TMX-SMX
• Resistant
o Penicillin
o Ampicillin
o Cefoxitin
o Erythromycin
o Clindamycin

33. Susceptibility Testing
S. pyogenes

34. Susceptibility Testing
Results
Streptococcus pyogenes
• Sensitive
o Penicillin
o Bacitracin
• Resistant
o Erythromycin
o Tetracycline

35. Indigenous Locations
S. aureus
and
S. pyogenes

36. Indigenous locations on the
Human Body
Staphylococcus aureus Streptococcus pyogenes
• Lower GI
• Skin
• Nose
• Pharynx
• Mouth
• Vagina
• Pharynx
• Mouth

37. References

38. References
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www.alilamedicalimages.com
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gallery.html#streptococcus
Blood agar (BAP) Bacterial Growth Medium. (n .d.) Retrieved http://www.scienceprofonline.org/microbiology/blood-agar-
bacterial-growth-medium-BAP.html
Cavanaugh, P., Lipsky, B., Bradbury, A., & Boteck, G. (2005, November 12).Treatment of diabetic foot ulcers. Retrieved
October 4, 2015, from http://www.thelancet.com/journals/lancet/article/PIIS0140-6736(05)67699-4/ppt
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Retrieved June 15, 2015. http://www.bacteriainphotos.com/bacteria-photo-gallery.html#streptococcus
Engelkirk, P., & Engelkirk, J. (2008). Chapter 9: Gram Positive Cocci. In Laboratory diagnosis of infectious diseases:
Essentials of diagnostic microbiology (pp. 213-237). Baltimore: Wolters Kluwer Health/Lippincott Williams & Wilkins
Engelkirk, P., & Engelkirk, J. (2008). Chapter 8: Introduction to medial Bacteriology. In Laboratory diagnosis of infectious
diseases: Essentials of diagnostic microbiology(pp. 185-209). Baltimore: Wolters Kluwer Health/Lippincott Williams &
Wilkins

39. ReferencesEngelkirk, P., & Engelkirk, J. (2008). Chapter 8: Introduction to medial Bacteriology. In Laboratory diagnosis of infectious
diseases: Essentials of diagnostic microbiology (pp. 185-209). Baltimore: Wolters Kluwer Health/Lippincott Williams &
Wilkins
Estridge, B., & Reynolds, A. (2012). 7-3 Culture Techniques for Bacteriology. In Basic clinical laboratory techniques (6th
Edition ed., pp. 703-710). Delmar
Estridge, B., & Reynolds, A. (2012). 7-3 Culture Techniques for Bacteriology. In Basic clinical laboratory techniques (5th
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Estridge, B., & Reynolds, A. (2012). 7-4 The Gram Stain. In Basic clinical laboratory techniques (5th ed., pp. 727-732).
Delmar CENGAGE.
Staphylococcus aureus (n.d.) Retrieved June 15, 2015; http://www.bacteriainphotos.com/bacteria-photo-gallery.html#saureus
Susceptibility testing. (n.d.). Retrieved June 15, 2015. http://cdn.iopscience.com/images/1367-
2630/14/7/073037/Full/nj424207f1_online.jpg