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Clinical Microbiology


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Wound Swab

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Clinical Microbiology

  1. 1. Wound Swab Ashely Hamilton Angela Thetford MLT-2010 Professor Tiffany Gill CSM June 15, 2015
  2. 2. Collection
  3. 3. Wound Swab
  4. 4. Wound Swab Swabs tipped with Dacron or Rayon polyester are optimum for recovery of infective bacteria.
  5. 5. Primary Set Up
  6. 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. 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. 8. Media and Slide Inoculation
  9. 9. Media/slide inoculation CDC,Atlanta
  10. 10. Gram Stain
  11. 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. 12. Morphology and Arrangement
  13. 13. Morphology and Arrangement Gram Positive Cocci , single, pairs and clusters Gram Positive Cocci, chains Gram Negative Bacilli, single and pairs Gram Negative Bacilli, pairs and chains
  14. 14. Pathogens
  15. 15. Pathogens • The 2 pathogens seen to cause wound infections: • Gram Positive Streptococcus pyogenes • Gram Positive Staphylococcus aureus and/or MRSA (Methicillin Resistant Staphylococcus aureus)
  16. 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. 17. Morphology S. aureus and S. pyogenes
  18. 18. Distinguishing Features Morphology Staphylococcus aureus Streptococcus pyogenes Gram positive Cocci Grape like clusters Gram positive Cocci Chains
  19. 19. Colonial Growth S. aureus and S. pyogenes
  20. 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. 21. Normal Flora or Pathogen
  22. 22. Normal Flora or Pathogen? Staphylococcus aureus and Normal Flora Streptococcus pyogenes and Normal Flora Contaminated Streptococcus pneumoniae plate with P disc
  23. 23. Virulence Factors S. aureus
  24. 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. 25. Virulence Factors S. pyogenes
  26. 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. 27. Differential/Selective Media
  28. 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. 29. Biochemical Testing S. aureus and S. pyogenes
  30. 30. Biochemical Testing Staphylococcus aureus Streptococcus pyogenes • Catalase positive • Coagulase positive • Bacitracin Resistant • Catalase negative • PYR positive • CAMP negative • Bacitracin Sensitive
  31. 31. Susceptibility Testing S. aureus and MRSA
  32. 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. 33. Susceptibility Testing S. pyogenes
  34. 34. Susceptibility Testing Results Streptococcus pyogenes • Sensitive o Penicillin o Bacitracin • Resistant o Erythromycin o Tetracycline
  35. 35. Indigenous Locations S. aureus and S. pyogenes
  36. 36. Indigenous locations on the Human Body Staphylococcus aureus Streptococcus pyogenes • Lower GI • Skin • Nose • Pharynx • Mouth • Vagina • Pharynx • Mouth
  37. 37. References
  38. 38. References Alila Medical Media | Cell, Molecular Biology & Genetics Images. (n.d.). Retrieved June 15, 2015. Bacitracin test for Streptococcus pyogenes. (n.d.). Retrieved June 15, 2015. gallery.html#streptococcus Blood agar (BAP) Bacterial Growth Medium. (n .d.) Retrieved 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 Culture Media - Blood Agar Plates and Hemolysis: Streptococcus and Other Catalase Negative Gram-Positive Cocci. (n.d.). Retrieved June 15, 2015. .
  39. 39. References 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 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 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 ed., pp. 711-722). Delmar
  40. 40. References 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; Susceptibility testing. (n.d.). Retrieved June 15, 2015. 2630/14/7/073037/Full/nj424207f1_online.jpg