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)
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
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. 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.
•
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
22. Normal Flora or
Pathogen?
Staphylococcus aureus and
Normal Flora
Streptococcus pyogenes and
Normal Flora
Contaminated
Streptococcus
pneumoniae plate
with P disc
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
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
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
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
36. Indigenous locations on the
Human Body
Staphylococcus aureus Streptococcus pyogenes
• Lower GI
• Skin
• Nose
• Pharynx
• Mouth
• Vagina
• Pharynx
• Mouth
38. References
Alila Medical Media | Cell, Molecular Biology & Genetics Images. (n.d.). Retrieved June 15, 2015.
www.alilamedicalimages.com
Bacitracin test for Streptococcus pyogenes. (n.d.). Retrieved June 15, 2015. http://www.bacteriainphotos.com/bacteria-photo-
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
Culture Media - Blood Agar Plates and Hemolysis: Streptococcus and Other Catalase Negative Gram-Positive Cocci. (n.d.).
Retrieved June 15, 2015. http://www.bacteriainphotos.com/bacteria-photo-gallery.html#streptococcus
.
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. 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; 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
Normal flora with S. pyogenes: http://lib.jiangnan.edu.cn/ASM/114-5.jpg
Normal flora with S. aureus: bacteria in photos
Contaminated S. pneumoniae plate-courtesy of CSM MLT program
www.alilamedicalimages.com
-Textbook page 218-219
www.alilamedicalimages.com
PEA-Phenlyethyl alcohol agar- Selective for Gram Positive cocci; staphylococcus would grow on agar. Inhibits Gram Negative bacteria
MSA photo: www.bacteriainphotos.com
