Post on 24-Jan-2017
Wound SwabAshely Hamilton Angela Thetford
MLT-2010Professor Tiffany Gill
CSMJune 15, 2015
Collection
Wound Swab
Wound Swab
Swabs tipped with Dacron or Rayon polyester are optimum for recovery of infective bacteria.
Primary Set Up
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
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)
Media and Slide Inoculation
Media/slide inoculation
CDC, Atlanta
Gram Stain
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
Morphology and Arrangement
Morphology and Arrangement
Gram PositiveCocci , single, pairs and clusters
Gram Positive Cocci, chains
Gram Negative Bacilli, single and pairs
Gram Negative Bacilli, pairs and chains
Pathogens
Pathogens• The 2 pathogens seen to cause
wound infections:•Gram Positive Streptococcus pyogenes•Gram Positive Staphylococcus aureus and/or MRSA (Methicillin Resistant Staphylococcus aureus)
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.
MorphologyS. aureus
andS. pyogenes
Distinguishing Features
MorphologyStaphylococcus
aureusStreptococcus
pyogenes
Gram positive CocciGrape like clusters
Gram positive CocciChains
Colonial GrowthS. aureus
and S. pyogenes
Distinguishing Features
Colonial GrowthStaphylococcus
aureusStreptococcus
pyogenesGolden yellowentire, smooth,convex colonieson BAP
Double zoneBeta hemolysison BAP
Opaque, pearly grey, smooth, flat colonies on BAP
Wide zone ofBeta hemolysis on BAP
Facultative anaerobes will grow in CO2 incubator
Growth period 18 to 24 hours @ 35 to 37C
Normal Flora or Pathogen
Normal Flora or Pathogen?
Staphylococcus aureus and Normal Flora
Streptococcus pyogenes and Normal Flora
Contaminated Streptococcus pneumoniae plate with P disc
Virulence Factors S. aureus
Virulence FactorsStaphylococcus
aureus• Protein A-major component of
cell walls, prevents opsonization from occurring
• Extotoxinso Exfoliatin-cause scaled skin
syndromeo Panton-Valentine-destroys
leukocyteso TSST-1-Toxic Shock
Syndrome• Exoenzymes
o Hyaluronidase-destroys tissues
o Hemolysins-destroy RBCo Coagulase-clot formationo Staphylokinase-dissolves
clots
Virulence FactorsS. pyogenes
Virulence FactorsStreptococcus
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
• Exotoxinso Streptolysin S and O: Beta
hemolytic componento Streptococcal pyrogenic
exotoxin: toxin that causes Toxic shock-like syndrome
o Hyaluronidase: spreading factor
o Streptokinase: digests fibrin
Differential/Selective Media
Differential/Selective Media
• MSA-Mannitol salt agaroSelective-bacteria that can tolerate
high salt concentrationsoDifferential-differentiates
Staphylococcus aureus from Staphylococcus epidermidis
Biochemical Testing
S. aureusand
S. pyogenes
Biochemical TestingStaphylococcus
aureusStreptococcus
pyogenes• Catalase positive
• Coagulase positive
• Bacitracin Resistant
• Catalase negative
• PYR positive
• CAMP negative• Bacitracin Sensitive
Susceptibility Testing
S. aureus and
MRSA
Susceptibility TestingResults
Staphylococcus aureus• Sensitive
o Ampicillin/Sulbactamo Amoxicillin/ Clavulanteo Oxacillino Naficillino Cefazolino Clindamycin
• Resistant o Bacitracino Penicillin
MRSA• Sensitive
o Vancomycino Moxifloxacino Doxycyclineo TMX-SMX
• Resistant o Penicillino Ampicillino Cefoxitino Erythromycino Clindamycin
Susceptibility Testing
S. pyogenes
Susceptibility TestingResults
Streptococcus pyogenes
• SensitiveoPenicillinoBacitracin
• ResistantoErythromycinoTetracycline
Indigenous LocationsS. aureus
and S. pyogenes
Indigenous locations on the Human BodyStaphylococcus
aureusStreptococcus
pyogenes• Lower GI• Skin• Nose • Pharynx•Mouth• Vagina
• Pharynx • Mouth
References
ReferencesAlila 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.sciencep
rofonline.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
.
ReferencesEngelkirk, 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
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ReferencesEstridge, 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