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

ed., pp. 711-722). Delmar

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