Staphylococcus aureus

1
Staphylococcus aureus

• Food Microbiology Laboratory

2
Two Methods

• Solid
• gt 100 S. aureus/g are expected
• Liquid
• lt 100 S. aureus/g are expected

3
Baird-Parker Agar

• Lithium Chloride Selective
• S. aureus can tolerate
• Glycine Selective
• S. aureus can tolerate
• Tellurite Selective and Differential
• Reduced to Tellurium (black)
• Egg Yolk Differential
• S. aureus Cleaves egg yolk and leaves clear zone

4
Solid Method - Plating

• MUST use SPREAD Plated
• Can NOT put 1 ml in plate
• Will not absorb into media
• MUST distribute 1 ml over 3 plates

0.3
0.3
0.4
5
Solid Method

• Incubate Inverted at 35 C for 45-48 h
• Count Plates with 20-200 Colonies
• Large Colonies
• Typical
• Grey/Black, Smooth, circular, convex and moist
• 2-3 mm diameter
• Light zone around colony

6
Liquid Method - MPN

• Media
• 10 NaCl Trypticase Soy Broth
• Use 3 Tube MPN
• Incubate at 35 C for 48 h
• Transfer Positives (loopful) to Baird-Parker Agar
• Positives TSA Agar and Catalase Test

7
Coagulase Test

• Determine if Staph agglutinates plasma
• BE SURE to
• USE POSITIVE CONTROL
• Coagulase is enzyme produced by S. aureus
• IF 4 - Likely to be S. aureus
• 1 to 3 - Further Testing

8
Ancillary Tests

• Anaerobic Fermentation of Mannitol and Glucose
• Carbohydrate Fermentation of media with 0.5
  mannitol/glucose (two separate tubes)
• Acid, NO gas Positive for S. aureus
• Catalase Test
• Take from Non-selective Slant (TSA)
• Put Culture on Slide
• Bubbles - Positive

9
Ancillary Tests

• Lysostaphin sensitivity
• Lysostaphin is an enzyme produced by OTHER Staph
  which causes lysis of S. aureus
• Lysostaphin added to phosphate saline buffer
• Incubate 2 hours at 35 C
• Positive Clear Tube (lysis)
• Negative - Turbid

10
Ancillary Tests

• Thermostable Nuclease Test
• Enzyme Produced by S. aureus
• Heat Stable
• Degrades DNA
• Boil a BHI culture for 10 min
• Streak onto Toluinine Blue Deoxynucleic Acid
  Agar
• Incubate 24 h at 35 C
• DNA Dye Blue (negative)
• No DNA Pink (positive)