Lee, Yoon-Hee Microbiology/FST 636.02 Spring 2005

Staphylococcus aureus Lee, Yoon-Hee Microbiology/FST 636.02 Spring 2005

☞ Properties of S. aureus ¤ Microscopically • Gram positive coccus (cluster) • ¤Produce • Enterotoxin • Thermonucalease • Coagulase • Catalase • ¤Hemolytic • ¤ Ferments mannitol anaerobically • ¤Can grow and survive at low water activity (Aw=0.86) and high salt (5-10 %)

☞ Intoxication vs. Infection • Intoxication: Ingestion of toxins produced by the m/o in food cause food borne illness • Infection: Ingestion of living cells leading to foodborne disease

☞ Intoxication with S. aureus toxin • Staphylococcal gastroenteritis * staphylococcal enterotoxin (enterotoxin) • Heat stable toxin • S. aureus growth > 106 CFU/ml or CFU/g • 1 µg toxin causes disease • Symptoms 4-6 hrs after ingestion: * vomiting * abdominal pain

Foods associated with S. aureus intoxication ¤ Salad ¤Fresh and cured meat products ¤Cream-filled bakery products ¤ Dairy products ¤Commonly found in your nose and throat • Proper hand-washing and sanitation is important

☞ OBJECTIVE • S. aureus enumeration • Isolation techniques using selective and differential media • Practice safe procedures for handling pathogens in the lab

☞ Period 1 • Stomach and dilute food sample • Spread plate 0.4, 0.3, and 0.3 ml (=1 ml total) of each dilution onto Baird-Parker plates (=9 plates) • Let plates sit 10 min • Streak positive (S. aureus) and negative (S. epidemidis) controls onto separate BPA plates

☞ Baird-Parker Agar (BPA) Selective/Differential ¤ Selective agent Lithium chloride and tellurite: suppresses other bacteria ¤Differential agent Potassium tellurite: reduced by S. aureus to produce a black colony Egg yolk: hydrolyzed by S. aureus to produce clear zone

☞ Period 2 ¤ Identify typical, suspect and non-typical S. aureus colonies from BPA Use positive and negative controls as a reference ¤Enumeration of typical S. aureus colonies - circular (2-3 mm in diameter) • gray/black surrounded by opaque halos and clear zones ¤Enumeration of suspect S. aureus colonies - gray/black without halos or clear zones

☞ Period 2 cont’d ¤ Streak 4 “typical” and 4 “suspect” colonies onto BHI and MSA ¤Inoculate BHI broth with 4 “typical” colonies BPA Inoculate Streak 1 2 1 1 2 2 1 2 3 4 1 3 4 4 3 3 4 4 BHI broth BHI agar MSA

☞ Period 2 cont’d ¤Controls • Streak a colony from the (+) control plate onto one BHI agar and one MSA plate. Repeat with negative control. • - Transfer a colony from the (+) control into a BHI broth tube. Repeat with negative control.

☞ Brain-Heart Infusion Media ¤Rich media -Variety of micronutrients and vitamins • Frequently used for cultivation of fastidious bacteria and fungi ¤pH 7.4 ¤Typical S. aureus colonies: - round - yellow

☞ Mannitol Salt Agar ¤7.5% NaCl (salt) - Selective for salt-tolerant m/o (S. aureus) ¤Phenol red (pH indicator) and mannitol (CHO) - Differentiation of species that can ferment sugar - Fermentation gives acid byproduct  yellow ¤Anaerobic conditions - Differentiation of species that can or can’t use mannitol anaerobically.

☞ Period 3 ¤ Confirm if food isolates are typical S. aureus ¤ Perform the coagulase test - We are NOT performing the thermonuclease test

☞ Period 3 cont’d ¤Inoculate “confirmed 1” isolates into plasma tube with ~0.3 ml from one BHI BROTH tube ¤ Inoculate “confirmed 2” isolates into plasma tube with ~0.3 ml from other BHI broth tube ¤ Similarly, inoculate “positive control” plasma tube with the (+) control BHI broth ¤ Inoculate “negative control” plasma tube with the (-) control BHI broth

☞ Period 3 cont’d Coagulase Test ¤Place plasma tubes in 35ºC waterbath ¤Gently tip the tube (~45º angle) ¤Coagulation indicates that the m/o produces coagulase ¤Should be able to see results within 20 min

☞ Period 3 cont’d Confirmed Count ¤Determine S. aureus CFU/g food - Confirmed counts only will be considered - Sum the confirmed counts on all three plates of the appropriate dilution - Follow counting rules: 20-200 range

Lee, Yoon-Hee Microbiology/FST 636.02 Spring 2005