EXERCISE 6 Detection and Isolation of Staphylococcus aureus In Foods Staphylococcus aureus first discovered in Aberdeen, Scotland in 1880 by Dr.

. Alexander Ogston who was a surgeon S. Aureus means the "golden cluster seed" or "the seed gold." It is also known as golden staph Gram positive Catalase positive Can ferment mannitol Form irregular or grape-like clusters and cocci pH range: 4.0-9.8, opt. at pH 6-7 Facultative anaerobe Mesophilic able to grow in a wide range of temperatures (7 to 48.5C with an optimum of 30 to 37C) Habitat and Reservoir Humans Resides in nasal cavity; also present in eyes, throat, intestinal tract, arms, hands and face esp. with boils, wounds and carbuncles Food Meat- Beef, pork and poultry Meat products- ham, salami, hotdogs Salads- ham, chicken, potato Cream-filled bakery products and dairy products- clair, cheese, milk Reason for detection of S. aureus in foods To confirm if causative agent of food borne illnesses To determine whether a food or food ingredient is a potential source of enterotoxigenic staphylococci To demonstrate post processing contamination, which is normally due to human contact with processed food or exposure of the food to inadequately sanitized food processing surfaces METHODOLOGY

1. Semi quantitative Enrichment Procedure Principles: Enrichment procedures are done to revive or repair the damaged cells This is recommended for foods with low counts of the desired microorganism Staphylococcus aureus is a non competitive organism Two types of Enrichment Techniques: 1. Selective Enrichment employs chemicals that are inhibaitory for non Staphylococcus organisms 2. Non selective Enrichment contains no selective agent; injured cells are revived Chemicals or Media: 0.1% Peptone Water (PW) not isotonic; prevents the rupture of the cells Giolitti and Cantoni Medium (GCM) selective enrichment medium that provides optimal condition for the growth of Staphylococcus but inhibits accompanying microorganisms and contaminants; recommended for detecting S. aureus in dried milk and infant foods Components and Mode of Action: 1. PW Peptone source of C, N, S, and energy 2. GCM Peptone (Casein) and Meat (Beef) Extract sources of C, N, vitamins, and minerals Yeast Extract supplies B complex to stimulate bacterial growth Lithium Chloride inhibits Gram negative bacilli growth by anaerobiosis; favors growth of Staphylococci D mannitol carbohydrate source Sodium Chloride inhibits non Staphylococcus organisms Glycine (Amino Acid) inhibits Gram positive bacteria; promotes growth of Staphylococcus Sodium Pyruvate stimulates growth of Staphylococcus

Tween 80 cell membrane integrity and repair of damaged ribosomes Potassium Tellurite acts with Glycine to inhibit other Gram positive bacteria by anaerobiosis

Results: Black coloration of the GCM Explanation: Tellurite is reduced to tellurium which indicates the growth of staphylococci. 2. Enumeration of Staphylococcus aureus Chemicals or Media: Baird Parker Agar (BPA) specified for the surface plate count of coagulase positive Staphylococci in foods Components and Mode of Action: 1. BPA Basal Medium source of C, H2O, and various salts Tryptone source of C, N, vitamins, and minerals Beef Extract source of C, N, vitamins, and minerals Yeast Extract - source of C, N, vitamins, and minerals Sodium Pyruvate selectively stimulates growth of Staphylococcus particularly stressed cells without destroying the selectivity Glycine selectively stimulates growth of Staphylococcus; acts as selective agent Lithium Chloride inhibits the growth of accompanying microflora; acts as selective agent Agar solidifying agent Potassium Tellurite - inhibits the growth of accompanying microflora; acts as selective agent Egg Yolk Emulsion makes medium yellow and opaque; acts as nutrient source; substrate to detect lecithinase production and lipase activity Results: Staphylococcus aureus colonies are black, shiny, convex, white edge surrounded by a clear zone.

Explanation: Blackening is due to the reduction of tellurite to tellurium. Clearing of egg yolk is due to the production of phospolipoprotein lipase by lipolysis. Staphylococci that produce lecithinase break down the lecithin in the egg yolk and cause clear zones around respective colonies. Opaque zone of precipitation may form due to lipase activity. 3. Confirmation and Detection of Coagulase Positive Staphylococcus aureus Principles: Confirmation is usually necessary because the reactions observed on plating media are not sufficiently specific Coagulase test is a done to confirm if the organism has the enzyme coagulase and identifies coagulase positive organisms Two types of Coagulase: 1. Free Coagulase extracellular enzyme produced when an organism is cultured in broth 2. Bound Coagulase Clumping factor; remains attached to the cell wall of the organism Many coagulase positive Staphylococci produce the enzyme staphylokinase which will cause lysis of the plasma clot, thus, a false negative reaction may occur Coagulase positive Staphylococci are pathogenic Chemicals or Media: Brain Heart Infusion Broth (BHIB) general purpose liquid nutritious buffered medium used in cultivation of fastidious and non fastidious organisms including aerobic and anaerobic bacteria; non selective enrichment medium which provide Staphylococcus aureus the essential energy and also for the enhancement of the coagulase enzyme Coagulase Plasma with Ethylene Diamine Tetraacetic Acid (EDTA) Gram Stain Reagents - Ammonium Oxalate Crystal Violet - Grams Iodine

95% Ethyl Alcohol Safranin

Safranin counterstain which gives color to decolorized cells; basic dye

Components and Mode of Action: 1. BHIB Infusion from Calf Brain clearer media with equivalent nutritional value Infusion from Beef Heart provide C, organic N, and vitamins Proteose Peptone or Polypeptone provide C, organic N, and vitamins Dextrose serves as carbohydrate Sodium Chloride maintains osmotic balance Disodium Phosphate 2. Coagulase Plasma with EDTA Desiccated or lyophilized rabbit plasma in vitro diagnostic use only Mannitol fermented by Staphylococcus aureus to have coagulation EDTA anti coagulant to prevent false positive reactions; prevents normal clot formation by Ca2+ so that the clot formation produced is only from the coagulase of S. aureus; clots fibrinogen (blood form) to fibrin (clot form) 3. Gram Stain Reagents Ammonium Oxalate Crystal Violet primary stain which color cell constituents; basic dye Grams Iodine mordant which enhance primary stain affinity 95% Ethyl Alcohol decolorizing agent which removes dye from stained cells

Results: Turbidity in BHIB Clot formation in Coagulase Plasma with EDTA *Actual Observation: No clot formation Purple in color, cocci in shape, and grape like clusters in arrangement in Gram Staining