EXERCISE 10 ISOLATION AND DETECTION OF Campylobacter jejuni

Components: Pancreatic digest of casein and Peptic digest of animal tissue nitrogen and carbon source Dextrose energy source (carbohydrates) Yeast Extract vitamin (Vit B complex) source Sodium chloride maintenance of osmotic environment Sodium bisulfite for growth enhancement

Campylobacter jejuni : Gram (-) bacteria Slender, spirally-curved motile rods , gull wings (s-shaped) Catalase (+) Oxidase (+) Microaerophile (requires 3 15% O2 and 210% CO2) Does not ferment carbohydrates Foodborne, mostly found in poultry

b. Campy-BAP Agar (Blasers Agar) Brucella Agar Distilled H2O Lysed Horse Blood Vancomycin Polymyxin B Trimethoprim lactate Amphotericin B Cephalothin

Campylobacter as an enteric pathogen: Campylobacter is the leading cause of gastroenteritis in the US It is believed to have caused more illnesses than Shigella spp. and Salmonella spp. Combined It is isolated usually from meat , milk and unpurified (non-chlorinated) water Campylobacteriosis (gastroenteritis) symptoms include: Watery to diarrhea that sometimes associated with blood-tinged stools Fever Abdominal cramps Headache Nausea Muscle pain Malaise

c. Campylobacter charcoal differential agar (CCDA) selective isolation and differentiation of Campylobacter species Components: Blood-Free Medium (charcoal was used instead of blood) Nutrient broth No. 2 general purpose medium Casein hydrolysate source of amino acids and essential nutrients; casein is added to aid in the growth of certain strains of nalidixic acid resistant thermophilic Campylobacter Sodium deoxycholate Ferrous sulfate and Sodium pyruvate reduces the aerotolerance of the medium by quenching photochemically generated oxygen derivatives Agar solidification of the medium Sodium cefopirazone selective agent to where Campylobacter is resistant

Media used: a. Brucella broth general purpose medium that supports the growth of fastidious bacteria such as Streptococcus spp., Neiserria spp. and for the cultivation of *Brucella spp. *intracellular parasite causing a zoonotic disease, brucellosis

d. Preston enrichment broth has rich basal medium to aid the revival/resuscitation of severely injured Campylobacter

Components: Nutrient broth No. 2 (oxoid) Distilled H2O Lysed horse blood provide essential growth factors Polymyxin B inhibits the growth of Gram (-) bacteria Rifampicin - selective against Gram (-) organisms Trimethoprim lactate inhibitor of some Proteus species and other Gram (-) bacteria Cycloheximide anti-fungal agent

Positive Reaction on Campy-BAP agar and CCDA:

Campylobacter colonies appear to be nonhemolytic, colorless to gray to an off white color or slight tan (watery) having a usual diameter of 1-2mm (up to 4-5mm); smooth, convex, round and glistening with a distinct edge

Colonies may also be flat translucent, shiny and

spreading with an irregular edge and confluent growth without distinct colonies may be observed.