R. Chuanchuen DVM, MS, PhD Department of Veterinary Public Health; Center for Antimicrobial Resistance Monitoring in Foodborne Pathogens (in cooperation with WHO) (CUARM) Faculty of Veterinary Sciences, Chulalongkorn University
Any microorganism that has a complex nutritional requirement and will only grow when specific nutrients are included in its diet. Example: Haemophilus somni, Actinobacillus pleuropneumoniae, Pasteurella multida The medium, QC procedures & interpretive criteria must be modified to fit each organism.
1. Chocolate Mueller-Hinton Muller-Hinton agar (38.0 g) Hemoglobin (10.0 g) Nutritional supplement (10.0 ml) Vitamin B12 (0.01 g) L-glutamine (10.0 g) Adenine (1.0 g) Guanine HCl (0.03 g) p-aminobenzoic acid (0.013 g) NAD (0.25 g) Thiamine pyrophophate (0.10 g) Ferric nitrate-8h 2 O (0.02 g) Thiamine HCl (0.003 g) L-Cysteine HCl (25.90 g) L-Cystine (1.10 g) Dextrose (100.00 g)
2. Test procedure The direct colony suspension procedure Incubate at 35 o C at an atmosphere of 5%+2%CO 2 for 20-24 h The innermost zone of obvious growth inhibition should be regards as the zone margin. 3. QC Testing Haemophilus somni ATCC 700025 A. pleuropneumoniae ATCC 27090
1. Agar Mueller-Hinton agar supplemented with 5% defibrinated sheep blood 2. Test procedure The direct colony suspension Incubate at 35 o C + 2 o C at an atmosphere of 5%+2%CO 2 for 20-24 h Penicillin & oxacillin disk diffusion testing is not reliable with viridans streptococci
1. Agar Mueller-Hinton agar supplemented with 5% defibrinated sheep blood 2. Test procedure The direct colony suspension Incubate at 35 o C + 2 o C at ambient air If the isolates cannot grow under the conditions, it should be tested by broth microdilution If the QC is out of range, it should be tested by broth microdilution
1. Agar MHA with 5% defribinated sheep blood 2. Test procedure Use direct colony suspension prepared from a blood agar plate at 36-37 o C for 48 h or 42 o C for 24 h Incubation at 36-37 o C for 24 h Atmosphere equivalent to 10% CO 2, 5%O 2 and 85% N 2 Due to a halogram effect, read the darkest & largest zone of inhibition
Example: Listeria spp., A. pyrogenes, Anaerobic bacteria, Enterococci Vet01-A4 Vet01-A4
3. QC Testing S. aureus ATCC 25923 is recommended for QC. M. haemolytica ATCC33396 may be used as a supplemental QC reference strain for ceftofur, tulathomycin and gamithromycin.
1. Chocolate MHA See VET01-S2 Table7 2. Standardizing the inoculum Direct inoculum suspensions must be used. Use colonies from an overnight (20-24 h) on chocolate agar culture The final inoculum conc. 5 x 10 4 CFU/spot a 5% + 2% CO 2, 20-24 h H. somni A. pleuropneumoniae
1. Agar MHA with 5% defribinated sheep blood 2. Test procedure Use direct colony suspension prepared from a blood agar plate at 36-37 o C for 48 h or 42 o C for 24 h Incubation at 36-37 o C for 24 h Atmosphere equivalent to 10% CO 2, 5%O 2 and 85% N 2 Due to a halogram effect, read the darkest & largest zone of inhibition
1. Cation-adjusted MHB Mueller-Hinton broth (22 g) Yeast extract (20.0 g) Lysed horse blood (20.0 ml) Supplement C c (or the equivalent) (20.0 ml) H. somni 2. Standardizing the inoculum Direct colony suspensions Use colonies from an overnight (20-24 h) on chocolate agar culture The final inoculum conc. 5 x 10 5 CFU/ml a 5% + 2% CO 2, 20-24 h A. pleuropneumoniae
CAMHB supplemented with final volume of 2.5% to 5% lysed horse blood Direct colony suspensions from an overnight (16-20 h) blood agar plate should be used.
May be used CAMHB and inocubated in a non-co 2 35 o C incubator with longer the incubation time (18-24 h) If fail to grow, may be retested using CAMHB + 2%-5% lysed horse blood Re-confirm if it is P. multocida? P. multocida M. haemolytica
1. Agar CAMHB supplemented with 2.5 to 5% v/v lysed horse blood 2. Test procedure Use direct colony suspension prepared from a blood agar plate at 36-37 o C for 48 h or 42 o C for 24 h Incubation at 36-37 o C for 24 h Atmosphere equivalent to 10% CO 2, 5%O 2 and 85% N 2
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