Study Of Paragallinarum Reference Millenium

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Materials and Methods
Overview
The whole study consists of four phases. To start with, the suitable media need to be identified for the growth of Av. paragallinarum. Secondly, the media used in disc diffusion and broth dilution testing need to be validated. This validation process is completed by performing disc diffusion and broth dilution testing of quality control strain on candidate agar or broth. Then, the antimicrobial sensitivity of Av. paragallinarum can be tested on validated media, by both disc diffusion and dilution method. Any antibiotic resistance shown in the tests will be recorded and analysed. Finally, PCR will be performed to identify any genetical reason underneath the resistance. This report only focus on growth media selection,
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They are also common antibiotics used in poultry industry. However, due to the absence of QC range for A. pleuropneumoniae in CLSI method, they were not used in method validation.
Growth Media Selection
Three overseas Av. paragallinarum reference strains (HP1, HP 90 and HP144) and two Australian Av. paragallinarum reference strains (HP14 and HP60) were selected for growth curve construction. Three broths were tested for their suitability of growth, Haemophilus Test Medium Broth (TMB), Cation-adjusted Mueller-Hinton Broth (CAMHB) with supplements and Veterinary Fastidious Media (VFM).
TMB was prepared with 1% Biosate Peptone (BD), 1% sodium chloride (Merck Millipore, Australia), 0.1% starch (Merck Millipore), 0.1% glucose (Univar, Ajax Finechem), 0.05% yeast extract (Sigma–Aldrich) and was supplemented with 0.0025% of NADH, 0.0005% of thiamine-HCl, 1% of heat inactivated chicken serum and 5% of O-A complex, which consists of 4.75% bovine serum albumin in normal saline (with the normal saline containing 0.06% oleic acid and 5% 0.05 N NaOH) (Sigma–Aldrich). It is the common media in our laboratory used for culturing hemophilic bacteria, including Av.
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For each repeat, the suspension was swabbed on a blood agar plate for sterility control. It is critical to use correct reading method in measuring inhibition diameter, as suggested by CLSI handbook. For some plates, multiple rings appeared, the reading of inhibition zone was based on the inner ring, which should be readily distinguished with unaided eyes (CLSI, 2013). The results of inhibition diameter was compared with QC ranges for each antibiotics provided in CLSI standards (2015). A media can be validated only if all tested antibiotics gave inhibition ranges that fall into the QC

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