Tuesday, January 17, 2006

McFarland standard <-> Optical Density

I've been having some difficulties on determining inoculum density as there's no golden standard for Campy. As the inioculu density may vary the final result of MIC significantly, we are supposed to control it consistently (and reasonably). Normally we can do "Total Count" to evaluate the total number of our bacteria in absorbance adjusted (0.5 absorbance at 540nm wavelength) and dilute it to particular concentration which is followed by loading 5-10 ul of it (triplex) on agar plate to see how many CFU there are. Here are two problems with my Campys. First, some of my Campys migrate on agar plates. This really make total count hard to achieve as several colonies might combine each other and it's impossible to count them. To overcome this I dilute the inoculum several more times so that there wouldn't be too many colonies on each observing unit (thus reduce the chance of merge). Second, and also the most annoying one, as Campys grow much slower than other cultures, I got to incubate quite a long time (2-3 days) to make the cell quantity achieves 0.5 absorbance. That really takes too long!! So, this time I modify my method: 1. Adjust inoculum concentration to 0.5 McFarland standard (~ 0.08 absorbance at 625nm wavelength) 2. Consider the antibacterial activity might reduce faster within aquatic condition (broth dilution method) than in semisolid condition (agar dilution), the incubation time is reduced to 1 day.

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