Tag Archives: colony forming units

Bacterial Dilutions – Determination of Colony Forming Units from Gigas Bacterial challenge (from earlier today)

All dilutions were performed with 1x LB+ 1%NaCl. 100uL were plated of all dilutions (see below) on 1xLB+1%NaCL plates. Plates were incubated O/N @ 37C. Colonies will be counted tomorrow to determine CFU for each sample.

Plated 100uL of:

V.vulnificus, t=0, 1:1,000,000 and 1:10,000,000

V.vulnificus H2O sample, t=1 & 3, 1:10,000 and 1:1,000,000

V.tubiashii, t=0, 1:1,000,000

Control H2O sample, t=1 & 3, Undiluted

Samples tubes containing bacteria and dilutions were stored @ 4C.

UPDATE 20110112:

Colony counts and calculations

V.vulnificus 0hr = Both dilutions produced a total lawn of bacteria. Uncountable. Will plate higher dilutions, but this will now only be a rough estimate due to the time that has passed.

—UPDATE—

New serial dilutions (90uL plated) of V.vulnificus 0hr were performed, down to 10^12. The only countable plate was the 10^12 dilution.

V.vulnificus 0hr = 10^12 dilution x 410 CFU = 4.1×10^14 CFU/90uL = 4.56×10^12 CFU/uL x 8×10^6uL (8L H2O in oyster tank) = 3.64×10^19 CFU total in oyster tank/8L = 4.56×10^18 CFU/L

V.vulnificus 1hr = 1:1,000,000 dilution x 48 CFU = 4.8×10^7 CFU/100uL = 4.8×10^5 CFU/uL x 8×10^6uL (8L H2O in oyster tank) = 3.84×10^12 CFU total in oyster tank/8L = 4.8×10^11 CFU/L

V.vulnificus 3hr = 1:1,000,000 dilution x 23 CFU = 2.3×10^7 CFU/100uL = 2.3×10^5 CFU/uL x 8×10^6uL (8L H2O in oyster tank) = 1.84×10^12 CFU total in oyster tank/8L = 2.3×10^11 CFU/L

Control H2O (no significant growth occurred O/N, just tiny colonies; continued incubation to allow colonies to increase in size for easier counting)

Control H2O 1hr = Undiluted 146 CFU/100uL = 1.46 CFU/uL x 8×10^6uL (8L H2O in oyster tank) = 1.168×10^7 CFU total in oyster tank/8L = 1.46×10^6 CFU/L

Control H2O 3hr = Undiluted 106 CFU/100uL = 1.06 CFU/uL x 8×10^6uL (8L H2O in oyster tank) = 8.48×10^6 CFU total in oyster tank/8L = 1.06×10^5 CFU/L

V.tubiashii 0hr = 1:1,000,000 dilution x 31 CFU = 3.1×10^7 CFU/100uL = 3.1×10^5 CFU/uL x 5×10^4 (50mL total volume of bacteria) = 1.55×10^10 CFU total V.tubiashii

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Gigas Bacterial Challenge – 1hr & 3hr Challenges with Vibrio vulnificus

400mL O/N culture (1x LB+1% NaCl, 37C, 150RPM, 1L flask) of V.vulnificus (STRAIN??) and V.tubiashii (Strain: RE22) were pelleted (4300RPM, 25C, Sorvall ST-H750 rotor). Supe was removed and pellets were each resuspended in 50mL sea water. 1mL was taken from each to use for dilutions to determine colony forming units (CFU).

Two containers were set up with each containing 16 C.gigas, and air stone and 8L of sea water. The entire 50mL of V.vulnificus was added to one of the containers. 8 oysters were sampled (gill and mantle tissue) from each container at 1hr and 3hrs after the addition of V.vulnificus culture and immediately frozen on dry ice. Samples were stored @ -80C in the “Gigas Vibrio Exposure 1,3hrs 1/11/11″ box. Additionally, 1mL samples of the water were taken at each time to determine CFU in the water.

In addition to the samples taken above, the following tissues were taken from 5 control oysters at the 3hr time point and treated/stored in the same fashion as the others, specifically for assessment of cyclooxygenase tissue distribution analysis: muscle, digestive gland/gonad (difficult to differentiate)

All oysters were measured. Morphometric data is here.

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