Tag Archives: bacterial challenge

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

Share

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.

Share

Vibrio challenge CONTINUED (from yesterday)

Significantly more bacteria in the container containing autoclaved oysters. Collected 2 x 50mL from each treatment. Collected ~750mL from each treatment. Cells were pelleted 4000RPM, 15mins, 4C. Supe was removed and pellets frozen @ -80C.

100uL of a 1:1,000,000 dilution of the control bacteria were plated on 1x LB + 1% NaCl plates and incubated O/N @ RT. 100uL of a 1:10,000,000 dilution of the exposed bacteria were plated on 1x LB + 1% NaCl plates and incubated O/N @ RT. Control colony count the next day = 563 colony forming units (CFU). Exposed colony count the next day = 367 CFU.

Control Calculations
563 CFU/100uL = 5.63 CFU/uL
5.63 CFU/uL x 1:1,000,000 dilution = 5.63 x 10^6 CFU/uL
5.63 x 10^6 CFU/uL x 1000uL/mL = 5.63 x 10^9 CFU/mL

Exposed Calculations
367 CFU/100uL = 3.67 CFU/uL
3.67 CFU/uL x 1:10,000,000 dilution = 3.67 x 10^7 CFU/uL
3.67 x 10^7 CFU/uL x 1000uL/mL = 3.67 x 10^10 CFU/mL

Share

Vibrio challenge CONTINUED (from yesterday)

500mL culture was split evenly between two containers containing 3L sterile sea water each. One container also contained 3 large, autoclaved C. gigas. Containers had an air stone to promote circulation. 4 x 1mL samples were collected from each container, pelleted @ 10,00RPM 1min. Supe removed and samples stored @ -80C. Samples will be collected @ t = 0, 0.5, 1.0 and 24 hrs. Containers were covered with aluminum foil to minimize splashing caused the by the air stone.

100uL of a 1:1,000,000 were plated on 1x LB + 1% NaCl plates and incubated O/N @ 37C. Colony count the next day = 399 colony forming units (CFU).

399 CFU/100uL = 3.99 CFU/uL
3.99 CFU/uL X 1:1,000,000 dilution = 3.99 x 10^6 CFU/uL
3.99 x 10^6 CFU/uL x 1000uL/mL = 3.99 x 10^9 CFU/mL
3.99 x 10^9 CFU/mL x 250 mL/container = 9.975 x 10^11 CFU/container

Share