One of the three starter liquid cultures from 20090706 wee used to inoculate 1L of Marine Broth + biphenyl. Incubated 200RPM @ 28C.

Inoculated 3 x 5mL Marine Broth + biphenyl crystals (UV sterilized) cultures from frozen stock in 50mL Falcon tubes. Incubated 200RPM, 28C. Will use these are starter cultures to inoculate larger cultures.

Inoculated 1x 1L of MB + biphenyl crystals from frozen stock. Incubated 28C, 200RPM. MB was autoclaved with the biphenyl crystals.

Inoculated 2 x 1L of 1x Marine Broth + biphenyl crystals (in 2L flasks) using 5mL of liquid culture from 20090501. Incubated at 28C 200RPM.

Started two 100mL cultures in 1x Marine Broth + biphenyl. One culture was inoculated from the original liquid culture (from 20090419) and the second was inoculated with a thick loopfull of C. pugetti from the plate (from 20090424). Cultures were incubated @ 28C, 200RPM.

Inoculated a total of 10, 5mL 1x Marine Broth in 50mL conicals. 5 tubes received 1mL of the original culture started on 20090419. 4 tubes received 1mL of the secondary culture (from 20090421). 1 tube was inoculated with a colony from the plate streaked on 20090424. Incubated all tubes @ 28C, 200RPM. Used a higher temp. to encourage faster/more robust growth.

Streaked C. pugetti onto marine broth plate + a few biphenyl crystals and incubated at RT over the weekend.

Results: No growth as of Monday, 20090427.

Transferred 1mL of the culture to a 50mL conical containing 4mL of 1x Marine Broth and a couple crystals of biphenyl. Kept the cap loosened and incubated at 20C with shaking at 200RPM. Kept the existing culture in the incubator as well. No apparent growth.

Rehydrated ATCC isolate according to directions. Added 5mL of 1x Marine Broth to a glass culture tube. Used 1mL of this to rehydrate ATCC sample and then added back into the culture tube. Added a few crystals of biphenyl, which had been exposed to UV for ~5mins prior. Incubated at 20C, no shaking. This was done at ~11:30AM.