Marine Bacteria Cultivation Michael Clement Dr' Stephen Giovannoni

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Marine Bacteria CultivationMichael
ClementDr. Stephen Giovannoni
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Purpose

• To culture and grow ecologically important marine
  bacteria from a complex marine environment
• Cultivation of marine microbes is difficult but
  necessary for phenotypic and genotypic analysis

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Background

• We know lots of different marine organisms exist
  because we can detect their DNA
• Most organisms we can detect we cannot grow in
  the lab

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Background

• SAR11 is abundant in marine waters worldwide
• SAR11 plays a large, but not well understood role
  in the carbon cycle
• SAR11 has been cultivated in the laboratory

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Growth Medium

• Oregon coast seawater filtered with a 0.1 micron
  filter
• Autoclaving vs. Not autoclaving

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Inoculation

• 24-well Teflon plates
• 5ml medium/well
• Inoculate at 2.5 cells/well
• Inoculated 144 wells
• 24 negative control wells
• Grew for three months

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Growth Assessment

• Guava flow cytometer
• DNA binding dye
• Assess cell density of culture

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Growth Assessment

• Positive wells determined by visual comparison of
  Guava reads to uninoculated wells

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Analysis

• PCR
• Gel electrophoresis
• RFLP
• Determine unique patterns

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Growth results

• 34 culturability
• Some co-cultures and some pure culltures
• Negative control wells had no growth

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RFLP Results
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gt7C6R_8294-27F_H03_009.ab1 Uncultured
Thiotrichales ACTTAACGCGTTAGCTTCGCCACTAAAGGGTAAATC
CCCCCAACGGCTAGTTATCATCGTTTACGGCGTGGACTACCAGGGTATCT
AATCCTGTTTGCTACCCACGCTTTCGTACCTCAGCGTCAGTATTGGTCCA
GAAAGCTGCCTTCGCCATTGATGTTCCTTCTGATATCTACGCATTTCACC
GCTACACCAGAAATTCCACTTTCCTCTACCATACTCTAGTTGACCAGTTT
CAAATGCAGTTCCCAGGTTAAGCCCGGGGCTTTCACATCTGACTTAATAA
ACCGCCTACGCACGCTTTACGCCCAGTAATTCCGATTAACGCTTGCACCC
TCCGTATTACCGCGGCTGCTGGCACGGAGTTAGCCGGTGCTTCTTCTAAA
GTTAACGTCAAGGCTAACGGTTATTAACCGCTAACTTTTCTTCACAATTG
AAAGTGCTTTACAACCCTCAGGCCTTCTTCACACACGCGGTATTGCTGGA
TCAGGGTTGCCCCCATTGTCCAATATTCCCCACTGCTGCCTCCCGTAGGA
GTTCGGGCCGTGTCTCAGTCCCGATGTGGCTGATCATCCTCTCAGACCAG
CTAAAGATCGTCGCCTTGGTAGGCTTTTACCCTACCAACAAGCTAATCTT
ACGCAGGCTCATCTGATAGCGTGAGGCTCGAAAGTCCCCCACTTTACTAC
GAATAGATTATGCGGTATTAATCCGAATTTCTTCGGGCTATCCCCCACTA
TCAGGCAGATTCCTACGCGTTACTCACCCGTCCGCCACTCGACGCCTACT
AGCAAGCTAGTATCGTTTCCGTTCGACTTGCATGTGTTAAGCATACCGCC
AGCGTTCAATCTGAGCCAT
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2 Uncultured Alphaproteobacteria
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1 Thiotrichales
2 plastid/ cyanobacteria
1 Uncultured Gammaproteobacteria
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Future Application

• Two mixed cultures are undergoing continuing work
• Uncultured Gammaproteobacteria (SAR 86?)
• Plastid or cyanobacteria
• Frozen stocks of cultures

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Conclusion

• Ongoing focus of the Giovannoni lab is to
  cultivate new species as well as diverse members
  of already cultivated species
• Filtering medium verses autoclaving it is a
  potentially viable means of cultivating rare or
  uncultured organisms

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Special thanks to

• Dr. Stephen Giovannoni For taking me into his
  lab
• Dr. Kevin Ahern
• For his wonderful organization of this program
• Paul Carini
• For all of his help day in and day out
• The Howard Hughes Medical Institute
• For their continued support of this program