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Practicum Grow Dicty

Practicum Grow Dicty. Stella Breslin MMP/ Cohort 3 Biosc 4. Purpose. The purpose of this experiment was to grow Dictyostelium discoideum and image the various stages of development using brightfield and fluorescence microscopy techniques. Question.

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Practicum Grow Dicty

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  1. Practicum Grow Dicty Stella Breslin MMP/ Cohort 3 Biosc 4

  2. Purpose • The purpose of this experiment was to grow Dictyostelium discoideum and image the various stages of development using brightfield and fluorescence microscopy techniques.

  3. Question • Would growing Dicty on plates with bacteria (E. coli ) that had been transformed with the pGLO plasmid to express GFP change the properties of cells under fluorescence? 5/15/2010 Nikon Captured10xTRITC.nd2 Mag: 10x Exp: TRITC 3.86s Dicty Fruiting Bodies – normal stock

  4. Protocol –Preparing Culture Stock Plates / Experiment Plates • Split Stock Cultures from Carolina to new plates depending on what type of growth • Growing plates for stock cultures - use nutrient agar • Growing plates for experiments - viewing life cycles – non-nutrient agar Life Cycle of Dictyostelium discoideum Image from http://www.ailab.si/supp/bi-visprog/dicty/dictyExample.htm

  5. Materials and Methods Materials from Carolina Supply • Dictyostelium discoideum • Escherichia coli B • Lactose-peptone agar • Non-nutrient agar • Bonner’s salt solution • Sussman’s medium Material from Laney Bio75 • pGLO transformed bacteria on LP/ampicillin/arabinose plate. • pGLO bacteria in LB/amp/ara solution. *adapted from Carolina Supply materials and http://dictybase.org/techniques/index.html

  6. Methods – Harvesting Cells / pGLO *adapted from Carolina Supply materials and http://dictybase.org/techniques/index.html

  7. Results – Original Stock 1st Growth – Peptone Plate Images from Carson Z-pix digital microscope ~26x 5/09/10 Images of fruiting bodies and aggregation - mounds Plates grown using stock culture protocol. Imaging done after + 4 days

  8. Results – Peptone Plates after 2 days Peptone Plate Development captured with Motic 352 on Zeiss STEM1 SV8 5/26/2010 Results – Images of stages of development present on plate Migrating pseudoplasmodium (slug) Aggregate, Tight Mound , Tipped Mound, Mexican Hat, Early Culminant ,Late Culminant. Some Fruiting Bodies present.

  9. Results – Non-nutrient agar plate after 2 days Non-nutrient Plate Development captured with Motic 352 on Zeiss STEM1 SV8 5/26/2010 Results - Images of stages of development present on plate The majority of development was at the fruiting body stage.

  10. Results – Fluorescence 5/15/2010 Nikon Captured 10x *.nd2 Overlay Mag: 10x Exp :BF 50 ms TRITC 3.86s FITC 5s Olympus 5/22/10 Dicty4xph.tif overlay – Phase/ Red/Green in ImageJ Mag : 4x Exp: ph/R/G 6.18ms/100.2ms/100.2ms Results – Fluorescent image capture attempted on non pGLO stock above and page 3. Nikon – TRITC and FITC channels – seemed to be the same. Olympus – Red Green Channels Autofluorescence?

  11. Results – pGLO Plate pGLO plate captured with Motic 352 on Zeiss STEM1 SV8 5/26/2010 pGLO plate captured Z-PIX – two weeks Results – capture of initial growth on pGLO bacteria plate shows mounds developing. After two weeks of growth – mounds, but no other stages present.

  12. Discussion Issues that arose during the experiment • Timing of purchasing Dicty stock cultures from(Carolina Biological Supplies) did not happen in time to use in Live Cell Imaging class. • Growing Dicty successfully at home proved to be challenging due to contamination issues and lack of centrifuge for rinsing cells. • Imaging Dicty in fluorescence – agar issues – need to find protocol for growing cells on cover slips. • Imaging Dicty in brightfield was hindered by lack of bulb in Zeiss dissecting scope. Pictures were taken with less than optimal lighting. • Finding glowing pGLO bacterium was delayed until Clara finished her practicum. Her leftover plates have now been partially spread with Dicty cells.

  13. Discussion • Non- nutrient plate development had reached fruiting body stage on majority of plate by 2 days. • On nutrient plates (peptone or LB) the abundance of food for bacteria made for development stages beginning later than on non-nutrient plate. • Dicty can be added to ampicillin growth plate and exhibit growth with no negative effects. • pGLO plate did initiate mound stage, but other stages not present after two weeks. Is there too much food present to initiate development? • Cultures need to be harvested from plates contaminated with extra microbial growth and new sterile plate growth started.

  14. Discussion Future experiments • Need to separate mounds from pGLO plate and test whether development cycle will start. • Harvested fruiting bodies or slugs from pGLO plates will be imaged under fluorescence to see if properties differ from regular stock. • Other fluorescent imaging to produce negative and positive controls – explore protocols to test for autofluorescence.

  15. Bibliography • Fey, P., Kowal, A. S., Gaudet, P., Pilcher, K. E., Chisholm, R. L. (2007) 'Protocols for growth and development of Dictyostelium discoideum.' Nat Protoc 2:1307-16 • General info and techniques http://dictybase.org/techniques/index.html • http://dictybase.org/teaching_tools/...docs/Dicty_life_cycle-Brazill.doc • Life cycle image from http://www.ailab.si/supp/bi-visprog/dicty/dictyExample.htm

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