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. Stock Culture Protocol 11-2 days prior to splitting cultures prepare broth culture of E. coli and medium – loop of bacteria + Sussman’s media 2Prepare Lactose- peptone plate 3Add bacterial suspension to plate (0.5 mL) 4From Carolina stock plate – use loop to collect spores 5Spread suspension and spores across plate 6Incubate at room temperature *adapted from Carolina Supply materials and http://dictybase.org/techniques/index.html

6. Methods – Harvesting Cells / pGLO Cell Harvesting Protocol 1From culture plate and harvest cells using Bonner’s salt solution (BSS). 2Spread BSS over Dicty culture plate 3Scrape cells off surface of plate 4Collect dislodged cells in centrifuge tube. 5Rinse cells to separate amoeba & bacteria Centrifuge tube /Discard supernatant/ Re- suspend pellet in BSS Repeat spin/discard/rinse x 3 *Rinse steps not performed in experiment due to lack of centrifuge at home. 6Pipette harvested cells on plate of choice pGLO Culture Protocol - Plate 1Perform cell harvesting protocol 2Spread solution on LB/amp/ara plate with pGLO bacteria 3Incubate at room temperature pGLO Protocol pGLO solution only 1Perform cell harvesting protocol 2Add pGLO bacteria solution on LB/amp/ara plate 3Add Dicty solution 4Spread bacteria & Dicty across plate 5Incubate at room temperature *adapted from Carolina Supply materials and http://dictybase.org/techniques/index.html

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

8. Results – Peptone Plates after 2 days 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. Peptone Plate Development captured with Motic 352 on Zeiss STEM1 SV8 5/26/2010

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

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

12. Discussion Issues that arose during the experiment 1.Timing of purchasing Dicty stock cultures from(Carolina Biological Supplies) did not happen in time to use in Live Cell Imaging class. 2.Growing Dicty successfully at home proved to be challenging due to contamination issues and lack of centrifuge for rinsing cells. 3.Imaging Dicty in fluorescence – agar issues – need to find protocol for growing cells on cover slips. 4.Imaging Dicty in brightfield was hindered by lack of bulb in Zeiss dissecting scope. Pictures were taken with less than optimal lighting. 5.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