Practicum Grow Dicty

Stella BreslinMMP/ 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

• 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 3.86sDicty Fruiting Bodies – normal stock

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

Materials and MethodsMaterials 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

1 1-2 days prior to splitting culturesprepare broth culture of E. coli and medium – loop of bacteria + Sussman’s media

2 Prepare Lactose- peptone plate

3 Add bacterial suspension to plate (0.5 mL)

4 From Carolina stock plate – use loop to collect spores

5 Spread suspension and spores across plate

6 Incubate at room temperature

*adapted from Carolina Supply materials and http://dictybase.org/techniques/index.html

Methods – Harvesting Cells / pGLOCell Harvesting Protocol

1 From culture plate and harvest cells using Bonner’s salt solution (BSS).

2 Spread BSS over Dicty culture plate

3 Scrape cells off surface of plate

4 Collect dislodged cells in centrifuge tube.

5 Rinse 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.

6 Pipette harvested cells on plate of choice

pGLO Culture Protocol - Plate

1 Perform cell harvesting protocol

2 Spread solution on LB/amp/ara plate with pGLO bacteria

3 Incubate at room temperature

pGLO Protocol pGLO solution only

1 Perform cell harvesting protocol

2 Add pGLO bacteria solution on LB/amp/ara plate

3 Add Dicty solution

4 Spread bacteria & Dicty across plate

5 Incubate at room temperature

*adapted from Carolina Supply materials and http://dictybase.org/techniques/index.html

Results – Original Stock 1st Growth – Peptone Plate

Images of fruiting bodies and aggregation - moundsPlates grown using stock culture protocol.Imaging done after + 4 days

Images from Carson Z-pix digital microscope ~26x 5/09/10

Results – Peptone Plates after 2 days

Results – Images of stages of development present on plateMigrating 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

Results – Non-nutrient agar plate after 2 days

Results - Images of stages of development present on plateThe majority of development was at the fruiting body stage.

Non-nutrient Plate Development captured with Motic 352 on Zeiss STEM1 SV8 5/26/2010

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 ChannelsAutofluorescence?

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

DiscussionIssues 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.

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.

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.

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