Preparation of Myxococcus xanthus Samples for Live-Cell Imaging

Samenvatting

Source: Schumacher, D., et al. Fluorescence Live-cell Imaging of the Complete Vegetative Cell Cycle of the Slow-growing Social Bacterium Myxococcus xanthus. J. Vis. Exp. (2018)

This video demonstrates the preparation and imaging of Myxococcus xanthus, a slow-growing soil bacterium. The culture is grown in a medium, applied to a coverslip with fluorescent microspheres, and overlaid with a pre-warmed agarose pad to support hydration and nutrient supply. This setup enables long-term, time-lapse live imaging of cellular processes during bacterial cell division.

Protocol

1. Preparation of Microscopy Samples

Note: Cells to be viewed by microscopy are placed on a microscope coverslip and then covered by an agarose pad containing nutrients. The coverslip is glued to a plastic or metal frame to provide mechanical support. In preparation for the microscopy, a large pad of 1% agarose/ TPM/0.2% CTT should be prepared in advance as described in steps 2.1 - 2.3. Please also refer to the Table of Materials for specific products used here.

1. Prepare 500 mL of TPM buffer (10 mM Tris-HCl pH 7.6, 1 mM KH₂PO₄ pH 7.6, 8 mM MgSO₄) and autoclave or filter sterilize using a bottle top filter.

NOTE: The sterile buffer can be stored for several months at room temperature.

2. Prepare 1% agarose microscopy solution containing 0.2% CTT (mix 1 g of agarose with 80 mL of TPM buffer and 20 mL of 1% CTT medium). Heat in a microwave oven until the agarose is molten.

NOTE: The 0.2% CTT is sufficient to allow cells to grow and prevent starvation. Higher concentrations of CTT in the microscopy medium will result in high background fluorescence.

3. Fill a Petri dish with the molten agarose to a thickness of 0.5 cm (for an 11.5 cm x 11.5 cm square Petri dish, approximately 60 mL of molten agarose is required) and let it cool down to room temperature.

NOTE: The agarose pad can be stored at 4 °C in a humid environment for up to 2 days.

1. Pre-warm the 1% agarose/TPM/0.2% CTT pad at 32 °C for at least 15 min prior to use.

NOTE: To prepare the cells for microscopy, follow steps 2.4 - 2.8.

4. Place a sterile glass coverslip (60 mm x 22 mm, thickness: 0.7 mm) on a plastic or metal frame that has a hole in the middle (Figure 1A); this frame serves as a mechanical support for the thin coverslip and helps to reduce drift during microscopy. Fix the coverslip to the frame with tape.

1. To prepare the frame, cut out a 75 mm × 25 mm frame from a 1 mm thick metal plate, then cut out an appropriately sized hole (20 mm × 30 mm in this experiment) in the middle.

5. Add 10 - 20 µL of exponentially grown M. xanthus cells on the coverslip.

6. Add fluorescent 0.5 µm microspheres as fiducial markers to the cells to simplify tracking of cells or proteins in time-lapse recordings.

1. Dilute the microspheres 1:100 in TPM buffer and store at 4 °C for up to several months. Shake thoroughly before use and add 5 - 10 µL of the diluted microspheres to the cells.

NOTE: Here, microspheres that are fluorescent in all common blue, green, yellow, and red fluorescent channels were used.

7. Cut out a small pad approximately the size of the coverslip of the large pre-warmed 1% agarose/TPM/0.2% CTT pad and place it on top of the cells (Figure 1B). Place a coverslip on top of the 1% agarose/TPM/0.2% CTT agarose pad to prevent evaporation and to maintain cells in a humid environment.

NOTE: The coverslip alone will prevent significant evaporation for at least 2 h. For longer time-lapse recordings, the 1% agarose/TPM/0.2% CTT pad and coverslip sandwich should be sealed with paraffin film to prevent evaporation.

8. Incubate the microscopy sample at 32 °C for 15 - 20 min to let the cells attach to the bottom of the agarose pad. Then start the time-lapse microscopy recordings.

Resultaten

Figure 1: Schematic of the experimental set-up used throughout this study. (A) A...

Materialen

Lijst van materialen gebruikt in dit artikel

Naam	Bedrijf	Catalogusnummer	Opmerkingen
DMI6000B with AFC	Leica microsystems	11888945	Automated inverted widefield fluorescence microscope with adaptive focus control
Universal mounting frame	Leica microsystems	11532338	Stage holder for different sample sizes
HCX PL FLUOTAR 100x/1.30 oil PH3	Leica microsystems	11506197	Phase contrast objective
Orca Flash 4.0 camera	Hamamatsu	11532952	4.0 megapixel sCMOS camera for picture aquisition
Filter set TXR ET, k	Leica microsystems	11504170	Fluorescence filter set, Ex: 560/40 Em: 645/75
Filter set L5 ET, k	Leica microsystems	11504166	Fluorescence filter set, Ex: 480/40 Em: 527/30
Filter set YFP ET, k	Leica microsystems	11504165	Fluorescence filter set, Ex: 500/20 Em: 535/30
ProScan III	Prior	H117N1, V31XYZEF, PS3J100	Microscope automation controller with interactive control center
EL 6000 light source	Leica microsystems	11504115	External fluorescence light source
Incubator BLX Black	Pecon	11532830	Black incubation chamber surrounding the microscope
Tempcontrol 37-2 digital	Leica microsystems	11521719	Automated temperature control for incubation chamber
Gentmycin sulphate	Carl Roth	233.4	Gentamycin
Oxytetracylin dihydrate	Sigma Aldrich	201-212-8	Oxytetracyclin
Kanamycin sulphate	Carl Roth	T832.3	Kanamycin
Filtropur BT25 0.2 bottle top filter	Sarstedt	831,822,101	Bottle top filter for sterilization of buffers
Deckgläser	VWR	630-1592	Glass cover slip (60 x 22 mm, thickness: 0.7 mm)
Seakem LE agarose	Lonza	50004	Agarose for microscopy slides
Leica Metamorph AF	Leica microsystems	11640901	Microscope control software and software for picture analysis
Tetraspeck Microsperes, 0.5 µm	ThermoFisher	T7281	Fluorescent microspheres
petri dish	Greiner Bio-one	688102	120 mm x 120 mm x 17 mm squared petri dish for agarose pads
BD Bacto Casitone	Becton Dickinson	225930	Casitone
Parafilm M	VWR	291-1213	Parafilm
Tris(hydroxymethyl)-aminomethane	Carl Roth	AE15.2	Tris
Magnesium sulphate heptahydrate	Carl Roth	P027.2	Magnesium sulphate
Potassium dihydrogen phosphate p.a.	Carl Roth	3904.1	Potassium dihydrogen phosphate
1% CTT medium: 1 % (w/v) BD Bacto™ casitone, 10 mM Tris-HCl ph 8.0, 1 mM potassium phosphate buffer pH 7.6, 8 mM MgSO₄			Cultivation medium for M.xanthus
TPM buffer: 10 mM Tris-HCl ph 8.0, 1 mM potassium phosphate buffer pH 7.6, 8 mM MgSO₄			Buffer for preparation of microscopy slides for M.xanthus