Time Lapsed Imaging of Microbial Cells
Overview
Its cool and at times informative to be able to watch cells divide. This procedure outlines how you can do so using optical microscopy.
Materials
rich media such as YEPD
minimal media such as Thorn Media, M9 Minimal Media + carbon source
2% agarose
glass bottom dish (Matek )
glass lid
parafilm
Important Safety Concerns:
Watch out for boiling over agarose!
Waste Disposal:
The bacteria containing agarose should be disposed of in the white biohazardous waste containers. The glass bottom dishes can be re-used.
Critical Steps:
The most likely thing to go wrong is that your field of view will drift over time and the focus will change. One reason the field of view changes is that the agarose pad shrinks over time as it cools or dries out. To avoid this, be sure to seal the imaging dish with parafilm & add a few small drops of media or water to avoid dehydration of the pad. Also be sure to let the pad cool sufficiently before starting to image.
Procedure:
Pad Preparation
1. Microwave 2% agarose (mix of low-melt and normal, to taste) in low-autofluorescence media (see below).
2. Apply 2mL to a 24×60 mm coverslip.
3. Cover with additional coverslip, creating a sandwich. Let cool for 30 minutes.
4. Once cooled cut a 5×5 mm square from the agarose pad.
Cell Preparation
1. Grow 5mL culture in media to OD 600 = 0.3. Back dilute if necessary.
2. Centrifuge at 2000 rpm for 2 minutes. Resuspend in 1mL Thorn media and transfer to a 1.5mL eppendorf tube.
3. Spin in a microfuge for 15 seconds to pellet. Resuspend in 100-200 µL Thorn media.
Apply 0.2 µL of cells to the top surface.
8. Invert square onto a glass-bottom imaging dish. Add a few drops of water to the dish (for moisture), and cover edges with parafilm.
9. Image.
For yeast, use
==Thorn Media==
Standard media except with low-fluorescence yeast nitrogen base.
(Yeast nitrogen base without riboflavin and folic acid.)
* 5 g/l (NH4)<sub>2</sub>SO<sub>4</sub>
* 1 g/l KH<sub>2</sub>PO<sub>4</sub>
* 0.5 g/l MgSO<sub>4</sub>
* 0.1 g/l NaCl
* 0.1 g/l Ca<sub>2</sub>Cl
* 0.5 mg/l H<sub>3</sub>BO<sub>4</sub>
* 0.04 mg/l CuSO<sub>4</sub>
* 0.1 mg/l KI
* 0.2 mg/l FeCl<sub>3</sub>
* 0.4 mg/l MnSO<sub>4</sub>
* 0.2 mg/l Na<sub>2</sub>MoO<sub>4</sub>
* 0.4 mg/l ZnSO<sub>4</sub>
* 2 µg/l biotin
* 0.4 mg/l calcium pantothenate
* 2 mg/l inositol
* 0.4 mg/l niacin
* 0.2 mg/l PABA
* 0.4 mg/l pyridoxine HCl
* 0.4 mg/l thiamine
(Sheff MA, Thorn KS. Optimized cassettes for fluorescent protein tagging in Saccharomyces cerevisiae. ”’Yeast”’ 2004; 21: 661–670.)