Madhadron:SpheroplastingSmegmatis

(Based on Udou et al, 1982)

1. Grow culture of smeg overnight
2. Add 20% glycine to final concentration 1.2%, and incubate for 16-20h.
3. Centrifuge at 1,200g for 15 minutes, decant supernatant. Resuspend in same volume SMM.
4. Centrifuge again. Resuspend in same volume of P medium.
5. Incubate for 16 to 20h
6. Centrifuge at 300g for 7 minutes and discard pellet.
7. Centrifuge supernatant at 1,200g for 15 minutes.
8. Wash twice with SMM

P medium

(Additives per L of water)

• 120g sucrose
• 10g glucose
• 12g L-glycine
• 1g (NH_4)_2 SO_4
• 0.25g K_2 SO_4
• 2.03g MgCl_2 times 6H_2)
• 3.68 CaCl_2 * 2H_2O
• 0.1g KH_2 PO_4
• 0.025M [N-tris(hydroxymethyl)methyl]-2-aminoethanesulfonic acid (TES; pH 7.2)
• 50mg lysozyme
• 30mg lytic enzyme no.2

SMM

0.5M sucrose + 20mM MgCl_2 + 0.02M maleate (pH 6.5)

Alternate Protocol

(based on Thacore et al, 1963)

• Spheroplasting agents: 100μg/mL lysozyme + 6.84mM EDTA
• Supportive agents: 0.34M sucrose, 1.1μg/mL Mg$^{2+}$

Just add to 7H9.

Notes

Need to measure mycolic acid concentration to make sure that they really have sloughed off cell wall (Mike Glickman is the local expert).

Make spheroplasts, then try centrifuging them, plating them, etc. How rough can I be, and how much does it matter? Can spheroplasted smeg grow? In the presence of lysozyme + EDTA? What is it's growth rate?

To Characterize Spheroplasts

Measure change in peptidoglycan concentration upon spheroplasty.

GAG medium: 7H9 + 0.02% Tween-80 + 1mM GlcNAc + 5mM glucose

• Grow culture overnight in osmotically supportive medium, with or without lysozyme + EDTA

Experiments

Spheroplast cells, check shape and viability

Materials

• 200 plates LB agar + osmotic support
• 24 slides
• 28 cuvettes
• PBS
• 5x 7H9
• 0.5M sterile EDTA
• 20% dextrose
• 20mg/mL lysozyme
• 0.83 (10%) MgSO₄ in water

Media: 5mL 5x 7H9 + 340μL 0.5M EDTA + 125μL 20mg/mL lysozyme + 7mL 20% dextrose + 275μL 0.83M MgSO₄ + 12.26 autoclaved water

Directions:

1. Grow M. smegmatis overnight in 7H9.
2. In the morning, wash the cells and inoculate new cultures to OD$_600$=0.1: 7H9, 7H9 + spheroplasting agents, 7H9 + osmotic support, 7H9 + spheroplasting agents + osmotic support. Make blank tubes for each of these media for use in the spec. Measure OD and plate, with and without centrifuging. Put on to shake.
3. Measure OD and plate every 2h for 10h, again with and without centrifuging. Heat fix samples on labeled slides for later observation.