Generating Protoplasts from the Gram-Positive Bacterium Bacillus megaterium

Abstract

Source: Figueroa, D. M., et al. Production and Visualization of Bacterial Spheroplasts and Protoplasts to Characterize Antimicrobial Peptide Localization. J. Vis. Exp. (2018)

This video demonstrates a step-by-step procedure for generating protoplasts from the Gram-positive bacterium Bacillus megaterium.

Protocol

1. Solution Preparation

1. Prepare 5 mg/mL lysozyme by dissolving 0.015 g lysozyme in 3 mL of dH₂O in a 50 mL flask. Sterilize by filtering through a 25 mm syringe filter with a 0.2 µm membrane. Aliquot solution into microfuge tubes and store at -20 °C.
2. Prepare 3% w/v Trypticase Soy Broth (TSB) by dissolving 30 g of TSB in 1 L of dH₂O in a 2 L flask. Aliquot the solution into flasks containing 100 mL TSB to be used in the preparation of B. megaterium protoplasts. Autoclave flasks to sterilize the liquid medium. Sterile TSB can be stored at either room temperature or 4 °C.
3. Prepare solution C (1 M sucrose, 0.04 M maleate, 0.04 M MgCl₂, pH 6.5) by dissolving 34.23 g sucrose (342.3 g/mol), 0.46 g maleic acid (116.07 g/mol), and 0.38 g MgCl₂ (95.21 g/mol) in 100 mL of dH₂O in a 250 mL flask. Adjust pH to 6.5. Sterilize by filtering through a 25 mm syringe filter with a 0.2 µm membrane and store in a conical tube at room temperature.
4. Prepare 200 mL protoplast medium by mixing 100 mL 3% w/v TSB and 100 mL solution C in a 1 L glass bottle. Autoclave to sterilize the solution and store at room temperature.

2. Preparation of Gram-positive B. megaterium Protoplasts

1. In a 250 mL flask, dilute the overnight culture 1:1,000 in 100 mL of 3% w/v TSB and incubate the bacterial solution at 37 °C, while shaking for approximately 4.5 h until the solution has reached an optical density of 0.9-1.0 at 600 nm. Measure the optical density using a spectrophotometer.
2. Pour the liquid culture into two 50 mL conical tubes and centrifuge at 2,000 x g, 4 °C for 10 min.
3. Using a serological pipette, discard the supernatant from both conical tubes. Resuspend each pellet in 2.5 mL protoplast medium and combine the resuspended solutions into a single conical tube. Pipette the combined resuspended solution into a 125 mL flask.
4. Add 1 mL of 5 mg/mL lysozyme and incubate for 1 h at 37 °C, while shaking.
5. Monitor the growth of protoplasts under 1,000x magnification using a light microscope, noting any irregularities, such as bacteria that appear as swollen rods instead of spheres (Figure 1). Using a serological pipette, transfer the solution to a 15 mL conical tube and centrifuge at 2,000 x g, 4 °C for 10 min.
6. Decant the supernatant and re-suspend the pellet in 5 mL protoplast media. Store protoplasts at -20 °C for up to a week or until they have gone through 3 freeze-thaw cycles.

Results

Figure 1: Representative images of B. megaterium. (<...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Magnesium chloride	Sigma	M8266
Sucrose	Sigma	S7903
Lysozyme	Sigma	L6876
BBL Trypticase soy broth	Fisher Scientific	B11768
Maleic acid	Sigma	M0375
Acrodisc 25 mm Syringe Filter w/ 0.2 μm HT Tuffryn Membrane	Pall Corporation	4192