1. Bacteria harvestingCAUTION: This step involves handling of pathogenic bacteria.<ol><li>Grow one colony of strains carrying either pCN51-P3-MS2-sRNA or pCN51-P3-MS2 plasmids in 3 mL of Brain Heart Infusion (BHI) medium supplemented with erythromycin (10 &#181;g/&#181;L) in duplicates.</li><li>Dilute each overnight culture in 50 mL (&#8776;1/100) of fresh BHI medium supplemented with erythromycin (10 &#181;g/&#181;L) to reach an OD600nm of 0.05. Use 250 mL sterilized flasks (5:1 flask-to-medium ratio). NOTE: Medium and growth conditions should be set according to the expression pattern of the studied sRNA.</li><li>Grow cultures at 37&#176;C with shaking at 180 rpm for 6 h.</li><li>Transfer each culture into a 50 mL centrifuge tube.</li><li>Centrifuge at 2,900 x g during 15 min at 4&#176;C. Discard the supernatant.</li><li>Keep pellets on ice and directly perform mechanical cell lysis or freeze and store pellets at -80 &#176;C.</li></ol>2. Mechanical cell lysisCAUTION: Following steps must be performed on ice and buffers must be at 4 &#176;C. Use gloves and take all precautions to protect samples from RNases.<ol><li>Resuspend pellets (step 1.6) in 5 mL of Buffer A (150 mM KCl, 20 mM Tris-HCl pH 8, 1 mM MgCl2 and 1 mM DTT).</li><li>Transfer the resuspended cells in 15 mL centrifuge tubes with 3.5 g of silica beads (0.1 mm).</li><li>Insert tubes in a mechanical cell lysis instrument. Run a cycle of 40 s at 4.0 m/s. NOTE: If one cycle is not enough to break cells, let the device cool for 5 min while keeping samples on ice. Then, repeat another cycle of 40 s at 4.0 m/s. The efficiency of cell lysis can be tested by plating the supernatant on BHI-agar plate.</li><li>Centrifuge at 15,700 x g for 15 min. Recover the supernatant and keep it on ice.</li></ol>

<table><tbody><tr><td>15 mL centrifuge tubes</td><td>Falcon</td><td>352070</td><td> </td></tr><tr><td>50 mL centrifuge tubes</td><td>Falcon</td><td>352051</td><td> </td></tr><tr><td>250 mL culture flask</td><td>Dominique Dutscher</td><td>2515074</td><td>Bacterial cultures</td></tr><tr><td>BHI (Brain Heart Infusion) Broth</td><td>Sigma-Aldrich</td><td>53286</td><td>Growth medium</td></tr><tr><td>Erythromycin (ery)</td><td>Sigma-Aldrich</td><td>Fluka 45673</td><td>Growth medium</td></tr><tr><td>Lysing Matrix B Bulk</td><td>MP Biomedicals</td><td>6540-428</td><td>Mechanical lysis</td></tr><tr><td>RNAPro Solution</td><td>MP Biomedicals</td><td>6055050</td><td>Mechanical lysis</td></tr><tr><td>FastPrep device</td><td>MP Biomedicals</td><td>116004500</td><td>Mechanical lysis</td></tr><tr><td>Spectrophotometer Genesys 20</td><td>ThermoFisher Scientific</td><td>11972278</td><td>Bacterial cultures</td></tr></tbody></table>

a procedure for bacterial harvesting and mechanical lysis

A Procedure for Bacterial Harvesting and Mechanical Lysis

Source: <a href='https://app.jove.com/v/61731/ms2-affinity-purification-coupled-with-rna-sequencing-in-gram-positive-bacteria'>Mercier, N., et al. MS2-Affinity Purification Coupled with RNA Sequencing in Gram-Positive Bacteria. J. Vis. Exp. (2021)</a>This video demonstrates a step-by-step procedure for harvesting bacterial cells and mechanically lysing them to release intracellular contents.

Source: Mercier, N., et al. MS2-Affinity Purification Coupled with RNA Sequencing in Gram-Positive Bacteria. J. Vis. Exp. (2021)This video demonstrates a ...

Take a culture of Staphylococcus aureus, a bacterium with a thick peptidoglycan cell wall.Dilute the culture in fresh media containing antibiotics and incubate it at a warm temperature with shaking to promote bacterial growth.Transfer the culture to a tube and centrifuge it to pellet the bacteria.Discard the supernatant and place the pellet on ice to protect the bacteria from damage.Resuspend the pellet in lysis buffer and transfer it to a tube containing silica beads.Insert the tube into a mechanical cell lysis instrument and initiate agitation.Rapid collisions between the bacteria and beads generate shear forces. This lyses the bacteria, releasing DNA, RNA, and RNA-protein complexes.Ions in the lysis buffer stabilize RNA structures, while reducing agents in the buffer inactivate RNA-degrading enzymes.Now, centrifuge the mixture again to pellet the debris.Collect the supernatant containing nucleic acids and proteins for further analysis.Grow one colony of strains carrying either pCN51 P3 MS2 sRNA or pCN51 P3 MS2 plasmids in three milliliters of BHI medium supplemented with 10 micrograms per microliter erythromycin in duplicates.Dilute each overnight culture in 50 milliliters of fresh BHI medium supplemented with erythromycin to reach an OD 600 of 0.05. Grow the cultures at 37 degrees Celsius with shaking at 180 RPM for six hours. Transfer each culture into a 50 milliliter centrifuge tube and centrifuge the tubes at 2, 900 times G for 15 minutes at four degrees Celsius, then discard the supernatant, freeze and store them at minus 80 degrees Celsius or keep the pellets on ice and directly perform mechanical cell lysis.To perform mechanical cell lysis, resuspend pellets in five milliliters of buffer A and transfer the resuspended cells to 15 milliliter centrifuge tubes with 3.5 grams of silica beads. Insert the tubes in a mechanical cell lysis instrument and run a cycle of 40 seconds at four meters per second. Centrifuge the tubes at 2,900 times G for 15 minutes, then recover the supernatant and keep it on ice.Make sure that the supernatant is clear, repeating the centrifugation if it is not.

a-procedure-for-bacterial-harvesting-and-mechanical-lysis

Research

JoVE Encyclopedia of Experiments

Microbiology

Bacterial Growth and Techniques

Isolation and Fractionation Techniques

789 Views. Take a culture of Staphylococcus aureus, a bacterium with a thick peptidoglycan cell wall.Dilute the culture in fresh media containing antibiotics and incubate it at a warm temperature with shaking to promote bacterial growth.Transfer the culture to a tube and centrifuge it to pellet the bacteria.Discard the supernatant and place the pellet on ice to protect the bacteria from damage.Resuspend the pellet in lysis buffer and transfer it to a tube containing silica be...

A Procedure for Bacterial Harvesting and Mechanical Lysis

In This Article

Abstract

Protocol

Materials

Tags

Name	Company	Catalog Number	Comments
15 mL centrifuge tubes	Falcon	352070
50 mL centrifuge tubes	Falcon	352051
250 mL culture flask	Dominique Dutscher	2515074	Bacterial cultures
BHI (Brain Heart Infusion) Broth	Sigma-Aldrich	53286	Growth medium
Erythromycin (ery)	Sigma-Aldrich	Fluka 45673	Growth medium
Lysing Matrix B Bulk	MP Biomedicals	6540-428	Mechanical lysis
RNAPro Solution	MP Biomedicals	6055050	Mechanical lysis
FastPrep device	MP Biomedicals	116004500	Mechanical lysis
Spectrophotometer Genesys 20	ThermoFisher Scientific	11972278	Bacterial cultures