Preparation of a Single-Cell Bacterial Inoculum

Abstract

Source: Niu, L., et al., Visualization of Macrophage Lytic Cell Death During Mycobacterial Infection in Zebrafish Embryos via Intravital Microscopy. J. Vis. Exp. (2019)

This video demonstrates the preparation of a Mycobacterium marinum single-cell suspension from a late-log phase culture for consistent use in infection assays and downstream applications.

Protocol

1. Mycobacterium marinum Single-Cell Inoculum Preparation (Figure 1)

1. Thaw Cerulean-fluorescent Mycobacterium marinum glycerol stock from -80 °C and inoculate a 7H10 agar plate with 10% (v/v) OADC (oleic acid, albumin, dextrose, catalase), 0.25% glycerol, and 50 μg/mL hygromycin. Incubate the plate at 32 °C for around 10 days.
2. Select a colony expressing positive fluorescence and inoculate 3 mL of 7H9 medium with 10% OADC, 0.5% glycerol, and 50 μg/mL hygromycin.
   1. Incubate the inoculation at 32 °C and 100 revolutions per minute (rpm) for 4–6 days until the culture reaches logarithmic phase (OD₆₀₀ = 0.6–1.0).
   2. Subculture (1:100) in 30 mL of fresh 7H9 medium with 10% OADC, 0.5% glycerol, 0.05% Tween-80, and 50 μg/mL until the OD₆₀₀ reaches ~1.0.
      NOTE: At this point, a subculture step is recommended for the highest culture quality. In our experience, adding the clone directly to a large volume of medium will lead to the formation of bacterial clumps.
3. Collect M. marinum cells as described below.
   1. Centrifuge at 3,000 x g for 10 min to collect the M. marinum as a pellet. Discard all but 300 μL of the supernatant and use it for resuspending the pellet.
   2. Add 3 mL of 7H9 medium with 10% glycerol to further resuspend the pellet, then sonicate the suspension in a water bath at 100 W at 15 s ON, 15 s OFF for a total of 2 min.
      NOTE: Sonication is used to achieve a single-cell homogenate for the inoculum, which prevents the microinjection needle from being blocked.
4. Transfer the bacterial suspension to a 10 mL syringe and pass it through a 5 μm filter to remove clumps.
5. Measure the optical density (OD) of the suspension using a spectrophotometer and dilute it to OD₆₀₀ = 1.0 with 7H9 media containing 10% glycerol. Divide the suspension into 10 μL aliquots and store at -80 °C freezer for future use.
6. Confirm the bacterial concentration of the inoculum (cfu/mL) by serial dilution and plating of the bacterial stock on a 7H10 agar plate containing 10% (v/v) of OADC, 0.5% of glycerol, and 50 μg/mL of hygromycin.

Results

Figure 1: Schematic diagram of single-cell bacteria preparation. Single-cell Cerulean-fluorescent ...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
0.05% Tween-80	Sigma	P1379
10 mL syringe	Solarbio	YA0552
10% OADC	BD	211886
5 μm filter	Mille X	SLSV025LS
50 μl/ml hygromycin	Sangon Biotech	A600230
7H10	BD	262710
7H9	BD	262310
Enviromental Chamber	Pecon	Temperature control 37-2 digital
Eppendorf microloader	Eppendorf	No.5242956003
Glycerol	Sangon Biotech	A100854
Incubator	Keelrein	PH-140(A)
Mycobacterium marinum			ATCC BAA-535
Sonicator	SCICNTZ	JY92-IIDN
Spectrophotometer (OD600)	Eppendorf AG	22331 Hamburg