Preparation of Bioluminescent Enterohemorrhagic Escherichia coli Inoculum

Abstract

Source: Kuo, C., et al. Detection of Enterohemorrhagic Escherichia Coli Colonization in Murine Host by Non-invasive In Vivo Bioluminescence System. J. Vis. Exp. (2018)

This video demonstrates the preparation of a luciferase-expressing enterohemorrhagic Escherichia coli suspension from frozen stock for use as an oral inoculum in animal models. This enables real-time, bioluminescent tracking of intestinal colonization during in vivo infection studies.

Protocol

1. Bioluminescent EHEC (enterohemorrhagic Escherichia coli) Bacteria Preparation for Oral Inoculation

NOTE: The timeline flowchart of the experimental procedures for EHEC preparation and mouse oral gavage is presented in Figure 1 to aid in experimental preparation.

1. Streak E. coli O157:H7 EDL933 bacteria harboring the luciferase plasmid onto an LB (Luria-Bertani) agar plate with 50 µg/mL kanamycin from -80 °C stock. Grow the bacteria for 16 - 18 h at 37 °C.
2. Pick a single colony from the overnight plate and culture in 3 mL LB medium with 50 µg/mL kanamycin for 16 - 18 h in a 37 °C incubator at 220 rpm.
3. Subculture the bacteria (1:100 dilution) into kanamycin (50 µg/mL)- containing LB broth for 2.5 - 3 h in a 37 °C incubator at 200 rpm. (For instance, add 2 mL overnight cultured bacteria to 200 mL LB medium with kanamycin (50 µg/mL) supplemented.)
4. Incubate the bacteria for 2.5 - 3 h and measure the optical density (OD) value at 600 nm (OD₆₀₀) until the value is between 0.9 to 1. The bacterial cell number is at a density of about 10⁸ colony-forming units (CFU)/mL.
5. Centrifuge the re-cultured bacteria at 8,000 x g for 30 min, 4 °C.
6. Discard the supernatant without agitating the bacteria pellet, and wash the pellet with 100 mL 0.9% sterile normal saline by gentle agitation.
7. Repeat steps 1.5 and 1.6 once.
8. After the wash, centrifuge the bacterial culture at 8,000 x g for 30 min, 4 °C, and discard the supernatant gently.
9. Condense the pellet 100-fold with 0.9% sterile normal saline. For instance, if 200 mL of bacteria are centrifuged, add 2 mL of normal saline to suspend the pellet.
10. Confirm the bacteria CFU (it should be approximately 10⁹ CFU/100 μL after condensation in normal saline) by plating the concentrated bacteria through a 10-fold serial dilution.

Results

Figure 1: Timeline of the experimental preparation flow chart.

Overview of the timing needed to prepare biolu...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Shaker incubator	YIH DER	LM-570R	bacteria incubation
Orbital shaking incubator	FIRSTEK	S300	bacteria incubation
Glycerol	SIGMA	G5516-1L	use for bacteria stocking solution
Sodium chloride	Sigma	31434-5KG-R	chemical for making LB medium, 10 g/L
Tryptone	CONDA pronadisa	Cat 1612.00	chemical for making LB medium, 10 g/L
Yeast Extract powder	Affymetrix	23547-1 KG	chemical for making LB medium, 5 g/L
Agar	CONDA pronadisa	Cat 1802.00	chemical for making LB agar
Kanamycin	Sigma	K4000-5G	antibiotics, use for seleciton
Streptomycin	Sigma	S6501-100G	antibiotics, eliminate the microbiota in mice
High-speed centrifuge	Beckman Coulter	Avanti, J-26S XP	use for centrifuging bacteria
Fixed-Angle Rotor	Beckman Coulter	JA25.5	use for centrifuging bacteria
Fixed-Angle Rotor	Beckman Coulter	JLA10.5	use for centrifuging bacteria
Centrifuge bottles	Beckman Coulter	REF357003	use for centrifuging bacteria
Centrifuge bottles	Thermo Fisher scientific	3141-0500	use for centrifuging bacteria
Eppendorf biophotometer plus	Eppendorf	AG 22331 hamburg	for measuring the OD600 value of bacteria