Quantification of Helicobacter pylori Load in an Infected Mouse Stomach

Abstract

Source: D'Costa, K. et al., Mouse Models Of Helicobacter Infection And Gastric Pathologies. J. Vis. Exp. (2018)

In this video, we describe a protocol to quantify Helicobacter pylori load in mouse stomach tissues post-infection to confirm its colonization.

Protocol

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.

1. Confirmation of Bacterial Colonization in the Stomach Post-infection

1. Viable Counting of H. pylori in the Stomach
   1. Supplement sterile HBA plates with additional antibiotics (200 μg/mL bacitracin and 10 μg/mL nalidixic acid) prior to performing colony counts from infected mouse stomachs.
   2. Homogenize stomach sections either manually, using autoclavable polypropylene micro pestles, or using a mechanical dissociatio....

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Bacteriological reagents
Oxoid Blood Agar Base No.2	Thermo Fischer Scientific	CM0271B	Dissolve in deinonized water prior to sterilization
Premium Defibrinated Horse blood	Australian Ethical Biologicals	PDHB100
Bacto Brain Heart Infusion Broth	BD Bioscience	237500	Dissolve in deinonized water prior to sterilization
CampyGen gas packs	Thermo Fischer Scientific	CN0035A/CN0025A
Antibiotics
Vancomycin	Sigma Aldrich	V2002-1G	Dissolve in deionized water
Polymyxin B	Sigma Aldrich	P4932-5MU	Dissolve in deionized water
Trimethoprim (≥98% HPLC)	Sigma Aldrich	T7883	Dissolve in 100% (absolute) Ethanol
Amphotericin	Amresco (Astral Scientific)	E437-100MG	Dissolve in deionized water
Bacitracin from Bacillus licheniformis	Sigma Aldrich	B0125	Dissolve in deionized water
Naladixic acid	Sigma Aldrich	N8878	Dissolve in deionized water
Equipment and plasticware
Oxoid Anaerobic Jars	Thermo Fischer Scientific	HP0011/HP0031
COPAN Pasteur Pipettes	Interpath Services	200CS01
Eppendorf 5810R centrifuge			Collect bacterial pellets by centrifugation at 2,200 rpm for 10 mins at 4°C
Parafilm M	Bemis, VWR	PM996
Eppendorf micropestle for 1.2 - 2 mL tubes	Sigma Aldrich	Z317314	Autoclavable polypropylene pestles used for stomach homogenization
GentleMACs Dissociator	Miltenyi Biotec	130-093-235	Use a pre-set gentleMACS Programs for mouse stomach tissue
M Tubes (orange cap)	Miltenyi Biotec	30-093-236
Qubit Fluorometer	Thermo Fischer Scientific	Q33216
Sterile plastic loop	LabServ	LBSLP7202