Quantification of Helicobacter pylori Load in an Infected Mouse Stomach

Overview

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 dissociation instrument (see Table of Materials).
   3. Prepare duplicate serial dilutions (10⁻¹ to 10⁻²) of the resulting gastric homogenates in sterile BHI.
      NOTE: Dilutions should be decided based on the typical bacterial loads obtained for a given H. pylori strain used for infection, as well as the duration of infection. Undiluted samples can also be used.
   4. Divide pre-dried HBA plates (NOTE: In order to ensure isolation of single colonies, HBA plates should be warmed and dried in a biological safety cabinet or 37 °C incubator for 10–15 min prior to use.) into three or four segments. Using an adaptation of the Miles and Misra technique, add 10–100 mL of each dilution onto a segment of the agar plate and spread using sterile plastic loops.
   5. Allow the plates to dry and then place them in an inverted position (lid side down) in anaerobe gas jars. To maintain humidity in the jars, include a Petri dish containing water.
   6. Incubate jars at 37 °C until colonies form (typically 4–7 days).
   7. Enumerate segment(s) containing between 10 and 100 isolated colonies.
      NOTE: H. pylori colonies and H. felis growth on plates can be distinguished from those of other members of the murine gastric microbiota using standard urease, catalase, and oxidase tests. H. pylori and H. felis are positive for all three tests.
   8. Calculate the bacterial loads as (CFU/g of tissue), using the following formula:
      [(Average number of colonies counted) × (dilution factor) x (volume plated)]/(stomach weight).

Materials

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