Quantification of Helicobacter pylori Load in an Infected Mouse Stomach

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).