This article details a method for quantifying Helicobacter pylori colonization in mouse stomach tissue. The process involves homogenizing gastric tissue and culturing the bacteria on specialized agar plates.
To quantify Helicobacter pylori colonization in an infected mouse stomach, begin with the flattened stomach tissue containing the body and antrum regions, suspended in nutrient media to support bacterial viability.
Homogenize to break down the gastric tissue with the mucus layer, releasing Helicobacter pylori from the tissue into media. Perform serial dilutions of the resulting gastric homogenate using media.
Obtain dried horse blood agar, HBA, plates, divided into multiple segments. The HBA plates are supplemented with a mixture of antibiotics. Transfer each homogenate dilution onto separate segments of the plate and spread it evenly.
Post-drying, position the plates in an inverted position within a humidified anaerobic culture jar with specialized gas mixtures. Incubate.
The microaerophilic environment provided by the anaerobic culture jar in conjunction with the HBA nutrients facilitates Helicobacter pylori to grow and form colonies. The antibiotics in HBA inhibit the growth of bacterial species from the endogenous gastric microbiota.
Count Helicobacter pylori colonies on HBA plate segments falling within a specific countable range.
Calculate the Helicobacter pylori load in the gastric tissue, indicating the extent of bacterial colonization within the tissue.
To count the number of viable H. pylori in the stomach post-infection, homogenize stomach samples stored in BHI, and perform duplicate serial dilutions of the resulting gastric homogenates in fresh sterile broth.
Divide pre-dried horse blood agar, or HBA plates, supplemented with additional antibiotics into three or four segments, and using an adaptation of the Miles and Misra technique, add 10 to 100 microliters of each stomach homogenate dilution onto a segment of each agar plate. Spread the homogenates with sterile plastic loops and allow the plates to dry. Then, place the plates in an inverted position in anaerobic gas jars containing a Petri dish of water and a gas pack, then, incubate the jars at 37 degrees Celsius for four to seven days.
When colonies can be observed, enumerate the segments containing between 10 and 100 isolated colonies.
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