First Steps: Phase 1 & 2

Phase 1: Collecting microbes 

The first step for finding effective probiotics is to capture frogs and sample their skin for bacteria in Madagascar.  After finding frogs, each individual is rinsed to get rid of soil debris and transient bacteria (shown here for a salamander - photo A).  After being rinsed, each individual is swabbed on the ventral region (its stomach) with a sterile cotton swab (photo B).

This swab is then either streaked directly onto an agar plate or stored in a glycerol stock solution and frozen for future processing. After streaking, the plates are incubated for approximately 2-3 days. At this point, many bacterial colonies appear on the plates (see photo on left). Each unique colony is then isolated into pure culture (see photo on right), and a sample of each isolate is frozen to preserve the bacteria in its original state. The plates are monitored for approximately 2 weeks to make sure all unique colonies, including slow growers, are isolated.  After all colonies are isolated, we can move on to phase 2.

Phase 2: Conducting Bd inhibition assays

The second step in identifying effective probiotics is determining if the bacterial isolates inhibit the fungal pathogen, Bd.  Each collected isolate will be tested for Bd-inhibition.  This is done in one of two ways: an agar-plate challenge assay or a cell-free supernatant challenge assay.  The agar plate challenge assay involves spreading the fungus across the surface of a tryptone (1%) agar plate and streaking the bacteria isolate across the plated fungus.  After incubation for approximately 72-96 hours, the plate is examined for a zone of inhibition.  If a clear zone with no fungal growth has formed around the bacteria, then it is inhibiting Bd by secreting inhibitory metabolites into the agar (see the right streak in the photo).  This zone is measured to compare the strength of inhibition of different isolates.

The second method involves obtaining the metabolites produced by each bacterial isolate in pure culture.  After growing a bacterial isolate in liquid culture, the culture is filtered to remove the cells and leave only the products produced by the bacteria.  These products include anti-Bd metabolites if the bacteria can produce them.  The Bd fungus is then grown with a suspension of this cell-free supernatant in a well in a microtiter plate (see photo).  A negative control and positive control are included for comparison, and replicate trials are conducted.  Using a spectrophotometer we can determine which bacteria inhibit Bd and the strength of inhibition of each bacterial isolate.  (More information on these techniques can be found in Harris et al. 2006 and Bell et al. 2013.)  The bacterial isolates that are found to be strongly inhibitory will be classified to the genus level using molecular methods and are probiotic candidates.