PCR Detection of Microbial Pathogens PCR Detection of Microbial ...

114 Englen et al.
3.1.3. Fecal Sample Enrichment
1. An inexpensive enrichment method to enhance recovery of Campylobacter utilizes
24-well tissue culture plates (29). Begin by dispensing 1.0 mL of Bolton’s
broth into the plate wells.
2. Dip sterile cotton-tipped swabs into the 1:4 fecal dilutions and inoculate wells
with the swabs.
3. Put the plates into zip-lock bags, gas as described for direct plating (see Subheading
3.1.2.), and incubate for 48 h at 42°C.
4. Noting the direct plates that were negative for Campylobacter, streak for isolation
only from those corresponding sample enrichment wells onto Campy-Cefex plates.
Bag and gas the plates as described in Subheading 3.1.2., and incubate for 48 h at
42°C. By limiting further isolation steps to only those samples that were negative
by direct plating, unnecessary duplication of the positive samples is avoided.
3.1.4. Presumptive Identification of Campylobacter
1. Prepare wet-mounts of Campylobacter as follows (see Note 8). Place a drop of
PBS or distilled water on a standard microscope slide.
2. Using a sterile loop, pick a portion of a colony to be examined. Suspend the cells
in the drop of PBS by mixing.
3. Place a cover slip on the cell suspension, using care to avoid trapping air under
the slip.
4. Add a drop of immersion oil on top of the cover slip, and observe the cells using
phase-contrast optics at 100×.
5. Campylobacter is easily identified by its characteristic narrow, curved, or spiral
rod shape and darting motility. Pairs of cells often resemble a gull’s wing or the
letter S. Campylobacters from cultures older than 24–48 h may become coccoid
in shape and are more difficult to identify.
3.1.5. Storage and Shipment of Campylobacter Isolates
1. For frozen preservation of Campylobacter cultures, begin by streaking isolates
onto HBA plates (see Note 9). Incubate the plates for 48 h at 42°C as previously
described.
2. Using a sterile cotton-tipped swab, transfer a heavy inoculum from a plate to a
2-mL cryovial containing 1 mL of freezing medium.
3. Store vials at –70°C. Cultures stored in this way will remain viable for 6–12 mo.
However, repeated thawing and refreezing may reduce viability significantly (see
Note 2).
4. For transport or shipment of Campylobacter cultures, Wang’s transport medium is
recommended. Transport tubes are prepared by adding 2–4 mL of medium to sterile
screw-capped glass vials or plastic 5-mL cryovials. Allow the soft agar to solidify.
5. Inoculate the vials by stabbing a loop of Campylobacter culture into the semisolid
media. Place vials in a clean rack, loosen the caps and put the rack of vials
in a zip-lock bag. Gas and seal the bags, and incubate for 24 h at 42°C.
6. Tighten caps, and ship according to local guidelines.