an epidemiological study of listeriosis in dairy cattle

5. 2. 2. Bacteriology :
a) Culture procedure: (Appendix 3)
A three stage procedure was used to isolate Listeria. This involved cold
enrichment followed by the use of selective broth culture and selective solid medium at
higher temperatures. This was adapted from the technique described by Fenlon (1985)
and Lewis and Corry (1991).
Samples were first cultured when fresh and then held at 4 0 C for a period of up to
7 weeks to enhance the probability of isolating Listeria by cold enrichment. Samples
which were negative at first culture were re-cultured at predetermined optimal times (at
the 3rd and 7th weeks of cold storage).
An aliquot of the sample was placed into Listeria Selective Enrichment Broth
(LSEB) (Lovett and others 1987) in a universal container to give a final concentration
of approximately 1/10 (w/v) and incubated for 24 or 48 hours at 30 0 C. One loopful of
this broth culture was streaked out on to Listeria Selective Agar (LSA) (Curtis and
others 1989a) and incubated at 30 0 C for 48 hours (Figure 5. 1).
Faecal samples collected in September (30 samples) and in October (70
samples) were cultured without cold enrichment. Listeria spp. were isolated from 3 of
the 100 samples.
To investigate the effect of cold enrichment on the number of isolates, 20
samples were collected in October 1994 and stored at 4 0 C and cultured weekly for 7
weeks and then again on weeks 13, 14 and 15.
The effect of storing the samples at 4 0 C in three liquid media, -saline, LSEB and
NB,- on the growth quantity was compared. The results obtained using these media
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