an epidemiological study of listeriosis in dairy cattle

6) Sugar tests: Listeria are capable of producing acid from some carbohydrates
(Rhamnose, Glucose, Xylose, Mannitol,) and reducing nitrate to nitrite. 1% sugar plates
containing different carbohydrates were prepared (Appendix 3) and divided into four
quarters. An isolate of Listeria was streaked out on each quarter and incubated at 37 0 C
overnight. A change in the colour of medium from red to yellow due to acid production
confirmed the presence of Listeria.
7) Storage of isolates: All isolates identified as Listeria spp. were placed on
preservative beads as instructed by the manufacturer (TSC, Lancashire, UK) and kept
at -20 0 C.
c) Investigation of the limit of detection:
The method of Miles-Misra (reviewed by Corry 1982) was used to determine the
detection limit of the culture method used in this study. Briefly a colony of L.
monocytogenes grown on SBA was inoculated into 10ml of LSEB and incubated
overnight at 30 0 C. Twelve tenfold dilutions were prepared. Four 20μl drops from each
dilutions were spotted on quartered LSA plates incubating at 37 0 C for 48 hours and
2ml into 10 g of faeces, previously autoclaved. The mixture was held at 4 0 C and
cultured weekly for three successive weeks and again at the 7th week to determine the
effect of cold enrichment on the limit of detection. The number of colony forming
units in the original suspension was calculated by selecting LSA plates on which
colonies could be easily counted. The weighted mean of the number of colonies in
each quarter was then calculated for 4 different dilutions of the starting inoculum
using the following formula:
cx + cx+1 + cx+2 .+ cx+3..
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