Novobiocin Selectatab™
Novobiocin Selectavial™
MS30, SV30 For the selective isolation of
Salmonella spp. by motility enrichment and the
selective enrichment of E.coli O157 in food and
faeces samples.
1. Motility enrichment of Salmonella spp.
1. Label Petri dishes using the self-adhesive
labels provided.
Introduction
Conventional procedures for the isolation of
salmonellae are time consuming and labour
intensive, often requiring the use of several
1
types of media. De Smedt and his colleagues
found modified semi-solid Rappaport-Vassiliadis
medium (MSRV), including novobiocin to inhibit
growth of competing organisms such as
Proteus, Escherichia coli and Pseudomonas,
was reliable for the isolation of Salmonella spp.
from food samples.
The majority of outbreaks and sporadic cases of
haemorrhagic colitis (bloody diarrhoea) and
haemolytic uraemic syndrome (HUS) in the
United Kingdom are associated with E.coli of
2
serogroup O157 (E.coli O157) . Outbreaks in
the UK have been associated with the
consumption of water and various foods, most
commonly of bovine origin, including, minced
3
beef, yoghurt and unpasteurised milk but,
isolation from suspect foods, notably red meat,
4
has only rarely been successful . Use of
tryptone soy broth, modified by the addition of
bile and buffer salts, and Novobiocin as an
enrichment medium has proved to be of value in
5
the examination of faeces samples .
Description
MAST has available supplements for the
selective enrichment of Salmonella spp. and
™
E.coli O157 - MAST Novobiocin Selelectatab is
designed for direct addition to 100ml of media.
™
MAST Novobiocin Selelectavial is designed for
addition to 2.25 litres of medium after
reconstitution with an approved diluent.
MS30
Content
™
Novobiocin Selectatab
Novobiocin
2.0mg
Concentration in
100ml medium
20mg/litre
SV30
Content
™
Novobiocin Selectavial
Novobiocin
45mg
Concentration in
2.25L of medium
20mg/litre
MAST is a Registered Trademark
Directions
2. Sterilise the medium, MAST MSRV
(Salmonella) medium (DM440), cool to 5055ºC and hold in a water bath at this
temperature.
™
3. Using sterile forceps add one Selectatab to
each 100ml of medium and label the bottle.
(For larger quantities reconstitute one vial of
™
SV30 Novobiocin Selectavial using 20ml of
sterile water and add to 2.25 litres of
medium).
4. Mix well, pour culture plates of normal
thickness (15-20ml per plate) and allow to
set.
5. Prepared culture plates may be used
immediately or stored in plastic bags at 2-8ºC
for up to one week before use.
In Use
Inoculate 3 drops (approximately 0.1ml each) of
an enriched culture in separate spots on the
surface of an MSRV plate. Allow the spots to air
dry and incubate the plates at 42ºC. After 24
hours examine the plates for migration and
confirm the identification of suspect colonies as
Salmonella by slide agglutination using MAST
™
ASSURE Antisera or biochemical methods.
2. Selective enrichment of E.coli O157
Food Samples
1. Sterilise the medium, MAST Modified
Tryptone Soy Broth (DM622) cool to 50-55ºC
and hold in a water bath at this temperature.
2. Reconstitute one SV30 Novobicin Selectavial
using 20ml of sterile water and add 2ml to
225ml of basal medium or one MS30
Novobiocin Selectatab per 100ml.
3. Mix gently, but thoroughly, to evenly distribute
the selective agents.
JAH 2/97 V0.2
-1
4. Prepare a 10 homogenate of food sample,
using either a stomacher or blender, by
homogenising 25g of sample in 225ml of
broth.
5. Incubate at 42ºC for 22 hours, preferably with
agitation, and subculture onto plates of CTSMAC medium (MAST DM491/SV48/SV49)
after 6 and 22 ± 2 hours. If immunomagnetic
separation techniques are used the broth
should be processed after 6 hours incubation
6. Incubate CT-SMAC plates at 37ºC for 24
hours and examine for the presence of nonsorbitol fermenting colonies.
7. Subculture five suspect colonies (or all visible
colonies if fewer than five) onto plates of
MAST MacConkey Agar (DM140) and
confirm the serotype of gram negative
lactose fermenting bacilli with suitable
™
antisera (MAST ASSURE Antisera M12030
for E.coli O157).
Faeces samples
References
1. De Smedt JM, Bolderijk RF, Rappold H,
Lautenschlaeger D. Rapid Salmonella
detection in foods by motility enrichment on a
modified semi-solid Rappaport-Vassiliadis
medium. J Food Prot. 1986; 49: 510-514
2. Sharp JCM, Ritchie LD, Curnow J, Reid
TMS. Incidence of haemorrhagic colitis due
to Escherichia coli O157 in one Scottish
town: clinical and epidemiological features.
J.Infect. 1994; 29: 343-350.
3. Bennett AR, Macphee S, Betts RP.
Evaluation of methods for the isolation and
detection of Escherichia coli O157 in minced
beef. Letters In Applied Microbiology. 1995;
20: 375-379
4. Chapman PA. Isolation, identification and
typing of verocytotoxin producing Escherichia
coli O157. PHLS Microbiology Digest. 1994;
11: 13-17.
5. Bolton EJ, Crozier L, Williamson JK.
Optimisation of methods for the isolation of
Escherichia coli O157 from beefburgers.
PHLS Microbiology Digest. 1995; 12: 65-70.
1. Sterilise the medium, MAST Modified
Tryptone Soy Broth (DM622) cool to 50-55ºC
and hold in a water bath at this temperature.
2. Reconstitute one SV30 Novobicin Selectavial
using 20ml of sterile water and add to 2.25
litres of medium or one MS30 Novobiocin
Selectatab directly to 100ml.
3. Mix gently, but thoroughly, to evenly distribute
the selective agents.
4. Aseptically distribute the medium, in 10ml
volumes, into previously sterilised containers.
5. Inoculate approximately 0.5g of faeces into
10ml of prepared broth.
6. Incubate at 37ºC for 18-22 hours and
subculture onto plates of CT-SMAC medium
(MAST DM491/SV48/SV49). Incubate the
CT-SMAC plates at 37ºC for 24 hours,
examine for the presence of non sorbitol
fermenting colonies and confirm as above.
MAST is a Registered Trademark
JAH 2/97 V0.2