SOP Bacteriological Collaborative Study VI (2002) page 1 of 8

CRL-6DOPRQHOOD, Bilthoven, The Netherlands

BACTERIOLOGICAL COLLABORATIVE STUDY VI (2002)
ORGANISED BY CRL 6$/021(//$
STANDARD OPERATING PROCEDURE FOR THE DETECTION OF
SALMONELLA IN THE PRESENCE OF COMPETITIVE MICRO-ORGANISMS.
1 Scope and field of application
This standard operating procedure (SOP) describes the procedure for the detection of
6DOPRQHOOD in the presence of competitive micro-organisms. For this purpose Reference
Materials (RMs) containing sublethally injured 6DOPRQHOOD Typhimurium or 6DOPRQHOOD
Enteritidis as prepared by the Community Reference Laboratory for 6DOPRQHOOD (CRL).
Furthermore poultry faeces is used. The application of this SOP is limited to the
bacteriological collaborative study for 6DOPRQHOOD described in this SOP.
2 References
International Standard ISO 6579: 2002(E)
Microbiology of food and animal feeding stuffs Horizontal method for the detection of
6DOPRQHOODVSS.
Beckers, H.J., Van Leusden, F.M., Meijssen, M.J.M., Kampelmacher, E.H. 1985.
Reference material for the evaluation of a standard method for the detection of 6DOPRQHOOD in
foods and feeding stuffs. J. Appl. Bacteriol., 59, 507-512.
3 Definitions
For the purpose of this SOP, the following definitions apply:
6DOPRQHOOD: micro-organisms which form typical colonies on isolation media for
6DOPRQHOOD and which display the serological and/or biochemical reactions described
when tests are carried out in accordance with this SOP.
SOP Bacteriological Collaborative Study VI (2002) page 2 of 8
CRL-6DOPRQHOOD, Bilthoven, The Netherlands
'HWHFWLRQRI6DOPRQHOOD: detection of 6DOPRQHOOD from reference materials in the presence
of competitive organisms, when the test is carried out in accordance with this SOP.
5HIHUHQFH 0DWHULDO: a gelatine capsule containing a quantified amount artificially
contaminated spray dried milk.
4 Principle
The detection of 6DOPRQHOOD involves the following stages:
a) Pre-enrichment
b) Selective enrichment
c) Isolation
d) Confirmation of typical colonies as 6DOPRQHOOD.
5 Culture media
Composition and preparation of the media and reagents are described in Annex B of the ISO
6579:2002(E).
5.1 Non selective pre-enrichment medium
Buffered Peptone water (Annex B.1)
5.2 Selective enrichment medium
Rappaport Vassiliadis medium with soya (RVS broth) (Annex B.2)
Muller Kauffmann tetrathionate-novobiocin broth (MKTTn) (Annex B.3)
Modified Semi solid Rappaport Vassiliadis (Newsletter,
This medium must be boiled to dissolve (instructions Vol.5, No.2,
manufacturer). After boiling the medium must be June 1999)
transparent blue. After cooling down to 50C the
supplement or the novobiocine has to be added.
The final concentration of the novobiocine in the
medium should be 0.01 g/l. Plates should be poured
with a volume of 15 to 20 ml.
Selective enrichment medium routinely used in your laboratory
SOP Bacteriological Collaborative Study VI (2002) page 3 of 8
CRL-6DOPRQHOOD, Bilthoven, The Netherlands
5.3 Solid selective media for first and second isolation
Phenol red/brilliant green agar (Annex B.4, ISO 6579993)
The medium must be boiled to dissolve
(instructions manufacturer). After boiling
the medium must be transparent red.
Plates should be poured with a volume of
30-40 ml (140 mm-plates).
Xylose-Lysine-Desoxycholate (Annex B.4)
This medium must be boiled to dissolve (instructions
manufacturer). After boiling the medium must be
transparent red. Plates should be poured with a volume
of 30-40 ml (140 mm-plates).
Third medium (optionally) (Paragraph 4.4)
5.4 Confirmation media
%LRFKHPLFDOFRQILUPDWLRQ
Triple sugar/iron agar (TSI agar) (Annex B.6)
Urea agar (Annex B.7)
l-Lysine decarboxylation medium (Annex B.8)
Nutrient agar (optionally) (Annex B.5)
6 Apparatus and glassware
The usual microbiological laboratory equipment. If requested, note specifications of the
apparatus and glassware on the test report.
Apparatus
Oven (for dry sterilisation) or autoclave (for wet sterilisation);
Incubator, capable of operating at 37C 1C;
Water bath, capable of operating at 41,5C 1C or incubator, capable of
operating at 41,5C 1C;
Water bath, capable of operating at 37C 1C;
Loops;
pH-meter; having an accuracy of calibration of 0.1 pH unit at 25C.
SOP Bacteriological Collaborative Study VI (2002) page 4 of 8
CRL-6DOPRQHOOD, Bilthoven, The Netherlands
Glassware
Culture bottles or jars with nominal capacity of 250 ml;
Culture tubes; 8 mm in diameter and 160 mm in length;
Micro-pipettes; nominal capacity 0,1 ml;
Petri dishes; small size (diameter 90 mm to 100 mm) and/or large size
(diameter 140 mm).
7 Procedure
Take the frozen faeces out of the freezer at the end of the working day and thaw the portions
frozen faeces overnight at 5 C.
7.1 Pre-enrichment
Allow the BPW to equilibrate to 37C for better dissolving of the capsules.
Record in test report (page 3) the requested data of the BPW. Take the numbered vials with
the 6DOPRQHOOD capsules and the control capsules out of the freezer one hour before they are
added to the BPW, to allow them to equilibrate to room temperature. Label 25 jars containing
225 ml of BPW from 1 to 25. For the naturally contaminated samples number 20 jars of BPW
from N1 to N20. Also label 12 jars of BPW from C1 to C12 (control capsules). One jar is a
procedure control (= C11) to which no capsule or faeces is added and one jar is a negative
faeces control to which only 10 gr. faeces is added (= C12). These control jars should further
be handled in the same way as the other jars.
After equilibration add to 35 labelled jars a gelatine capsule from the vial with the
corresponding label number. Do not open the gelatine capsule and do not shake the BPW to
dissolve the capsule more rapidly. Place the jars with the capsules in the 37 C incubator for
30 minutes for dissolving of the capsules. Record the temperature and time at the start and at
the end of this period in the test report (page 3). After 30 minutes add the thawed faeces to the
jars according to the following scheme:
Add 10 grams of faeces from portion A to jars labelled 1-25 and C12,
Add no faeces to jars labelled C1 - C11,
Add 25 grams of faeces from portion B to jars labelled N1-N20.
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SOP Bacteriological Collaborative Study VI (2002) page 5 of 8
CRL-6DOPRQHOOD, Bilthoven, The Netherlands
Place all jars in the 37 C incubator for 16 h to 20 h. Record the temperature and time at the
start and at the end of the incubation period and other requested data on page 3 of the test
report.
If PCR is performed, fill in all requested data in the test report page 13. Results of PCR can
be written in the test report Table 5 (page 26).
7.2 Selective enrichment
Allow the selective enrichment broths to equilibrate to room temperature, if they were
stored at a lower temperature. Dry the surface of the MSRV plates in a Laminair Air
Flow cabinet. Record (page 4-7) the requested data of the selective enrichment broths
and MSRV plates in the test report. Label 25 jars/tubes/plates of each selective
enrichment broth from 1 to 25. Also label 20 selective enrichment jars/tubes/plates
from N1 to N20 and 12 jars/tubes/plates from C1 to C12. All selective media are
incubated for 24 h and later on for another 24 h.
After equilibration:
Transfer 0.1 ml of homogenised BPW culture to each tube containing 10 ml RVS
medium. Incubate at 41,5C 1
o
C for 24 h 3 h and later on another 24 h 3 h;
Transfer 1 ml of homogenised BPW culture to each tube containing 10 ml
MKTTn medium. Incubate at 37C 1
o
C for 24 h 3 h and later on another 24 h
3 h;
Inoculate the MSRV plates with three drops of BPW culture, with a total volume
of 0.1 ml. Incubate (not upside down) at 41,5C 1
o
C for 24 h 3 h and later on
another 24 h 3 h;
Inoculate the routinely used selective medium/media (other than those mentioned
above), with the corresponding BPW culture (note the inoculation volume of BPW
used and the volume of the selective medium/media on test report). Incubate at the
temperature routinely used.
Place the jars/tubes in the appropriate incubator(s)/waterbath(s) and record the
temperature and time for the different enrichment media at the start and at the end of
the incubation period and other requested data in the test report (page 4-7).
7.3 Isolation on media (first and second isolation)
1RWH In the case that you do not have large dishes (140 mm) at your disposal use two
small (90-100mm) dishes, one after the other, using the same loop.
Record in the test report (page 8-10) the requested data of the isolation media used.
Label 25 large petri dishes of the isolation media from 1 to 25, label 20 large petri
dishes from N1 to N20 and label 12 large petri dishes from C1 to C12.
SOP Bacteriological Collaborative Study VI (2002) page 6 of 8
CRL-6DOPRQHOOD, Bilthoven, The Netherlands
First isolation after 24 h
,QRFXODWLRQ:
Inoculate, by means of a loop, from all selective enrichment cultures and from suspect
MSRV plates, the surface of an isolation medium in a large size petri dish with the
corresponding label number (see also QRWH at the beginning of section 7.3). The
following isolation media will be used:
1) Phenol red/brilliant green agar;
Place the petri dishes with the bottom up in the incubator set at 37C 1
o
C
(record the temperature and time at the start and at the end of the incubation and
other requested data in test report, page 8).
2) Xylose Lysine Desoxycholate agar
Place the petri dishes with the bottom up in the incubator set at 37 C (note the
temperature and time at the start and at the end of the incubation and other
requested data on test report, page 9).
3) Optionally: selective isolation medium/media routinely used in your laboratory.
Only if media used are different from those mentioned above.
Incubate the medium/media at the temperature routinely used (record temperature
and time and other requested data in test report, page 10).
After incubation for 24 h 1
o
C, examine the petri dishes for the presence of typical
colonies of 6DOPRQHOOD.
Second isolation after 48 h
After a total incubation time of 48 h 3h of the selective enrichment media, repeat the
procedure described above (First isolation after 24 h).
7.4 Confirmation of colonies from first and second isolation
For confirmation take from each petri dish of each selective medium at least 1 colony
considered to be typical or suspect (only use well isolated colonies). Store the plates at
5
o
C 3
o
C. Before biochemical confirmation, optionally, streak the typical colonies
onto the surface of nutrient agar plates with the corresponding label numbers, in a
manner which allows to develop well isolated colonies. Record on test report (page
11) the requested data of the nutrient agar. Incubate the inoculated plates at 37C
1oC for 24 h 3h.
SOP Bacteriological Collaborative Study VI (2002) page 7 of 8
CRL-6DOPRQHOOD, Bilthoven, The Netherlands
If the selected colony is not confirmed as 6DOPRQHOOD, test at maximum another 5
typical colonies. Report the number of colonies tested and the number of colonies
confirmed as 6DOPRQHOOD for each dish in Table 1 (isolation using RVS), Table 2
(isolation using MKTTn), Table 3 (isolation using MSRV) and Table 4 (isolation
using own enrichment) on test report page 14-25. For the results of detection of
6DOPRQHOOD using PCR fill in Table 4 on test report page 26.
Biochemical confirmation
By means of a loop, inoculate the media specified below with the colony selected as
described above. Optionally inoculate other media which are routinely used for
biochemical confirmation. Record in test report (page 11+12) the requested data of the
media.
TSI agar
Urea agar
l-Lysine decarboxylation medium
Nutrient agar (optionally)
Interpretation of the biochemical tests
6DOPRQHOOD generally show the reactions given in Table 1 of ISO 6579:2002(E) on
page 9).
TSI agar:
%XWW -yellow by fermentation of glucose;
-black by formation of hydrogen sulfide; and
-bubbles or cracks due to gas formation from glucose
6ODQW -red or unchanged
Urea agar: rose pink to cerise
l-Lysine decarboxylation medium: coloured purple
8 Test report
The test report will contain all information, that might influence the results and is not
mentioned in this SOP. Some incidents or deviations from the specified procedures
will also be recorded. The test report will include the names of the persons, who are
carrying out the work and will be signed by these persons.
SOP Bacteriological Collaborative Study VI (2002) page 8 of 8
CRL-6DOPRQHOOD, Bilthoven, The Netherlands
Schedule of the adapted ISO 6579: 2002(E) method
Day Topic Description
1 Pre-enrichment 1 capsule to 225 ml BPW
Do not shake
30 min. at 37 C
Add 10 or 25 gr. faeces to BPW
Incubate 16-20 h at 37 C
2 Selective enrichment 0.1 ml BPW culture in 10 ml RVS
1 ml BPW culture in 10 ml MKTTn
0.1 ml BPW culture on MSRV plate
Other selective enrichment medi(um)(a)
Incubate 24 h at 41,5 C
3 First isolation after
24 h
Inoculate from RVS, MKTTn, suspect MSRV plates
and other medi(um)(a)
phenol red/brilliant green agar
Xylose Lysine Desoxycholate agar
other selective medi(um)(a)
Incubate 24 h at the specified temperature
3 Continue selective
enrichment
Incubate RVS and MSRV medium (see day 2) another 24
hours at 41,5 C
4 Second isolation
after 24 h
Inoculate from RVS, MKTTn,suspect MSRV plates and
Other medi(um)(a)
phenol red/brilliant green agar
Xylose Lysine Desoxycholate agar
other selective medi(um)(a)
Incubate another 24 h at the specified temperature
4 Biochemical
confirmation
Inoculate the media from first isolation media (day 3) for
biochemical identification and incubate 24 h at the
specified temperature
5 Biochemical
confirmation
inoculate the media from second isolation media (day 4)
for biochemical identification and incubate 24 h at
the specified temperature