17.9.
08
AOAC Official Method 975.54
Salmonella in Foods
Fluorescent Antibody (FA) Screening Method
First Action 1975
Final Action 1977
A. Precautions
Method is screening test for presence of Salmonella; it is not
confirmatory test, because conjugate will react with some other
members of Enterobacteriaceae.
Enrichment broths from test portions positive by FA method must
be streaked on selective media as in 967.26B (see 17.9.02) and
typical or suspicious colonies identified as in 967.26C (see 17.9.02),
967.27 (see 17.9.03), and 967.28 (see 17.9.07).
Method must be fol lowed rig or ously be cause errors in
preparation of test portion, smears, conjugate, and other reagents
can lead to invalid results. Microscopic observation of stained
smears must be performed with critically aligned and properly
functioning equipment.
Visual estimation of degree of fluorescence of stained cells is
somewhat subjective and should be conducted by analyst with prior
training or experience in both FA methodology and in cultural
technique for detection of Salmonella.
If test portion prep a ra tion does not nor mally in clude
pre-enrichment step (as with meat, poul try, and cer tain
environmental specimens), 4 h post-enrichment incubation period
may not be sufficient for development of number of Salmonella cells
re quired for de tec tion by FA method. There fore, in clude
pre-enrichment step or extend post-enrichment incubation time. In
some cases when pre-enrichment step is not used, test portion is not
adequately diluted and carryover of debris into post-enrichment
broth may interfere with observation of FA stained cells.
B. Apparatus
(a) Multiwell coated slides.—Clean thin (1.0–1.2 mm) slides
thoroughly with detergent and rinse with distilled H2O and alcohol.
Apply double row of 4 separate drops of glycerol (8 drops total) to
each of series of slides and spray with fluorocarbon-coating material.
After few min, rinse off each slide individually under tap and then
with distilled H2O, and stand on end in rack to dry. (Prepared slides
are available from Cel-Line Associates, PO Box 35, Newfield,
NJ 08344, USA, and Clinical Sciences, Inc., 30 Troy Rd, Whippany,
NJ 07981, USA.)
(b) Flu o res cent mi cro scope.—With ex citer fil ter with
wavelength transmission of 330–500 nm and barrier filter with
wavelength reception >400 nm.
C. Reagents
(a) Phosphate-buffered saline (PBS) solution.—pH 7.5; 0.01M;
0.85% NaCl. Dis solve 12.0 g an hy drous Na 2 HPO 4 , 2.2 g
NaH2PO4×H2O, and 85.0 g NaCl in H2O and dilute to 1 L. Dilute
100 mL of this solution to 1 L with H2O. Adjust pH to 7.5 with 0.1M
HCl or 0.1M NaOH, if necessary.
(b) Carbonate buffer.—pH 9.0. Mix 4.4 mL 0.5M Na2CO3 (5.3 g
in 100 mL H2O) with 100 mL 0.5M NaHCO3 (4.2 g in 100 mL H2O).
pH should be 9.0; if not, adjust by addition of 0.5M Na2CO3.
(c) Glycerol saline solution.—pH 9.0. Mix 9 mL glycerol with
1 mL carbonate buffer, (b). pH decreases on storage; prepare
weekly.
( d ) S a l m o n e l l a p o l y v a l e n t f l u o re s c e n t a n t i b o d y
conjugate.—Fluorescein isothiocyanate-labeled Salmonella OH
globulin, polyvalent, containing antibodies for all antigens within
Salmonella O groups A–S, and meeting specifications of Centers for
Disease Con trol and Pre vention, At lanta, GA 30333, USA.
(Available from BD Biosciences, 7 Lorton Circle, Sparks, MD
21152, USA [FA Salmonella Poly; No. 3187]; Clinical Sciences,
Inc., 30 Troy Rd, Whippany, NJ 07981, USA). Before use, titer each
lot to determine appropriate routine test dilution (RTD). Use pure
cultures of Salmonella representative of several somatic groups.
Prepare 5 dilutions (1:2, 1:4, 1:8, 1:16, and 1:32) of conjugate in
PBS solution, (a). Stain duplicate smears from cultures with each
dilution and determine intensity of fluorescence. RTD is that
dilution one less than highest dilution giving 4+ fluorescence with
rep re sen ta tive Sal mo nella cul tures. Store stock (un di luted)
conjugate of known titer frozen, and dilute when needed. Diluted
conjugate can be stored at 4°C for few weeks as long as control
cultures remain positive.
D. Determination
(a) Pre-enrichment.—Pre-enrich product in noninhibitory broth
to initiate growth of Salmonella spp. Methods used vary with
product as in (1)–(9). In all cases loosen jar caps ¼ turn and incubate
24 ± 2 h at 35°C. Except where selenite cystine and tetrathionate
broths, 967.25A(b)(1) or (2) and (c) (see 17.9.01), respectively,
have already been used [(2)(b) and (5)], transfer 1 mL incubated
mixtures to selenite cystine broth and tetrathionate broth for
selective enrichment as in 967.26B(a) (see 17.9.02). Where these
broths have already been used [(2)(b) and (5)], proceed directly to
post-enrichment, (b).
(1) Dried yeast (inactive).—Weigh 25 g into sterile, wide-mouth,
screw-cap, 500 mL (pt) jar, add 225 mL sterile Trypticase (tryptic)
soy broth, 967.25A(t) (see 17.9.01), and mix well to form smooth
suspension. Cap jar se curely and let stand 60 min at room
temperature. If pH is <6.6, adjust to 6.8 ± 0.2 with 1M NaOH.
(2) Meats, animal substances, glandular products, and fish
meal.—(a) Heated, processed, and dried products.—Weigh 25 g
into ster ile blending jar, add 225 mL ster ile lac tose broth,
940.36A(f) (see 17.1.02), and blend 2 min at 8000 rpm. If product is
powdered, ground, or comminuted, blending may be omitted.
Transfer aseptically to sterile, wide-mouth, screw-cap, 500 mL (pt)
jar and adjust pH to 6.8 ± 0.2 with 1M NaOH. If product contains
large amount of fat, add 2.2 mL steamed (15 min) Tergitol Anionic 7
(sodium heptadecyl sulfate, Dow Surfactants). (b) Raw and highly
contaminated products.—Weigh duplicate 25 g test portions into
separate sterile blending jars. Add 225 mL selenite cystine broth to
one jar and 225 mL tetrathionate broth to other, and blend 2 min.
Transfer aseptically to sterile, wide-mouth, screw cap, 500 mL (pt)
jars. (c) Raw frog legs.—Aseptically place 2 legs into single sterile,
wide-mouth, screw cap, 500 mL (pt) jar containing 225 mL sterile
lactose broth, 940.36A(f) (see 17.1.02).
(3) Dry whole milk.—Weigh 25 g into sterile, wide-mouth, screw
cap, 500 mL (pt) jar, add 225 mL sterile distilled H2O, and mix well.
Adjust pH to 6.8 ± 0.2 with 1M NaOH, if necessary. Add 0.45 mL
1% aqueous brilliant green solution and mix well.
(4) Dried whole eggs, yolks, and whites; pasteurized liquid and
frozen eggs; prepared powdered mixes (cake, cookie, donut, biscuit,
and bread); and infant formula.—If product is frozen, thaw rapidly
at 45°C for 15 min or overnight at 5°–10°C. Weigh 25 g into sterile,
wide-mouth, screw cap jar. Add 225 mL lactose broth, little at a time
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with mixing, cap jar, and let stand at room temperature 60 min. Mix
well and adjust to pH 6.8 ± 0.2 with 1M NaOH or HCl.
(5) Nonpasteurized frozen egg products.—Thaw as in (4). Weigh
duplicate 25 g test portions into separate sterile, wide-mouth, screw
cap, 500 mL (pt) jars. Add 225 mL selenite cystine broth to one jar
and 225 mL tetrathionate broth to other, and mix well. Adjust pH to
6.8 ± 0.2 with 1M NaOH.
(6) Egg-containing foods (noodles, egg rolls, etc.).—Proceed as
in (2)(a).
(7) Coconut.—Proceed as in (2)(a), using Tergitol Anionic 7, but
omitting blending.
(8) Candy and candy coatings.—Weigh 25 g into sterile blending
jar. Add 225 mL sterile reconstituted nonfat dry milk, 967.25A(v)
(see 17.9.01), but without brilliant green dye, and blend 2 min.
Adjust pH to 6.8 ± 0.2 with 1M NaOH, if necessary. Add 0.45 mL
1% aqueous brilliant green solution and mix well.
(9) Nonfat dry milk.—Examine as in 967.26A(f) (see 17.9.02).
(b) Post-enrichment.—Transfer 1 mL incubated selenite cystine
en rich ment broth to 10 mL sel e nite cy stine broth as
post-enrichment. (Other volumes may be used if 1:10 dilution ratio
is main tained.) Take aliquot from up per third of se lec tive
enrichment cultures to minimize product carryover. Similarly,
transfer 1 mL incubated tetrathionate enrichment broth to 10 mL
selenite cystine broth. Incubate 4 h in 35°C H2O bath.
(c) Staining.—Transfer 0.0075 mL of each post-enrichment
medium with sterile 2 mm loop into separate wells of multiwell
coated slide, and dry thoroughly in air at room temperature. Fix by
immersion in bath of alcohol–CHCl3–formalin (60 + 30 + 10) 3 min.
Rinse 2 or 3 times in alcohol, and air-dry at room temperature.
Change alcohol periodically to prevent cell carryover (250 mL
alcohol will rinse 5–10 slides). Slides may also be fixed and rinsed
by flooding. Apply solutions to one end of slide and allow to flow
into wells.
Cover dried smears with titered Salmonella polyvalent FA
conjugate and let stain in moist chamber 15–30 min. FA conjugate
must not dry on smear. (Covered plastic Petri dish containing piece
of filter paper moistened with H2O is excellent staining chamber.)
Drain excess conjugate by standing slide on edge few seconds.
(Avoid mixing conjugate from one well on slide to another.)
Immediately rinse slides in PBS solution, C(a). Then soak slides
ã 2005 AOAC INTERNATIONAL
10 min in fresh PBS solution and rinse briefly with H2O. Air-dry
smears again at room temperature and then mount by placing drop of
glycerol saline solution, C(c), directly onto each smear and covering
with No. 1 glass cover slip. Add enough glycerol saline solution to
smear to ensure adequate, but not excessive, coverage of all wells
after cover slips have been placed. Do not trap air bubbles under
cover slip.
(d) Ex am i na tion.—Ex am ine smears with flu o res cent
microscope. Scan entire smear using 40–50´ oil immersion
objective to locate fluorescent cells. When found, change objective
to 100´ oil immersion lens for definitive determination of cell
morphology and fluorescence. Objectives with iris diaphragm for
adjusting numerical aperture are helpful for control of contrast
between cells and background. Estimate degree of fluorescence of
cells on a scale from negative to 4+ as follows:
4+ = Maximum fluorescence; brilliant yellow-green; clearcut cell
outline; sharply defined cell center.
3+ = Less brilliant yellow-green fluorescence; clearcut cell
outline; sharply defined cell center.
2+ = Definite but dim fluorescence; cell outline less well defined.
1+ = Very subdued fluorescence; cell outline indistinguishable
from cell center in most instances.
- = Negligible or complete lack of fluorescence.
Typical positive smears for Salmonella spp. Exhibit ³2 short to
medium rod-shaped cells per field, using 100´ objective. Cells
should be dis trib uted through out en tire smear. In ten sity of
fluorescence should be in range of 3+ to 4+. Occasionally cells are
observed with proper morphology and cell distribution, but
fluorescence is rated 2+. Sometimes 3+ to 4+ fluorescence is
observed, but distribution is poor and not all fields contain cells, due
to improper processing of slides. Score both cases positive and
subject to confirmatory tests.
Each time test portions are tested, carry culture of known
Salmonella strain through all cultural, staining, and observation
steps as control.
Report (1) morphological characteristics of fluorescent cells;
(2) number of typical cells per field under 100´ oil immersion
objective; and (3) degree of fluorescence of cells (1+ to 4+).
Reference: JAOAC 58, 828(1975).