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, since conjugate will react with some other members of Enterobacteriaceae.
Enrichment broths from samples 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), 967.28. (see 17.9.07)
Method must be followed rigorously since errors in preparation
of sample, 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 sample preparation does not normally include pre-enrichment
step (as with meat, poultry, and certain environmental samples), 4 h
post-enrichment incubation period may not be sufficient for development of number of Salmonella cells required for detection by FA
method. Therefore, include pre-enrichment step or extend post-enrichment incubation time. In some cases when pre-enrichment step
is not used, sample 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 (1.01.2 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
(Fluoroglide, Ace Scientific Co., Inc., 1420 E Linden Ave, Linden,
NJ 07036). 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 and Clinical Sciences, Inc., 30 Troy Rd, Whippany, NJ
07981.)
(b) Fluorescent microscope.With exciter filter with wavelength transmission of 330500 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. Dissolve 12.0 g anhydrous Na2HPO4, 2 . 2 g
NaH2PO4H2O, and 85.0 g NaCl in H2O and dilute to 1 L. Dilute 100
mL this solution to 1 L with H2O. Adjust pH to 7.5 with 0.1N HCl
or 0.1N 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) Salmonella polyvalent fluorescent antibody conjugate.
Fluorescein isothiocyanate-labeled Salmonella OH globulin, poly-

valent, containing antibodies for all antigens within Salmonella O

groups AS, and meeting specifications of Centers for Disease
Control and Prevention, Atlanta, GA 30333. (Available from Difco
Laboratories [FA Salmonella Poly] [No. 3187]; Clinical Sciences,
Inc., 30 Troy Rd, Whippany, NJ 07981). 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 representative
Salmonella cultures. Store stock (undiluted) conjugate of known titer
frozen, and dilute when needed. Diluted conjugate can be stored at
4 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 1/4 turn and incubate 24
2 h at 35. 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 securely and let stand 60 min at room temperature. If pH is <6.6, adjust to 6.8 0.2 with 1N NaOH.
(2) Meats, animal substances, glandular products, and fish
meal.(a) Heated, processed, and dried products.Weigh 25 g
into sterile blending jar, add 225 mL sterile lactose 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 1N NaOH. If product contains large amount of
fat, add 2.2 mL of steamed (15 min) Tergitol Anionic 7 (sodium
heptadecyl sulfate, Union Carbide Corp.).
(b) Raw and highly contaminated products. Weigh duplicate
25 g samples into separate sterile blending jars. Add 225 mL of
selenite cystine broth to one jar and 225 mL of 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 1N 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 for 15 min or overnight at 510. Weigh 25 g into sterile,
wide-mouth, screw cap jar. Add 225 mL lactose broth, little at time
with mixing, cap jar, and let stand at room temperature 60 min. Mix
well and adjust to pH 6.8 0.2 with 1N NaOH or HCl.

Copyright 1998 AOAC INTERNATIONAL

(5) Nonpasteurized frozen egg products.Thaw as in (4). Weigh

duplicate 25 g samples 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 1N 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 1N 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 of incubated selenite cystine enrichment broth to 10 mL of selenite cystine broth as post-enrichment. (Other volumes may be used if 1:10 dilution ratio is
maintained.) Take aliquot from upper third of selective enrichment
cultures to minimize product carryover. Similarly, transfer 1 mL of
incubated tetrathionate enrichment broth to 10 mL of selenite cystine
broth. Incubate 4 h in 35 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 510 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 1530 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, 975.54C(a). Then soak slides 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), 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) Examination.Examine smears with fluorescent microscope. Scan entire smear using 4050 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 scale of 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 2 short to
medium rod-shaped cells per field, using 100 objective. Cells should
be distributed throughout entire smear. Intensity 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 samples 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).

Copyright 1998 AOAC INTERNATIONAL