17.9.03 Table 967.27A.

Characteristics of Salmonella
AOAC Official Method 967.27 a
Test or substrate Results
Salmonella in Foods
Urease, B(a) – (Orange-red)
Identification
First Action 1967 Lysine decarboxylase, C(a) + (Alkaline; purple
Final Action 1968 throughout medium)
b
Phenol red dulcitol broth, C(b) + (Yellow and/or gas)
A. Cultures
KCN broth, C(c)(1) – (No growth)
Pure cul tures on triple sugar iron (TSI) are re quired for c
inoculation of biochemical test media. Malonate broth, C(c)(2) – (Unchanged green)
(a) Pure cultures.—Proceed to B. Indole test, C(c)(3) – (No red color)
(b) Mixed cultures.—Streak any culture that appears to be mixed Polyvalent flagellar test, B(b) and + (Visible agglutination)
on MAC, 967.25A(q) (see 17.9.01), XLD, 967.25A(d) (see C(d)
17.9.01), or HE, 967.25A(e) (see 17.9.01). Incubate 24 ± 2 h at Polyvalent somatic test, C(f) + (Visible agglutination)
a
35°C. + = ³90% positive in 1–2 days; – = ³90% negative in 1–2 days.
b
(c) Appearance of Salmonella colonies.—(1) On Majority of S. arizonae cultures are negative.
c
MAC.—Typ i cal col o nies ap pear trans par ent and col or less, Majority of S. arizonae cultures are positive.
sometimes with dark centers. Salmonella will clear areas of
precipitated bile caused by other organisms sometimes present in
medium. (2) On XLD.—See 967.26B(b)(1) (see 17.9.02). (3) On
HE.—See 967.26B(b)(2) (see 17.9.02). 17.9.01), and incubate 24 ± 2 h at 35°C or inoculate rapid urea broth,
Pick with needle 2 typical or suspicious colonies and inoculate 967.25A(k)(2) (see 17.9.01) with two 3 mm loopfuls of growth from
TSI slants by streaking the slant and stabbing the butt as in each presumptive-positive TSI slant culture, and incubate 2 h in H2O
967.26C(a) (see 17.9.02). Retest purified cultures as in 967.26C(b) bath at 37° ± 0.5°C. Discard all cultures that give positive test (purple-red
(see 17.9.02), and proceed with identification. color). Salmonella spp. are urease negative (no change in orange color of
As alternative to conventional tube system for Salmonella, any medium).
one of the AOAC-approved commercial biochemical kits can be
used for pre sump tive ge neric iden ti fi ca tion of foodborne (b) Serological flagellar (H) screening test.—To reduce number
Salmonella. See 978.24 (see 17.9.04), 989.12 (see 17.9.05), and of pre sump tive pos i tive TSI agar cul tures car ried through
991.13 (see 17.9.06). identification tests, perform serological flagellar (H) screening test
by transferring one 3 mm loopful of each urease-negative TSI agar
B. Subcultures culture to either: (1) Brain–heart infusion broth, 967.25A(r) (see
(a) Urease test.—Subculture small amount of growth from 17.9.01), (for test on same day) and incubate at 35°C until visible
presumptive positive TSI agar culture to urea broth, 967.25A(k)(1) (see growth occurs (ca 4–6 h); or (2) Trypticase soy–tryptose broth,

Table 967.27B. Biochemical and serological reactions of Salmonella

a
Test or substrate Positive Negative Salmonella reaction
Glucose (TSI), 967.26C(b) Yellow butt Red butt +
H2S (TSI), 967.26C(b) Blackening No blackening +
Urease, B(a) Purple-red No color change -
Lysine decarboxylase broth, C(a) Purple Yellow +
b
Phenol red dulcitol broth, C(b) Yellow and / or gas No gas; no color change +
KCN broth, C(c)(1) Turbidity No turbidity -
c
Malonate broth, C(c)(2) Blue No color change -
Indole test, C(c)(3) Violet at surface Yellow at surface -
Polyvalent flagellar test, B(b), C(d) Agglutination No agglutination +
Polyvalent somatic test, C(f) Agglutination No agglutination +
c
Phenol red lactose broth, D(a) Yellow and / or gas No gas; no color change -
Phenyl red sucrose broth, D(b) Yellow and / or gas No gas; no color change -
Voges-Proskauer test, D(c)(1) Pink to red No color change -
Methyl red test, D(c)(2) Diffuse red Diffuse yellow +
Simmons’ citrate, D(d) Growth; blue No growth; no color change v
a
+ = ³90% positive in 1–2 days; – = ³90% negative in 1–2 days; v = variable.
b
Majority of S. Arizonae cultures are negative.
c
Majority of S. Arizonae cultures are positive.

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967.25A(s) (see 17.9.01), (for test on following day) and incubate reaction throughout broth (final color is slightly darker than original
24 ± 2 h at 35°C. purple color of medium). Sometimes tubes which have yellow color
To 5 mL of each of the 6 broth cultures, add ca 2.5 mL after 8–12 h of incubation change to purple later. Negative test is
formalinized phys i o log i cal sa line so lu tion, 967.25B(g) (see permanent yellow color throughout broth. If medium appears to be
17.9.01). Se lect 2 formalinized broth cultures and test with dis col ored (nei ther pur ple nor yel low), add few drops of
Salmonella flagellar (H) antisera, 967.25B(j) or (k) (see 17.9.01), 0.2% bromocresol purple dye, 967.25B(o) (see 17.9.01), and reread
as in 967.28C or D (see 17.9.07). tube reactions.
If selected formalinized broth cultures are positive, perform (LIA is incubated loosely capped so that aerobic conditions are
additional tests on these cultures, beginning with C, except step maintained, while lysine decarboxylase broth is incubated tightly
C(d) may be omitted. closed to exclude air.)
If both formalinized broth cul tures are neg ative, perform ( b ) P h e n o l re d d u l c i t o l b ro t h , 9 6 7 . 2 5 A ( p ) ( 1 ) ( s e e
serological test on the 4 additional broth cultures [B(b)(1) or (2)] to 17.9.01).—Incubate 48 ± 2 h at 35°C. Examine at least every 24 h.
obtain, if possible, 2 positive cultures for additional testing, C. Most Salmonella spp. give positive test indicated by gas formation
If all urease-negative TSI cultures from test portion are (displacement of liquid in inverted tube) and/or acid reaction
Salmonella serological flagellar (H) test negative, then perform (yellow). Negative test is alkaline reaction (red) and no gas
additional tests, beginning with C, on these cultures. formation.
C. Testing Urease-Negative Cultures [Purple broth base with dulcitol, 967.25A(p)(2) (see 17.9.01),
Using needle, transfer portion of presumptive positive TSI culture may be substituted. Positive test is acid reaction (yellow) and gas.
to LIA medium and small amount of growth from the TSI culture to Negative test is alkaline reaction (purple).]
each of other media: (c) Tryptophane broth, 967.25A(h) (see 17.9.01).—Incubate
(a) Lysine iron agar, 967.25A(m)(1) (see 17.9.01).—Stab butt 24 ± 2 h at 35°C and test as follows: (1) Transfer 3 mm loopful,
twice and then streak slant. Replace tube cap loosely and incubate excluding all solid particles, to KCN broth, 967.25A(o) (see
24 ± 2 h at 35°C. Most Salmonella spp. give purple color of alkaline 17.9.01). Heat rim of tube to form good seal when restoppered.
re ac tion through out medium (final color is slightly darker than Incubate 48 ± 2 h at 35°C. Salmonella spp. do not grow in this
original purple color of medium). If H2S is produced, butt of broth as shown by lack of turbidity (negative test). (2) Transfer
medium is blackened. Negative test is purple or red slant and yellow 3 mm loopful to malonate broth, 967.25A(l) (see 17.9.01), and
butt. If LIA test, 967.26C(a) (see 17.9.02), was satisfactory, it need incubate 48 ± 2 h at 35°C. Salmonella spp. give negative test as
not be re peated. Use lysine de car box yl ase broth for fi nal shown by green color (unchanged). Positive test (alkaline reaction)
determination of lysine decarboxylase if culture gives doubtful LIA is shown by blue color. (3) Transfer 5 mL to empty test tube and add
reaction. 0.2–0.3 mL Kovacs reagent, 967.25B(a) (see 17.9.01). Positive test
If liq uid me dium is pre ferred, in oc u late tube of lysine for indole is shown by deep red color in reagent on surface of broth.
decarboxylase broth, 967.25A(m)(2) (see 17.9.01). Close tube cap Most Salmonella spp. are indole negative.
tightly after inoculation and incubate at 35°C for 48 ± 2 h. Examine
(d) Brain–heart infusion broth, 967.25A(r) (see 17.9.01), or
at least every 24 h. Salmonella spp. give purple color of alkaline
Trypticase soy–tryptose broth, 967.25A(s) (see 17.9.01).—Incubate
brain–heart infusion broth until visible growth occurs (ca 4–6 h) or
Table 967.27C. Criteria for discarding non-Salmonella cultures incubate Trypticase soy–tryptose broth 24 ± 2 h at 35°C. To 5 mL
broth culture, add ca 2.5 mL formalized physiological saline
Test(s) or substrate(s) Results solution, 967.25B(g) (see 17.9.01). Refrigerate formalized broth at
Urease test, B(a) Positive (purple-red) 5°–8°C if test is to be performed on another day. Perform Salmonella
Indole test, C(c)(3) Positive (red/or violet at surface) serological flagellar (H) test, 967.28C (see 17.9.07), or
Polyvalent flagellar test, B(b), Negative (no agglutination) “Spicer-Edwards” flagellar (H) test tube test, 967.28D (see 17.9.07),
C(d), or Spicer-Edwards flagellar
using formalized broth culture as flagellar (H) antigen to be tested.
(H) test, 967.28D
(e) Tests indicating absence of Salmonella.—Discard, as not
Lysine decarboxylase test, Negative (yellow)
C(a) Salmonella, cultures that show either: (1) Positive indole test (red)
KCN broth, C(c)(1) Positive (growth) and negative Salmonella serological flagellar (H) test. (2) Positive
Phenol red lactose broth ,
a
Positive (yellow and/or gas)
b KCN broth test (growth) and negative lysine decarboxylase test
D(a) (yellow).
b
Phenol red sucrose broth, Positive (yellow and/or gas) (f) Testing of TSI agar cultures.—Use Salmonella serological
D(b) somatic (O) test, 967.28A (see 17.9.07).
KCN broth, C(c)(1) Positive (growth) (g) Classification.—Classify as Salmonella spp. cultures that
Voges-Proskauer test, Positive (red) have all characteristics shown in Table 967.27A. If one TSI culture
D(c)(1)
Methyl red test, D(c)(2) Negative (yellow)
from 25 g test portion is classified as Salmonella spp., further testing
a of other TSI cultures from same 25 g test portion is unnecessary.
Malonate broth positive cultures are tested further to determine if they are
Salmonella Arizonae, (D)(2). (h) Spe cial cases.—Cul tures that con tain de mon stra ble
b
Do not discard positive broth culture if corresponding LIA cultures give Salmonella antigens as shown by positive Salmonella serological
typical Salmonella reactions; test further to determine if they are somatic (O) test and positive flagellar (H) test but do not have
Salmonella spp. See 967.26C(a) (see 17.9.02).
biochemical characteristics of Salmonella spp. should be purified as
in A(b) and retested, beginning with B.

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D. Additional Biochemical Tests by transferring 5 mL culture to test tube and adding 5–6 drops
Perform additional tests on cultures that do not give identical test methyl red solution, 967.25B(e) (see 17.9.01), and read results
results as in Table 967.27A and do not classify as Salmonella spp. immediately. Salmonella spp. give positive test (red). Negative test
Transfer one loopful of culture from each unclassified TSI agar slant is indicated by yellow color.
to each of following media: (d) Simmons citrate agar, 967.25A(j) (see 17.9.01).—Inoculate
( a ) P h e n o l re d l a c t o s e b ro t h , 9 6 7 . 2 5 A ( p ) ( 1 ) ( s e e by streaking slant and stabbing butt. Incubate 96 ± 2 h at 35°C.
17.9.01).—Incubate 48 ± 2 h at 35°C. Examine inoculated broth at least Salmonella spp. usually give positive test shown by growth and
every 24 h. Positive test is shown by gas formation (displacement of color change from green to blue (alkaline). Color change usually
liquid in inverted tube) and acid reaction (yellow color). Most appears first on slant and then spreads through medium. Negative test
Salmonella spp. give negative test shown by alkaline reaction (red is indicated by no or very little growth and no change in color of
color) and no gas formation. medium.
Discard, as not Salmonella, cultures that give positive phenol red (e) Classification.—Classify cultures according to results listed
lactose broth test, except: (1) Cultures described in 967.26C(b) (see in Table 967.27B. If one TSI culture from 25 g test portion is
17.9.02), and (2) cultures that also give positive malonate broth test. classified as Salmonella spp., further testing of other TSI cultures
Cultures that are phenol red lactose broth positive or negative and from same 25 g test portion is unnecessary. (1) Salmonella
malonate broth positive are tested further to determine if they are spp.—Cultures that have reaction patterns of Table 967.27B. (2)
S. Arizonae, (e)(2). [Purple lactose broth, 967.25A(p)(2) (see S. Ar i zonae spp.—Cul tures that have re ac tion pat tern of
17.9.01), may be substituted. Positive test is acid reaction (yellow Ta ble 967.27B, ex cept foot note re ac tions b and c. (3)
color) and gas. Negative test is alkaline reaction (purple color) and Non-Salmonella spp.—Discard, as not Salmonella, cultures that
no gas formation.] give results listed in any one subdivision of Table 967.27C.
( b ) P h e n o l re d s u c ro s e b ro t h , 9 6 7 . 2 5 A (p )(1) (se e
E. Summary of Classification of Non-Salmonella Cultures
17.9.01).—Incubate and read as in (a). Discard, as not Salmonella,
cultures that give positive test, except cultures described in 967.26C(b) Classify, by performing additional tests described in Edwards and
(see 17.9.02). [Purple sucrose broth may be substituted and read as in Ewing’s Identification of Enterobacteriaceae, 4th Ed. (1986) W.H.
(a).] Ewing (Ed.), Elsevier Science Publishing Co., Inc., New York, NY,
(c) Buffered glucose broth [MR-VP medium, 967.25A(i) (see USA, any culture that is not clearly identified as Salmonella spp. or
17.9.01)].—Incubate 48 ± 2 h at 35°C. (1) Perform Voges-Proskauer S. arizonae by classification schemes in Tables 967.27A and B or not
(VP) test at room temperature by transferring 1 mL 48 h culture to eliminated from these groups by test reactions listed in Table 967.27C.
If neither of 2 TSI cultures carried through biochemical tests, C
test tube and adding 0.6 mL a-naphthol solution, 967.25B(b)(1) (see
and D and Tables 967.27A–C, confirms as Salmonella, perform
17.9.01), and 0.2 mL 40% KOH solution (w/v), 967.25B(b)(2) (see
17.9.01). Shake after each addition. To intensify and speed reaction, biochemical tests, beginning with C, on remaining urease-negative
add few creatine crystals to test medium. Read results 4 h after TSI cultures from same 25 g test portion.
adding reagents. Positive VP test is development of eosin pink color. References: JAOAC 50, 753(1967); 51, 870(1968); 52, 455(1969);
Salmonella spp. give negative test. (2) Incubate remainder of 56, 1027(1973); 59, 731(1976); 62, 499(1979);
MR-VP medium additional 48 ± 2 h at 35°C. Perform methyl red test 64, 893(1981); 64, 899(1981); 65, 356(1982).

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