JOURNAL OF CLINICAL MICROBIOLOGY, Mar. 1996, p. 686–688 Vol. 34, No.

3
0095-1137/96/$04.0010
Copyright q 1996, American Society for Microbiology

Comparison of Five Plating Media for Isolation of Salmonella

Species from Human Stools
JOAQUÍN RUIZ,1* MARIA-LUZ NÚÑEZ,1 JULIÁN DÍAZ,1 ISABEL LORENTE,1
JERÓNIMO PÉREZ,1 AND JOAQUÍN GÓMEZ2
Laboratory of Microbiology1 and Section of Infectious Diseases,2 Hospital Virgen de la Arrixaca,
El Palmar, 30120 Murcia, Spain
Received 11 July 1995/Returned for modification 20 September 1995/Accepted 27 November 1995

A comparative study was carried out to evaluate the performances of different culture media for the recovery
of Salmonella spp. from 1,000 routine samples of human stools. By direct plating we tested Salmonella-Shigella
agar (SS), Hektoen enteric agar (HE), bismuth sulfite agar (BS), novobiocin-brilliant green-glycerol-lactose
agar (NBGL) and SM-ID medium (SM), and after selenite enrichment, we tested all of the media except HE.
C8-esterase and oxidase tests were used for the screening of Salmonella spp. on SS and HE. The total number
of Salmonella isolates from direct culture was 74, with respective sensitivities and positive predictive values
(PPVs) of 78.4 and 61%, 64.9 and 18.7%, 36.5 and 34.2%, 55.4 and 20.7%, and 39.2 and 43.9% for NBGL, SS,
HE, BS, and SMID, respectively. After enrichment, the total number of Salmonella isolates was 88. The
respective sensitivities and PPVs obtained were 90.9 and 62.5%, 92 and 17%, 90.9 and 32% and 93.2 and 71.3%
for NBGL, SS, BS, and SM, respectively. According to our results, NBGL in direct plating was the medium with
the highest sensitivity with respect to the sensitivities of the other media, with significant statistical differences
(P < 0.05). Likewise, the PPV for NBGL was also the highest (61%). After enrichment in selenite broth, the
sensitivities of the four media tested were similar, with the best PPV obtained with SM (71.3%); this was
followed by NBGL (62.5%). When C8-esterase was used on SS or HE, the PPVs improved from less than 40%
to about 100%.

The isolation of Salmonella spp. from stool samples requires routine protocol and obtain the largest number of isolates with
the use of culture media which fulfill two attributes: an inhib- the minimum amount of work and cost.

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iting effect on the growth of the largest possible amount of
saprophytes and a discriminating capacity which allows it to be
recognized among the other species which are also capable of
growth on the medium.
Besides the numerous classic media, new ones have just
become available, including novobiocin-brilliant green-glucose
agar (NBG), novobiocin-brilliant green-glycerol-lactose agar
(NBGL), Rambach agar, and SM-ID medium (SM; bio-
Mérieux. Montalieu-Vercieu, France). The results of several
studies showing the usefulness of Rambach agar have already
been published (5, 9, 15, 16). However, we were not able to
include it in the present study because of a lack of funds, since
this medium, at least in Spain, is expensive (.$1 per plate).
For NBGL (11), whose forerunner could be considered NBG
(4), the detection of Salmonella spp. is based on the production
of H2S. Other bacteria, e.g., Citrobacter spp., are not able to
grow on NBGL because of the high acidity caused by the
fermentation of the lactose and/or glycerol contained in the
medium. On the other hand, SM is based on the formation of
acid from the glucuronate and on the absence of b-galactosi-
dase. The different serotypes of Salmonella, including S. typhi,
produce pinkish red colonies, while the coliforms have other
colors (green, blue, colorless, etc.) (14).
The purpose of the study described here was to evaluate
three classic culture media, Salmonella-Shigella agar (SS),
Hektoen enteric agar (HE), and bismuth sulfite agar (BS), and
two new ones, NBGL and SM, for their abilities to isolate
Salmonella from stool samples so that we could optimize our
* Corresponding author. Mailing address: Jaime I 1, 38 izda, 30.008
Murcia, Spain. Phone: 34-68-237099.
MATERIALS AND METHODS
HE, SS, and BS were supplied by Difco (Detroit, Mich.). BS was used accord-
ing to the manufacturer’s instructions. In SS and HE, all of the colonies that had
a black center (H2S positive and that were C8-esterase positive were investigated
together with the transparent ones, which were also oxidase negative. In this way
we would be able to detect not only the usual H2S-producing strains but also
those which do not produce H2S or those which produce it slowly and weakly
(e.g., S. paratyphi type A and S. typhi). According to our previous experience
(unpublished data), the test does not work on the other media. In particular,
Monnery et al. (9) have already indicated that it does not work on SM.
SM was obtained in the form of commercially prepared plates. In this medium,
Salmonella spp., which were the only species investigated, appear as pink colo-
nies.
NBGL is not available commercially and was prepared in our laboratory with
40.0 g of Trypticase soy agar (369-17-6; Difco), 1.5 g of ferric ammonium citrate
(3762; Merck), 5 g of sodium thiosulfate (6518; Merck), 10.0 g of lactose (156-
17-3; Difco), 10 ml of glycerol (4094; Merck) 7 mg of brilliant green (1374;
Merck), 10 mg of novobiocin (1628; Sigma), and 1,000 ml of distilled water,
according to the formula proposed by Poisson (11). Only H2S-positive colonies
were investigated with this medium.
The C8-esterase test (MUCAP test; Biolife, Milano, Italy) consists of an
eight-carbon-atom ester conjugated with 4-methylumbelliferone. This substrate
interacts with the Salmonella C8-esterase, leading to the release of umbellifer-
one, which is strongly fluorescent at 365 nm. A total of 5 ml of the reagent was
added to two to three suspect colonies, and the test was considered positive if a
blue fluorescence of the colony was observed under a Wood lamp within 5 min
(1, 10, 17, 18). The cytochrome oxidase test was obtained from Difco.
One thousand stool samples received consecutively in our laboratory for cul-
ture were inoculated onto five media (NBGL, SM, SS, HE, and BS) directly and,
after enrichment for 16 to 18 h in selenite broth, to four media (all of the media
given above except HE). On direct plating, each plate was inoculated with a
different sterile swab which had previously been submerged in a suspension of
the stool sample in saline. Selenite broth was inoculated with 0.5 ml of this latter
suspension. Subcultures were carried out as described for the original cultures.
The data from these two stages of the study were computed independently since
different inocula were used. The plates were incubated at 378C for 24 h. The
colonies which were suspected of being salmonellae were subcultured in Kliger
agar and were then identified biochemically by using the API 20E system (bio-
Mérieux) and serologically with Difco antisera.

686
VOL. 34, 1996 DIFFERENT MEDIA FOR ISOLATION OF SALMONELLA SPECIES 687

TABLE 1. Recovery of Salmonella spp. by different selective media TABLE 3. Recovery of Salmonella spp. by different selective media
by direct platinga after enrichment in selenite brotha
No. of colonies No. of true No. of false Sensitivity PPV No. of colonies No. of true No. of false Sensitivity PPV
Medium Medium
investigated positives positives (%) (%) investigated positives positives (%) (%)

SM 66 29 37 39.2 43.9 SM 115 82 33 93.2 71.3

BS 198 41 157 55.4 20.7 BS 250 80 170 90.9 32
HEb 79 27 52c 36.5 34.2c SS 474 81 393b 92 17c
SSb 256 48 208c 64.9 18.7c NBGL 128 80 48 90.9 62.5
NBGL 95 58 37 78.4 61 a
A total of 88 isolates were recovered.
b
a
A total of 74 isolates were recovered. On SS 390 colorless colonies were investigated. None of them was C8-
b
On SS and HE 316 and 160 colorless colonies were investigated, respectively. esterase positive.
c
None of them was C8-esterase positive. By the C8-esterase test on SS, there was one false-positive result (P. vulgaris)
c
By the C8-esterase test on HE and SS, there were no false positives and the and the PPV improved to 98.8%.
PPV improved to 100%.

Once the study was finished, all of those strains that had not grown in one of
the media were subcultured onto the media to count the organisms from a
suspension in saline by inoculating 3 3 102 CFU per plate. An identical inoculum
was seeded onto a Mueller-Hinton plate, which was used as a control to compare
the number of colonies obtained.
The costs of a culture plate prepared in our laboratory with Difco dehydrated
medium are as follows: SS, $0.24; HE, $0.42; BS, $0.26; a tube of selenite, $0.15.
A plate of NBGL prepared with the ingredients and prepared as described above
costs $0.28. Because SM was kindly provided by bioMérieux in prepared plates,
we do not know its exact price. We suppose that it would depend on demand.
Five microliters of MUCAP test reagent costs approximately $0.10.
Comparisons were evaluated statistically by the x2 test, which was modified for
paired series when necessary.
RESULTS
Table 1 provides the number of isolates, the sensitivity, and
the positive predictive value (PPV) obtained after direct cul-
ture. A total of 74 Salmonella strains were isolated from the
(61%) were obtained with NBGL. With SS, although the sen-
sitivity was also good (64.9%), the PPV was very low (18.7%).
However, when the C8-esterase test was used, we were able to
eliminate the 208 false-positive results and thus obtained a
PPV of 100%. Identical PPV results were obtained with HE by
the same method.
Table 3 shows the results obtained with the four inoculated
media after enrichment in selenite. The sensitivities obtained
with the four media were very similar (greater than 90%). SM
presented the highest PPV (71.3%); this was followed by
NBGL (62.5%). Likewise, when C8-esterase was used to elim-
inate the false-positive results obtained with SS, the PPVs
increased from 17 to 98.8%. There was only one false-positive
result, which corresponded to a strain of Proteus vulgaris.
No S. typhi strains or other H2S-negative Salmonella spp.
were recovered during the study.
All of the strains which were not isolated in one of the media
grew after inoculating 3 3 102 CFU of the pure strain into the

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1,000 stool samples in direct culture, and 88 strains were iso-
lated after selenite enrichment. The distribution according to medium in question, with no apparent reduction in the number
serotypes (number of isolates) was as follows: S. enteritidis (n 5 of colonies recovered when Mueller-Hinton agar was used as a
34), S. typhimurium (n 5 23), S. virchow (n 5 9), S. newport (n control. Thus, we were able to confirm that the failure in the
5 5), S. hadar (n 5 4), S. derby (n 5 3), S. heidelberg (n 5 3), recovery was not due to the inhibiting effect of the medium.
S. muenchen (n 5 2), S. ohio (n 5 2), S. bredeney (n 5 1), S.
worthington (n 5 1), and S. infantis (n 5 1). No relation was DISCUSSION
found between the serotypes isolated and the sensitivities of
the media. In the present study, the highest sensitivity was obtained
Table 2 provides the results of statistical comparisons be- with NBGL by direct sampling, with significant statistical dif-
tween the methods by the x2 test. Significant statistical differ- ferences in relation to all other media tested being obtained.
ences were obtained when the sensitivity of NBGL was com- The PPV was also the highest with NBGL (61%), although if
pared with those of the other media tested (P , 0.05) and growth in SS or HE was combined with the C8-esterase test,
when those of SS and BS were compared with those of HE and the PPVs of these media rose to 100%. The usefulness of this
SM (P , 0.05). On the other hand, we did not find any differ- test has already been established (1, 10, 17, 18). The excellent
ences in sensitivity between the pairs HE-SM and SS-BS. The performance of NBGL coincides with the results of Poisson
best results with regard to both sensitivity (78.4%) and PPV and colleagues (11, 13), which gave average sensitivities and
PPVs higher than those of SS and HE. On the other hand,
these results contradict those of Dusch and Altwegg (6), who
TABLE 2. Statistical differences with respect to sensitivity between found that NBGL performed poorly. It is interesting that the
five selective media by direct plating by the x2 testa number of isolates that we obtained on SS and HE were dif-
ferent compared with those obtained in other studies, in which
P valueb both media performed similarly (12, 13). In our study, HE
Medium SM BS HE SS NBGL allowed the growth of high levels of accompanying flora, which
(n 5 29) (n 5 41) (n 5 27) (n 5 48) (n 5 58) reduced the possibility of isolating Salmonella spp. Also, on
this medium, we obtained very few black colonies compared
SM ,0.05 NS ,0.001 ,0.0001
BS p,0.05 ,0.05 NS ,0.005
with the number obtained on SS.
HE NS ,0.05 ,0.001 ,0.0001 With BS we obtained an intermediate sensitivity (55.4%)
SS ,0.001 NS ,0.001 ,0.05 and a poor PPV (20.7%), which meant that the number of
NBGL ,0.0001 ,0.005 ,0.0001 ,0.05 colonies being tested was very high, considerably increasing the
a
workload. The sensitivity and differential properties of this
A total of 74 Salmonella isolates were isolated with all media by direct plating
(sensitivity, 100%).
medium can change as a result of preparation and storage
b
Values in parentheses are the number of salmonellae isolation each medium. conditions (3). Nevertheless, since it does not contain lactose it
NS, no statistical difference. allows the isolation of Salmonella spp. which are capable of
688 RUIZ ET AL.
fermenting that sugar (2). Several investigators have already
pointed out the lack of selectivity of SM in direct sampling (5,
6, 8, 9). In our study, although we used a new formula pro-
posed by the manufacturer, the selectivity did not improve
appreciably, overlooking in many cases the isolation of salmo-
nellae. Nevertheless, this medium together with BS has the
advantage of being an excellent alternative for the isolation of
S. typhi (6), contrary to NBGL, in which this serotype is not
detected (4, 13), and other media like SS, in which S. typhi can
go undetected in some cases (4).
The use of enrichment broth improved the recovery, as it did
in other studies, so we think that it would be interesting to use
it on a routine basis. After enrichment of the samples in se-
lenite, the sensitivities of all culture media tested were very
similar (above 90%). This fact is in contrast to the results
obtained by Poisson and colleagues (11, 13) in studies in which
NBGL was also superior to SS and HE. The best PPV results
were obtained with SM, thus revealing how easy it is to work
with this medium at this stage. Slightly lower PPVs were ob-
tained with NBGL, but the PPVs were way above those ob-
tained with the other media.
Conclusions. In light of the results of the present study, we
believe that it would be interesting to use NBGL in direct
sampling (high sensitivity and PPV), in addition to SS with the
C8-esterase test, which would also allow us to detect H2S-
negative strains. After enrichment, SM might be the ideal
medium since it is based on a different differential test and
allows a high degree of performance with minimum effort.
The cost of this method would be economical on direct
plating ($0.28), and in the subcultures, the choice would de-
pend on the price of SM or a different medium if the savings of
other supplementary tests did not compensate for the differ-
ence.
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