The document describes identifying Salmonella typhimurium from a mixed culture using differential tests. A pure culture of the Gram-negative bacteria was obtained and tested on five types of differential media. The results identified the bacteria as S. typhimurium based on its ability to ferment glucose and perform mixed acid fermentation, utilize citrate, reduce sulfur and be motile on SIM media. Standard differential testing using affordable media can successfully identify bacterial species from mixed cultures.

1. Identification of Salmonella typhimurium from an unknown mixture
Jeff Mackey
April 12, 2015
Teaching Assistant: Nagmeh Hassanzadeh Khayyat
Unknown #8
Purpose
The purpose of this study is to demonstrate that an unknown species of the class
Enterobacteriacaea—in this case, Salmonella typhimurium—could be successfully identified
from a mixed culture. This identification between a variety of common species may be
accomplished by means of a small number of standard, easily performed tests using differential
media.
Infection by pathogenic strains of Enterobacteriae is responsible for many of the enteric
diseases which are responsible for approximately 2.2 million deaths each year among humans
worldwide, and pose a particular threat to the lives and welfare of residents of poor and less-
developed nations. (Bublitz, et al., 2014) S. typhimurium posesses virulence factors that can lead
to gastroenteritis, but does not typically cause potentially deadly infections, yet its close relative,
Salmonella typhi, produces the toxin that causes typhoid fever, a disease made more problematic
since it can be spread by asymptomatic carriers like Mary Mallon, a cook in early-20th century
New York who was dubbed “Typhoid Mary” after infecting dozens of people who ate the food
she prepared. (Stebbins, 2013)
Advanced molecular typing methods such as multiplex polymerase chain reaction now
allow for rapid and specific identification of Salmonella and other enteric bacteria (Ali, et al.,
2009), but the high cost of the equipment used to perform these procedures make it prohibitive
for diagnostic use in the emerging world where it is most needed. If an enterobacterial infection I
is suspected, the use of valdiated, well-established differential tests using affordable media and

2. equipment, as described here, may make it possible to determine the specific species according
to its revealed biochemical traits and, therefore, recommend the best course of treatment for a
patient.
Fig. 1 – Analytical Process
Gram-negative colonies from a sample are isolated to obtain a pure culture, then further
analyzed using five types of differential media. The entire process takes around
six days, with three 48-hour incubation periods.
Since samples may contain multiple bacteria, the process used to identify S. typhimurium
from a mixed culture (Fig. 1) incorporated Gram staining of isolated colonies grown from the
sample and distinguished by variations in their morphology to eliminate the Gram-positive
strain, since Enterobacteria are Gram-negative. After obtaining a pure culture, five types of
differential media were inoculated to characterize the organism according to its ability or
inability to perform mixed acid and/or 2,3-butanediol fermentation (Methyl Red/Voges-
Proskauer broth); glucose, lactose, and/or sucrose fermentation (Phenol Red broth); citrate
metabolism (Simmons Citrate agar); urea hydrolysis (Urea broth); and sulfur reduction and/or
indole production, with or without apparent motility (SIM medium). Aseptic techniques were
employed at all stages of the process.
Results
A nutrient agar plate was inoculated by quadrant streaking with a portion of the mixed
sample of unknown species. After incubation at 37°C for 48 hours, isolated colonies were
observed and differentiated by their distinct morphologies: both round and whitish, though some
colonies were smooth, translucent, convex, and mucoid, while the others were rougher and more
opaque. Samples from both types of colonies were subjected to Gram staining, and those from

3. the former (mucoid) stained red, indicating that it was the Gram-negative species, while the latter
stained violet (Gram-positive). Examination of the Gram-negative sample under bright-field light
microscopy (100X/1.25 oil) revealed short rod-shaped bacteria (bacilli) exhibiting some apparent
clustering and chaining. A second nutrient agar plate was quadrant-streaked with a sample of the
colony from which the Gram-negative species was obtained to obtain a pure culture; after 48
hours of incubation at 37°C, the growth was of uniform morphology, with no anomalies to
indicate contamination.
The pure Gram-negative culture was used to inoculate media for the differential tests
described above, and as detailed in our laboratory manual. (Leboffe & Pierce, 2012) Following
another 48 hour/37°C incubation period and, where necessary, the addtion of reagents, the results
were observed, as summarized in Table 1 and shown in Figure 2.
Table 1
Results of the differential tests performed on the unknown Gram-negative bacterial species.
Test Visible Result
1a Phenol Red (sucrose) Pink color/no gas (K)
1b Phenol Red (lactose) Pink color/no gas (K)
1c Phenol Red (glucose) Yellow color/gas (A/G)
2a Methyl Red Red color (+)
2b Voges-Proskauer No color change (-)
3 Citrate Blue (+)
4 Urea Hydrolysis Orange (-)
5a SIM (Sulfur) Black color (+)
5b SIM (Indole) No color change
5c SIM (Motility) Black color throughout media

4. Fig. 2
Photographs of results for 1a) Phenol Red sucrose, 1b) Phenol Red lactose, 1c) Phenol Red glucose,
2a) Methyl Red, 2b) Voges-Proskauer, 3) Citrate, 4) Urea, and 5a/b/c) SIM differential tests.
The Phenol Red tests—in which samples of the pure culture were mixed with broths
containing Phenol Red and differing sugars—produced a yellow color and a bubble in the
Durham tube (Fig. 2, 1c) only for the broth containing glucose. Phenol Red broths containing
sucrose and lactose (Fig. 2, 1a and 1b) turned pink, with no bubble in the Durham tube.
The Methyl Red test, with a sample again mixed into the broth, produced a a strong red
color (Fig. 2, 2a) upon addition of Methyl Red, while the Voges-Proskauer test, performed on a

5. portion of the same inoculated medium, showed no color change (to red) within 60 minutes of
addition of the reagents.
The Citrate test, in which a sample of the pure culture was streaked along the surface of a
Simmons Citrate agar slant, showed a change in the medium from a green color to blue (Fig. 2,
3). The Urea Hydrolysis test, with a sample stirred into the broth, produced a pale orange color
(Fig. 2, 4). Finally, the SIM agar, stab-inoculated with a pure culture sample, resulted in a black
color throughout the medium; Kovacs' reagent showed no color change when added to the test
tube (Fig. 2, 5a/b/c).
Discussion
According to the results of the Phenol Red tests, the organism is capable of fermenting
glucose, producing acidic (since Phenol Red is yellow below pH 6.8) and gaseous end products,
and it is incapable of fermenting sucrose and lactose but can deaminate peptone amino acids in
the broth, producing alkaline NH3 (Phenol Red is pink above pH 7.4) with no gas production.
The Methyl Red test demonstrated that the unknown species is capable of performing
mixed acid fermentation, lowering the pH in the phosphate-buffered medium to 4.4 or lower
(Methyl Red is orange or yellow at higher pH values). The Voges-Proskauer test showed that the
organism cannot convert the acid products of glucose fermentation to acetoin and 2,3-butanediol.
The Citrate test offers evidence that the unknown bacterium can utilize citrate as a carbon
source, producing alkaline NH3 and NH4OH from the ammonium dihydrogen phosphate in the
medium, raising the pH sufficiently to cause the included bromthyol blue dye to turn from green
to blue. However, the lack of color change of the Phenol Red in the Urea Hydrolysis test shows
that the organism either does not produce urease or is not able to grow in the broth medium.

6. Finally, the black color seen throughout the SIM medium indicates both that the organism
is able to reduce sulfur to H2S which reacts with iron to produce a black ferric sulfide precipitate,
and that it posesses motility, since the color suffuses the medium. The lack of a color change to
red in the Kovacs' reagent added to the tube shows that tryptophan is not hydrolized to form
indole.
Table 2
As seen in Table 2, which lists the differential test results for a range of Enterobacteria,
the ability to ferment only glucose, as shown by the Phenol Red tests, rules out all species except
Proteus mirabilis, Shigella flexneri, and S. typhimurium. The positive Citrate test eliminates
Shigella, and the negative Urea Hydrolysis test likewise eliminates Proteus, leaving S.

7. typhimurium as our now-known organism, further confirmed by the results of the Methyl Red,
Voges-Proskauer, and SIM tests.
The exact series of tests performed here may not be effective in every situation. The
bodies of humans and other animals carry so many bacteria as to make simple isolation difficult
for further testing. Further tests may also be needed to more specifically identify a species of
interest—for instance, these procedures may not be able to distinguish S. typhimurium from S.
typhi. The use of media that is both selective and differential—such as MacConkey, Eosin
Methylene Blue, and Hektoen Enteric agars—could assist in identification by suppressing Gram-
positive bacteria while narrowing down the exact organisms present in a sample. Even in
analyzing only the microbes in Table 2, certain test results are unique enough to identify a
species on their own, such as the positive Indole production test for Escherichia coli or the
negative Methyl Red result for Enterobacter aerogenes.
As this experiment shows, however, it is possible to identify an unknown species of
bacteria from a mixed culture using standard differential test media, offering advantages for both
research and diagnostic laboratories, particularly those with limited funding.

8. Bibliography
Ali,A.,Haque,A.,Haque,A.,Sarwar,Y., Mohsin,M., Bashir,S.,& Tariq, A.(2009, January).Multiplex PCR
for DifferentialDiagnosisof EmergingTyphoidal PathogensDirectlyfromBloodSamples.
Epidemiology and Infection,137(1),102-107.
Bublitz,D.,Wright,P.,Bodager,J.,Rasambainarivo,F.,Bliska,J.,& Gillespie,T.(2014, July1).
Epidemiologyof PathogenicEnterobacteriainHumans,Livestock,andPeridomesticRodentsin
Rural Madagascar. PLoSOne,1-2.
Leboffe,M.J.,& Pierce,B.E. (2012). Microbiology Laboratory Theory and Application,Brief Edition.
Englewood,CO:MortonPublishing.
Stebbins,C.E.(2013, July18). Bacteriology:ToxinsinTandem. Nature,499,293.