Unknown Project Report
- 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.