Unknown Identification 4



Labinstructors name:



Identifyingan unknown Bacterium – Unknown 11c.

Determiningand identifying the strain or characteristics of an unknown organismin a given sample is crucial in microbiology. This is achievedthrough performing relevant tests to the unknown sample in order toascertain the presence of a suspected organism or bacterium as inthis study. In microbiology and medical circles, it is the basis ofdiagnosis and later on, treatment of the affected persons. In thisstudy, methods learned for identifying an unknown bacterium wereapplied to identifying specimen 11c.


Thepurpose of this study is to determine the presence of a given speciesof bacteria in a mixed culture labeled 11c. In this report, severaltests were carried out aiming at confirming the presence ofSalmonellatyphimurium.The mixed culture is for a bacterial family called Enterobacteriaceaeof which Salmonellais one of the genuses. The general characteristic of the family isthat they are small, gram negative, non-sporing, straight rods inappearance [ CITATION Nat14 l 1033 ]. It is important to discern each suspected micro organism in a samplebefore a curative or improved mode of action is taken. This is donethrough performing several biochemical and serological tests whichare used for identifying the bacterium [ CITATION Pub141 l 1033 ].

Itis therefore necessary to determine the presence of some of thespecies present in the culture labeled 11c and our study aims atdetermining whether Salmonellatyphimuriumis present in this culture or not. Our hypothesis would be thatSalmonellatyphimuriumis present in the culture labeled 11c. This culture was selectedrandomly from samples given to us by the instructor.


Toascertain the presence of Salmonellatyphimuriumspecies in the culture the following biochemical tests were carriedout in the laboratory following the steps laid down in the labmanual: Gram stain, Oxidase test, anaerobic test, Lactosefermentation test, indole test, Urease test, motility test, HydrogenSulfide test, endospore test, sucrose test, glucose test, Mrvp testand citrate test.

Gramstain test

Thegram test gave a pink color on rod shaped organisms which indicatedthat the bacteria were gram negative.


Thepresence of cytochrome-c oxidases was tested using redox indicator. The indicator did not show any color changes.

Lactosefermentation test

Phenolred broth that had lactose in it was inoculated into sample tubeswith the organism culture. After twenty four hours, there was nocolor change in the tubes from the initial red color.


Atest tube with the culture was inoculated with SIM medium and Kovacsreagent. There was no color change.

Motilitytest and H2Stest

Asample of the culture was inoculated with SIM medium and observedafter some time. It was noted that the culture travelled in themedium in an outward manner and the sample gave a dark color.


Asample of the culture in a test tube was inoculated with urea brothand allowed to stay for 24 hours. There was no color changeobserved.


Therewere no endospores seen when the culture was observed under amicroscope.


Thecolor did not change but remained pink hence the result is negative


Ayellow color was observed in this test showing that there is acid andgas present in the culture.


mrvptest showed a red coloration indicating that the test is positive andthe vp test is negative.


Ablue color was observed for this test indicating that the culture ispositive for the test.

Asubculture of the specimen was made using trypticase soy agarincubated at temperature of 40C,200Cand 370C. It was noted that there was better growth in cultures incubated at200Cand 370Cshowing that they are mesophiles.


Table1 below shows a summary of results obtained after carrying out thetests on the sample 11c.

Table1: Summary of tests



Mediums &amp reagents used



Gram stain

To determine if the sample is gram +ve or gram -ve

Crystal violet, grams iodine,95% ethanol, safari

Pink rod shapes

Gram negative bacillus

Anaerobic test

To determine the organisms ability to thrive with and without the presence of oxygen

Anaerobic jar, TSA plates

Growth in both aerobic and anaerobic conditions

Facultative anaerobes

Oxidase test

To determine the presence of cytochrome c

TSA plate, oxidase indicator, (Tetramethylphenylene)

No color change within 20 seconds


Lactose fermentation

To determine the ability to ferment lactose

Phenol red broth with lactose

No color change after 24+ hours


Indole test

Determine the ability of an organism to split indole from tryptophane

SIM medium, Kovacs reagent

No color change after reagent added


Urease test

To determine the presence of the urease enzyme

Urea broth

No color change after 24+ hours



To determine the organisms ability to ‘travel’ away from the stab line

SIM medium

SIM medium turned almost completely purple – black



To determine the presence of hydrogen sulfide

SIM medium

SIM medium turned purple black away from the stab line


Endospore test

To determine the presence of endospores


No endospores seen


Sucrose test

To determine whether sucrose is used in fermentation

No color change


Glucose test

To determine production of acid from glucose

Color changed to yellow

Acid/ gas present

Mrvp test

To determine production of acid


Color changed to red


Citrate test

To determine the use of citrate

Citrate slant

Color changed from green to blue


Theresults obtained indicate that Salmonellatyphimuriumis present in the mixed bacterial culture. These results aresummarized also in the flow chart below:-


Thetests performed confirmed that Salmonellatyphimuriumis present in the sample labeled 11c that was tested during thisstudy. Therefore, the hypothesis was confirmed to be true. Salmonellatyphimuriumis a pathogenic gram negative bacterium of the familyEnterobacteriaceae. The genus Salmonellahas two species thus Salmonellaentericaand Salmonellabongari. Salmonellatyphimuriumis a serotype in the entericasub species [ CITATION Foo11 l 1033 ]. Salmonellatyphimuriumis pathogenic and the intestines provide a good environment for thebacteria. Human beings infected with the bacteria suffer fromsalmonellosis and gastroentetiritis characterized by diarrhea. Inthe intestines, the bacterium is protected from destruction by theouter lipopolysaccharide membrane surrounding it. Foods contaminatedby Salmonellatyphimuriumprovide a sure way of infection particularly the ready-to-eat (RTE)foods [ CITATION Pub14 l 1033 ].

Consequently,a precise confirmation of the bacteria through such biochemical andserological tests will be very useful in arriving at an accuratediagnosis in medical circles and hence making correct decisions. Scientists or Laboratory technicians will be presented with aspecimen with unknown organisms and therefore such microbiologicalidentification techniques for unknown organisms are very important indiscerning microorganism that are present in the specimen. Thismeans that the chemicals, reagents and all equipment used in testingmust be of high quality and standardized so as to give accurateresults. Wrong results can lead to wrong diagnosis and conclusion,resulting in detrimental or lethal decisions by the concernedscientist. Therefore, accurate identification will be limited by thequality and standard of reagents and equipment used. Development ofelectronic or digital tests can improve the accuracy and precision oftests for unknown bacteria and hence result in correct and accuratedecisions by stakeholders.

Innormal cases, Salmonellatyphimuriumcan be cleared by the immune system of our body save for those whoseimmunity is affected in one way or the other.


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