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Free «Determining the Identity of Two Unknown Bacteria» Essay Sample


The aim of the experiment was to identify two unknown bacteria, tube N and tube 15.  Multiple tests were carried out to demonstrate biochemical reactions, fermentation abilities, and the presence of enzymes. Results found are compared with known bacteria identification key to aid in identification of the unknown. The results found in this experiment suggested that tube N was Enterobacter aerogenes and tube 15 Bacillus subtilis. These bacteria are pathogenic and are common cause of illness in human beings.

Identification of unknown bacteria has many practical applications and this makes it crucial in research if diversity of bacterial is to be dealt with. The applications range from identifying a causative agent of a disease in order to design treatment to knowing the correct bacterium in manufacturing foods and antibiotics.

Materials and methods

Two unknown bacteria were obtained from a bacteria culture while aseptic technique was ensured throughout the procedure.  They were subjected to various tests to enable their identification; Identification key was used in identification of unknown bacteria.



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Gram staining was done to identify whether the bacteria were gram positive or gram negative.

Gram staining procedure

A drop of psychological saline was placed on a microscope slide and then  bacteria colony from each tube were inoculated in two separate slides using a sterile wire loop. The smears were fixed by passing the slides over a Bunsen burner for about three times. A drop of crystal violet was placed in each slide and left for one minute; this was then washed under running distilled water (Benson-Brown, 2001).

Grams iodine was flooded on the slides and left for one minute then it was washed away with distilled water. 95% acetone was used to rinse the slides then Safranin stain was added to the slides and left for one minute; this was then washed away under running distilled water (Benson-Brown, 2001). The slides were then observed under a microscope.

Colonies from each tube were then streaked in MacConkey Agar and incubated at 370 for 48 hours; this was done to determine lactose fermentors and non-lactose fermentors (Harley, 2003).

A triple sugar iron agar (TSI) test was used to detect production of hydrogen sulphide by the bacteria; this was done by streaking a bacteria colony from each tube on the agar; production of the gas was indicated by formation of bubbles and a black precipitate in the test tube (Harley, 2003).

Indole test was performed on the bacteria to determine whether the bacteria contain tryptophanase.  This was performed by preparing 1% typtone and Kovacs reagent to make a broth. The unknown bacteria were then placed in different tubes containing the broth. Tryptophanase activity was determined by formation of a red ring on the top layer of the broth (Harley, 2003)

Urease test was performed whereby colonies from the two tubes were inoculated in tubes with phenol red and urea and incubated for 24 hours at 370 (Holt & Wilkins 2000). Color change was checked to determine urease activity; positive results show color change of phenol red due to acid production (Harley, 2003)

Simmon citrate test was performed to determine the ability of the bacteria to produce citrase; Bromo thymol blue was used in this test as the indictor whereby positive results are indicated by color change of the indicator from green to blue (Harley, 2003).

Catalase test was also performed to determine catalase activity; in this test, bacteria colony was picked from tube 15 and tube N and spread separately on a microscope slide. Two drops of hydrogen peroxide were place on the slide; catalase activity was demonstrated by production of bubbles (Holt & Wilkins 2000).

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Colonies from tube 15 were gram stain positive, it tested positive on catalase test, and tested negative for indole test. In a glucose fermentation broth, colonies from tube 15 were identified as non-glucose fermentors and they were also non-lactose fermentors. The identification of bacteria in tube 15 was determined to be Bacillus subtilis.

Colonies fro tube N were gram stain negative, tested positive in Simmon Citrate test, and in catalase test. The colonies were negative in urease test, negative in TSI agar and negative in Indole test. The colonies fermeted lactose in Maconkey’s agar. The plausible identification of bacteria in tube N was Enterobacter aerogenes. 

Test Unknown tube 15 Unknown tube N
Gram stain + -
MacConkey Agar Non-lactose fermentor Lactose fermentor                   
Glucose broth Non-glucose fermentors Glucose fermentor
TSI Agar + -
Indole test - -
Urease test + -
Citrate test + +
Catalase test + +
Identity Bacillus subtilis Enterobacter aerogenes


Enterobacter aerogenes is a bacterium which appears as gram negative straight rods (bacillus) under a microscope (Madigan & Clark, 2009). It is a falcultative anaerobe causing a wide range of diseases in human including osteomyelitis and infections of respiratory tract as well as those of urinary tract (Madigan & Clark, 2009).

Bacillus subtilis are gram positive rods obligate aerobe (Madigan & Clark, 2009) capable of living in different temperatures thus it is even capable of surviving in human body although their virulence is very low. However, capable of causing food poisoning and are preferred in many industrial applications such as in the production of antibiotics and antifungal (Madigan & Clark, 2009).

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Catalase test done confirmed that the colonies from tubes 15 and tube N were Bacillus species (Holt & Wilkins 2000). The tests which can help distinguish between the two bacillus include gram staining, MacConkey agar, glucose broth and TSI agar.


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