Identify Unknown Microbes

NTRODUCTION Often in the study of microbiology, the need to identify an unknown microbe may arise. This aids in the recognition of which microbes are harmful or pathogenic as well as those that may be beneficial to us in some way. This report will detail how a similar project was performed in order to identify two unknown micobes using the techniques that were learnt during the course of the semester. We embarked on the project with the knowledge that we would have to identify a Gram(+) as well as a Gram(-) from the species that were given and the experiment proceeded as follows.
METHODS
The two streak plates made from the mixture of unknown bacteria showed two different colony morphologies. Colonies of Bacteria A were translucent in color, circular in form, with entire margins and a smooth surface. Colonies of Bacteria B were cream colored, irregular in form, with undulate margins and an umbonate surface. Two different morphologies allowed Gram stains to be performed on each of the bacterium.
Gram staining of Bacteria A revealed pink, rod-shaped cells signifying that the bacterium was Gram-negative and rod-shaped.
Next, an oxidase test was performed to eliminate one or more of the bacterium choices. P. aeruginosa was eliminated as the identity of the bacterium when the oxidase test results were negative. An EMB plate of the bacteria was examined to identify whether the bacterium was S. typhi or E. coli. The growth on the EMB plate was shiny and metallic green, meaning that it was positive for lactose fermentation. S. typhi exhibits intermediate lactose fermentation with pink or purple growth on an EMB plate, so it was eliminated as the possible identity of Bacteria A. To confirm the identity of Bacteria A as E. coli, an Enterotube test inoculated with Bacteria A was observed. The Biocode confirmed the identity of Bacteria A as E.
coli. Gram staining of Bacteria B revealed purple, rod-shaped cells signifying that the bacterium was Gram-positive and rod-shaped. The shape of the bacterium eliminated cocci-shaped S. aureus and S. epidermidis as possibilities for the identity of Bacteria B. Next, an endospore stain of the bacteria was examined to identify whether the bacterium produced spores or not. The stain showed red cells with green spores, indicating that the bacterium did produce spores. The results of the endospore stain confirmed the identity of Bacteria B as B. subtilis. Table 1 lists all of the biochemical tests, their purpose and results. The results are also shown in flow chart form in Table 2.
RESULTS
Test
Purpose
Reagents
Observations
Results
Gram Stain
To determine the gram reaction of bacteria
Crystal violet, Iodine, Alcohol, Safranin
Unknown A: pink rods
Unknown B: Purple rods
Unknown A: gram negative bacilli
Unknown B: gram positive bacilli
Oxidase Test
To determine the presence of oxidase
Oxidase paper
Unknown A: yellow
Unknown A: negative for oxidase
Lactose Fermentation Test
To determine if bacteria ferments lactose
EMB plate
Unknown A: shiny, green, metallic colonies
Unknown A: positive for lactose fermentation
Endospore Stain
To determine if bacteria produces endospores
Malachite green, Steam, Safranin
Unknown B: red cells with green spores

Unknown B: Positive for producing endospores
Enterotube
To identify gram negative enterobacteriaceae
Enterotube
Unknown A: code 24160
Unknown A: E.coli
Table 1: Biochemical tests performed on Unknown A and Unknown B.