Microbiology Unknown

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Ron Williams


Bio 205

Prof. Curlee

Fall 2005


There are many reasons for knowing the identity of microorganisms. The reasons range from the knowing the causative agent of a disease in a patient, so as to know how it can be treated, to knowing the correct microorganism to be used for making certain foods or antibiotics. This study was done by applying all of the methods that have been learned so far in the microbiology laboratory class for the identification of an unknown bacterium. Hypothesis

By slowly narrowing down the bacteria by each test, I will be able to conclude the unknown bacteria by its special characteristics.


An unknown labeled as #54 was given out by the lab instructor. The methods that have been learned thus far for identifying bacteria have been applied to this unknown. Procedures were followed as stated in the course laboratory manual, unless otherwise noted. The first procedure that needed to be done was to streak the unknown out on a Trypticase Soy Agar plate, using the T streak method. This needed to be done in order to test the purity of the unknown. After the plates were incubated and grown, the morphology was observed and recorded and a Gram stain was performed. After determining the Gram reaction, specific biochemical tests were performed. The biochemical tests were chosen from the unknown identification tables that were in the lab manual. Since the unknown #54 was determined to be a Gram positive rod, a thyoglycollate test was performed to determine aerobic activity. Table 1 lists the test, purpose, reagents and results. All of the following tests were performed on this unknown:

1Colony Appearance
3Gram Stain
4Thyoglycollate – aerobic vs. anaerobic
5Innoculate T soy slant
6Optimum growth temperature 37o vs. 42o
7Nitrate test
8Methyl Red
9Carbohydrate fermentation
10Catalase Test
11Oxidase test
12Starch hydrolysis
13Indole test
14Urease test
15Citrate Utilization test


Unknown 54 had the following morphology on a TSA plate:large sized opaque cream colored colony. After determining that it was a Gram positive rod, a test was performed using Thyoglycollate where the unknown #54 was inoculated. Table I lists all of the biochemical tests, their purpose and results. The results are also shown in a flow chart form. Table 1: Biochemical Test Results

Gram stain To determine the Gram reaction of the bacterium Crystal violet, Iodine, Alcohol, Safranin Pink rods Gram positive rods Thyoglycollate To determine the aerobic vs. anaerobic
Acid ProductionTo determine the ability of a bacterium to ferment a specific carbohydrate

lactose mannitol None redNegative lactose & mannitol fermenter
sucrose & glucose yellowPositive sucrose & glucose fermenter Carbohydrate fermentation
Gas Productionlactose mannitol glucoseBubblesGlucose, lactose and mannitol positive Carbohydrate fermentation
Gas Productionlactose mannitol sucrose glucoseno bubbleSucrose negative

Indole Test To determine the ability of an organism to split indole from tryptophane Kovac's added to broth Red Ring at top of broth Negative indole test Oxidase test

To determine the presence of oxidase which can oxidize reduced cytochrome
Oxidase solutionPink to maroon from purple Negative oxidase test

Urease Test
This test is used to differentiate organisms based on their ability to hydrozye urea with the enzyme urease. This test is particularly useful in distinguishing the genus Proteus from other enteric bacteria.

ureaChange to pink from yellowNegative for urease will not produce NH3 Nitrate testThis test detects the ability of an organism to reduce nitrate (NO3) to nitrite (NO2) or some other nitogenous compound, such as molecular nitrogen (N2), using the enzyme...
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