Microbiology Unknown
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UNKNOWN LAB REPORT
UNKNOWN NUMBER 54
Ron Williams
7-28-05
Bio 205
Prof. Curlee
Fall 2005
Purpose
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.
MATERIALS AND METHODS
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:
Colony Appearance
Motility
Gram Stain
Thyoglycollate – aerobic vs. anaerobic
Innoculate T soy slant
Optimum growth temperature 37o vs. 42o
Nitrate test
Methyl Red
Carbohydrate fermentation
Catalase Test
Oxidase test
Starch hydrolysis
Indole test
Urease test
Citrate Utilization test
RESULTS
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
TEST
PURPOSE
REAGENTS
OBSERVATIONS
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
growth
Acid Production
To determine the ability of a bacterium to ferment a specific carbohydrate
lactose mannitol
None
Negative lactose & mannitol fermenter
sucrose & glucose
yellow
Positive sucrose & glucose fermenter
Carbohydrate fermentation
Gas Production
lactose mannitol glucose
Bubbles
Glucose, lactose and mannitol positive
Carbohydrate fermentation
Gas Production
lactose mannitol sucrose glucose
no bubble
Sucrose negative
Indole Test
To determine the ability of an organism to split indole from tryptophane
Kovacs 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 solution
Pink 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.
Change to pink from yellow
Negative for urease will not produce NH3
Nitrate test
This test detects the ability of an organism to reduce nitrate (NO3) to nitrite (NO2) or some other nitogenous compound, such as molecular