Microbiology Unknown

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:

1 Colony Appearance

2 Motility

3 Gram Stain

4 Thyoglycollate - aerobic vs. anaerobic

5 Innoculate T soy slant

6 Optimum growth temperature 37o vs. 42o

7 Nitrate test

8 Methyl Red

9 Carbohydrate fermentation

10 Catalase Test

11 Oxidase test

12 Starch hydrolysis

13 Indole test

14 Urease test

15 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 red 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 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 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.

urea 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