Uknown Microorganism Lab Report

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31 October 2011
Identification and observations of Citrobacter Freundii
Citrobacter Freundii is a species of bacteria that can be potentially harmful to humans. It is known to cause meningitis by protruding into the brain and replicating itself (1). The Citrobacter species has also been found as a cause of some urinary tract infections, diarrhea, and even gastrointestinal diseases and symptoms (3). C. Freundii can be located in a wide variety of soils and water (3). Lastly, it is also the cause of many nosocomial infections due to its presence in water (1). Identifying C. Freundii holds importance in order to determine infection in gastrointestinal and urinary tracts and in the brain. These infections will often be noticed through pain in these areas. The main purpose of this laboratory experiment was to utilize microscopic and macroscopic observation, biochemical tests, and antibiotic and disinfectant sensitivity tests to identify an unknown bacterium. Materials and Methods

Microscopic and Macroscopic Observations
All samples and observations of the unknown bacterium were taken after 48 hours of incubation at 35° Celsius. Microscopic observations were performed on four different microscope slides. Three of these observations were gram stains and the last observation was of an endospore staining. The unknown bacteria samples for the endospore stain were taken off of a nutrient agar plate and stained using the Bartholomew and Mittwer’s method. Gram stain samples were taken off of tryptic soy agar and stained using the gram stain procedure that is listed in the Willgoh and Bleakly manual (2). Physiological Tests

All biochemical tests are shown in table 4. Each of these tests required an incubation time of 48 hours at an incubation temperature of 35° Celsius with an exception to the gelatin test that required a 30 minute refrigeration period before observing results. All tests were performed according to the procedures listed in the Willgoh and Bleakly lab manual (2). Antibiotic and Disinfectant Tests

The sensitivity or resistibility of C. Freundii to antibiotics and disinfectants was tested using the steps outlined in the Willgoh and Bleakly lab manual (2). The four tested antibiotics include: penicillin G, 10 U; chloramphenicol, 30 μm; tetracycline, 30 μm; and ampicillin 10 μm. The four disinfectants that were tested on the unknown bacteria include: Lysol, Basic G, Clorox, and Assist QT. Susceptibility of resistibility of the unknown bacteria to these antibiotics and disinfectants were tested and measured using the chart on page 125 and the Kirby-Bauer test procedures in the Willgoh and Bleakly lab manual as a reference (2). These results are shown in table 3. Data

The following results show that C. Freundii had the following colonial morphology when observed in an isolated pure culture (Table 1). ObservationC. Freundii Colonial Morphology

The following results indicate that C. Freundii is a facultative anaerobe and a gram negative rod shaped bacterium that is endospore negative and motile (Table 2).
ObservationsStain, Oxygen, and Motility Test Results
Gram StainPink and rod shapedGram negative rods
Endospore StainNo green spotsEndospore negative
Thioglycollate TestGrowth in both areasFacultative anaerobe MotilityFuzzy, red growthMotile bacterium

The next results indicate the susceptibility or resistibility of C. Freundii to antibiotics and disinfectants (Table 3). Antibiotic/DisinfectantGrowth Inhibition Diameter
P103mm R
TE300mm R
C3020mm S
AM1013mm S
Lysol0mm R
Basic G2mm R
Clorox8mm R
Assist QT4mm R
(P10=Penicillin G, C30= Chloramphenicol, TE30=Tetracycline, AM10=Ampicillin, S=susceptible, R=Resistant)
Table four can be found on the next page and it shows the biochemical test results for C. Freundii. All results are consistent with the...
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