31 October 2011 Identification and observations of Citrobacter Freundii Introduction 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 ? ; tetracycline, 30 ? m; and ampicillin 10 ? m. 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 FormCircular ElevationRaised MarginUndulate SizeMedium ColorBeige TextureMucoid OpacityOpaque 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 documented results found in the Willgoh and Bleakly lab manual (2). TestObservationResult GlucoseYellow w/ bubblePositive SucroseRedNegative

LactoseRedNegative MannitolYellow w/ bubblePositive Indole Yellow ringNegative Methyl RedRedPositive VPBrownNegative Citrate HydrolysisBluePositive UreaseYellowNegative Gelatin SolidNegative CatalaseBubbles on slantPositive (VP=Vogues-Proskeur) Results The unknown bacteria formed colonies that appeared circular, undulate, medium in size, raised in elevations, opaque, mucoid in texture, and beige in color as shown in table 1. Table 3 shows Gram stain results that indicated C. Freundii as a gram negative bacterium in rod shapes scattered in singles and some in pairs.

Each gram stain produced the same results. The Bartholomew and Mittwer method of endospore staining indicated that C. Freundii tested negative for endospore formation. Table 4 shows the biochemical test results of the unknown and the official test results for comparison. The Kirby-Bauer antibiotic tests indicated that the unknown is susceptible to chloramphenicol and ampicillin and resistant to penicillin G and tetracycline. The disinfectant sensitivity tests indicated that C. Freundii is resistant to all four of the disinfectants. Discussion

The biochemical tests, the Gram stain, the Bartholomew and Mittwer’s stain, the microscopic and macroscopic observations, and chemical sensitivity tests helped to identify the unknown bacteria as Citrobacter Freundii. Each biochemical test result maintained consistency with the official recorded results in the Willgoh and Bleakly lab manual (2). Chemical tests showed which antibiotics would be most effective in treating a C. Freundii infection. Penicillin and Tetracycline would be poor choices for treating C. Freundii infection due to the fact that C. Freundii is resistant to these antibiotics. However, C.

Freundii is incredibly susceptible to Chloramphenicol and also significantly susceptible to Ampicillin. These antibiotics would therefore be effective in treating a C. Freundii infection in a patient. Disinfection of C. Freundii should not be performed using any of the disinfectants that were tested. This is due to the fact that C. Freundii is resistant to Lysol, Basic G, Clorox, and Assist QT since none of these disinfectants created a large growth inhibition zone. If one of these four disinfectants had to be chosen, Clorox would be the most effective as it produced the largest growth inhibition zone, but C.

Freundii is still considered resistant to Clorox. Literature Cited 1)Badger, Julie L. , Stins, Monique F. , Kim, and Kwang Sik. 1999. Citrobacter Freundii Invades and Replicates in Human Brain Microvascular Endothelial Cells. American Society for Microbiology. 67: 4208-4215. 2)Willgohs, J. A. and B. Beakley. 2009. Isolation and identification of an unknown bacterium. In Laboratory manual for general microbiology, 9th edition. Pearson Custom Publishing, New York, NY. 3)Gill, M. A. , and Schutze, G. E. 1999. Citrobacter urinary tract infections in children. The Pediatric Infectious Disease Journal. 10: 889-892.