lwalsh90 Posted April 29, 2011 Posted April 29, 2011 (edited) Hi everyone, I'm a sophomore biochemistry, cell, and molecular biology student currently enrolled in a microbiology course. We have been assigned an unknown bacteria and must identify it's taxonomy, genus and species based on a series of tests. My tentative identification of the bacteria currently is Alcaligenes Faecalis. However, a few of the test results are misleading me from my tentative ID, but I'm not sure what else it can be. Below is the list of possible unknowns (some on the list are definitely not possible being they are highly pathogenic). I have also listed my current test results and any relevant attached pictures. (All images taken under the microscope are at 1000x magnification under oil immersion.) Everything seems to agree with A. Faecalis besides the positive test I'm getting for urease and also after having just done an enterotube, the glucose chamber seemed to turn orange indicating fermentation. However, my glucose test using a Phenol red carbohydrate fermentation was alkaline giving a test result of "-", which corresponds to A. Faecalis. Any input would be greatly appreciated!! Thank-you! Unknown list, Biology 250 Acetobacter aceti Acidovorax sp. Acinetobactercalcoaceticus* Actinomyces israelii Agrobacterium tumefaciens Alcaligenes faecalis Alcaligenesviscolactis Arthrobacterglobiformis* Azotobacterchrococcum* Bacillus brevis* Bacillus circulans Bacillus coagulans Bacillus megaterium Bacillus stearothermophilus Bacillus subtilis Bacillus thuringensis Bacteroides fragilis Bartonellahenselae Bordetellasp. Borreliaburgdorferii Borreliarecurrentis Branhamellacatarrhalis Brucellasuis Burkholderiacepacia Burkholderiapseudomalii Buttiauxellaagristis Caulobactercrescentus Campylobacterjejuni Cedecealapagei Cellulomonassp. Chlamydiapsittaci Chlamydia trachomatis Chromobacterium sp. Citrobacter fruendii Clostridium perfringens Clostridium sporogenes Corynebacterium diphtheriae Coryebacterium hofmanii Corynebacterium pseudodiphtheriticum Diplococcus pneuomoniae(a/k/a Streptococcus pneumoniae) Edwardsiellatarda Enterobacteraerogenes Enterobacteragglomerans Enterococcusfaecalis Enterococcusfaecium Edwardsiellatarda Enterobactercloacae Enterobacterhafnia Erwiniaspp. Escherichia coli Escherichia fruendii (fkaCitrobacter fruendii) Ewingellaamericana Flavobacteriumcapsulatum Francisellatularensis Gardnerella vaginalis Gluconobacter oxydans Haemophilus ducreyi Haemophius influenzae Hafnia alvei Hafnia spp. Helicobacter pylori Klebsiella oxytoca Klebsiella ozaenae Klebsiella pneumoniae Kluyvera cryocrescens Lactobacillus acidophilus Lactobacillus lactis Lactococcus lactis Legionella pneumophila Listeria monocytogenes Micrococcus luteus Morganella morganii Moraxella catarrhalis Mycobacterium phlei Mycoplasma hominis Neisseria catharrsis Neisseria perflava Neisseria sicca Neisseriasubflava Nocardiaasteroids Pasteurellamultocida Plasmodiumvivax Proteusmirabilis Proteuspenneri Proteusvulgaris Providenciaalcalifaciens Pseudomonasaeruginosa Pseudomonascepaceae Pseudomonasfluorescens Pseudomonassolanacearum Pseudomonasstutzeri Pseudomonassyringae Rhanellaaquatilis Rhizobiumleguminosarum Salmonellaparatyphi SalmonellaGroup C Salmonellaarizonae Salmonella enteritidis Salmonellatyphimurium Salmonellatyphi Sarcinalutea Serratiamarscescens Serratiaplymuthica Shigellaflexneri Shigellasonnei Sporosarcina urea Streptococcus bovis Streptomyces albus Streptomyces antibioticus Staphylococcus aureus Staphylococcus epidermisa/k/a Staphylococcusepidermidis Streptococcus faecalis Streptococcus lactis Streptococcus mitis Streptococcus mutans Streptococcus pneumoniae(a/k/a/ Diplococcus pneumoniae) Streptococcus pyogenes Streptococcus viridans Tatumella ptyseos Thermus aquaticus Thermus flavis Treponemasp. Ureaplasmaurealyticus Vibriofischeri Vibrioanguillarum Vibrioangulara Vibriocholerae Vibrioparahemolyticus Wuchereriabancrofti Xanthomonasmaltiphila Xanthomonas campestris Yersinia enterocolitica Yersinia paratyphi* Yersinia pseudotuberculosis The first unknown broth I made from the unknown TSA plate had a mixture of gram positive cocci and gram negative rods evidenced after doing a Gram stain. I inoculated a new TSB from the original TSA plate I received for the assignment, carefully inoculating the loop with a single isolated pinprick colony and I've since isolated the gram - rods, as can be seen with the second gram stain below. Test results for unknown organism to date: G - Rods (Medium to Long) Good turbidity in 48 hours, optimal growth at 37 degrees C Hektoen - Blue/Green Growth MacConkey - Colorless Growth EMB - Colorless Growth (after 24 hrs), Blue Growth (after 48 hrs) Lactose - -/- Glucose - K/- Sucrose - K/- Oxidase - Positive Catalase - Positive Starch - Negative Citrate - Positive Nitrate - Negative MR - Negative VP - Negative Phenylalanine - Negative DNAse - Negative Indole- Negative Urease- Positive Gelatinase- Negative (possible inconclusive) Casein - Negative (possible inconclusive) Ampicillin- Resistant Tetracycline - Susceptible Penicillin - Resistant Motility - Moderate Radiation (should be positive) SIM - Negative for Sulfur and Indole, Indeterminable for motility Hanging Drop - some movement, positive Oxidation-Fermentation - Negative (N) Blood Agar - Growth with no clear or green halo (Gamma hemolysis) Chocolate Agar- Growth Enterotube II (picture attached below for results with uninoculated control below it) Reagents not yet added to enterotube so disregard the results for chambers requiring addition of reagent Pictured below is my first gram stain (contaminated) from a TSB culture inoculated from my original TSA plate. Someone else in my lab had the exact same problem with their first gram stain with their unknown. We both have the exact same results so far so I think we have the same unknown organism. (I made the broth by taking a streak of bacteria growing on the original plate I received on the day the assignment began. At that point there definitely shouldn't have been contamination especially because I made sure to follow proper procedure. Nevertheless, the next gram stain from my second broth culture looks better!) (1000x) Pictured below is a gram stain after making a new TSB culture from my original TSA plate with the unknown organism. I made a point to inoculate the loop from a single colony and NOT from a streak of organism on the plate. My buddy in lab who very likely has the same organism was also able to isolate the rods with the same result as below. Therefore, the unknown should be gram - rods. (1000x) Enterotube II Results. Bottom tube is uninoculated control. No reagents added yet so disregard chambers requiring addition of reagents. Growth of Organism on Chocolate and Blood Agar Bile Eschulin Plate Results (S. Bovis on right hemisphere and unknown organism on left) It's been a great learning process so far. Again I think I've narrowed it down to A. Faecalis but one or two tests don't seem to agree (urease should be negative and blood agar is supposed to have green halo?). I would greatly appreciate any advice! Edited April 29, 2011 by lwalsh90
nataliya Posted April 30, 2011 Posted April 30, 2011 hi, wow what a nice post to describe biochemistry.thanks very informative
Horza2002 Posted May 1, 2011 Posted May 1, 2011 Would it not be easier to sequnce its 16s DNA and identify it that way?
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