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!
