lwalsh90

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!