Unknown Bacteria 228Adisney PinoProfessor Dowding Miami Dade CollegeSection T/R 7:05PM              Introduction             Enterobacter aerogenes is rod-shaped bacteria that belongs in thefamily of Enterobacteriaceae. This rod-shaped bacterium is gram negative whichmakes it a facultative anaerobe which grew perfectly in 37-degree temperature.This species was motile and therefore it can synthesize an enzyme known asornithine decarboxylase.

This bacterium is prevalent in the intestines of animals,there presence in the intestines of animals makes it easier to get in the soil,water and sewage. In humans, this bacterium is known as an opportunisticpathogen which can cause multiple conditions which include, meningitis,bacteremia, pneumonia, endocarditis and urinary tract infections.             Manytimes, exposure to this bacterium is the result of hospital stays and nursinghomes.

Long hospitalizations and invasive procedures increase your chances ofgetting this bacterium.   Recently some Enterobacter organisms have become drug resistant which is makingit harder to make a treatment to fight the infection. The usual treatment for Enterobacter infections consist ofantimicrobial therapy. Some of these treatments include Aminoglycoside, Fluoroquinoloneand Cephalosporin, however in some recent cases the enzyme that is produced by thisbacterium when exposed too much to these drugs will cause the bacteria tobecome resistant to the treatment.

Therefore, newer and more modern treatmentsare being put into place, such combining multiple therapies at once to forcethe bacteria to go away.             Somesigns and symptoms one might have when infected include, fever, hypotension,tachycardia, and even hypothermia when it comes to respiratory tractinfections. This bacterium is diagnosed by performing laboratory studies suchas urine analysis, fluid analysis (cells and differential analysis for example)and electrolyte evaluation.

Furthermore, Enterobacterare capable of nitrogen fixation. Some species in specific E. cloacae, this bacterium has been found to be involved insymbiotic nitrogen fixation in plants and have even been separated from theroots of certain types of plants.  Materials            Thematerials used to experiment on this unknown bacterium were as follows: Microscope, agar slants, Phenol red broth, Durhamtubes, NH3, Iodine, Bromothymol blue dye, Kovac’s reagent, Enterotube 2, transferpipette, loop, Bunsen burner, agar plates, slants, distilled water, tube racks,tape, sharpie, metric ruler, Methyl Red reagent, test tubes, hydrogen peroxide,microscope slides, Nutrient agar, cotton tipped applicator, zinc powder,nitrate powder, gelatin stabs, Simmons citrate slants. Methods            Thefollowing steps were taken to discover what bacteria was in the originalrandomly given plate.

When the original agar plate was given, the first stepwas to look at the bacterial colony features. Here we looked for the elevation,margin and the way the colony looked. After the agar was observed, a sample wastaken from the original plate and it was put in different temperatures. Twosamples went in a nutrient broth and two more samples went in a slant.

The twosamples of the nutrient broth went on different temperatures, one went to 25degrees Celsius, while the other went at 37 degrees Celsius. The following lab,we observed the slant with the best growth. This slant became the “workingstock” which was used as a sample for the rest of the labs and the slant withthe least growth was left as a backup in case of contamination. With this verysame samples, we made a gram stain. For this gram stain, we heat fixed ourunknown bacteria and covered it with crystal violet stain for a minute, afterthis we rinsed the slide with distilled water and removed the excess of stain.

After, we covered the smear with Iodine stain and after a minute we rinsed theexcess of stain and then we decolorized the smear using acetone. This procedurewas done until most of the stain was gone, then we used the counterstain safraninfor another minute and once the minute passed we rinsed it with distilled waterand used bibulous paper to dry the smear and after it was fully dried weobserved the smear under the microscope.             Thenext step done was to inoculate broths with our unknown organism and usingFluid Thioglycolate tubes we inoculated three broths with our unknown organism.Then we stored these away at 37 degrees Celsius, the next class we looked atthe growth pattern and decided if it was aerobe or facultative anaerobe. Afterthis procedure, we tested our unknown bacteria in glucose, lactose, mannitoland arabinose broths. Once again, we inoculated each broth with our unknownorganism and when done we put it away at our optimal temperature of 37 degreesCelsius. The broths used for this test were PR broths, some of this had asmaller tube inside to test for gas. Once done with the PR broths you will obtainthree MR-VP broths, and we will inoculate two broths with our cultures, we didnot inoculate the control which was the third tube.

The following lab day wegrabbed test tube one (Methyl Red Test) and we added three drops of Methyl Redagent and we observed it for red color change, this change had to happenimmediately.             Thenext tube was for the VP test and we added 15 drops of VP reagent A, we thenvortexed the tube and added 5 drops of VP reagent B. Once again, we vortexedand we observed for red color formation, this process took 10 minutes. Afterthese tests were done we looked at the tables in our lab manuals and dependingon the color change we had, we decided if we had fermentation or not. On thenext lab date, we used test tubes we had arranged with our unknown bacteria andgrabbing a good portion of bacteria we dropped a mass on an oxidase slide test.A color change had to happen within the first 20 seconds, after this we grabbedthe same tube and added hydrogen peroxide directly into the test tube and weobserved the tube for bubbles/reaction.

This lab we also got four Nitratebroths and inoculate the broths and after 48 hours in our optimal temperaturewe examined the tubes for evidence of gas production. After the observationswere done, we added eight drops of reagent A and reagent B to each test tubeand using our control we observed and compared it to our control. The followingstep done was to grab a starch agar plate, a milk agar, and a Tributyrin agar wherewe spot inoculated the agar plates and then incubated them at our optimaltemperature.             Thefollowing lab date we observed the growth on the starch agar and then addediodine and looked for a halo, after that we picked up the milk agar and lookedfor a halo without adding any agent.

Then, we looked at the Tributyrin agar andlooked for a clearing/halo around the bacteria. After this procedure, weinoculated three urea broths and after storing it at our optimal temperature for48 hours we brought it out and examined them for color change. After thesetests had been run and observed we grabbed three nutrient gelatin stabs, and weinoculated two tubes, this time we stabbed the gelatin once with an inoculatingneedle and left one more nutrient stab for control purposes. The following labwe observed the gelatin, if it was solid or soft and watery. We also watchedfor any formation where we stabbed the gelatin. After these observations weredone, we obtained a Simmons Citrate tubes, and made a zigzag pattern on theslant, lightly inoculating with a needle and incubated them all at our optimaltemperature. The following lab we will watch the slant for any color change andgrowth. The following lab date we had a Phenylalanine Agar slant and inoculatedheavily doing a zigzag motion and the lab class after that we added 2 or 3drops of 12% ferric chloride solution to the tube and observed for colorchange.

The reaction we were looking for should have happened immediately.             Additionally,we grabbed a SIM tube and inoculated three tubes with the unknown organism andwe stabbed the tube using a needle, wherever originally stabbed we had to tryand stay in the same line. We then inoculated these tubes. The following labclass we examined the tubes for spreading away from the stab line and if anyformation of black precipitate in the medium had formed.

We then added Kovac’sreagent to each tube and waited for about 5 minutes and observed for any redcolor formation at the top, by now we were able to make a presumptiveidentification of our unknown bacteria. The following test would then haveproved or disproved our identification. We grabbed an Enterotube 2 and removedthe blue and white cap, being careful not to contaminate the sterile wire. Thenwe grabbed our original plate and removed a lar amount of growth from one ofthe colonies in my agar and then slowed turned the wire through the Enterotube2 compartment. We continued to turn until we reached the bottom where we willthen roll it back inside and leave the tip of the needle in the Glucosecompartment and break of the bottom part that is left out. This was then getstored at our optimal temperature where the following lab we compared theresults from this tube and the ones we had been getting throughout the class.  Results and ConclusionsIn order to get the exact bacteria in ourunknown agar plate, we had to run a series of test and observe what the reactionswere.

We started with the characteristics of the bacteria, for example we did agram-negative stain and noted that unknown bacteria 228 was a gram-negativebacterium. We then did an oxygen test where we watched for an aerobe orfacultative anaerobe and my unknown bacteria tested facultative anaerobe,furthermore 37 degrees Celsius was this unknown bacteria’s optimal growthtemperature because when the test tubes were observed it showed the most growthbetween the two temperatures. This bacterium was also a motile bacterium becauseafter doing a wet mount the bacteria although difficult to tell, was movingaround the slide which made it motile and the growth pattern on the nutrientbroth was sediment like.

The next couple of test would have given us thisunknown bacteria’s fermentation and respiration. We did a series of test withglucose, lactose, mannitol and arabinose this was done to be able todifferentiate members of Enterobacteriaceaeand to distinguish them from other gram-negative rods. The glucose testtested positive for both acid and gas, as did the mannitol and arabinosehowever the lactose tested positive for acid but negative for gas. These testswere done using a series of PR broths that had an inverted Durham tube insideto test for gas production, when these tests were compared to our controls wewere able to know whether these organisms had gas production and to see ifthere was any acid production.

 On thesevery same lab days we ran a Methyl red test and a Voges-Proskauer test, whichfor our bacterium tested for negative on both tests. Furthermore, we ran acatalase and oxidase test and the catalase once the hydrogen peroxide was addedit immediately formed bubbles making it a positive test. Yet, for the oxidaseslide test it tested for negative since no color change happened within thefirst 20 minutes.

The next step was to run a nitrates reductase test which forour bacterium was a positive test. Additionally, the next series of test wereHydrolysis test and we tested for starch, urea, casein, gelatinase and lipasepresence. The starch and urea tested negative however the casein and gelatin aswell as the lipid test all tested positive. By now we could assume what ourbacteria might have been. The next couple of biochemical test would havedefined what our bacteria truly was. Here we tested for citrate permease and wegot a positive reaction there, the next test was a Phenylalanine deaminase testand our results were negative as well as our hydrogen sulfide test and indoletest.

By now we could make a presumptive assumption that our bacterium was an Enterobacter bacterium.