Through the weeks of our Mircrobiology Lab we conducted many many tests to determine our bacteria. Finally, with all our results in hand, we followed a flow chart to finally discover the name of this elusive bacteria.  And by the end we discovered that it was Micrococcus Roseus.


Micrococcus roseus is a gram positive bacterial cell that grows in the tetrad arrangement. The normal habitat for this Micrococcus species is skin, soil, and water.  It derives its name from the carotenoid pigment that it secretes. Isolated colonies on a TSA plate are circular, 1.0-1.5μg in size, slightly convex, smooth, and pink in color. Optimal growth temperatures range from 25-35 degrees Celsius. Micrococcus roseus is a strictly aerobic organism.


And thus we come to the end of our journey, and have learned, through much patience and dedication, the identity of our bacteria. Nice to meet you Micrococcus Roseus.

The 7th week of lab we continued to come closer to determining what our bacteria was.

We performed the Starch Hydrolysis Test next. This test is to distinguuish among bacteria in terms of their ability to hydrolyze (digest) starch.
     Plants store clucose as bery large molecules called starch. Starch is made up of amylose, a long unvranched bolymer of several hundred glucose subunits, and amylopectin, a branched polymer. When plants die, bacteria in the soil digest the starch in the plant cells. The bacteria use an extracellular enzyme called amylase to hydrolyze the bonds that link the glucose subunits. The products glucose and maltose are transported across the bacterial plasma membrane, where they are used for energy and construction of other biomolecules.
     Starch in a medium is detected by adding an iodide/iodine solution. When iodine reacts with starch, a purple-black color develops. If bacteria growing on starch agar produce amylase, all of the starch around the growth will be consumed after incubation. When iodine is added, no color change around the bacterial growth means that starch is no longer present. This is a positive test for starch hydrolysis.

Our result was negative, as evidenced by the lack of a clear zone around the bacteria.

Next we performed the Casein Hydrolysis Test to distinguish among bacteria in terms of their ability to hydrolyze casein, the major protein in milk.
     Many bacteria secrete enzymes to hydrolyze proteins in their immediate surroundings. The resulting amino acids and peptides are then transported across cell membranes where they are used to construct bacterial proteins, to serve as carbon and nitrogen sources, and to serve as an everygy source.
     Casein, the predominant protein in milkm can be used as a substrate to assess the production of proteinases by certain bacteria. When mixed with agar, casein forms a white colloid with calcium ions. When bacteria secrete caseinase, a specific protease, casein molecules are digested an the area around the bacterial growth becomes clear. This clear zone is a positive test. If the bacteria do not secrete caseinase, the medium remains white around the bacterial growth.

Our bacteria tested negative to this test.

Next was the Fat (Triglyceride) Hydrolysis Test. The purpose of this test was to determine the ability of our bacteria to hydrolyse a tryglyceride, a type of lipid.
     The test medium for triglyceride hydrolysis is tributyrin agar. Tributyrin is an oily triglyceride that is emulsified with melted agar. The cooled medium appears cloudy. When bacteria growing on the medium secrete lipases that hydrolyze tributyrin, a clear zone appears around the groth. THis represents a positive test for lipid hydrolysis.

Our bacteria tested negative, as evidenced by the lack of a clear zone.

Next we performed the Gelatin Hydrolysis Test. This was to determine if our bacteria could hydrolyze gelatin.
     Our bacteria tested negative, the gelatin was still solid, with no liquid.
Our bacteria also tested negative to the Triple Sugar Iron Agar Test.
It tested negative to the Litmus Milk Reactions test, as evidenced by a lack of color change.
It tested negative to the Methyl Red Test: It was clear, pale, no color.
In the Fermentation of Carbohydrates test: it tested negative to the Sucrose test, negative to the Lactose test, and Positive to the Glucose test.
It also tested negative to the Voges-Proskauer Test (Butanediol Fermentation)

Next, was the Urea Test to which our bacteria tested positive; meaning it is able to hydrolyze urea.

Next our bactera tested negative to the Indole (Tryptophan Degradation) Test. Which means that our bacteria does not use tryptophan as a source of energy. 

It also tested negative to the Citrate Test, it does not utilize citrate.

Finally, we determined through the Kirby-Bauer Technique, which is used to determine the sensitivity of our bacteria to several antibacterial medicines.  Our bacteria was determined to be sensitive to all of them.

     As our 6th week of lab rolled around, we were getting closer and closer to being able to identify this unknown bacteria. Our 6th week focused on discovering our bacteria's oxygen requirements. This week we also said a fond farewell to our environmental bacteria. It had served its purpose in giving us another type of bacteria to experiment our staining skills with, but now it was time to focus on our unknown.

     First, we did the Nitrate Reduction Test. The purpose of this test was to determine if our bacteria was able to reduce nitrate ions to either nitrite ions or to nitrogen gas.
     In anaerobic respiration, bacteria add electrons that have been passed along the electron transport chain to an inorganic substance that is not oxygen. One such final electron acceptor is the nitrate ion. The enzyme complex nitrate reductase facilitates this reduction of nitrate ion. For some bacteria to do this, nitrate ions are reduced to nitrite ions.
     Other bacteria that use nitrate reduction during anaerobic conditions for energy production are able to reduce nitrate completely to molecular nitrogen. This is called denitrification.
     To detect nitrite ions that are products of nitrate reduction by bacteria inoculated in nitrate broth, two reagents are added: sulfanilic acid and dimethyl-1-naphthylamine. If nitrite is present, the medium turns pink or red. This is a positive test for nitrate reduction.
     But the absence of a color change cannot be interpreted as a negative test. Either the nitrate ions were not reduced to nitrite ions (a true negative test) or the nitrate ions were completely reduced to molecular nitrogen (a positive test). To distinguish between these two possibilities, a small amount of zinc is added to the tube, which already contains the two reagents. Zinc will reduce nitrate ions to nitrite ions. If the bacteria did not reduce nitrate ions, zinc will do this. Therefore the appearance of a pink or red color after addition of zinc is interpreted as a negative result. No color means the test is positive for nitrate reduction. No nitrate ions remain in the brother because they were completely reduced to molecular nitrogen.

Our unknown bacteria tested positive in the first step; turning a red/pink color. Therefore, nitrite ions are present, and our bacteria can reduce nitrate ions to nitrite ions.

     Next, we preformed the Oxidase Test. This test is to determine if bacteria have cytochrome oxidase, a participant in electron transport during respiration. When cytochrome oxidase adds electrons to the oxidase reagent, the reduced for turns dark blue to purple within seconds.

As you can see, our bacteria tested positive.

     Next we performed the Catalase Test. The purpose of this test was to detect the presence of catalase, an enzyme that degrades hydrogen peroxide.
     In aerobic respiration, hydrogen peroxide is a reactive intermediate that forms as oxygen is reduced. H2O2 damages DNA and cell membranes and alters the active sites of some enzymes. Bacteria that tolerate oxygen or require the gas for metabolism use the enzyme catalase to quickly break down H2O2 into water and O2.
     When drops of a dilute solution of H2O2 are added onto bacteria containing catalase, bubbles of O2 rapidly appear. These bubbles denote a positive test.

Our bacteria tested positive. 

We also discovered our bacteria to be obligate aerobic. 

During our 5th week of lab we learned about three more stains: the acid fast stain, endospore stain, and capsule stain.
Since our unknown was gram-positive, we could forsee what the results of the acid fast staining would be, but we did the test anyways for practice.
The purpose of this staining is to distinguish between gram positive and gram negative bacteria based on the lipid content in their cell walls: acid fast results in red colored stain, and non-acid fast results in a blue stain.
If done correctly, the staining should result in gram-positive being blue, and gram negative being red.

Gram negative bacteria contain a waxy lipid, mycolic acid, in there cell wall.  This lipid makes the cells more durable.  Acid fast cell walls are so durable that the stain (carbol fuschin) must be driven into the cells with heat.  The cells are then decolorized with acid-alcohol, all other cells will decolorize with this strong solvent, but acid fast bacteria will not.  Other cells are then counterstained with methylene blue. Thus the acid fast are left stained red by the carbol fuschin. 

This is the result of acid fast staining on our unknown, which we already knew to be gram positive, so this result was appropriate.

This is the result of the acid fast stain on our environmental bacteria which we knew to be gram negative, so this result too was appropriate. 

Next the endospore stain of our unknown revealed that our bacteria did not have endospores.

Finally, we did a capsule stain of our unknown and environmental bacteria. 

The capsule stain  selectively stains external capsules surrounding bacterial cells.  First, we prepared a smear of the bacteria in nigrosin, making a negative stain.  After allowing the smear to dry we covered it with safranin, and after letting it set, washed it off with water, blotted it dry and examined our slide.

Our result was that neither the unknown nor the environmental samples had capsules.

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