Introduction: The purpose of this experiment is to isolate and differentiate between two bacterial species using Gram's stain, microscope visualization and different biochemical tests. Bacteria can be classified and differentiated based upon different morphological characteristics, staining reaction, nutritional needs, cultural characteristics, physiology, cellular chemistry, and biochemical tests results for specific metabolic end products. Identification of unknown bacteria is useful in many situations. It can determine if bacterial contamination is present in food products, blood or tissue samples, water or cosmetics. Then go on to determine what type of contamination is present and give clues as to what type of steps should be taken to solve the problem.

Identification of unknown bacteria is also used to screen materials for the presence of new antibiotic, vitamin, solvent and enzyme producing organisms. This experiment first isolates the gram positive and gram negative bacteria based upon their ability to grow on phenylethyl alcohol agar and MacConkey agar respectively. MacConkey agar inhibits the growth of gram-positive bacteria because it contains crystal violet. MacConkey agar also differentiated between enteric bacteria based on its ability to ferment lactose. Coliform bacteria will ferment lactose to produce acid which turns the media red while dysentery, typhoid and paratyphoid bacilli do not ferment lactose and therefore there is no color change in the media. The phenylethyl agar is a partial inhibitor of gram-negative bacteria and therefore grows gram-positive bacteria in more abundance.

Next discrete isolated colonies from both dishes are separately Gram stained to verify that the bacteria are indeed gram positive and gram negative for the respective colonies on the appropriate dishes. The gram stain uses crystal violet as the primary stain, then uses gram's iodine as a mordant to increase the absorption of the primary stain. Next a 95% alcohol wash is used as a decolorizing agent and finally safran in is used as a counter stain. If the bacteria has a thick peptidoglycan wall on the outer surface then the crystal violet will become trapped in this matrix and will remain purple from the primary stain and thus will be gram positive. If the bacteria has a lipid layer outside of a thin peptidoglycan layer, then the primary stain will be washed off and the counter stain will be visible as pink and the bacterial species is gram negative.

The samples are then viewed using a light microscope to see the microscopic morphology. At this time stock cultures are made from the same colonies that were Gram stain tested. The experiment then goes through a tree like system of tests, first based on the microscopic shape and gram type of the bacteria. Then the bacterium is tested until it exhibits all of the traits characteristic of only its self.

At this point the identity of the bacteria species can mostly be concluded. Data: Original tube of mixture of gram positive and gram negative bacteria: tube #34 Table #1: Results for the Gram-Positive Bacterial Isolate Morphology Purple cocci bacteria arranged in grape like clusters Catalase test (+) bubble formation, gas formation Mannitol salt agar (+) Grows and turns agar around growth yellow Table#2: Results for the Gram-Negative Isolate: Morphology Pink bacilli bacteria Lactose fermentation test (+) Acid and gas products, turns broth yellow Indole test (-) no rose / pink color change with addition of Barritt's reagent Methyl red test (-) no red color change with the addition of methyl red Urea test (-) no peach / pink color change of media Conclusion: The gram positive bacteria was cocci in shape when viewed with a light microscope so a catalase test was performed. The bacteria was able to break down hydrogen peroxide upon its addition into water and gaseous oxygen which created bubbling and indicated a positive result. A sample was then inoculated on a mannitol salt agar plate. After incubation growth was present and the red media had turned yellow around the growth as a result of high levels of acid production. The data suggests that the gram positive bacteria was Staphylococci aureus because it was gram positive, was catalase positive with the production of O 2, and grew yellow on the selective and differentiating mannitol salt agar plate.

S. aureus can be pathogenic to humans when it produces coagulase that clots the fibrinogen in plasma and isolates it from the host's defenses. It is responsible for diseases such as boils, abscesses, bacteremia, endocarditis, food poisoning, pharyngitis and pneumonia. Typically it grows in large opaque-golden colonies, is cocci in shape, produces acid in a litmus milk test, ferments lactose, dexterose and sucrose, does not produce H 2 S, reduces NO 3, cannot degrade tryptophan into indol, is able to oxidize glucose (methyl red positive), cannot use citrate as a carbon source, cannot degrade urea into ammonia, can break down H 2 O 2 in to water and gaseous oxygen, can break down cytochrome (oxidase positive), cannot hydrolyze starch, and can hydrolyze lipids.

The gram negative bacteria was bacilli in shape when viewed with a light microscope so a lactose test was performed. The bacteria was able to use lactose as a carbon source and both acid (a yellow color change) and gas were produced in the Durham tube. A sample was then inoculated on SIM agar and upon the addition if Kovac's reagent no red color change occurred showing the inability to degrade tryptophan into indol. Next the methyl red test was performed which showed the bacteria unable to oxidize glucose as no color change occurred because no acid end products were present.

Lastly a urea test was performed to test for the bacteria's ability to breakdown urea into ammonia via the enzyme urease. Since no dark peach / pink color change occurred the urea test was negative. The data suggests that the gram negative bacteria is Enterobacter aerogenes because it was gram negative (because it grew on MacConkey agar and was pink when Gram stained), was lactose positive with the production of acid and gas, gave negative results to both the indol and the Methyl Red tests by producing no red color and gave negative results to the urea test by producing no peach or pink color change. E.

aerogenes is a coliform bacteria that is part of the natural flora of the human intestine. It is a facultative anaerobe and is non spore forming. Typically it grows in large thick glistening white colonies, is a rod in shape, produces acid in a litmus milk test, ferments lactose, dexterose and sucrose while producing gas, does not produce H 2 S, reduces NO 3, cannot degrade tryptophan into indol, is unable to oxidize glucose (methyl red negative), can use citrate as a carbon source, cannot degrade urea into ammonia, can break down H 2 O 2 in to water and gaseous oxygen, cannot break down cytochrome (oxidase negative), and cannot hydrolyze starch or lipids. This experiment incorporated a lot of the different methods of isolation and differentiation that we have learned through out this semester.

This lab proves that bacteria can be isolated and identified upon their different characteristics and enzymatic abilities and also that no two species of bacteria are identical.