"F" revealed!

Today was an exciting day in lab! After extensive testing, we finally realized the identity of "F"!

Urea Hydrolysis Test: 

Recall from the previous blog that this broth was a orange/yellow color. As evident now, this broth is now a bright magenta! This color change means that our test was positive for the presence of the enzyme urease. The color change occurred because of the ammonia product in result of the reaction.

Indole (Tryptophan Degradation) Test


After the incubation period, we added 5 drops of Kovac's Reagent to the broth.

If our bacteria was able to break down the amino acid Tryptophan, indole would be one of the three biproducts. To see if indole is present it is necessary to add Kovac's Reagent. If a red layer quickly appears on the top of the tube, indole is present. Since this did not happen in our broth, "F" tested negative for the enzyme Tryptophanase and cannot break down this amino acid.

Nitrate Reduction Test


After incubation we added 5 drops of sulfanilic acid and 5 drops of dimethyl-α-napthylamine. 

Since there was a red color change within the broth, nitrate has been reduced to nitrite. This proves that our bacteria has the enzyme nitrate reductase. Even though our bacteria tested positive, we were instructed to add Zinc to the broth, for extra assurance. 

Since our broth continued to deepen in red color, this helped to prove that our bacteria had the presence of nitrite. If there was no color change, ammonia would have been present. 


Citrate Utilization Test

Recall from the last blog that the agar in this tube was green prior to incubation. Since there was no color change our bacteria does not have citrate permease and therefore cannot utilize citrate as a source of carbon and energy. 


Oxidase Test

This test is designed to determine if a certain bacteria has the enzyme cytochrome oxidase. Cytochrome oxidase collects electrons at the end of the ETC in aerobic respiration. When adding a reagent known as N, N, N, N tetramethyl-p-phenylenediamine, cytochrome oxidase is detected by a color change. If a deep purple color is present at the end of the reaction within 10 seconds, the test is positive. If it is positive after this it is due to the oxygen in the air. 


As seen above, our bacteria tested negative since it had no color change. 


Antibiotic Spread Plate: 

Although it was difficult to see growth, there is some bacterial growth underneath the center antibiotic disk. Since there was no growth throughout the entire plate, our bacteria proved to be sensitive to a wide variety of antibiotics including Penicillin, Vancomycin, Novabiocin, Tetracycline, and Chloramphenical. It was not completely sensitive but had an intermediate sensitivity to Erythromycin and Neomycin. Sensitivity was determined by measuring the diameter of the area without growth surrounding a particular antibiotic disk. This area is called "Zone of Growth Inhibition". This area differs for each particular antibiotic and is calculated in millimeters. 


Laboratory Differentiation of Streptococci

There are many different groups of streptococci bacteria. Group A consists of S. pyogenes. As seen above this bacteria is extremely sensitive to many antibiotics including Penicillin, Bacitracin, and Vancomycin. However, it is not sensitive to Neomycin. We know it is sensitive to these antibiotics due to the bright red color surrounding the three antibiotic disks. This bright red color indicates that the red blood cells in the blood agar were not lysed as a result of S. pyogenes. Next time you get Strep have no fear! It is essentially sensitive to every antibiotic!


and now a drumroll please...the moment you have all been waiting for... THE IDENTITY OF "F"!


To determine "F" we had to go back to the beginning: the staining process. Recall that during the gram staining procedure "F" took a purple cocci appearance. Because of this we know that "F" is both gram positive and cocci. After we referred to the catalase test, where "F" tested positive for the enzyme catalase. Because of this we know that "F" cannot be of the Streptococcus genus. Catalase is found in two main cocci genuses: Staphylococcus and Micrococcus. Because of this, we referred to the Mannitol Salt Agar test. Our bacteria tested negative for fermentation during this test. Recall that we previously noticed that there was no green sheen surrounding our bacteria when it was growing on the salt agar, proving that it could not ferment Mannitol. This negative result provoked us to believe there were then 2 possible identities. We now know that our "F" is Staphylococcus epidermidis  or Staphylococcus saprophyticus. Because we did not complete a Fructose test during our experiments, we are unable to identify "F" any further. 


It's been grand, but don't leave us just yet! Join us next week for an exciting new Environmental Project! A true scavenger hunt of the world unseen!