Hmmm....

The results of all our tests seem a little strange considering that we believed that our bacteria was gram-negative.

For our blood agar plate, we determined that our bacteria do not secrete enzymes that completely dismantle the red blood cells, also known as gamma-hemolysis.

For the Eosin Methylene Blue (EMB) plate, we found that since our bacteria is gram-negative, our bacteria grew a little bit.

For our Mannitol Salt Agar, we found out that our bacteria cannot live in a high salt environment since our bacteria did not grow on the agar plate. This means that our bacteria are not halophile. 

For our MacConkey Agar plate, we determined that our bacteria can not ferment lactose because they are not able to grow on this medium. 

For the Phenylethyl Alcohol (PEA) Agar, we found that our bacteria grew a little bit on the agar plate, which was a little weird considering we thought our bacteria are gram-negative and PEA inhibits the growth of gram-negative bacteria.

In our thioglycollate broth tube, our bacteria grew in a pretty cool way where it grew right in the middle of where oxygen is not present in the tube. Our bacteria are anaerobic but it is amazing to see our bacteria floating in the middle of our tube!

For our DNA Hydrolysis test, we needed to flood the plate with 1N HCl (hydrochloric acid) to determine if our bacteria are able to hydrolyze (digest) DNA.

After adding 1N HCl since there is no clear area around our bacteria growth we determined that our bacteria are not able to hydrolyze (digest) DNA.

Conclusion: 

After we looked over all of the results, we found that some of the results came out weird considering that we thought our bacteria was gram-negative. So our professor told us to do another gram-stain of our bacteria. 

After doing another gram-stain of our bacteria, we found that our bacteria has both gram-negative and gram-positive stains (as shown above). We showed the gram-stain to our professor and he determined that our bacteria are more gram-positive than gram-negative BUT! our bacteria are able to take on both stains, which makes it gram-variable also. Therefore, our results were a little weird because our bacteria are gram-positive to gram-variable. Our bacteria seems to not like to make up it's mind! 

Test upon test upon test!!!

We did so many more tests in lab!

These experiments included a Catalase test, culturing Anaerobic bacteria, an DNA Hydrolysis test and seeing how our bacteria grew on a blood agar plate, an Eosin Methylene Blue (EMB) Agar, a Mannitol Salt Agar, a MacConkey Agar, a Phenylethyl Alcohol (PEA) Agar.

First our professor had us do a streak plate of our bacteria on a nutrient agar plate. At first we did not know what to do with it but then we found out that we would do a catalase test with that streak of bacteria. With the catalase test, we added a few drops of 3% of hydrogen peroxide to our streak of bacteria. It was really cool to see that our bacteria use the enzyme catalase to quickly break down H2O2 into water and O2 (as shown below)

We then obtained a thioglycollate broth to inoculate with our bacteria.

By inoculating a thioglycollate broth we will be able to cultivate bacteria that cannot survive in the presence of oxygen.

Next we used the aseptic technique to inoculate all of these agar plates with an inoculating loop full of our bacteria. We had a little bit of fun by writing our own initials or the initials of our school on the agar plates.

We first inoculated our blood agar plate.

Now we just have to incubate the plate for two days and see if our bacteria are able to lyse red blood cells.

Now onto the Eosin Methylene Blue (EMB) Agar plate. We again did the aseptic technique with an inoculating loop full of bacteria.

We then put the EMB plate into the incubator for two days and after two day we will see if our bacteria are gram-negative enteric bacilli.

Next is the Mannitol Salt Agar.

For this test we will be able to isolate our bacteria based on their salt tolerance and differentiate among these isolates for mannitol fermentation.

Then we inoculated a MacConkey Agar.

If you can see we had a little by putting the initials of our school on the agar plates. Anyways for this test we are able to detect if our bacteria are gram-negative enteric bacilli, based on their ability to grow on the medium and to ferment lactose.

Our next agar plate that we inoculated was a Phenylethyl Alcohol (PEA) Agar.

For this test we will be able to isolate gram-positive bacteria if our bacteria has a mixture of gram-positive and gram-negative bacteria.

Finally we inoculated a DNA agar plate for a DNA Hydrolysis test.

For this test we will be able to see if our bacteria are able to hydrolyze (digest) DNA.

We incubated all of these plates and broth tube at 37 degrees Celsius and we will see what our bacteria can and cannot do!

Urea Hydrolysis Test

For the Urea Hydrolysis test, we can determine the ability of our bacteria to hydrolyze urea.

We obtained a urea medium tube and used the aseptic technique to inoculate the urea medium tube with a loopful of bacteria from our agar slant culture





















We then incubated the inoculated tube at 35 degrees Celsius for about two days.

Upon reviewing the results we found out that our bacteria did not utilize urea. 

Urea is excreted in urine to eliminate excess nitrogen from the body. Bacteria who do utilize it contain the enzyme urease to quickly degrade urea into carbon dioxide and ammonia. The presence of the carbon dioxide and ammonia make the medium more alkaline, causing it to turn more red. 

Since our's had no appearance of reddish or pinkish color we could determine that it does not utilize urea.  

Citrate Utilization Test

The Citrate Utilization test determines if our bacteria can utilize citrate as its sole source of carbon and energy.

First we obtained a Simmons citrate slant tube and used an aseptic technique to inoculate the agar slant with a loopful of bacteria from our agar slant culture.





















We then incubated the inoculated slant at 35 degrees Celsius for about 2 days.
When we reviewed the results, we found out that out test was negative. Meaning that our bacteria does not utilize nitrate.
The color of the agar slant remained green instead of changing a bluish color which would have indicated that our bacteria utilized nitrate.
We repeated this test again later when trying to determine our bacteria, but we still obtained the same results.

Indole (Tryptophan Degradation) Test

In the Indole test, we can determine the ability of our bacteria to split the amino acid tryptophan into indole and pyruvic acid.

We first used the aseptic technique to inoculate the tryptone broth tube with a loopful of bacteria from our agar slant culture. 

Then we incubated the inoculated tube at 35 degrees Celsius for 2 days. 

When we came back after two days we added 5 drops of Kovac's reagent to the inoculated tube.

As you can see there is a darker yellow that has formed on top of the lighter yellow broth. This means that the test came out negative. Therefore, our bacteria are not able to split the amino acid tryptophan into indole and pyruvic acid.

Nitrate Reduction Test

The Nitrate Reduction test determines if our bacteria is able to reduce nitrate ions to either nitrite ions or nitrogen gas.

We first obtained a nitrate broth tube and used the aseptic technique to inoculate the nitrate broth tube with a loopful of bacteria from our agar slant culture.

We then incubated the inoculated tube at 35 degrees Celsius for 2 days.

After two days, we added 5 drops of reagent A (sulfanilic acid) and 5 drops of reagent B (dimethyl-alpha-naphthylamine) to our inoculated tube. We made sure to wear gloves when we added the nitrate reagents.

After one to two minutes, we did not see a color change to pink or red color which means that we now need to add a small amount of powdered zinc to the tube.

After about 10 minutes we saw a small color change to our broth. This means that our bacteria are not able to reduce nitrate ions to either nitrite ions or to nitrogen gas.

Methyl Red Test and Voges-Proskauer Test

The Methyl Red test determines the ability of some bacteria to ferment glucose due to mixed-acid fermentation. While the Voges-Proskauer test determines the ability of some bacteria to ferment glucose via butanediol fermentation.

First we used the aseptic technique to inoculate the MR-VP (methyl red/Voges-Proskauer) broth tube with a loopful of bacteria from our agar slant culture.

Then we incubated the inoculated tube at 35 degrees Celsius for two days. 

After two days we came back to lab and divided the MR-VP broth into two tubes so that we could use the broth to use for another test. For one of the tubes we added 6 drops of the methyl red to the tube. We then gently swirled the tube to mix the broth culture and the pH indicator

After we mixed the broth culture and the pH indicator we read the reaction immediately and we found that our test was positive.

Then we took the other tube of the MR-VP broth and added 15 drops of Barritt's reagent A (alpha-naphthol) and 5 drops of Barritt's reagnet B (KOH) to the tube.






















We then tapped the bottom of the tube vigorously so that the oxygen in the air aerates the medium.

Afterwards we put the tube back in our rack for about 30 minutes. After 30 mines we read our results and found that our bacteria was positive which means that our bacteria used the butanediol fermentation pathway.