Culturing Anaerobic Bacteria!

In a previous test we found out that our bacteria was anaerobic, but we were not yet sure if it was facultative or a strict anaerobe. But we were going to find out.
A strict anaerobic bacteria is one that cannot grow in the presence of oxygen.
To determine this we used the GasPak Anaerobic system. Everyone in the class made a smear plate of their bacteria and put it into the container.

We then put some thermo-packs into the container, and an anaerobic indicator strip so be could see when it turned blue that there was no more oxygen left in the container.
Then we sealed the container with a screw cap, closing it tightly so no oxygen could get in.
We then incubated our bacteria at 37 degrees centegrade, and waited to see what our results might be.

When we took the GasPak out of the incubator and obtained out bacteria samples from it, we found out that our bacteria was not a strict anaerobe, because it did not grow in the container.

We instead determined that our bacteria was facultative anaerobic. 

Just swabbing eachother's noses!!

Swabbing a nose, seems pretty gross right. Especially when it is your own nose. You are totally self conscious about what they are going to find in there.
But for a nursing student, something like this is super exciting!!
So to begin we partnered up with another lab group. We divided our mannitol salt plate into four quadrants, one for each of us. We all got our swabs, in a sterile package so as not to be contaminated by any other bacteria.
In doing this test we were looking for the bacteria Staphylococcus aureus. Now lets swab some noses!!

Because the nose isn't as moist of an environment as our throat we first have to dip the swab in a saline solution so we can pick up the bacteria more easily.
After we dipped the swab in saline, we then put it right inside the nose, but towards the front (not far back) and putting some pressure we circled it around the walls of the nose to ensure we obtained a pure sample.
Then we are spread the swab on our quadrant of the mannitol salt plate, and put it in the incubator, at 37 degrees centigrade, which is our body temperature, and waited to see what results we would obtain.

Here are the results that we obtained!! 

Three out of the four are safe, and the bacteria cultures are not turning a yellowish culture so they are free of Staphylococcus aureus. But one of the nose samples did contain Staphylococcus aureus, we can tell because the bacteria turned a yellowish color in the mannitol salt plate. 

It was really cool to do this test to practice skills that we will be utilizing in the hospital once we are real nurses. 

Our Field Trip to the Water Treatment Plant!

Yay, another microbio field trip!
So for this field trip we got to become a drop of water travelling through the water treatment plant. The process is a lot more extensive than I would have thought. First they must remove all the large waste, this is stuff that we put there from littering or flushing things down the toilet that do not belong there. We learned to never flush any sort of unsafe chemical or medicines or drugs down the toilet, because these can be very hard to remove from the water and can therefore be very harmful to the environment. We also discover that sometimes little animals get caught in this process, we even saw a dead beaver in some of the water storing vats...gross. Anyways then the water goes onto further purification. Then it is put in the vats allowing the different algae and other contaminants to rise to the top, this is then filtered out and the sludge as they call it is used for a different purpose, not merely disposed of. Water treatment plants can sell their sludge to farmers to use as fertilizer. But they first must examine the amounts of microbes present within it. There are different classifications, with a higher rating correlating with less microbes in the sludge. In order to kill some of the thermophile microbes, the sludge must be heated to high temperatures, and we were able to see the furnace that they used to heat this in. Currently they are producing grade 2 sludge, but would like to in the near future make arrangements to make their sludge grade 1.
Before they are ready to dump the water back into the river they add the chemical citrate acid to purify the water even more, killing unwanted microbes, trying to prevent any further contamination of the environment.

Many people left after this, but a few of us stayed to see what took place within the lab. Every day, multiple times a day they must test the water and sludge for multiple different things in order to determine the microbes in them and the quantity of them. They are only allowed to release a certain amount of microbes back into the environment, and are held under strict rules to make sure they do not go over those numbers.

It was a really cool day, and so awesome to be able to learn all the microbiology that goes behind treating water!

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.