Last post I forgot to mention that Dr. P. gave us a bacteria and had us spread it all over a petri dish. Then he gave us a virus and we wrote our initials in the plate. Today we found this:

So the clear lines in the bacteria is where the virus infected it. This virus is a bacteriophage.

So now back to our unknown H. So we had to redo our acid-fast stain because of missing a step. The results were the same though. It was negative meaning that our bacteria is not a mycobacteria.

The results

Next we did did the endospore stain. This will show us if a few or if many of our bacterial cells have spores.

Again we heated the slide and added stain to it for five minutes then removed. This stain was malachite green.

Once the remaining stain had been washed off a we used safranin and stained it. This time just over the sink.

That was a pretty simple process compared to others don't you think :) 

Results time!

All of the green portions are the spores so our bacteria has quite a few. I think this was one of the coolest pictures yet.

Now it's the end of the week so I'll recap the facts we have on our unknown H bacteria.

1. It is gram positive
2. it has a capsule
3. is not a mycobacteria because of the negative acid-fast stain
4. It has quite a few spores

Well, That all we know so we will have to continue our investigation next week. See you then!

Mycobacteria or Microbacteria

So its the start of week 5 here and we are on the second week of tests on our unknown bacteria H. This is where we stand. We know that our bacteria is gram positive and that it has endospores. Today we tested to see if it is a mycobacteria which is a much smaller bacteria. To find this out we used an acid fast stain. Here's how we did it.

First we started with fixing the bacteria on the slide so that we didn't lose it during the staining process. Then we put the slide over a beaker of boiling water. A small piece of paper was put on the sample and saturated with Ziehl-Neelsen carbolfuchsin.

The fixed sample

First stain

Once that stain had been on there for 4 minutes we removed the slide from over the beaker, through away the paper, and let the slide cool. Then after rinsing the excess stain off, we decolorized it with acid alcohol and rinsed it again immediately. Its not as interesting to read about but it was really fun and cool in lab I promise!!

Acid!! 

Finally the last stain methylene blue is added.

The action shot

methylene blue

Now for the best part. The results!! This is what it looked like:

Now, after lab I realized that we had missed a step in the staining process. While the slide was over the beaker we were suppose to continue to add the stain so that the paper never dried out. Well.....we didn't do that. So even though these results look to be negative for mycobacteria, we need to do it again just to make sure. 

Day 8: Negative Stain

Thursday, September 19

Went on a much needed coffee break! :) 

Today we completed a Negative stain of unknown bacteria H.

 It was cool to see the bacteria against the dark contrast of the negative stain. 

 We looked at our unknown bacteria under the microscope and then noticed its shape and size.

We determined under the oil immersion lens that the bacteria appeared tube- like.

Day 7: Staining the Unknown

Today we made a new sample of our unknown bacteria. We stored our bacteria at 25*C. 

Tuesday, September 17

We looked at our unknown bacteria and determined that it had a filiform shape! We then proceeded to do a gram stain to our unknown H bacteria. After completing the stain, we were able to determine that our unknown bacteria is gram positive. It looks pretty cool! 

Second Try

After all our work on Tuesday, Dr. P. thought we should do the staining again and maybe get a clearer result. We ran out of time though, so today we tried it again.

The culture

Second Try: Hope it works!!

This time we flew through the staining process in under 10 minutes.

Staining

Our results were great!! The stain was really good and the sample was very clear. Dr. P. even called the stain beautiful! Our bacteria we thought was gram-positive on our first sample, but today's test showed us clearly that it is gram-negative. 

First Try on Tuesday

Second Try: Today

After everyone was done, Dr. P. gave us each different samples of bacteria to grow for next week. He knows what each bacteria is, but our job this semester is that we are going to be testing it in all different ways to find out what it is. Then for the last 45 minutes we started watching the movie Contagion. I am now officially freaked out. That movie has now made me slightly paranoid about germs. I hope this paranoia won't last to long. 

Gram-Positive or Gram-negative? That is the Question

So it is the start of another week and we started by checking on the swab plate we made last Thursday.

Our culture

Today we did a staining process which involved a lot more steps and a three different stains. Before the we start staining, we have to fix the sample again just like last week. So we started with a water droplet. Then we take a small sample of bacteria, place it in the water on the slide, and spread it out over a good portion of the slide so as to get a very thin layer.

Spreading the bacteria

Once this air dries we seal the sample to the slide by heat. Now let the staining begin!

Ready for some color!

Well, that was a bit to much color

I caused a bit of a mess right at the start. One of the stains I was opening splashed everywhere and all over one of my hands! With lots of rubbing alcohol and lots of rubbing I managed to have only a few faint purple spots left. The table however was not as lucky. Anyway, back to the staining process. First we used crystal violet stain, covering the sample for 20 seconds. Using water (always deionized) Jessica washed off the stain and placed it back on the rack for the next color.

Violet stain after rinsing

 Next was Gram's iodine which has a yellowish color to it. This one sat for a minute. (The excitement is building!)

Gram iodine stain

Again, this was rinsed, but in between that and the last staining we decolorized the slide using 95% ethanol. This was rinsed and finally the last stain, safranin, was applied for a minute

Finally that last stain!!

The final rinse

Now we can see if everything we did was correct! This is what we saw:

The majority of the sample was gram-negative and it also contained many endospores. We may have also over heated it, so in Thursday we are going to redo the staining and see what results we get. See you then!!

Day 4: Simple Staining!

Thursday, September 4, 2013

Today we practiced simple staining of bacteria. 

We gathered our pure culture from the incubator and then proceeded to practice the simple staining. We used crystal violet as our stain. We covered our bacteria with the violet stain and then rinsed the slide with DI water. We then blotted it with the bibulous paper. 

We took pictures of our bacteria, and little purple bacteria showed up! 

So precious, look at how cool they are!! That’s all for now!

More Growing

Tuesday, September 3, 2013

Today in lab we looked at our cultures that we collected the previous Thursday. Our sample from the river rock grew a lot of tan and bright orange bacteria. 

We proceeded to take a sample of the culture and spread it into a new dish. This allowed for a pure sample to be collected. We spread the bacteria in each quadrant of the dish, and sterilized the hook after each spread.

After this was completed we labeled our sample and put it in the incubator. We stored it at air temperature. Today we also looked at the bacteria under the microscope and took pictures of the bacteria.

Afterwards, we put our old samples into the refrigerator to preserve our bacteria. We then proceeded to discuss Canvas. That’s all for now!