Tuesday, October 8th, 2013

*This blog does not reflect Franciscan University in any way*

Dear Blog and Blog Readers,

Today in microbiology lab we checked the all of our tests from the previous class and here is what we found:

Casein Hydrolysis Test

This was our skim milk plate. If you look closely you can see a clear area around the white stripe (which is our bacteria). This indicates a positive test result and that our bacteria secretes caseinase which is a type of protease that digests casein molecules. 

Starch Hydrolysis Test

To test to see if our bacteria was positive or negative for this test we flooded the plate with iodine. This revealed a clear area around the the bacteria growth which is a positive test result. Therefore, along with caseinase, this bacteria also produces amylase that digests starch.

Gelatin Hydrolysis Test

This is what the tube looked like when we removed it from the 25 degree incubator. Afterwards, we placed it in the refrigerator for 15 minutes. The gelatin was still very liquid-y. This indicates that the test is positive. Therefore, our bacteria produces gelatinase which hydrolyzes gelatin. 

 Litmus Milk Test

Our test tube had a small layer of white liquid on top with a hard, purple curd throughout the rest of the tube. The white liquid indicates that a little bit of acid was produced. The purple, hard curd indicates that the litmus was not reduced the the casein was completely digested, confirming our knowledge that the bacteria produced caseinase.

Finally, we innoculated test tubes with sucrose, glucose, lactose and TSI (Triple Sugar Iron) with our bacteria "S" and placed them in the 25 degree celsius incubator.

That's all for now!

Thursday, October 17th, 2013

*This blog does not reflect Franciscan University in any way*

Dear Blog and Blog Readers, 

Today we checked the tests we had inoculated last class: indole, urea, nitrate, methyl-red, citrate. 

Citrate Test

Since the color of the agar slant was Persian Blue this test is positive and the bacteria does utilize 

citrate. 

Urea Test

The tube on the right is an uninoculated tube and the tube on the left is the tube inoculated with S. This test turned out to be partially negative because the color of the inoculated turned slightly but not completely pink. 

Methyl-Red Test

The first step was to pour half of the MV-VP test tube into another test tube so we could do an oxidase test with it. 

Max pouring the broth.

Next in one of the test tubes we dropped 6 drops of methyl red .

Sam dropping Methyl Red into the MV- VP test tube.

Immediately our reaction was as follows:

Right test tube: MV-VP with Methyl-Red

Left test tube: MV-VP without Methyl-Red

This indicated a slightly positive test  and therefore is it evident that S can ferment glucose via mixed-acid fermentation. 

Voges-Proskaur Test

In the other MV-VP test tube we dropped 15 drops of reagent A (alpha-naphthol) and 5 drops of reagent B (KOH).

Max dropping reagent A and B.  We shook the test tube and then let it sit for 30 minutes.

This indicates a positive test because of the red color; therefore, the bacteria does ferment glucose via butanediol.

Nitrate Reduction Test

We dropped 5 drops each of reagent A and B (sulfanic acid and dimelthy-alpha-napthylamine, respectively) in the nitrate broth tube and this was our result. 

This indicates a strongly positive test; therefore, S can reduce nitrate to nitrate ions or nitrogen gas.

Oxidase Test

We took our agar slant test tube labelled T and swabbed some of the growth.

We then dropped a few drops of oxidase reagent on the test tube. The swab turned purple immediately which is indicative of a positive test. 

To make sure we dumped the rest of the reagent in the test tube labelled T.

The bacteria growing there quickly turned purple, confirming our positive result.

Indole Test

Max dropping 5 drops of Kovac's reagent into the Tryptophan broth. 

This test was negative due to a yellow layer on top instead of red. This indicated that the bacteria S does not split tryptophan into indole and pyruvic acid.

Lastly, we observed Rhizopus fungus growing on a sandwich under the microscope.

We also made another streak plate of our bacteria "S" to use next class. 

Tuesday, October 15th, 2013

*This blog does not reflect Franciscan University in any way*

Dear Blog and Blog Readers,

Today in Microbiology Lab we checked what we had previously inoculated test tubes filled with glucose, sucrose, lactose, and TSI with our unknown "S".

Sucrose

The yellow color indicates a positive

Lactose

This is a slight positive test since there is some color change. 

TSI

Since the slant is partly red this indicates that S hydrolyzes glucose to come extent and uses sucrose. Therefore S is preferential for glucose. 

The yellow butt indicates that S produces acid. 

Glucose

The yellow color indicates a positive test; therefore, S digests glucose. 

Then we inoculated tubes with indole, urea, nitrate, methyl-red, citrate, and oxidase.  Come back next time to see the results of these tests!

Thursday, October 3, 2013

*This blog is in no way related to Franciscan University*

Dear Blog and Blog readers,

Today  in lab, we checked our results for our motility test that we completed last lab. We determined that our bacteria is non-motile because there was no movement for the original point of insertion as can be seen from the following image.

Next, we conducted multiple lab procedures beginning with the
litmus milk reaction. This reactions will tell us what our bacteria is producing, whether it be acid, renin, proteolytic enzymes, proteases, or deaminases. We started by inoculating the Litmus milk tube with our unknown bacteria "S2." Then, we placed the tube in the 25 degree celsius incubator until next class.

After we finished with the Litmus milk, we began a test involving Casein, which will tell us if our bacteria is able to hydrolyze casein, which is a major protein in milk. First, we obtained a sample of our unknown bacteria. We then made a short streak of our bacteria on the surface of the Casein.  We placed the plate in the 25 degree incubator until next class.

Making a streak of bacteria on the Casein.

Once we completed preparing the Casein plate, we obtained another loop of bacterium from our unknown sample. Next, we inoculated our gelatin tube with this sample of bacteria in order to determine our bacteria's ability to hydrolyze gelatin. We placed the tube in the 25 degree incubator until next class.

Following our procedure with the gelatin, we made another loop full of bacteria and made a streak on our starch plate. This was conducted in order to determine if out bacteria is able to hydrolyze starch. We placed this plate into the 25 degree incubator and waited until next class.

Finally, we began making our hanging droplet slide . . .


We started with obtaining our bacteria culture broth.

Then, we applied petroleum jelly to the edges of our slide cover from the edges of Sam's hand.

Next, we placed the coverslip jelly side up on a paper towel as we used a flame inoculating loop to place a loop full of bacteria on the coverslip.

Then, we placed the coverslip over the depressed side of the slide. Droplet side down.

We placed the hanging drop slide under the microscope, and we determined that we had non-motile bacteria due to the fact that we could see no movement under the microscope. Our results matched the results of our Gel-motility test which also said that our bacteria was non-motile.

Tuesday, October 1st 2013

*This blog does not reflect Franciscan University in any way*

Dear Blog and Blog Readers, 

Today we prepared an endospore stain of our unknown "S2". The purpose of this was the view bacterial endospores under the microscope and to see the location of them in a cell. 

First, we prepared a fixed smear of the "S2" bacteria. (see previous blog posts). 

Then we placed the slide with the fixed smear on a beaker with boiling water.

We then placed a piece of bibulous paper on top of the slide. 

We then saturated the paper with malachite green

We allowed the stain to sit for 6 minutes while continuously adding stain as it evaporated. 

We then removed the slide with forceps and placed the paper in a biohazard bag. We allowed the slide to cool then rinsed off the slide for 30 seconds with DI water to remove the excess dye.

We then covered the smear with safranin for 90 seconds--the purpose of this was to act as a counterstain so that we would be able to see the endospores inside of the cells.

We then rinsed off the safranin.

Then we blotted the slide with bibulous paper to remove water.

We looked at the slide under the oil immersion microscope and here are the images we saw.

We determined that  we had no spores because  in the cells stained with safranin we saw no malachite green which was supposed to stain the endospores.

We also did two inoculations to determine the motility of the bacteria "S2". 

We labeled a tube with a motility test medium agar with S2, our initials, and the date.

We then sterilized the inoculating needle under a flame. 

We took a small sample of our unknown bacteria. 

Then we inoculated it into the tube by inserting the inoculating needle straight down into it and immediately pulling it straight out. 

We then sterilized the inoculating needle and took another sample of the unknown bacteria. This time we inserted it into a labelled test tube with a broth culture. When we inserted the needle we shook the needle a bit to make sure the bacteria came off of it.

We placed both test tubes in the incubator which the sample was originally grown at--25 degrees Celsius.

If the bacteria in the the motility test tube only grows within the stab, we will be able to determine that it is not motile. If it spreads from the stab we will see that it is motile. We predict that it will not be motile since in the images of it under the microscope we have not seen any flagella. 

We will be able to tell characteristic features of the bacteria from it's growth in a broth culture such as sediment (only growing at the bottom), pellicle (only growing at the top), ring (a ring around the top), or flocculent (speckled throughout). (see citation).

Citation

Lammert, John M. "Characteristic Features of Bacterial Growth in Culture."Techniques in Microbiology: A Student Handbook. San Francisco: Pearson Benjamin Cummings, 2007. 62.