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.