Staining Bacteria: Preparing a Smear and Preparing an Unknown Sample

After four days in the incubator, our single colony grew rampant. Look at the bacteria we uncovered!

Here are the results of our pure cultures:
First is the bacteria found on the outside door handle of the bathroom.  The picture on the left is the initial sample, and on the right is the pure culture of bacteria. We chose to isolate the yellow bacteria on the original plate; that is why there is a huge color difference between the two samples.

Second, we have the bacteria that was found on the spout of the drinking fountain.  The results of the pure culture can be observed in the right agar plate.  We had to make a second pure culture of the bacteria today because there were no individual colonies (single bacteria living in isolation on the plate) present.  Although at this time we do not know the exact identity of the bacteria, our professor hypothesized that there may be spores evident upon examination of this bacteria sample.

After viewing the bacteria and making a second pure culture for the latter sample, we practiced a smearing technique for the bacteria.

Preparing a Bacterial Smear:
First, we took a clean slide and put a small drop of distilled water onto the center of the slide. This was most easily done with the help of an inoculating loop.

As usual,  our next step was to light the Bunsen burner -- our main source of sterilization throughout the procedure. With the sterilized loop (which we put through the flame until it glowed red), we touched the pure culture briefly and smeared the small sample of bacteria on the slide with the water.

After the bacteria was sufficiently spread on the slide (thinly and evenly), we waited for the sample to air dry. The sample was dry when an opaque area was seen on the slide.

After our samples air-dried, we had to heat-fix the bacteria onto the slide. In order to do this we had to briefly hold the slide in the flame of the Bunsen burner for three quick motions. It is necessary to heat-fix the bacteria to the slide so the bacteria sticks.  This is needed to view the sample on a microscope.

Next, we had to do the actual staining of the slide!  Now the fun begins! Next, we added a drop of 95% Methanol to the dried smear, which was suspended over the sink on a slide rack. We had to be careful to not stain anything other than the slide with the 95% Methanol because it will permanently stain anything that it comes in contact with. We made sure to cover the entire surface of the dried smear with the 95% Methanol. After, we let it air dry for one minute.

 In order to make sure the entire smear was covered, we had to gently shift the slide in a circular motion.

In order to keep staining to a confined area, we placed the staining slide on a drying rack over the sink.

After waiting one minute, we rinsed the 95% Methanol off of the slide with distilled water. Then, to clean off excess water, we dabbed it dry with Bibulous Paper.

Then we put the slide away for further drying and examination next class after clearly marking it with a China marker.

Preparing Our Unknown Sample:

We have been given a task to identify an unknown sample of bacteria. However, before we can do any identifying we must make a pure culture of the bacteria. At this point, all we know is that our Bacteria is labeled F. "Well, F, we are going to get to know each other really well."

We had to create two slants of "F" using the aseptic technique. As a review of this process, we first sterilized the inoculating loop, and then we carefully uncapped "F" and sterilized the top of the tube. After, we carefully used the inoculating loop to get a small sample of "F" to spread in our slants.

We spread the sample of "F" in the two slants so that it could grow for further examination next class.


After our slants were created, we made sure to sterilize the equipment and put our slants away in the proper incubator. "F" required 37degrees Celsius, "oh picky 'F.'"


What new mysteries will "F" bring us? What will our newly stained slides depict? The microscopic world is unpredictable. Nobody will know until next class. Join us!