Week 4

Week 4, Day 1:
9/17/13

A New Test Tube, A New Chapter

This week, we received a test tube with a new colony of bacteria to work with. Since this bacteria was growing on an agar slant, our first step in identifying the bacteria was to analyze the growth pattern in the test tube. The bacteria we received was growing in what looked like a filiform, or even, pattern.

After observing this growth pattern, we used a sterilized inoculating loop to transfer a small amount of the new bacteria to another test tube. This tube was then placed in the incubator at 37' C to grow over the next few days. 

Using aseptic technique, we then took a loopful of bacteria from the original test tube and mixed it with a drop of distilled water on a new glass slide. We let this mixture air-dry, then passed it through the propane torch three times to heat-fix it, creating a bacterial smear.

Our first step in identifying this bacteria was to determine whether it was gram-positive or gram-negative. To do this, we performed a gram stain on our bacterial smear. We put the slide with the smear on a rack over the sink, covered the smear with crystal violet for 20 seconds, and rinsed the slide with distilled water.

We then covered the smear with Gram's iodine for 1 minute, before rinsing the slide again with distilled water.

After rinsing, we decolorized the smear by dripping 95% ethanol on the slide at a 45' angle until the color from the stain stopped running, then rinsed with distilled water.

We covered the smear with safranin for 1 minute, then rinsed the slide and blotted it with bibulous paper.

By observing the stained smear under the oil immersion lense of our microscope, we saw that our bacteria were gram-negative. Because of their thin peptidoglycan layers, the bacteria had been unable to hold the crystal violet stain, and came out safranin-pink instead.

Week 4, Day 2
9/19/13

After determining last Tuesday that our new bacteria was gram-negative, we spent Thursday's lab preparing negative and capsule stains on both our gram-positive environmental sample bacteria and the gram-negative test tube bacteria we received in class.

To prepare a negative stain on the test tube bacteria, we took two clean slides and placed a small drop of nigrosin at the end of one. Then, using aseptic technique, we used our inoculating loop to transfer a loopful of test tube bacteria to the slide. We then mixed the transferred bacteria with the nigrosin on the slide.

After transferring the bacteria, we took the second (spreader) slide and touched it to the nigrosin/bacteria drop at a 30-45' angle. Once the drop had spread across the length of the spreader slide, we quickly pushed the spreader slide across the first slide, spreading the bacteria/nigrosin mixture across the first slide.

Once this smear had air-dried completely, we examined it under the oil-immersion lense of our microscope. The negative stain allowed us to view the bacteria against a dark background, making it easy to see their shape. This method can also be used to observe bacteria that do not absorb other dyes, or bacteria that are too fragile to undergo heat-fixation when preparing a bacterial smear.

We also repeated this procedure on our environmental sample bacteria. Both bacterial strains appeared to have a spherical shape.

Once we had prepared a negative stain for our environmental and test-tube bacteria samples, we used the negative stains as a foundation for preparing a capsule stain of each type of bacteria. The capsule stain would allow us to view bacterial capsules (the outer coating of a bacteria cell) or slime layers.

To prepare a capsule stain, we took the negative stain slides for the environmental and test-tube bacteria samples, and placed them on a rack over the sink. We covered each slide with safranin for 1 minute, then rinsed off the excess stain with distilled water.

After blotting the slides with bibulous paper, we observed that our bacteria did not have any capsules. However, upon examination, we began to doubt our initial belief that our bacteria was spherical in shape.

We did another bacterial smear and determined that our bacteria was, actually, rod-shaped