Day 2: Streaks and Stains

We arrived bright and early (although not very awake) for our second day of lab, still unsure of what we should expect. Once we made it in the door (after washing our hands of course), we examined the bacteria samples from yesterday's hand washing experiment. The results were surprising: our 'after washing' fingerprints had significantly more bacteria growth than the 'before washing' prints.

From this, we concluded that the Steubenville water and/or the paper towels we used were harboring bacteria. As a nurse, it will be important to recognize the presence of bacteria in the environment. Washing hands frequently is incredibly important, but hygienic hand dryers and sterile gloves are good extra steps to take, especially around patients with fragile immune systems.

Stocks and Streak Plates

Our next adventure started with more aseptic technique practice. Once we proved that we had this down, we graduated to real samples (materials: bunsen burner, inoculating loop, 2 agar slants, 1 nutrient broth tube, 2 agar plates, agar slant with unknown bacteria sample, broth tube with liquid sample, pipettor, glass spreader, ethanol, test tube rack). 

We learned about making stock cultures, particularly working and reserve stocks for short-term storage, and then got to try it for ourselves. Procedure: We transferred our sample to a nutrient broth test tube and to two agar slants for our working and reserve stocks using the aseptic technique that we learned yesterday. These are now incubating so we can observe bacteria growth later.

We also discussed streak plates and spread plates and tried our hand at these as well. Procedure: Our unknown sample was transferred to a streak plate by streaking the sample across all four quadrants of the agar plate with an inoculating loop, which was flamed for sterilization between quadrants. Spread plates were made from liquid samples transferred by fancy schmancy pipettors and spread across the agar by sterilized glass rods. Both of these plates are incubating as well.  

Dr. P also made sure to teach us not to dip our fingers in ethanol and then light them on fire.

Learning how to isolate individual bacterium on streak plates is not necessarily something we would do as practicing nurses, but if we go into disease research later on this will be a very valuable skill. However, the fact that there can be many different strands of bacteria present in such small samples is a good thing to be aware of as a nurse. Also, knowing the specific, isolated bacteria that is causing a patient's sickness will help in devising treatment plans.

Preparing and Viewing Gram Stains

After a short lunch break, we came back to the lab for a lecture about different types of microbes. We learned that viruses (nanometers) are smaller than bacteria (micrometers), which are smaller than fungi and protozoa (millimeters). Next we discussed different types of prokaryotic cells (bacteria), differentiating between gram-positive and gram-negative cell walls.  After going over this a few times, we were ready to make and stain bacterial smears to find out whether our samples were more gram-positive or gram-negative.

Materials: clean slide, china marker, bacteria sample, inoculating loop, wash bottle with distilled water, bunsen burner, slide clamp, staining rack, crystal violet stain, Gram's iodine, 95% ethanol, safranin stain, immersion oil, microscope, bibulous paper

Procedure: we prepared slides of our bacteria sample by putting a small drop of distilled water on our labeled slide and using the inoculating loop that we touched to our sample to smear the water and sample around the slide. Next, we used heat from the bunsen burner to fix our slide so that the stains wouldn't wash it away. Once it cooled, we were ready to begin staining. The first step in this process was to cover the smear with crystal violet. After 20 seconds, we rinsed the slide with water before covering the smear with Gram's iodine. This stayed on for 1 minute before rinsing. Next, 95% ethanol was added to the slide drop by drop to remove excess stain. The slide was rinsed again and covered with safranin, which was rinsed off after 1 minute.

Results: After drying our slide, we viewed it under the microscope and got to use the oil immersion lens for the first time. We discovered that the large majority of the remaining stain was purple, which means that [conclusion:] most of the bacteria in our sample have gram-positive cell walls (gram-negative cell walls would be stained orange from the second stain - safranin). We could also see that our bacteria were round and found mostly in clusters.

Gram stains are something we might encounter as nurses when dealing with patients who have respiratory infections. Doctors might order a Sputum Gram Stain, which is a laboratory test done to help understand if a bacterial infection of the respiratory system is present. This test is often performed if pneumonia is suspected because the rapid test results allow for speedy treatment. This test detects the thickness of bacterial cells walls, and knowing the thickness helps to identify the type of bacteria and how to treat it. Gram stains are also used to diagnose meningitis from samples of cerebrospinal fluid, which is an extremely important test to catch a life-threatening disease.

Environmental Samples, Round 2

Since our first environmental sample, from the cafeteria table, did not yield any impressive or helpful results, we wrapped up our second day in the lab with a trip outside to gather another round of samples.  This time, we chose the surface of a fountain knowing that it is covered in bacteria and hoping that some of them will grow in our agar plate. We swabbed the wet surface with a sterile cotton swab and applied the sample to an agar plate, which is now incubating. Hopefully tomorrow will bring more exciting results.