Day 4: Results and Antibiotics

Inoculation Results

From the moment we made it into the lab today, we were kept plenty busy checking for and interpreting the results of yesterday's many inoculations. Some of these required a few more experimental steps to really see results, but soon we had a lot more information about our unknown bacteria.

For the starch test, which had already been incubated, we flooded the starch agar plate with Gram's iodine. Since the iodine remains brownish-orange on top of areas of the plate that contain starch, a clear area around the squiggle of bacteria growth would indicated that the bacteria had catabolized the starch in that area. Our plate didn't have this clear area, so this was a negative starch hydrolysis test and we can conclude that our bacteria does not use starch as an energy source.

The casein test uses the same concept of a clear zone around the bacteria growth indicating a positive result - in this case, hydrolysis of casein. Our plate showed negative results, which would mean that our bacteria does not use casein, but we reincubated this plate and will check again tomorrow to be sure.

We checked for triglyceride hydrolysis in the same way. A clear zone would indicate that the bacteria produces lipase to break down lipids, which would be a positive test. Our plate had no clear zone, so we recorded it as a negative test and placed it back in the incubator for another day.

The test for gelatin hydrolysis was slightly more complicated than checking the color of an agar plate. When we removed our gelatin tube from the incubator, it was a liquid rather than a solid. This could either be because our bacteria produced gelatinase (and therefore uses gelatin as an energy source), or because it was at a high temperature and the liquidation was only a state change. To test this, we put our tube in the refrigerator for 15 minutes. If it was still a liquid after this, we could consider it a positive test and conclude that our bacteria produces gelatinase. However, our tube was full of solid gelatin after 15 minutes, so we recorded this as a negative test and returned it to the incubator for another day to be double-checked later.

The durham tubes for carbohydrates checked for two different results. Each one had a special tube inside that tested for the presence of gas bubbles, but none of our sugar tubes produced any gas bubbles. Each durham tube was also colored to act as a pH indicator - a change in color means that acid is produced as a result of the bacteria using the particular sugar that the tube is testing for. Our bacteria tested positive for sucrose, lactose, and glucose use and negative for mannitol.

The MR-VP tube that we inoculated yesterday was split into two separate tests today.  We poured half of the liquid into a clean test tube so we could use one tube for the MR test and the other for the VP.

For the MR (methyl red) test, we added 6 drops of methyl red to our designated MR tube and swirled. The methyl red acts as a pH indicator for the broth - the presence of a red color is a positive test, which means that the bacteria is able to ferment glucose through mixed acid fermentation. This test is significant for healthcare because it recognizes the presence of E. coli. Our MR test was negative, so we can conclude that our bacteria does not use mixed acid fermentation and is not E. coli.

The VP (Voges-Proskauer) test had two major steps: we first added 15 drops of VP reagent A and then 5 drops of VP reagent B to our designated VP tube. We shook the tube to aerate it and then let it sit. If the top had turned pink or red, this would be a positive test and we could conclude that the bacteria used the butanediol fermentation pathway. Our test came back negative (no color change), so our bacteria does not use this pathway.

For the results of the citrate test, we looked for color changes from the slant's original green. Ours was negative (no color change), so we returned it to the incubator for another 24 hours. If it is still negative, we can conclude that citrate is not our bacteria's sole carbon source.

Our next job was to add 10 drops of Kovak's reagent (very carefully!) to our tryptophan tube. Since no red layer appeared after this, we can conclude that our bacteria does not convert tryptophan to indole.

Then came the nitrate test. We added 7 drops of reagent A (sulfanilic acid) and 7 drops of reagent B (dimethyl-alpha-naphthylamine) to our incubated nitrate broth tube. We shook the tube and waited a few minutes to see if a pink or red color would develop. Since no color appeared, this first stage of the test was negative for nitrate reduction (negative for presence of nitrite). This could be either because nitrate was not used or because nitrate was reduced to nitrite which was then reduced further. To test which was the case for our bacteria, we added a small amount of powdered zinc to the test tube. The broth eventually turned red, so this test was negative for nitrate reduction - our bacteria does not use nitrate.

The triple sugar iron test has significance in healthcare because it checks for stomach bacteria. There were no noticeable color differences in our slant after 24 hours of incubation, which suggests a negative test. However, since this test checks for the use of glucose, sucrose, and lactose, all of which the carbohydrate tests showed that our bacteria used, we returned the test tube to the incubator and will check back tomorrow to hopefully find a positive result.

Our urea test was decidedly positive, as evidenced by a beautiful deep pink color. This means that our bacteria uses urea, breaking it down to produce ammonia and carbon dioxide.

The litmus milk test checked to see if our bacteria uses lactose and/or produces gas. The liquid started out purple, but it would turn white if the bacteria oxidized lactose. We didn't observe any color change, so this tube is also incubating until tomorrow. This extended incubation should also tell us if there is any curd formation, which could be due to either acid or renin.

The next group of tests checked for growth in aerobic and anaerobic conditions. We already knew that our bacteria is capable of growing in aerobic conditions, so we wanted to find out if it is facultative (if it can grow under both aerobic and anaerobic conditions), or if it is an obligate aerobe.  Bacterial growth in the GasPak (anaerobic conditions, shown on left) showed that unknown bacteria F is facultative.

Results from our thioglycollate test (right) confirmed this conclusion. We inoculated this tube yesterday by stabbing the broth from top to bottom with an inoculating needle. After incubation, we could see where the bacteria was able to grow. Since ours grew both at the top (where there is oxygen) and at the bottom of the tube (where there is no oxygen), we can further conclude that it is facultative.


To see the results of the oxidase test, we added a few drops of oxidase reagent to a small piece of filter paper. We transferred a visible amount of bacteria from an agar plate to the reagent-infused filter paper using a sterile plastic tip and watched to see if the paper/reagent would change color. Since there was no color change within 10 seconds, the test was negative. We can conclude that our bacteria does not have cytochrome oxidase (which can be used for nitrate reduction when oxygen is not available). It's still safe to say that our bacteria is facultative.

For the catalase test, we added hydrogen peroxide to the inoculated and incubated catalase plate. The quick appearance of bubbles indicated a positive test. We can conclude that our bacteria uses the enzyme catalase to degrade the hydrogen peroxide that results from oxygen reduction.

Our motility stab tube showed that our bacteria is non-motile. We saw bacterial growth along the line of our inoculation stab, but no movement away from the line of inoculation. This is a negative result, but we'll incubate it longer to be sure.

While day-to-day nursing routines aren't likely to involve motility stabs and Kovak's reagent, it will be important to understand that different types of bacteria can be identified by their different, testable properties. Effective treatment will require the recognition that not all bacteria act in the same way.

Differential and Selective Plates

Once we finished going through all the inoculation results from yesterday, we moved on to differential and selective plates. We inoculated blood agar, phenylethyl alcohol agar, MacConkey agar, and mannitol salt plates. These select for salt-loving bacteria and differentiate one from another. We inoculated all of these using aseptic technique and our unknown bacteria and placed them in the incubator until tomorrow.

Antibiotics

Another blood agar plate was used to culture throat swabs. This was an exciting experiment because it's something we will do in healthcare to check for strep throat, and we can even perform this test in the lab now instead of waiting for expensive results from another doctor's office. We used sterile cotton swabs to take a sample from the back of each of our volunteers' throats and applied them to the blood agar plate after marking whose bacteria would be on each side. We added an antibiotic disc to each side of the plate as well. If strep bacteria is present, it will kill the red blood cells in the plate but won't go near the antibiotic disc. This is incubating until tomorrow.

The other lab group did a nasal swab instead of a throat swab. This tested for MRSA, which colonizes in the nasal cavities. Since MRSA is methicillin-resistant, it won't grow around the methicillin antibiotic disc.

Next, we tested a variety of antibiotics against our unknown bacteria sample. We smeared our bacteria all over the surface of two agar plates and divided each plate into four quadrants, labelled 1 through 8, with a china marker. We used sterilized tweezers to place an antibiotic disc in each quadrant:

1. penicillin
2. novobicin
3. neomycin
4. streptomycin
5. tetracycline
6. erythromycin
7. amoxicillin
8. oregano

Since the amoxicillin and oregano were not in disc form, we placed plain paper discs in their quadrants and added 5M of each to their respective disc by pipette. Both plates are incubating until tomorrow, when we will be able to see our bacteria's response to the various antibiotics.

Making Yogurt

Our final adventure for Day 4 was to try our hand at making our own yogurt. Dr. P swore that he knew the most delicious way to do this, so we had to test it out in spite of a little skepticism. To do this, we heated milk and then cooled it to 37 degrees Celsius before adding a small amount of pre-made Greek yogurt and putting the concoction in an incubator overnight (this time it went in a specified yogurt incubator to stay away from all our bacteria).  Our sample of the Greek yogurt was quite sour, so hopes aren't too high, but we'll know more tomorrow...

While we probably won't spend our time as nurses personally making yogurt for our patients, it will be important to know and to tell patients that eating yogurt has many health benefits. The probiotics found in yogurt are important to consume while taking antibiotics so the gastrointestinal tract doesn't get overly irritated. These probiotics also work to prevent yeast infections because they create an acidic environment, and yeast grows well in alkaline conditions.

Day 3: Inoculations!

Results from Day 2

By the time we made it into the lab for day three, yesterday's samples were ready to view. Results varied - our streak plate worked well, and we could see several different colonies (pure cultures), one in each quadrant. The spread plate, though, was less successful. There was too much of the bacteria sample on everyone's plates, so no one could see any individual growth other than a solid layer of bacteria on the agar plate.

We looked at our streak plate under a dissection microscope to determine the colony's characteristics. This investigation showed us that our bacteria are cream and rose-colored in terms of pigmentation and circular in terms of colony form. The colonies were also raised and convex. These characteristics will help us identify our unknown bacteria later in the week.

Our second environmental sample was about as successful as our first. There was no visible bacteria growth, which could indicate a problem with either the swabbing or incubation, since this time we made sure to choose an environment that we knew would have bacteria. We returned the sample to its incubator and will check for growth tomorrow.

Next we looked at the stocks we had prepared, which were more successful overall. Our working and reserve stocks grew bacteria, and we were able to use our working stock throughout the rest of the day for our inoculations. Our broth culture did not appear to yield any bacteria growth, so we prepared another one that is now incubating so we can use it to test our sample's motility tomorrow.

Since our unknown bacteria grew in the slant tubes and agar plates, which are environments with oxygen, we can conclude that our bacteria is either an obligate aerobe or facultative. Further tests will determine if it is also anaerobic. Differentiating between aerobic, anaerobic, and facultative bacteria is important

More Stains

After a short coffee break, we started preparing various stains to further examine our bacteria samples. Materials: working stock with unknown bacteria, inoculating loop, clean slides, bunsen burner and lighter, slide tongs, bibulous paper, wash bottle with distilled water, staining rack, sink, stains (nigrosin, safranin, malachite green, Ziehl-Neelsen carbolfuchsin, methylene blue), droppers, china marker, microscope and immersion oil, hot plate, large beaker, filter paper, acid-alcohol

Procedure: the first step in most of the stain preparations was to make a slide with a fixed smear of bacteria. To do this, we drew a large circle on a clean slide to delineate the smearing area and placed a small drop of distilled water inside the circle. We then touched the inoculating loop to the bacteria in our working stock (using the aseptic technique, of course) and then smeared this around with the water on the slide. After allowing the smear to dry, we ran it quickly over the bunsen burner flame a few times to fix the smear, being careful not to get the slide so hot that the bacteria were damaged.

The first stain we prepared was a negative stain. The beginning procedure was a bit different for this one - we started by placing a small drop of nigrosin on one end of a clean slide and smearing our bacteria directly into that, being sure to keep it on only a small portion of the slide. Next, the edge of a second clean slide was used to spread the nigrosin/bacteria along the length of the first slide so that we ended up with a thin film that trailed off at the end of the slide (we tried this a few times to get it right). After this dried completely, we were able to view it under the microscope with the oil immersion lens.

After viewing the negative stain, we used it as a starting point for preparing a capsule stain. We placed the smeared slide on a staining rack over the sink and covered it with safranin. This was then gently rinsed off with distilled water and blotted with bibulous paper before being viewed with the oil immersion lens.

Our next trick was to prepare an endospore stain. We started with a slide that we had fixed a bacteria smear to and placed it on a staining rack over a beaker of boiling water. We covered the smear with filter paper and saturated the paper with the malachite green stain. This stayed on for 5 minutes as we added more stain to keep the paper from drying out as the original evaporated. Once time was up, we removed the slide from the heat, disposed of the paper, and let the slide cool before rinsing it for 30 seconds. The smear was then covered with safranin for 90 seconds, then rinsed and blotted so it could be viewed with the oil immersion lens.

Finally, we prepared an acid-fast stain. This also started with a fixed smear of our bacteria sample placed over a beaker of boiling water. This time, the filter paper cover was saturated with Ziehl-Neelsen carbolfuchsin for 4 minutes. The slide was then removed from the heat, cooled, and rinsed. After rinsing the slide with distilled water, we decolorized it with acid-alcohol (drop by drop until the color stopped running). This also had to be rinsed off before the slide could be covered with methylene blue, which was rinsed off after 2 minutes. After blotting with bibulous paper, we were able to study our slide under the microscope.

Results and conclusions: by observing both our negative stain and capsule stain, we were able to conclude that our bacteria does have a capsule. The bacteria looked smaller after the capsule stain than it did with only the negative stain, which suggests that there is a capsule present in the extra space colored by the capsule stain.

The other stains showed that our bacteria has no endospores and is not acid-fast (because only the blue stain was visible).

Significance: while we probably won't use these particular techniques in a nursing setting, it will be important to recognize that different types of bacteria have different properties like capsules and endospores that necessitate different treatment plans if the bacteria are to be destroyed.

Inoculations

To wrap up our third day in the lab, we prepared a multitude of inoculations to test various properties of our unknown bacteria.

Matierials: bunsen burner and lighter, inoculating loop, inoculating needle, working stock with unknown bacteria, starch agar plate, skim milk plate, spirit blue plate, nutrient gelatin deep tube, sugar tubes with gas and pH indicators (lactose, glucose, sucrose, mannitol), methyl red/Voges-Proskauer (MR-VP) broth tube, citrate agar slant, tryptone broth tube, nitrate broth tube, plain agar plates, triple sugar iron agar slant tube, urea broth tube, litmus milk tube, tube with gelatinous motility test medium, thioglycollate broth tube, GasPak anaerobic system, incubator (35 degrees Celsius), test tube rack

Procedure: this whole process involved little more than inoculating plate after plate and tube after tube with bacteria from our working stock:

The starch hydrolysis, casein hydrolysis, triglyceride hydrolysis, and gelatin hydrolysis tests will show us what digestive enzymes our bacteria uses. The first three involved inoculating plates while the gelatin test used an inoculating needle to stab a gelatin tube. These are all incubating and will be checked for results tomorrow.

The sugar (lactose, glucose, sucrose, and mannitol) tubes, MR-VP test, and citrate test will tell us what carbohydrates are used by our bacteria. We inoculated the sugar and MR-VP tubes with the sterilized loop and the citrate agar slant with the inoculating needle. These are also incubating overnight.

The tryptophan (indole) test in the inoculated tryptone broth tube will provide information about our bacteria's degredation of amino acids.

The nitrate reduction and catalase tests, as well as the GasPak, will tell us about our bacteria's respiration. We inoculated the nitrate broth with the sterilized loop to test nitrate reduction. For the catalase test, we . The GasPak test started with everyone placing their inoculated agar plates in an anaerobic jar (agar side up). The GasPak envelope was shaken and placed inside the jar, and the lid was attached to the jar by screw clamp. All these tests are incubating as well.

The other miscellaneous tests (triple sugar iron, urea, litmus milk, and motility) will also help us to identify our unknown bacteria. The urea and litmus milk tubes were inoculated with the sterilized loop and the triple sugar iron and motility tubes were stabbed (the slant portion of the TSI tube was then inoculated by drawing a wavy line with the bacteria-covered needle). These are also incubating until tomorrow.

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.