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
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.
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-alcoholProcedure: 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.
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).
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.
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.
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