User:Alyssa Lillian/Notebook/Biology 210 at AU

February 10, 2016

Purpose: The purpose of this lab was to quantify, observe, and identify the bacteria present in the Hay infusion through morphological characteristics, motility, and a gram stain.

Materials & Methods: Wet Mount Procedure: The Agar plates (both normal and Tet+) were inspected for the presence of bacterial colonies. Once a colony had been located, a small sample was removed using a sterilized loop and was placed on a microscope slide. A small drop of water was added to the slide and was mixed with the bacterial sample. The wet mount was then observed using a compound microscope to find and identify any bacteria present. This process was repeated until four microorganism samples (two from the normal Agar plates and two from the Tet+ plates) were observed.

Gram Stain Procedure: Another sample was taken from the same bacterial colonies used for identification of microorganisms using a sterilized loop. The bacterial sample was mixed with a drop of water on a microscope slide and the area underneath the sample was circled with a wax pencil. Once the slide had dried, it was passed, bacterial side facing upward, through a flame three times. The bacterial smear was then covered with crystal violet dye for one minute; the dye was washed off with water after a minute had passed. The bacterial smear was then covered with Gram's iodine solution for one minute and the solution was washed off with water after the minute. The bacterial smear was then decolorized by flooding it with a solution of 95% alcohol for approximately 10-20 seconds. A safranin stain was used to cover the smear for approximately 20-30 seconds and then the smear was rinsed with water. After the slide was given time to air dry, it was observed under a compound microscope to determine whether it was gram positive (retained the dye; blue color) or gram negative (did not retain the dye; pink color).

PCR Procedure: Of the two normal Agar plates and the two Tet+ Agar plates that bacteria was taken from, one of each type was selected. Two PCR tubes were obtained and each was filled with 20 μL of a primer/water mixture and then the tube was mixed until the PCR bead dissolved. A small sample of the same bacterial colony previously used was taken from the Agar plate and placed into the corresponding PCR tube -- this was done twice, once for each of the two samples being tested. The tubes were then placed into the PCR machine.

Data & Observations: It is evident from table 1 that there was a difference in number of colonies counted between the nutrient Agar plates and the nutrient and Tet+ plates: the nutrient plates showed significantly more colonies than the Tet+ plates. With the exception of the 10^-3 Tet+ plate, the other Tet+ plates contained only one colony. This indicates that Tetracycline is effective at neutralizing bacteria; in fact, only 21 different species were unaffected by the tetracycline and able to grow on the plates. Tetracycline works by inhibiting protein synthesis by "preventing the attachment of aminoacyl-tRNA to the ribosomal acceptor (A) site"(Chopra et al 232). Tetracycline is effective against both gram positive and gram negative bacteria as well as "atypical organisms such as chlamydiae, mycoplasmas, and rickettsiae, and protozoan parasites"(Chopra et al 232). My group had difficulty finding bacteria, so only two, rather than four, observations were made. One was from the Tet+ 10^-3 plate and the other was from the Tet+ 10^-7 plate. The colony observed from the Tet+ 10^-3 plate was circular with a curled edge, a smooth and glistening surface, and a convex elevation; it was approximately 1 cm in diameter and a mustard yellow color (see fig. 16). It's cells were non-motile, spirochete, and layered on top of one another with a few separated from the others (see fig. 20). The colony observed from the Tet+ 10^-7 plate was circular with an entire edge, a dry and powdery surface, and had a slightly raised elevation; it was approximately 3 cm in diameter and a tan/brown color (see fig. 18). There was a single organism which was non-motile, filamentous, and curled slightly around itself (fig. 21). The gram staining procedure did not yield conclusive results for our bacterial samples; however, pictures of gram positive and gram negative stains were taken from another group (see fig. 22 and 23, respectively).

Conclusions & Future Directions: Since we had so much trouble finding bacteria from our samples, I think it's important to ensure that when we go back to the transect we try and obtain a wide variety of samples so that any lack of microorganisms cannot be attributed to error on our part. Nevertheless, it appears that Transect 1 does not have many microorganisms currently living in it (though that could be a result of the recent weather).

100-fold Serial Dilution Results Table 1. Chart showing the type of Agar plate used and how many bacterial colonies were identified on those specific plates, and the conversion used to determine the number of colonies/mL

Normal 10^-3 Plate Fig. 12. Picture showing the bacterial colonies present in the normal 10^-3 Agar plate.

Normal 10^5 Plate Fig. 13. Picture showing the bacterial colonies present in the normal 10^-5 Agar plate.

Normal 10^-7 Plate Fig. 14. Picture showing the bacterial colonies present in the normal 10^-7 Agar plate.

Normal 10^-9 Plate Fig. 15. Picture showing the bacterial colonies present in the normal 10^-7 Agar plate.

Tet+ 10^-3 Plate Fig. 16. Picture showing the bacterial colonies present in the Tet+ 10^-3 Agar plate.

Tet+ 10^-5 Plate Fig. 17. Picture showing the bacterial colonies present in the Tet+ 10^-5 Agar plate.

Tet+ 10^-7 Plate Fig. 18. Picture showing the bacterial colonies present in the Tet+ 10^-7 Agar plate.

Tet+ 10^-9 Plate Fig 19. Picture showing the bacterial colonies present in the Tet+ 10^-9 Agar plate.

Tet+ 10^-3 Bacterial Cells Fig. 20. Bacterial cells from the Tet+ 10^-3 plate under 40x using compound microscope.

Tet+ 10^-7 Bacterial Cells Fig. 21. Bacterial cells from the Tet+ 10^-7 plate under 40x using compound microscope.

Gram Positive Stain Fig. 22. Picture showing the gram positive bacterial cells after staining under 40x using compound microscope.

Gram Negative Stain Fig. 23. Picture showing gram negative bacterial cells after staining under 40x using compound microscope.

Works Cited: Chopra, I., & Roberts, M. (2001). Tetracycline antibiotics: mode of action, applications, molecular biology, and epidemiology of bacterial resistance. Microbiology and molecular biology reviews, 65(2), 232-260.

-AL

January 20, 2016

The Hay Infusion was made by placing approximately 12 grams of the transect sample (made up of 50% soil and 50% plant matter) in a plastic jar along with 500 mLs of Deerpark water and 0.1 g of dried milk. The lid was placed on the jar and it was shaken to mix the contents. After mixing, the lid was removed from the jar and it was set aside for seven days. A week later, the hay infusion was examined for the presence of protists. Initially, the foul smell was the first thing noticed. While there was no mold or green shoots seen on top of the liquid, there was a a clear film on the top of the mixture (see fig. 5). The lid was placed on the jar and it was thoroughly mixed and then samples were taken from the top, middle, and bottom of the jar (see fig. 6 and 7, respectively). From the top layer of the hay infusion we found algae that we identified as Oedogonium (see fig. 8). The Oedogonium was identified through being autotrophic, non-motile, having a grass-green color with the pigment contained in its chloroplasts, and having chloroplasts that form a network that extends from both ends of each cell. It's approximate size is 160 μM. From the middle layer, we found protozoa that we identified as Colpidium (see fig. 9). It was identified as such through its characteristics of being heterotrophic, colorless, motion by means other than sliding or floating, having cilia that covered the entirety of its body, and having a small (approximately 15 μM) oval shaped body with a small mouth. We were unable to locate any protists in the bottom layer of our hay infusion so we looked at an organism found by a different group from the top layer of their hay infusion. The protozoa found was identified as Peranema through its physical characteristics of being a heterotrophic, single colorless cell that moves by means other than sliding or floating through the use of a single flagella and appears to be vibrating while in motion yet appears to be plastic while stationary (see fig. 10). It is approximately 38 μM in size. All of the identifications of the protozoa were made using Ward's Free-Living Protozoa dichotomous key and the identification of algae was made using a dichotomous key from Connecticut Valley Biological Supply Company. The different protists found in each layer may be related to their location to plant matter (i.e. close to or far away from) depending on if they are autotrophs or heterotrophs. If the Hay Infusion culture was allowed to "grow" for another two months, I would expect to see many more organisms present assuming that all of the requirements necessary for the organisms to thrive were met. Some selective pressures that may decrease the survival ability of the organisms would be lack of space since the jar that was used for the Hay Infusion wasn't overly large, and access to sunlight (for the autotrophs) since the hay infusion is kept indoors. To determine the bacteria present in the Hay Infusion, a serial dilution of a sample taken from the infusion was performed and then the dilutions were placed onto agar plates for further investigation next week (see fig. 11 for diagram of this process).

Hay Infusion Top View Fig. 5. An picture showing an aerial view of the top layer of the hay infusion

Hay Infusion Middle View Fig. 6. A picture showing the middle layer of the hay infusion

Hay Infusion Bottom View Fig. 7. A picture showing the bottom of the hay infusion

Oedogonium Under Microscope Fig. 8. Algae identified as Oedogonium under 40x magnification

Colpidium Under Microscope Fig. 9. Protozoa identified as Colpidium under 40x magnification

Peranema Under Microscope Fig. 10. Protozoa identified as Peranema under 40x magnification

Serial Dilution Process Fig. 11. Diagram of the procedure followed to make serial dilutions and agar plates

-AL

January 13, 2016

Transect 1: Biotic Factors -- trees, plants, animals (squirrels and chipmunks); all seen on the ground/growing from the ground Abiotic Factors -- wind, sunlight, rocks, temperature, soil; the rocks and soil were found on the ground

Transect 1 is comprised mostly of factors that make up a wooded terrain -- trees, leaves, soil, plants and is located right outside the East side of Hurst. The majority of the transect is on a small hill and is dominated by one large tree as well as other smaller trees (see fig. 3 and 4 for location, respectively). As a result of the recent weather (cold and snowy) much of the plant life is no longer living. According to a sign placed on the transect, there is Kerria japonica 'Pleniflora' present, a plant typically found in China, Japan, and Korea (see fig. 2). On the day when the samples were taken from the transect (01/13), the high was 32°F and the low was 21°F and there was no precipitation. Although the transect was visited at night and sunlight was not present -- the only light present came from the light pole -- it is reasonable to assume that sunlight plays a significant role in the plant and animal life seen on the transect.

Aerial View of Transect: Fig. 1. Aerial Drawing of Transect 1

Kerria japonica 'Pleniflora' Sign Fig. 2. Sign identifying the plant Kerria japonica 'Pleniflora'

Transect 1 Tree Fig. 3. Picture showing the one large tree dominating Transect 1

Transect 1 Fig. 4. Picture showing the light present in the transect as well as some other smaller trees

-AL