User:Darrell A Humphries Jr/Notebook/Biology 210 at AU

Exercise VI - Embryology and Development [ Zebra Fish ]

Purpose: Zebra fish are an important model system, they are easy to observe and are vertebrates. In this lab the zebra fish are exposed to a chemical and the effect of it on development and viability is tracked. Me and my partner chose caffeine as the chemical choice for experiment.

Materials and Methods: To carry out this study 20mLs of Deer-park water is set up for the control and the treated/experimental (20mLs of Deer-park water containing treatment) petri dishes. 24 healthy translucent embryos are selected and placed in a dish- A dropper is used to transfer the eggs. The Zebra fish embryos are about 18 to 36 hours post fertilization (hpf) when this experiment is set up on day one. On day 4-5 25mLs of fresh water is added to the wells, the zebrafish are fed 1 microliter of shrimp once a day. Using a dissecting scope and depression slides, the developmental stages at each time point in the experiment were able to be determined.

Data/ Results: [1] [2] [3] [4] [5]

Media:Zebra_Fish_Experiment.docx‎

Discussion:At the end of this experiment it is observed that all fish in both the control and caffeine groups died. The caffeine group showcased inhibited movement and growth throughout the experiment while the control remained with normal behavior. The zebra fish were fed shrimp on an inconsistent basis- this could have had a notable impact of the unprecedented death observed in the control groups. From this experiment it is observed that caffeine indeed does inhibit development and viability. In conclusion, caffeine-induced reduction of loco-motors activity, malformation of muscle fiber alignment and delayed+deformed development.

16S PCR

Purpose: Analyzing 16S Sequences from the Bacterial Colonies

Materials and Methods: primers and PCR are used to selectively amplify the 16S rRNA gene. This gene sequence is diverse and the sequences are specific to each species. Using a sterile toothpick, a small amount of a bacterial colony wished to characterize is picked up then then placed in a tube and placed in the PCR machine. The PCR products is on an agarose gel then sent off to be analyzed. The returned DNA sequences were analyzed using the nucleotide BLAST program from the National Center for Biotechnology Information. BLAST is a suite of programs provided by NCBI for aligning query sequences against those present in a selected target. database.

Data & Tables:

[6]

[7]

- 16s - TNNCNNNCCNANCGGTAGANATNTNNNNNNNNNGNNAGCGGCGTACGGGTGAGGNNNNA NNNNGNNTGCCTGANAGGGGGATGGCTTTCNNNAGGANNANNAATACCCCATAATATTTTAAGTGGNATCACTTGNAATT GAAAACTCCGGTGGATAAAGATGGGCACGCNCCAGATTAGATAGNTGGTAGGGNNANGGCCTACCANGTCTNTGATCTTT AGGGGGCNTGANAGGGTGATCCCCCACACTGGTACCGANACACGGANCANACTCCTACNGCACGCNCCAGTGAGGAATAT TGGACAATGNGTGAGANCCTGATCCCNCCNTCCCGCGTGAAGGACGACGGCCCTCCGGGTNGTAAACGTCTTTTGTATAN GGATAAACCTNGACACCTGTCNGAAGCTGAAGGTACTATTTNAATAAGCACCGGCTAACTCCGTGCCCACATCCGCGGNA ATCCNNAGGGTGCCACCGTTATCCCCATTTACTGCGTTTAAATGCNCCNTAAGCGGAGNGTCTCAAAGTGGTGANNACTC ACAGNTCNNATGTGAAACTGCCACCTAAACNGCGTGTCTTGNTTGATAANAACTGNNGNAATNAGTAGTGNNNTGATGAA GTGCGTANATATTATGCAGATCACCAATTGCGAAGGCAGGTTAGTATTTNTANCTGATCNNGACGGACCAAANTGTGANN AGNNNAANGATTANATCGCGGCCAGTGNCGCTGCAACGATGCTAACTCGNNTTNGNTCNNNNNNGANTANGAGNTCNCNN NANTGANAAGTNANCNCGNGNNGAGCNCNGTCGNAAGNACACACNNCACNGNCTAGGANNGGCCCCCGCTCCAACCCTGN ACTATCGGGNNCNNNNCCNCGAGCACGNNGGGATCGAGACCANGGACANNGNGGCCANNNGANCCGNNANNNACCAGCTC ANNCNTGNNNANNNCNNCCNCNGN

Exercise V - Invertebrates and Vertebrates

Purpose: The purpose of this lab is to study the soil invertebrates form our transect. Most soil invertebrates are Arthropods; within this phylum there are large and small organisms, several kinds fuch as worms, spiders, ants. We will Observe examples of these organisms from the Berlese funnel.

Materials and Methods: Observation of the invertebrates that reside within the transect are made using the Berlese Funnel method. To set up the berlese funnel, 500 grams of leaf litter were placed in a funnel connected to 25 mL of 50:50 ethanol/water solution that is transferred into the 50mL conical tube. Screening material is placed into the bottom of the funnel to constrain leaf litter from falling into the preservative. The funnel is set up on a ring stand so that it is held into the tube with the ethanol. To force the invertebrates down into the ethanol preserving solution, the berlese funnel is placed under a 40 watt light bulb and covered with tin foil. The invertebrates move away from the light and ultimately fall into the ethanol in the flask. The Berlese funnel was left to sit for a week before the invertebrates found in the transect were identified. To analyze the invertebrates collected within the Berlese Funnel, the apparatus was carefully broken down and the invertebrates were poured into a petri dish to be examined under a microscope.

Results: [8] [9]

Exercise IV - Plantae and Fungi

Purpose: Plants and Fungi are critically important as autotrophs and decomposers in any ecosystem. The purpose of this lab is to observe the characteristics of a moss, Mnium, and an angiosperm, Lilium, as well as five(5) plant samples from the transect. These samples will be characterized for three(3) general features.

Methods and Materials: We obtained (3) ziploc bags and obtained a leaf liiter sample weighing 500g from our transect. This leaf litter is used to set up the Berlese funnel for collecting invertebrates. Next we obtained (5) representative samples of plants in our transect. No flowers were present in this transect. With these obtained samples we were able to examine the leaves of the moss and the plants under a dissection scope and a low magnification microscope.

---

Results: Table 1 Characteristics of Plants Collected from the Transect

---

[10] [11]

4/4 Exercise III Microbiology and Identifying Bacteria

Purpose: The purpose of this lab is to identify species of bacteria based on motility, gram stain, colony morphology and sequencing of the 16s ribosomal subunit gene.

Methods & Materials: Using the Agar plates inoculated from the hay infusion culture, observation and quantifying present microorganisms with use of a microscope and the naked eye, we were able to count the total numbers of colonies on each plate. The Prokaryotes are observed with a microscope because they range in small size from 1 to 10 um. The Wet mount procedure and Gram stain procedure were used to determine the cell shapes and if the organisms are motile, and determination of gram-positive vs gram-negative bacteria.

Results: The state of the Hay Infusion Culture me and my lab partners observed, show notable physical change from the first time the Hay Infusion Culture were observed. A stronger, darker odor is emitting from its container (sewer-like), while evidence of more bacteria growth have become present. The turbidity of the water has increased, it has more difficult to see through the water as it is much more 'cloudy' than before, possibly due to an increase in particles and bacteria.

Dilution |Agar Type | # Colonies on plate | Conversion Factor | Colonies/mL |

---

10^-3 |nutrient | 756 | x10^3 | 756,000 |

---

10^-5 |nutrient | 64 | x10^5 | 6,400,000 |

---

10^-7 |nutrient | 2 | x10^7 | 20,000,000 |

---

10^-9 |nutrient | 0 | x10^9 | 0 |

---

10^-3 |nutrient + tet | 70 | x10^3 | 70,000 |

---

10^-5 |nutrient + tet | 0 | x10^5 | 0 |

---

10^-7 |nutrient + tet | 0 | x10^7 | 0 |

---

10^-9 |nutrient + tet | 0 | x10^9 | 0 |

---

• This table indicates that the antibiotic Tetracycline was effective with inhibiting bacteria growth on the Agar Plates; so effective that our last three(3) agar plates recorded zero(0) bacteria colonies on the Agar Plate. Dilution 10^-7 without the tetracycline recorded 20 million colonies/mL of bacteria, while dilution 10^-7 with tetracycline recorded zero(0) colonies/mL of bacteria.

Mechanisms of action for tetracycline:Citation -> Antimicrobial Research Centre and Division of Microbiology, School of Biochemistry & Molecular Biology, University of Leeds, Leeds LS2 9JT, United Kingdom,1 and Department of Pathobiology, School of Public Health and Community Medicine, University of Washington, Seattle, Washington 98195-72382 <- \Mechanisms of action for tetracycline/

• inhibit protein synthesis by preventing the attachment of aminoacyl-tRNA to the ribosomal acceptor (A) site

Types of Bacteria effected:

• Tetracyclines are broad-spectrum agents, exhibiting activity against a wide range of gram-positive and gram-negative bacteria, atypical organisms such as chlamydiae, mycoplasmas, and rickettsiae, and protozoan parasites.

---

Colony Label | Plate Type| Colony Description + Cell Description | Gram + or Gram - | Additional Notes |

    1       |     -     |        Orange, white like              |                  |
                            pigments. Circular & irregular.      |      negative    |
                            Flat & raised elevation. Filamentous |                  |
                                     Colony.                     |                  |

---

    2       |     -     |  Yellow, orange, white, smooth,        |                  |
                           some convex elevation. Rhizoid        |   positive       |  
                              colony and circular                |                  |

---

    3       |     +     | Mixture of circular, irregular         |                  |  
                         & filamintous colonies. Lobat &         |    positive      |  gram positive 1um- pink circular bacteria 
                           entire edge. Convex elevation,        |                  |
                          smooth surface, jell-like.             |                  |

---

    4       |     +     |       No Bacteria Growth               |     negative     |

[12] [13] [14] Here in this image of colony label,there is a mixture of circular, irregular and filamentous colonies. Yellow, orange, white.