For Discussion (2-3 paragraphs) ⢠What was the concentration of your pre-diluted NPs? ⢠Determine the effect of NPs on brine shrimp lethality. ⢠Determine the effect of NPs on cell viability. ⢠What is (are) the control(s) in this experiment? What is its purpose? ⢠Based on your data in this lab, what is a safe concentration for brine shrimp Threshold Level of Toxicity (TTL)? ⢠Often indicator species are used to study the overall health of an ecosystem. If you were to study an ecosystem containing brine shrimp, would you use it as an indicator species? Why or Why not? Explain your reasoning. ⢠What possible sources of error were present in this experiment?
Introduction
LD50 is a commonly used term to describe acute toxicity: LD stands for Lethal Dose and 50 is used because the dose could kill 50% of the animals exposed to the chemical. For LD50 tests, the animals are given specific amounts of the chemical, orally, by injection or dermally. The value is then reported along with the method of administration and the type of animal used in the test. LD50 is typically reported in milligrams or grams per kilogram of body weight of the animal. The lower the LD50 is, the higher the toxicity of the chemical would be. This information is always provided in Safety Data Sheet (SDS) of chemicals in the âtoxicity dataâ category. A similar measure, the LC50 is usually used to describe the ecological hazards of chemicals. LC stands for lethal concentration that could kill 50% of the animals after 24-96 hours. This value is always provided in Safety Data Sheet (SDS) of chemicals in the âEcological Dataâ category. In this lab, you will be measuring LC50 or LD50 of iron oxide nanoparticles using brine shrimp. Iron oxide nanoparticles are magnetic. Application of magnetic nanoparticles ranges from magnetic storage media to medical industry where they can be used for targeted drug delivery or as contrast agents in MRI. Thus, measuring environmental and biological toxicity of these nanoparticles is crucial. To test toxicity of any such reagents on small organisms, Daphnia and brine shrimps (also known as Artemia) can be used. Artimia are found in salt lakes around the world. They are important live food for aquarium fish. In this lab, you will use brine shrimp to measure toxicity of the nanoparticles. Trypan blue is a stain used to assess cell viability. It is able to diffuse through cell membranes of dead cells but is unable to permeate live cells. This stain can be used to assess cell survival 1 Fall 2017 following exposure to chemicals, nanoparticles and experimental drugs. The ratio of live: dead cells can be used as a preliminary assessment of cytotoxicity and translated to tissue viability.
Methods Part A: Serial Dilution of NP Solution 1) Label 5 test tubes as 0, 1:1, 1:10, 1:100, 1:1000 2) Add 2mL of seawater to tube 0 3) Add 2mL of specific group stock solution to tube 1:1 and add 2mL of seawater to it. 4) Transfer 1mL of stock solution to tube 1:10 and add 9mL of seawater to this tube 5) Transfer 1mL of 1:10 solution to the tube labeled as 1:100 and add 9mL of seawater to â¨this tube 6) Transfer 1mL of 1:100 solution to the test tube labeled as 1:1000 and add 9mL of â¨seawater to this tube Part B: Preparation of test environments 1) Label 5 wells of 6-well plate as control (0), 1:1, 1:10, 1:100 and 1:1000 2) Label 3 petri dishes as: control (0), 1:1 and 1:1000 â¨NOTE: Be sure to include your initials on all plates 3) Transfer 2mL of each solution from the corresponding test tube to the respective wells on your six-well plate and each petri dish Part C: Preparing specimens for the Trypan blue cell viability test 1) Transfer 2 brine shrimp into each of the pre-labeled petri dishes containing the solution of NPs and seawater (0, 1:1 & 1:1000) and set a timer for 30 minutes. 2) Transfer a single brine shrimp into the unlabeled petri dish with a drop (~50ul) of seawater and use the dissecting microscope to make observations related to the behavior and morphology of untreated brine shrimp.â¨Make observations in lab notebook of all samples Part D: Preparation of specimens for the toxicity assay 1) 2) 3) 4) 5) Add 10 brine shrimp into each well in the 6 -well plate Make observations in lab notebook Using a dissecting microscope (if needed), count the number of brine shrimp in each well (alive shrimps).â¨Record your data in your notebook using the graphic below as a template. Close the plate and store it for next observation.â¨Observations must be done within 24h-96hâ¨Note: There will be times allocated and a sign up paper for observations Part E: Trypan blue viability test 2 Fall 2017 1) Label 3 glass slides to match the petri dishes prepared in Part C. 2) Transfer 1 brine shrimp from each petri dish onto the corresponding slide. â¨Make observations in your lab notebook 3) Add a drop (~60 μl) of PBS on top of each specimen 4) Add 60 μl of Trypan blue to your shrimp and incubate for 1-2 minutes at room â¨temperature. 5) Wick away excess liquid 6) Add 100 μl PBS to sample and observe (while alive!), looking for blue cells, using the â¨dissecting scope â¨Make observations in your lab notebook 7) Label 3 microfuge tubes to align with each sample. 8) Transfer each brine shrimp from the slide into the corresponding microfuge tube 9) Add 50 μl of PBS to the tube and use the mechanical disruption tool to homogenize the â¨sample for ~2 minutes until a cell suspension has been generated. 10) Load 30-50 μl of your sample onto a new glass slide and add a cover slip. 11)Observe the sample on high power and use the scale below to qualitatively assess cytotoxicityâ 0-no blue 1-some blue 2-fairly blue 3-totally blue.
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