Biology 2290F/G Study Guide - Final Guide: Dichlorophenolindophenol, Light-Dependent Reactions, Chlorophyll

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28 Jun 2016

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Dean unit (22 marks, 5 questions: (similar to practice question 1 and 2) (a) the first question is basically using the formula a*(100/34. 5), which is provided in the paper. Ho(cid:449) (cid:272)al(cid:272)ulations: an absorption spectra was provided with 3 solutions. Solution c was similar to either solution a or b with a different concentration. (a) ask you it"s a or b and reason. Find number of cells, using ratios or c1v1=c2v2, similar to tables in labs. Total volume is given, make sure each tube add up to that: buffer question, make a ph 7. 0 buffer. 3 components are provided with it"s kpa (cid:448)alue. (a) ask you to choose one component and explain why you choose it or not choose the other (2 marks) My answers: (1) within ph1 (2) substances are compatible (b) find [base]/[acid], using formula ph=pka+log[base]/[acid] Give two reasons: write a conclusion for the experiment given, e. coli with pgreen (ampr) was plated on a lb plate and incubated.

Related Questions

You have been provided with 100 grams of a yogurt culture. You are asked to perform a plate count to determine the number of viable organisms present in the sample. You estimate that there may be as many as 10^8 cfu/g in the culture. Understanding that a countable number of cfu on a plate is 30 t0 300.....

1. Which of the following dilution series is appropriate for this essay if 1.0 mL was plated from the last dilution tube? (circle the correct answer and show your reasoning)

a) (0.1 gram of sample): 9.9 - 1 mL - 9 - 1 mL - 9 - 1 mL - 9 - 1 mL - 9

b) (10 grams of sample): 9 - 1 mL - 9 - 1 mL - 99 - 1 mL 9

c) (10 grams of sample): 90 - 10 mL - 90 - 10 mL - 90 - 10 mL - 90

d) (1 gram of sample): 9 - 1 mL - 99 - 1 mL - 99 - 1 mL - 9 - 1 mL - 9 - 1 mL - 9

2. Referring to the diargram/scheme you chose above, assume that 90 cfu/g grew on the plate from the last dilution. Calculate the total number of bacteria (cfu) in the entire yofurt culture. Show all work, including the determination of the bacterial concentration in the original sample.

3. In the above example, if 0.1 mL had been plated from the last tube of the dilution series instead, how many cfu would you anticipate on the plate? Show your work.

4. Looking at the tubes in row C, calculate the final overall dilution in the last tube of the series if each tube contained 49 mL of diluent and 1 mL was transferred between each tube. Show your work.

For Discussion (2-3 paragraphs) â¢ What was the concentration of your pre-diluted NPs? â¢ Determine the effect of NPs on brine shrimp lethality. â¢ Determine the effect of NPs on cell viability. â¢ What is (are) the control(s) in this experiment? What is its purpose? â¢ Based on your data in this lab, what is a safe concentration for brine shrimp Threshold Level of Toxicity (TTL)? â¢ 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. â¢ 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 Î¼l) of PBS on top of each specimen 4) Add 60 Î¼l of Trypan blue to your shrimp and incubate for 1-2 minutes at room â¨temperature. 5) Wick away excess liquid 6) Add 100 Î¼l 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 Î¼l 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 Î¼l 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.

ultramarinepig261

yeast population dynamics

Procedure

1. Work in pairs on this lab, so 12 tubes per pair of students. And share a tube rack with one other pair of students

2. Turn on your spectrophotometer. It needs at least 15 minutes to warm up to give you good readings.

3. Add 5 mL of yeast extract solution (YECM) to each of 12 tubes. (The yeast extract provides vitamins and amino acids for yeast growth and will be the same for all cultures). The tubes should be labeled with your initials, treatment, and tube number. Tape or Parafilm down the lids of 3 tubes, and label them âCONTROLâ.

Do not touch the insides of the tubes or lids! Try to keep these as sterile as possible!!

4. Add 50 mL live yeast culture to each of the remaining 9 tubes.

5. Add the varying volumes of sugar and/or ethanol using Table 1 below.

6. Use Parafilm to close the tops of each tube, making sure the Parafilm is tight and no air can get in, and label each tube with the following:

Amount of sugar added (mL) Amount of ethanol added (mL)

Name of your group Tube number

Table 1: setup yeast tubes (remember, 1 mL = 1000 mL)
Tube number	Yeast culture medium? (5 mL)	Live yeast culture? (50 mL)	Sugar added (mL)	Ethanol added (mL)
1 â control	YES	NO	0	0
2 â control	YES	NO	0	0
3 - control	YES	NO	0	0
4	YES	YES	0	0
5	YES	YES	0.25	0
6	YES	YES	0.5	0
7	YES	YES	0	0.25
8	YES	YES	0.25	0.25
9	YES	YES	0.5	0.25
10	YES	YES	0	0.5
11	YES	YES	0.25	0.5
12	YES	YES	0.5	0.5

Procedure for measuring absorbance (in absorbance units, or AU)

7. Calibrate the spectrophotometer:

Turn on the spectrophotometer and let it warm up for 15 minutes. You will get erroneous results if you donât let it warm up first.

Be sure the spectrophotometer is set to read at the wavelength of 550 nm

With no tube in the spectrophotometer and the lid closed, use the left-hand knob to adjust the reading to 0% Transmittance/push zero button to calibrate

Insert a CONTROL tube (making sure it is clear, without bacterial contamination which would make it cloudy), and use the right-hand knob to readjust the spectrophotometer to 100% Transmittance.

When reading the absorbance, be sure to line up the needle on the spec with its reflection.

8. Immediately before reading any tube, vortex the tube so that the spinning column reaches the bottom of the tube for several seconds. This is critical! The yeast cells are heavy and will tend to sink to the bottom of the tube, so you must vortex the tubes to resuspend them: otherwise, your spectrophotometer readings will be erroneously low. If the vortex is not enough to suspend the pellet of yeast cells at the base of the tube, take a piece of Parafilm and cover the top of the tube, then cover this with your thumb and shake the tube vigorously. The pellet should dislodge and the yeast cells should be easily resuspended after doing this. Use a Kimwipe to wipe down the outside of each tube, to remove fingerprints and other smudges that could affect the absorbance reading. (COULD BE A POTENTIAL ERROR)

9. Record the absorbance (in absorbance units, AU) for the tube on your data sheet.

10. Repeat steps 5 and 6 for every tube.

11. Leave the spectrophotometer turned on for the next user.

Figures you should include are:

Average absorbance vs. time for the no ethanol (0 mL) treatment

Average absorbance vs. time for the 0.25 mL ethanol treatment

Average absorbance vs. time for the 0.50 mL ethanol treatment

Sugar added vs. average carrying capacity (K). Use different symbols to denote each of the three alcohol concentrations

A. After a limit, the increasing concentration of sugar decreases the carrying capacity and growth rate. This is because at higher sugar concentrations, the medium becomes hypertonic and the yeast cells loss water towards the medium.With increasing concentration of the ethanol, the carrying capacity and the growth rate decreases. Why does this happen?

B. Is there any interaction between the effects of adding sugar and alcohol on yeast?

C. why do some cultures not reach K?

D. What are the potential sources of error and assumptions made in this experiment?

E. What do these results mean in a more general (non-yeast) context?

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Biology 2290F/G Study Guide - Final Guide: Dichlorophenolindophenol, Light-Dependent Reactions, Chlorophyll

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