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Name: Pre-Lab 4 Worksheet Counting Cells Students will be counting cells using an automated cell counter called the Countess. However, determining total cell number and cell viability has traditionally been accomplished using a Hemocytometer and the Trypan Blue Assay. Learning and understanding his type of calculation also informs you of how the automated cell counter (Countess) determines the final numbers for your experiments. Hemocytometers have a grid to help you not lose your place as you count the cells. There are nine "big squares", each 1 mm x 1 mm (see image to the right). To begin, count all the cells in the middle square and the four corner squares (i.e. A, B, C, D, and E in the diagram). Each "big square" has a surface area of 1 mm x 1 mm, plus it is 0.1 mm deep. Therefore, the volume of liquid that covers that big square (0.1 mm3) is 0.0001ml or 1.0 x10-4 ml because there are 1000 mm3 in 1 ml. Apoptotic (dying) and dead cells are more permeable than those of healthy cells Trypan blue can cross the plasma membrane of these apoptotic and dead cells. Therefore, cells that turn blue after the trypan blue assay are considered unhealthy or nonviable. Once the cell pellet has been re-suspended, we typically remove 100ul of cell suspension and place it into a microcentrifuge tube. Then we add 100ul of trypan blue to the cell suspension for a 1:1 dilution of the cells. This is a dilution factor of 2. We can use the trypan blue exclusion assay to determine cells per mL, total cells in the suspension, percent viable and percent nonviable cells. Use the equations below in the practice problems. 1) the equation to calculate total cells per mL a. Solve for X (Cells per mL) = # of cells you counted x 1x104 x 2 (dilution factor of Trypan Blue) / number of big squares 2) the equation to calculate total cells in cell suspension (in the tube) a. Solve for X (Total cells in cell suspension) = cells per mL x total volume of cell suspension 3) the equation to calculate percent viable cells: a. Solve for x (percent viable ) = total viable cells counted / total cells counted (viable + nonviable) x 100 4) the equation to calculate percent nonviable cells: a. Solve for x (percent nonviable ) = total nonviable cells counted / total cells counted (viable + nonviable) x 100
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Use the following information to answer the next 5 questions: The Hawaiian Islands are home to honeycreepers, a diverse group of birds with curved beaks that feed on the nectar of different flowers. The Iiwi and Apapane are two species of Hawaiian Honeycreepers. The Iiwi is endangered. To protect the species, conservation planners are trying to engage students of Hawaiian high schools to grow and plant more lobeliid flowers. They set up an experiment to test the best conditions to grow lobeliid flowers. The high school students are very engaged in this project and apply their knowledge of plant growth to create a successful experiment; however, Dan the Beagle is on vacation in Hawaii and wants to participate. He gets some lobeliid seedlings and puts them in a dark closet where he waters them regularly. 1. According to the 2012 Christmas bird count there were 38 Iiwi in an area of 50 square miles of Haleakala National park on the Island of Maui. What is the population density of Iiwi? A) 0.76 liwi per square mile B) 1.35 liwi per square mile C) 7.6 liwi per square mile D) 135 liwi per square mile E) 1850 liwi per square mile 2. The carrying capacity of Apapane in Haleakala National Park is 100. If there were 85 Apapane at Christmas 2012 and r = 0.6, how many Apapane should there have been at Christmas 2013? A) 8 B) 51 C) 93 D) 100 E) 185 3. Given the information in the previous question, you would correctly predict that... A) Apapane show exponential growth. B) Apapane show logistic growth. C) dN/dt equals zero. D) K will decrease over time. E) Apapane show density independent growth. 4. If a volcanic eruption occurred on a Hawaiian island and killed many Iiwi and Apapane you would consider this A) coevolution B) a founder effect C) adaptive radiation D) density dependent event E) a density independent event 5. Which of the following graphs below correctly shows the relationship between temperature and metabolic rate in Iiwi, the honeycreeper, in Hawaii?
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