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Whatcom Community College

Human Anatomy and Physiology I


Anatomy Laboratory

Laboratory Exercise: Erythrocyte Osmosis

NOTE: You, the student, should provide your own goggles or wrap-around safety glasses. WCC will supply a biohazard lab coat which is NOT to ever leave the lab.

Adapted from Cunningham, K., Lancaster, D., and Erickson, M. (2002). Human anatomy and physiology laboratory manual. Bellingham, WA: Whatcom Community College. In this lab you will be using erythrocytes as a model of a typical body cell. The movement of water across a semipermeable cell membrane is called osmosis. Water will osmose in response to diffusion gradients between the intracellular fluid (ICF) and the extracellular fluid (ECF). The cells of your body are very sensitive to fluid concentrations and have developed extensive mechanisms to maintain isotonicity between the ICF and ECF. You will investigate how cells respond when placed in saline solutions of varying concentrations that may or may not overwhelm the cells ability to maintain isotonicity. Objectives: After completing the lab exercise and answering questions contained herein, the student should be able to do the following. a. b. c. d. e. Aseptically obtain several drops of blood from a finger stick. Use the universal precautions when dealing with bodily fluids. Explain what a solution is and name the two components of any solution. Explain what a saline solution is. Define the terms: 1) hypotonic, 2) hypertonic, and 3) isotonic. Explain what happens to erythrocytes when they are immersed in the three types of saline solutions listed under e., above. Determine if an unknown saline solution is isotonic, hypotonic, or hypertonic on what happens to RBCs when placed into that solution. Determine the percent (w/v) strength of an ideal saline tissue irrigation and that particular strength is ideal.

f. g. based h. tell why

i. closely j. fluids.

Calculate how many tsp of salt can be mixed with 1 pt of water to make a approximate isotonic saline solution. State what the general milliosmolarity is of any solution isotonic to tissue

Lab Exercise: Erythrocyte Osmosis, continued

PRE-LAB PREPARATION
1. BEFORE COMING TO LAB, read this entire procedure and study the introductory sections in this manual about the Lab. Introduction and the Universal Precautions. Also review your notes from the lab safety lecture. Answer the PRE-LAB questions beginning on page 151 before coming to lab. You must answer the PRELAB QUESTIONS BEFORE COMING TO LAB. The instructor will check to see that you have answered the prelab questions. If you have not answered the questions, you will be asked to leave the lab for the day and you will NOT be able to participate in the investigation and will receive zero points for the entire lab.

2.

PROCEDURE
1. You will work in tables of four students for this lab, but each person will do the procedure. Each table should have the following supplies which you will need to obtain: a. 4 test tubes, each containing one of the following saline solutions (You will need to label each test tube with one of the following % solutions.): 1) 0.30% saline solution 2) 0.89% saline solution You will get these solutions from 3) 2.0% saline solution stock solution bottles. 4) unknown % saline solution b. 4 disposable transfer pipettes (one in each test tube for each saline solution) c. 16 new microscope slides (4 per student) d. 16 cover slips (4 per student) e. One grease pencil f. About 16 tooth picks (4 per student) PLEASE dispose of toothpicks in the Sharps Box. g. 4 masks (1 per student) h. 8 cotton balls (2 per student) i. 4 alcohol pads (1 per student) j. 4 lancets (1 per student)

k. 4 band-aids (1 per student) l. A prepared blood smear slide (#1 from slide boxes) m. 4 sheets of white paper (1 per student) to put individual slides and supplies upon. n. 1 bottle of ethanol (EtOH) o. 2 microscopes (1 per pair of students)

Lab Exercise: Erythrocyte Osmosis, continued Each student should get the following a. Gloves (obtain from glove containers on the counter). Do not put gloves on until ready to work. b. Goggles or wrap-around safety glasses (supplied by you) c. Biohazard lab coat. Do not put a lab coat on until ready to work. Put gloves on before putting a lab coat on. 2. Each student, using a grease pencil, marks a circle on each of four microscope slides. Number each slide.

The number on the slide will correspond to the appropriate % saline solution. 3. Each student should lay out, on their white piece of paper, the following so they are ready and handy to use before continuing: Alcohol pad Cotton ball Band-aid Lancet 4. Saline test tubes Marked slides (4) Cover slips (4, one for each slide)

Apply eye protection, mask, and gloves. Leave the hand to be stuck ungloved.

As long as blood is being used in lab, EVERYONE will use respiratory and eye protection as well as gloves. (You may remove eye protection when looking through the microscope.) EACH student will do the following procedures. 5. Put two drops (2 gtt) of one of the saline solutions in the circle on a slide. Repeat this for each saline solution so that each slide has a different solution. Therefore

slide #1 will have 2 drops (gtt) of 0.30% saline, slide #2 will have 2 gtt of 0.89% saline, slide #3 will have 2 gtt of 2.0% saline in its circle, and slide #4 will have 2 gtt of unknown saline. 6. Clean the stick site on your finger with an alcohol pad, allow the alcohol to dry, stick the site as described by the instructor, and drop one SMALL drop of blood into the saline in each circle on each of the slides. Wipe the stick site with a cotton ball between slides so saline solutions do not become cross contaminated. Work quickly so blood will not coagulate.

7.

Using a toothpick, gently mix the blood and saline within each circle. Use a separate toothpick for each circle. Be careful to not contaminate the contents of one circle with the contents of another. Gently place a coverslip over the blood saline solution. Lab Exercise: Erythrocyte Osmosis, continued 8. 9. Bandage your finger. Discard your used gloves. Apply new gloves. Try very hard to NOT touch blood.

10. Wait 2 minutes. While waiting, observe a prepared blood slide under all dry magnifications so you know what normal erythrocytes look like. You will NOT observe under oil immersion. Note the shape and appearance of the normal RBCs on the prepared slide. 11. Observe your saline slides under all dry magnifications (NOT oil) until you see definite changes. 12. Periodically compare your slides and record your observations in the data tables. 13. Did your observations support or disprove your hypotheses? 14. Clean up: Into sharps containers: Slides Toothpicks Cover slips Transfer pipettes Lancets Biohazard bucket with red plastic biohazard lining Gloves Alcohol pads Cotton balls Other blood contaminated soft debris Broken glass container Test tubes - Unused saline solutions are dumped into sink. - Test tubes are rinsed then discarded into the broken glass box.

15. Wash your hands. Apply new gloves. 16. Completely disinfect your microscopes with alcohol. Pay particular attention to the objective lenses, stage, condenser lens, and knobs touched by your hands. Properly store microscopes in cabinet. 17. Disinfect your table and your assigned bench area to include sink handles. 18. Complete the data table and finish answering the questions. 19. Wash your hands again before leaving the lab.

ERYTHROCYTE OSMOSIS LAB INVESTIGATION


The following pages contain the PRE-LAB QUESTIONS, the DATA TABLE, and the POST-LAB QUESTIONS. EACH STUDENT will submit a set of prelab questions, their own data, and post lab questions. Only this page and the following pages need to be turned-in. Your pre-lab questions, data table, and post lab questions are due one week after completion of the lab. Student name:______________________________________

PRE-LAB QUESTIONS
PRE-LAB QUESTIONS MUST BE COMPLETED PRIOR TO COMING INTO LAB. Before submitting this lab paperwork, make a copy for yourself for study purposes. 1. During this lab, you will be obtaining blood by finger stick. Blood is, of course, a bodily fluid. In only two or three sentences for each disease, name the causative organism, and describe the following diseases and how they are transmitted. a. HIV infection (AIDS).

b.

Hepatitis C.

Lab Exercise: Erythrocyte Osmosis PRE-LAB, continued 2. Answer the following questions about safety measures you will use to prevent cross contamination of each other with bodily fluids. Refer to the Universal Precautions handout, Lab. Introduction hand-out, this lab procedure, and to the safety lecture given at the beginning of the quarter. a. List the five universal precaution steps.

b. Describe the hand washing procedure. When will you do it? (hint: at least three times)

c.

Other than gloves, describe what personal protection barriers you will use.

d.

How and when will you disinfect surfaces?

e. How will you dispose of gloves, cotton balls, and other soft, contaminated debris?

f.

How will you dispose of slides, cover slips, toothpicks, lancets, and transfer

Lab Exercise: Erythrocyte Osmosis PRE-LAB, continued 2. g. How will you disinfect and store the microscope?

3.

Define the term solution.

4.

List and describe the two components of any solution.

5.

Define a saline solution?

6.

Name and describe the process whereby water gets in and out of a cell across that cells membrane. (For this experiment we will assume NaCl is NOT crossing the membrane.)

7.

Define the following terms: a) isotonic

b)

hypertonic

Lab Exercise: Erythrocyte Osmosis PRE-LAB, continued 7. Define the following terms, continued: c) hypotonic

d)

crenation

e)

lysis

f)

hemolysis

g)

normal saline

8.

In your mind predict what will happen with RBCs in each known saline solution and write a falsifiable, working hypothesis for each of the solutions based your predictions. Hypothesize what the cells will look like. 1) 0.30 % solution.

2)

0.89 % solution.

3)

2.00 % solution.

RBC Osmosis DATA TABLE


Table for data obtained during lab. (Each student will complete a table,) Note: times are only approximate. Write in the correct time lapses. Student Name: % NaCl 0.30% 2 minutes Appearance of RBCs after applying blood 10 minutes 20 minutes

0.89%

2.00%

unknown %

Lab Exercise: Erythrocyte Osmosis POST-LAB Answer the following questions after completing the lab exercise. Each student will complete and submit one set of pre-lab questions, a data table, and one set of post-lab questions. Your pre-lab questions, data table, and post lab questions are due one week after completion of the lab. 1. a. Which known % saline solution produced crenated cells?

b.

Was the solution on the slide with crenated cells hypo-, hyper-, or iso-tonic?

c. Explain what was happening to the RBCs on the slide with crenated cells. In other words, what actually is happening to cause crenation?

2.

a.

Which known % solution produced hemolysis?

b.

Was the slide with hemolysis hypo-, hyper-, or iso-tonic?

c.

Explain what was happening to the RBCs on the slide with hemolysis.

3.

Was your unknown saline solution isotonic, hypertonic, or hypotonic? How do you know?

4.

Which percent saline solution would be best for irrigating tissues? Why?