BIL 266: PHYSIOLOGY LABORATORY

COURSE INTRODUCTION AND SYLLABUS FOR FALL 2001

1 CREDIT

This is a 1-credit introductory laboratory on the instrumentation and fundamentals of physiology. The laboratory experiments are designed to introduce students to common techniques and concepts in physiology as well as an opportunity to observe phenomena described in lectures and texts. Students should have completed or be currently enrolled in General Physiology (BIL265) The laboratory should demonstrate the complex and varied factors that influence physiological response and adaptation. Students are expected to have completed general biology, general chemistry, organic chemistry and at least a semester of calculus prior to taking this course.

COURSE INSTRUCTOR:	Dr. K. Sulllivan Sealey Office: Rm 25 Cox Science Building (basement) Phone 305/ 284-3013 e-mail: ksealey@miami.edu
GRADUATE TEACHING ASSISTANT:	Mr. Mark Gallagher Office: Rm 205 Cox Science Building e-mail: mgallagher@miami.edu
OFFICE HOURS/ HELP SESSION	WEDNESDAY AFTERNOONS 2 PM to 5PM Physiology Laboratory, Room 207 Cox Science Building
COURSE TEXTS	Ø UNIT Handouts from Instructor Ø Zao, P. 2000. PhysioEx: Laboratory Simulations in Physiology Ø Each student should have a General Physiology text book for reference in the laboratory course
LABORATORY SECTIONS	TUESDAY AFTERNOON - RX 1:40PM - 4:30PM THURSDAY AFTERNOON - RY 1:40PM - 4:30PM
FINAL EXAM SCHEDULE	TUESDAY RX and THURSDAY RY Section “R” exams Thursday, 6 December 2 to 4:30 PM

Course Expectations

Animal Care Protocols and Procedures

Physiology is the functioning of living organisms, and the laboratory material to be studied must, by necessity, be alive. The right to invade the living body is not to be granted lightly, there are certain responsibilities and ground rules that each student needs to understand and accept.

Special care is to be taken that animals suffer no pain. A sincere regard for the health and well-being of animals housed in the laboratory is to be maintained throughout the semester. Details of handling and caring for animals will be presented with each laboratory, but it is the student's responsibility that during the performance of an experiment that no pain is being inflicted. Disregard for animal care and handling protocols will result in immediate dismissal from the course and a failing grade. All students must take an active role in the animal handling and preparations.

Laboratory protocols and care of instrumentation

Any damage to laboratory property and equipment will be sustained by the student or students responsible. If you are not sure how to use equipment, be sure to consult your instructor or teaching assistant. Although the laboratory is conducted in an informal manner, your highest priority at all times should be learning. You are expected to attend all laboratory sessions, and stay for the entire time allocated.

You will need the following things for the laboratory:

· a bound laboratory notebook (provided)

· a dissection kit with extra scalpel blades (scissors, probe, forceps and scalpel – we suggest you purchase these at the medical school book store).

· lab coat or lab apron (provided by the instructor at the beginning of the lab period).

When you enter the laboratory, keep books and backpacks off the counters, wash your hands, and be ready promptly to start the laboratory session with your lab notebook, laboratory handouts, pens, pencils and a calculator. For your safety and protection, you need to wear a lab coat or apron. THIS COURSE WILL REQUIRE SIX TO EIGHT HOURS OF WORK PER WEEK OUTSIDE OF THE LABORATORY, maybe even more time with the preparation of your independent project report. This is the nature of directed learning in laboratories. Each exercise has three parts:

· the collection of information,

· the analysis of data, and

· the interpretation of results.

Each week, you are expected to READ your laboratory exercise before class, and have an understanding of general concepts to be covered that week. At the beginning of each laboratory session, your instructor will give a brief overview, then you will carry out the laboratory experiments in pairs. Organize the materials needed for the lab on your counter space as efficiently as possible. You may have a quiz over the lab or reading assignments.

You are required to MAINTAIN a laboratory notebook to record your lecture and procedural notes, original data and background research. This notebook will be vital for preparing your laboratory report. This notebook will contain notes, rough sketches, diagrams, and data tables made during the laboratory session. The course syllabus provides you with a schedule of assigned reports. You are required to COMPLETE laboratory reports on time and in the prescribed format.

LABORATORY REPORT FORMAT FOR GENERAL PHYSIOLOGY LAB

Laboratory reports will often be written at the end of a unit, and can include several weeks of experiments and exercises. The laboratory report is made up of five parts:

§ Statement of objectives, and expected results based on physiological principles – this section should only be a few sentences. – less than 100 words

§ Description of methods (using a flow diagram) and a presentation of your data and observations

§ Data analysis, including any statistical tests needed, graphs or charts

§ Background research and interpretation – what did you measure and why? Cite references of other studies with similar objectives or methods. You will be given some references with each exercise to help you get started in looking for background material on these experiments.

§ Literature cited or other resources used in your interpretation – all information, your text book, lab handout, articles, or websites used in writing the report need to be cited in the text, and listed at the end of the report in a standard journal format.

Laboratory reports should not exceed 4 –5 pages of text (exclusive of tables, and graphs). You need to include a table of the laboratory data collected (from your lab notebook). Reports should be type-written, single spaces in 12 point font.

DO NOT REPEAT INFORMATION PRESENTED IN YOUR LABORATORY HANDOUTS. You are bound as a student to the University of Miami Honor Code. Be sure you know how to use and cite references.

A guide to keeping a laboratory notebook

The ability to organize and record your laboratory work is a critical skill for any scientist. Taking the time to keep good notes on the protocol and recording your results in a logical manner will greatly facilitate writing the laboratory report. You will be given a bound laboratory notebook. Each week, make a general flow diagram of the methods in your notebook, note what steps come first, second, etc. Each entry in your notebook should be dated and initialed. The organization and completeness of this notebook will be evaluated for the final course grade. In a laboratory notebook, all information should be recorded in PEN, not pencil. If you make a mistake or want to re-write a data table, simple make one cross-out and initial this change. This is part of learning good laboratory technique. In this notebook, you want to keep your laboratory notes, diagrams of methods, data and observations from the laboratory as well as notes from any background research that you do to interpret your results.

When you read your laboratory assignment prior to lab, try to understand what information you will be collecting. Will you be making observations or measurements? Can you identify a hypothesis to be tested? What are the dependent and independent variables involved? Structure a table or chart in your notebook for recording that data.

NEW GUIDELINES ON INTEGRITY IN SCIENCE STIPULATE THAT YOU NEED TO SIGN (INITIAL) AND DATE EACH ENTRY TO YOUR LABORATORY NOTEBOOK. Your teaching assistant will be reviewing your notebooks with you on a regular basis, your initials and date are verification that the information is truthful and accurately reported to the best of your knowledge. All information must be recorded in SI units, but be sure you understand the quantity you are measuring. There are detailed conversion tables in the laboratory if you need references. The following table can be used as a handy guide:

Table 1: Common quantities, fundamental dimensions and SI units used in Physiology
QUANTITIES	DIMENSIONS	SI UNITS
Length, distance	L	Meter - m
Area, Surface	L^2	Square meter - m^2
Volume	L^3	Cubic meter - m^3
Time	T	Seconds - s
Velocity, speed	LT^-1	Meter per second - ms^-1
Acceleration	LT^-2	Meter per second squared - ms^-2
Mass	M	Kilogram - kg
Force	MLT^-2	Newton - N or kg m s^-2
Density	ML^-3	Kilogram per cubic meter - kg m^-3
Work	ML^2T^-1	Joule - J or Nm
Power	ML^2T^-3	Watt - W or J s^-1
Pressure, shear stress	ML^-1T^-2	Pascal - Pa or N m^-2 Atmospheres used in oceanography - Atm
Dynamic viscosity	ML^-1T^-1	Pascal Second - Pa s or N m^-2 s
Kinematic viscosity	L^2T^-1	Square meter per second - m^2 s^-1

Your ability to carefully carry out new methods and protocols is dependent on your attention to detail. Get all the equipment and glassware you will need for the experiment out on your lab bench. Check the glassware to be sure it is clean. There is a standard procedure for washing glassware to prevent contamination in an experiment.

Table 2: Standard procedure for the washing and storing of laboratory glassware

§ You are responsible for your own glassware used in the laboratory. Check that glassware is clean at the start of the lab experiment, and clean up after yourself

§ Do not mix glassware to be used with live organisms with glassware that has held fixatives or formalin. Glassware contaminated with formalin requires a special dichromate bath to decontaminate, or it would be best to isolate contaminated glassware in another part of the laboratory.

§ Standard glassware washing should be carried out in four steps:

1.) hot water scrub with glassware soap,

2.) Rinse at least four times with de-ionized water,

3.) Rinse glassware with 5% Hydrochloric acid (HCl) v/v ^† solution, repeat rinse (4x) with de-ionized water

4.) final rinse with distilled water, check for residues, and repeat washing as necessary. Beware of cleaners and solvents used in the laboratory that can contaminate counters and lab surfaces.

§ Glassware should be stored upside down on lint-free cloth in a closed cabinet.

§ If you are unsure of the status of glassware or pipettes, wash again prior to your experiment.

§ Broken, cracked or chipped glassware should be immediately reported to the teaching assistant, and discarded appropriately.

§ Pipettes should never be allowed to dry with reagents inside, soak all contaminated pipettes, and maintain used pipettes in vertical stands. Never mouth pipette. Use bulbs or pumps to avoid ingesting reagents.

Remember, your results depend on attention to detail. Each student is responsible for the condition and cleanliness of glassware used in their work.

^{† -} v/v means a "volume to volume" mixture of 5 ml. HCl made up to 100 mls with de-ionized water.

Table 3: Use of analytical and top-loading balances

The area around the balance should be kept clean and clear of reagents. Balances are expensive and delicate instruments; special care should be taken to

¨ Keep reagents from getting contaminated by using a clean spatula to remove the necessary amount. Never put reagents back into bottles, keep lids closed tightly and store reagents appropriately (reagent shelf, refrigerator or dessicator)

¨ Never put any reagents directly on the balance, use a weigh boat of the appropriate size for the amount of reagent you need.

¨ Start by checking your balance, and follow instructions to be sure the balance is LEVEL and READS ZERO with nothing on the pan

¨ Tarr your weight boat or reagent paper

¨ Slowly add your reagent to reach the amount needed. Double check your calculations for amounts needed for preparation of "volume:volume", "weight:volume" or molar solutions

¨ Immediately record all weights in your lab notebook, and clean all equipment you used, RE-ZERO the balance for the next user.

Laboratory Protocols

Follow laboratory protocols, especially preparing reagents and solution, using the balances, and washing glassware. Several rules and procedures are necessary in the lab to provide a safe and productive environment. Please review the following protocols with your teaching assistant:

§ No food or beverages should be brought into the laboratory. A good practice would be to wash your hands thoroughly at the beginning and end of each session. Eating or drinking in the lab will expose you to the risk of contamination from chemicals and pathogens. Your comfort and safety are essential for learning. You are required to wear a lab coat or apron.

§ Glassware and laboratory equipment is to be used and stored appropriately - see Table 2 for glassware handling. Your results depend on attention to details like clean glassware.

§ Know how to use and maintain laboratory equipment. Your results will also depend on the operation and maintenance of equipment - make sure you know how to use this equipment and report any malfunctions immediately to your teaching assistant. IF you are using the equipment for the first time, ask your instructor or teaching assistant for help.

§ Understand the University Honor Code and the definition of plagerism. You will be asked to summarize or evaluate material from books, the internet or research articles, be sure you understand how to correctly attribute information to the original author. You can consult with classmates, but you can not copy their work. CITE ALL RESOURCES YOU USED IN YOUR LAB REPORT.

Grading

Students that work consistently throughout the semester will be rewarded. Grades are based on the following criteria, final total percentages will be graded on a standard curve (+93% = A, >89% = A-. >86% = B +, etc).

CRITERIA FOR GRADING	DUE DATE		PERCENT OF FINAL GRADE
6 LABORATORY REPORTS - Typed lab report completed in required format, original data and lab notes in laboratory notebook	UNIT 1 UNIT 2A UNIT 2B UNIT 3 UNIT 4A UNIT 4B	25 or 27 Sept. 9 or 11 Oct. 23 or 25 Oct. 6 or 8 Nov. 13 or 15 Nov. 21 Nov.	50%
8 LABORATORY PROBLEM SETS – 2 hand outs and six exercises from the PhyioEx CD	See Class Schedule		24%
QUIZZES and attendance	Throughout Semester		6%
Methods presentation and paper	27 and 27 November		10%
FINAL EXAM	6 December		10%
TOTAL			100%

There are several reference handouts to be reviewed during the first week. Keep these together with your UNIT handouts, and refer to them throughout the semester:

Ø Introduction to Instrumentation – spectrophotometers and electrodes

Ø Introduction to Statistical tests

Ø Conversion tables for SI units

Ø “Fish as Model Systems” article

Ø Animal Rights Statement

Ø Handout on using automated pipettes

CLASS SCHEDULE FOR PHYSIOLOGY LABORATORY

DATES	LABORATORIES AND PROBLEM SETS	REQUIRED READINGS AND ASSIGNMENTS
UNIT 1: ACID –BASE BALANCE AND RESPIRATION
Week 1 28 Aug & 30 Aug	Introduction to Physiology Laboratory, · Laboratory Overview · Making reagents and standard buffers · Review of pipetting,, use of analytical balances · Glass washing, and lab protocols	READ: Syllabus, Laboratory Handouts
Week 2 4 & 6 Sept.	Acid Base Balance: · Use of pH meter , · Titrations and and Buffering Capacity	READ: UNIT 1 HANDOUT DUE: PHYSIOEX 5B Cell Transport Mechanisms
Week 3 11 & 13 Sept.	Ammonia Excretion: · Spectrophotometric assays, · Standard curves · Closed system respirometry, · Osmo-regulatory strategies	READ: UNIT 1 HANDOUT DUE: PHYSIOEX 49B Renal Systems Physiology
Week 4 18 & 20 Sept.	Aquatic Respiration – · Oxygen electrodes, · Closed and open system respirometry, · Hypoxia tolerance	READ: UNIT 1HANDOUT DUE: PROBLEM SET #1 – Oxygen Availability DECIDE: selection of paper and topic for presentation /report.
UNIT 2: ENERGETICS AND LOCOMOTION
Week 5 25 & 27 Sept	Protein and water content of muscle · Proximate analysis of tissue · Spectrophotometric assays · Standard curves	READ: UNIT 2 HANDOUT DUE: UNIT 1: EXCRETION/ RESPIRATION LAB REPORT
Week 6 2 & 4 October.	Buffering Capacity and performance of muscle · Definition of buffering capacity · Use of pH meters and titrations · Calculating buffering capacity	READ: UNIT 2 HANDOUT DUE: Problem Set #2 – Sample Size Determination
Week 7 9 & 11 Oct	Digestion: Lipid Assays Visceral Tissue · Gravimetric vs. spectrophotometric assays · Detection range of methods · Classes of lipids	READ: UNIT 2 HANDOUT DUE: UNIT 2A: MUSCLE PROTEIN AND BUFFERING CAPACITY LAB REPORT
Week 8 16 & 18 Oct	Digestion: Redox Reactions and Enzyme activity	READ: UNIT 2 HANDOUT DUE: PHYSIOEX 40B: Chemical and Physical Processes of Digestion
UNIT 3: TEMPERATURE ADAPTATION
Week 9 23 & 25 Oct	Temperature effects of muscle enzymes	READ: UNIT 3 HANDOUT DUE: PHYSIOEX 33 Cardiovascular Dynamics DUE: Unit 2B: DIGESTION LAB REPORT
Week 10 30 Oct – 1 Nov	Respiration and Q-10 effects	READ: UNIT 3 HANDOUT DUE: PHYSIOEX48B Respiratory System Mechanics
UNIT 4: NEURO AND SENSORY PHYSIOLOGY
Week 11 6 & 8 Nov	Neurophysiology - Giant nerve axon in crayfish - Dr. Evoy · Preparation of nerve · Use of oscilloscope · Nerve signal propagation	READ: UNIT 4 HANDOUT DUE: Unit 3: TEMPERATURE ADAPTATIONS LAB REPORT
Week 12 13 & 15 Nov	Muscle action - Frog preparation - Dr. Evoy · Preparation of isolated muscle · Muscle action · Interpretation of osilloscope	READ: UNIT 4 HANDOUT DUE: PHYSIOEX 16 B Skeletal Muscle Physiology DUE: Unit 4A: CRAYFISH NEUROPHYSIOLOGY LAB REPORT
Week 13 20 & 22 Nov.	No Labs this week – Thanksgiving Holiday	DUE BY 5 PM WEDNESDAY Nov. 22: UNIT 4B: FROG MSUCLE ACTION LAB REPORT
Week 14 27 and 29 Nov.	PHYSIOLOGY RESEARCH METHODS PRESENTATIONS‡	DUE: Methods Paper – not to exceed 4 pages in length, cite references.
December 3 and 4	READING DAYS, FINAL REVIEW SESSION
FINALS WEEK 6 DEC.	Final comprehensive examination on physiology laboratory techniques and experiments	TUESDAY RX and THURSDAY RY Section “R” exams Thursday, 6 December 2PM to 4 PM

‡ You will be assigned current research paper discussing a topic in physiology. This article will provide background information on a research question. Your job will be to find out information on the instrumentation and protocols used in this area of physiology. You will produce in a 3 to 4 page report including:

- experimental objectives

- outline of experiment, with flow diagram of protocols

- list of equipment needed, with description of instrumentation

Each student will have 10 minutes to make a class presentation to teach each other about the protocols and instrumentation used in physiological research.