ROOM (TBA), SCHULER (CS) BUILDING, ANNANDALE CAMPUS, 8333 LITTLE RIVER TURNPIKE, ANNANDALE, VA

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1 NAS 162 Pre-Lab Northern Virginia Community College ROOM (TBA), SCHULER (CS) BUILDING, ANNANDALE CAMPUS, 8333 LITTLE RIVER TURNPIKE, ANNANDALE, VA Professor: Dr. Raj Bawa Phone: Northern Virginia Community College, 2009 NAS 162 NOVA 2009 Page 1

2 I. General Laboratory Information 1. The laboratory is located in room (TBA) of CS building on the Annandale Campus. Click here for the Annandale Campus Map. 2. Parking on the Annandale campus is not free. You may park in a visitor lot or purchase a parking sticker for the semester from the campus security office. 3. The laboratory will promptly begin at 10:00 A.M. on Saturday and Sunday and will take the better part of each day. There is no make-up or deferral for this lab. If you are more than 15 minutes late for the lab, there will be an automatic 5 point from your final lab score. Furthermore, if you are more than 30 minutes late, you will be given an F for the lab. 4. You must bring a lab coat, man s old shirt, smock or similar protective clothing that can be washed in bleach following the lab. 5. Shoes must cover your entire feet (i.e., no sandals or flip-flops). 6. You may not work in the lab without an instructor or instructional assistant present. 7. You are responsible for full compliance with safety regulations as set forth herein and other instructions given by Dr. Bawa. 8. You must leave our work area in clean condition and ready for use by the next class. All equipment and glassware must be left clean and in proper working order. Please notify Dr. Bawa if any of the equipment is inoperable or fails during use. 9. The cafeteria is not open so you must either bring food and drinks or else purchase them from machines on the campus. There will be a lunch break each day. 10. Your final lab grade will be based on the following point distribution: Quiz for the Pre-Lab (on your Blackboard site) Lab Participation Lab Examination on Day 2 (Exam 8) Total 10 points 20 points 70 points 100 points NAS 162 NOVA 2009 Page 2

3 II. General Safety in the Laboratory Safety is of utmost importance in the laboratory. Each student must learn and apply the safety rules as set forth below and all other safety precautions as instructed during the day of the lab. 1. Identify the location of and learn how to operate safety equipment available in the laboratory. 2. Learn two routes for evacuating the laboratory/building. 3. Learn the location of material safety data sheets (MSDS) and consult them for information about hazardous chemicals. 4. Report all accidents and spills immediately to Dr. Bawa. 5. Pipetting by mouth is prohibited. Use a mechanical pipetting device to transfer fluids. 6. Dispose of all chemicals in a safe and acceptable manner as instructed. 7. When dealing with body fluids (i.e., blood, saliva, urine, etc.) only work with your own or wear disposable surgical gloves and avoid skin contact. Bring your own gloves to the lab. 8. Wear protective clothing and understand and use aseptic technique in handling body fluids or microorganisms. 9. Disinfect all work surfaces before and after working with body fluids or microorganisms. 10. Discard all items contaminated with body fluids or microorganisms in designated biohazard containers. 11. No eating or drinking will be permitted in the laboratory. 12. Wash your hands thoroughly with soap and water before leaving the laboratory. Additionally, know and follow these rules during lab: 1. Keep all objects out of your mouth (pencils, pens food, drink, etc.) 2. A lab coat or similar type of protective clothing must be worn over street clothes. 3. Anchor your hair so that you do not touch or wipe your face or eyes at any time during lab. 4. Wash your hands before culturing and before leaving the lab. Pay particular attention to nail beds. 5. Begin laboratory work by placing all books, purses, etc. in a central area or on top of the carrels. 6. Thoroughly clean your working area with disinfectant before beginning work and after final cleanup. 7. Place all used broth cultures, old agar plates, etc. in the central disposal area near the sink before leaving lab. 8. Fire is a tool, not a toy. Keep the burner at least one foot from the table s edge. Turn it off when you are finished with relevant work. 9. Restrict unrelated conversation. Mistakes and accidents are likely to occur when you re not paying attention. NAS 162 NOVA 2009 Page 3

4 10. If you should knock over a culture, DON T PANIC! Cover the liquid with paper towels and then liberally pour disinfectant over the entire area. Wait 15 minutes before wiping up. Notify Dr. Bawa immediately! NAS 162 NOVA 2009 Page 4

5 III. The Light Microscope Microscope Podcast: click here Directions: 1. After the Web site opens, click on Launch BCC on itunesu 2. Follow the directions, it will open the itunes program on your computer. If you don t have itunes installed on your computer, you need to download it from 3. After your itunes opens, click on Medical Laboratory Technology 4. Watch and listen to the movies 1-10 listed there. A. Obtaining the Microscope 1. Obtain the microscope assigned to you by your instructor and remove the microscope cover. 2. Pick up the microscope and carry it by placing one hand under the base while grasping the arm with the other hand. B. Parts of the Microscope 1. Read the material in the laboratory text on the microscope (in the preface). 2. Locate all of the microscope parts. C. Readying the Microscope for Use 1. Unwrap the cord and plug in the microscope. 2. Turn the light intensity switch on. 3. Clean the ocular lenses with lens paper. 4. Clean the 40x objective lens with lens paper. The 100x objective should also be cleaned before and after use. 5. Put a slide on the stage. 6. Move the mechanical stage until the specimen on the slide is centered over the opening in the stage. 7. Make sure the substage condenser is all the way up and the diaphragm is open. 8. Rotate the scanning objective (4x, red band) in line with the oculars. Be sure it is locked in place by the spring lock on the revolving nosepiece. 9. Adjust the interocular distance as necessary for the interpupillary distance of your eyes. You should be able to see clearly with both eyes. D. Focusing 1. While watching the objective from the side, use the coarse adjustment knob to raise the stage until it stops. NAS 162 NOVA 2009 Page 5

6 2. Look into the oculars and turn the coarse adjustment knob so the stage moves downward slowly until the specimen is in focus. 3. If necessary, fine focus by turning the fine adjustment knob. E. Magnification Glass lenses in the oculars and objectives form an enlarged (magnified) image of the specimen being observed. The magnification of a typical ocular is ten times (10x). The magnification of an objective is indicated by a number (4x, 10x, 40x or 100x) stamped on it. Since the enlarged image formed by the objective in transmitted to the oculars and is further magnified, total magnification is determined by multiplying the magnification of the ocular (10x) times the magnification of the objective. For example, using the scanning objective (4x), the total magnification would be 40x (4x x 10x = 40x). F. Resolution A compound light microscope such as the one you are using in the laboratory will magnify objects effectively up to 1000 times. Any further magnification loses resolution, the ability to see details. The greater the resolution, the closer two points (objects) can be and still be perceived as separate points (objects). Points closer than 0.2 µm (200 nm) cannot be resolved with the compound light microscope. With the invention of the electron microscope during the first half of the twentieth century our ability to study cellular structure took a giant step forward because of its greater magnifying and resolving power. Limit of Resolution Eye 0.1 mm = 100 µm Compound Light Microscope 0.2 µm = 200 nm Transmission Electron Microscope 0.5 nm = 5 A G. Switching Objectives to Obtain Greater Magnification The microscope you are using is parfocal. Once the specimen is in focus under a low power objective, no further use of the coarse adjustment knob is necessary. Proceed as follows: 1. Center the specimen in the field of vision. 2. Carefully rotate the revolving nosepiece to move the next higher power objective into place while watching it to be sure it clears the slide. 3. If necessary, fine focus. 4. Repeat steps 1 through 3 each time you progress to a higher power objective. H. Contrast Cells and parts of cells are often quite transparent. To enhance the degree of difference between the lightest and darkest parts of a specimen, cells and/or parts are stained or the light intensity is changed. The light intensity can be varied by: 1. Adjust the diaphragm lever. 2. Alternatively, if adequate contrast is not obtained by adjusting the diaphragm lever, a. Raise or lower the sub-stage condenser using the sub-stage condenser adjustment knob, or b. Turn the light intensity switch to a higher or lower setting. I. Returning the Microscope NAS 162 NOVA 2009 Page 6

7 Turn the light intensity switch off. Rotate the scanning objective in line with the oculars. Remove and return the slide to its proper location. Turn the coarse adjustment knob to move the stage to its lowest position. Unplug the cord by grasping the plug and wrap the cord around the cord wrap. Return the microscope to its proper storage location and cover it. NAS 162 NOVA 2009 Page 7

8 IV. Respiratory Volumes The total capacity of the lungs is divided into various volumes. It is necessary that you become familiar with these volumes in order to understand the respiratory process. These volumes are determined by the use of a spirometer. (Also, review these concepts in Hole s Anatomy and Physiology) NAS 162 NOVA 2009 Page 8

9 V. ABO and Rh Blood Typing Blood Handling Safety Procedures: When using human or animal blood samples, you must protect yourself from any blood-borne infectious disease (hepatitis, HIV, etc.) Wear gloves and safety goggles while performing blood tests. Place all blood slides and toothpicks in a biohazardous container according to your instructor s directions. Wash tabletops thoroughly with 10% bleach solution at the end of the lab. Wash your hands with soap and water before leaving the lab. If a blood spill occurs, cover the spill with paper towels soaked in 10% bleach solution and immediately inform your instructor. Review the ABO blood groups: Click Here (Also, review these concepts in Hole s Anatomy and Physiology) Blood groups arise because of the expression of antigens (agglutinogens) on the extracellular surface of RBC membranes. In some cases these antigens are found only on RBCs; however, in many cases they are also found in other tissue types. A transfusion reaction occurs when antibodies (agglutinins) in a recipient s plasma interact with the RBC agglutinogens, leading to clumping (agglutination) of the donor RBCs and haemolysis. The first blood group to be described in detail was the ABO system, which gives rise to A, B, AB and O blood groups. The ABO system is currently the only system in which individuals naturally possess antibodies to non-self. Currently, there are 29 recognized blood group systems, including the Rhesus system, which is responsible for haemolytic disease of the newborn. An understanding of blood group systems has implications beyond transfusion medicine into areas such as transplantation, autoimmunity and population biology. (From: Anaesthesia & Intensive Care Medicine Volume 8, Issue 5, May 2007, Pages ) The main method of classifying human blood is on the basis of the inherited properties of red blood cells (erythrocytes) as determined by their possession or lack of the antigens A and B, which are carried on the surface of the red cells. Persons may thus have type A, type B, type O, or type AB blood. Blood containing red cells with type A antigen on their surface has in its serum (fluid) antibodies against type B red cells. If, in transfusion, type B blood is injected into persons with type A blood, the red cells in the injected blood will be destroyed by the antibodies in the recipient s blood. In the same way, type A red cells will be destroyed by anti-a antibodies in type B blood. Type O blood can be injected into persons with type A, B, or O blood unless there is incompatibility with respect to some other blood group system also present. Persons with type AB blood can receive type A, B, or O blood, as shown in the table below: The ABO and Rh groups in transfusion system recipient type donor red cell type donor plasma type ABO A A* or O A or AB ABO B B or O B or AB ABO O O only O, A, B, or AB ABO AB AB*, A*, B, or O AB Rh positive positive or negative positive or negative Rh negative negative or positive**, *** negative or positive** *Not if the patient s serum contains anti-a1 (antibody to common type A red cell in subgroup A patients). **Not if the patient is a female less than 45 years old (childbearing possible), unless life-threatening NAS 162 NOVA 2009 Page 9

10 hemorrhage is present and transfusion of Rh-positive blood is lifesaving. ***Not if the patient s serum contains anti-d (antibody to positive red cells), except under unusual medical circumstances. Blood group O is the most common blood type throughout the world, particularly among peoples of South and Central America. Type B is prevalent in Asia, especially in northern India. Type A also is common all over the world; the highest frequency is among the Blackfoot Indians of Montana and in the Sami people of northern Scandinavia. The ABO antigens are developed well before birth and remain throughout life. Children acquire ABO antibodies passively from their mother before birth, but by three months infants are making their own it is believed the stimulus for such antibody formation is from contact with ABO-like antigenic substances in nature. Erythroblastosis fetalis (hemolytic disease of the newborn) is a type of anemia in which the red blood cells of the fetus are destroyed by the maternal immune system because of a blood group incompatibility between the fetus and its mother, particularly in matings where the mother is type O and the father type A. NAS 162 NOVA 2009 Page 10

11 VI. Genetics Problems NAS 162 NOVA 2009 Page 11

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15 Karyotyping Exercise: Click Here ( NAS 162 NOVA 2009 Page 15