BIOLOGY IN FOCUS MAINTAINING A BALANCE Chapter 2 Transport dissolved nutrients and gases Estimating the size of red and white blood cells perform a first-hand investigation using the light microscope and prepared slides to gather information to estimate the size of red and white blood cells and draw scaled diagrams of each Guided method for estimating the size of red blood cells 1. Estimate the field of view under low power. Place the mini-grid (or transparent ruler) on the microscope stage and view under the 10 objective. This gives a magnifi cation of 100 times (eyepiece objective = 10 10 = 100). Use the grid or ruler to estimate the diameter of the fi eld of view in mm and μm (1 mm = 1000 μm) (see Fig. 2.4, step 1 and 2). For example, if this fi eld of view has a diameter of approximately 1.6 mm, it is 1600 μm. If you are using a grid slide, each of the larger grids is 1 mm and the smaller are 0.1 mm (see Fig. 2.4). Linked to page 38 Figure 2.4 Sequence of steps to estimate the size of the field of view 1 grid line at edge of field Focus on large grid under 100. field of view diameter of field of view 2 Focus under 400 cannot see grid, therefore need to calculate. 1 mm 0.6 mm grid lines on ruler or minigrid slide Mag. 100 (low power) Field = 160 m Mag. 400 (high power) 160 m Field = = 400 m diameter 4 1
2. Calculate the field of view under high power. Swing the high power objective into place. You will no longer see the distance from one grid line to the next and so you need to calculate the fi eld of view. If your high power lens has a magnifi cation of 40, everything you look at will appear four times bigger than under the previous ( 10) lens, and the diameter of the fi eld of view that you can see will be four times less. The fi eld of view can therefore be calculated as follows (see step 2 of Fig. 2.4): low power = 100; high power = 400 high power fi eld of view = 100/400 = ¼ the size of your low power fi eld of view. 3. View a prepared slide of blood and estimate the size (length) of red and white blood cells. Place the prepared slide of a blood smear on the microscope stage under low power. Once it is in focus, rotate the high power objective into place. (Do not change the focus before doing this, as the school microscopes are parfocal they remain in focus while you rotate objectives and will not crush your slide.) Distinguish between the numerous small red blood cells and the few, larger white blood cells (the nucleus in the white blood cells takes up a stain and appears dark blue or purple in colour). The nuclei in white blood cells may vary in shape (see Fig. 2.3b on the previous worksheet). Estimate the size of a red blood cell. Count or estimate the approximate number of red blood cells that would fi t across the diameter (imaginary centre line) of the fi eld of view (using 400 magnifi cation). Using this number and the known diameter for the fi eld of view, calculate the size of each blood cell. (See Fig. 2.5.) For example, if about 50 red blood cells fi t across the fi eld of view and the fi eld of view = 500 μm, then each cell is 500/50 = 10 μm. 4. Improve accuracy and validity. Repeat this process three times and fi nd an average size for red blood cells. Figure 2.5 Estimating the size of a red blood cell Place blood smear under low power then high power. Estimate number of blood cells across diameter. EXAMPLE ONLY (Do not use these figures in your practical) Count: ±55 red blood cells 400 m (diameter of field of view) 55 (number of red blood cells) = 7.3 m = estimated size of one red blood cell 5. Estimating the size of white blood cells Since there are so few white blood cells, it is not possible to count the number of white cells across the diameter and therefore much more diffi cult to estimate how many would fi t across the diameter. Another method of estimating their size is to compare their proportions with that of red blood cells. For example, is the white blood cell half the size or twice the size i.e. how much bigger or smaller is the white blood cell compared with the red cell? Use this estimate to calculate their size. To increase accuracy and validate the conclusion based on the data gathered, repeat the process with three different white blood cells and obtain an average. 6. Draw a scale diagram of each type of blood cell as follows. (a) Draw a line of a particular length (e.g. 1 cm or 2 cm). This will be your scale bar that represents 10 μm. (b) Using this scale, draw a circle on your paper to represent the average size of each cell. 2
(c) Now add the detail to the cells, for example: red blood cell: represent the cytoplasm, cell membrane and use appropriate shading to show its biconcave shape white blood cell: draw the nucleus, cell membrane and cytoplasm. (d) Label all parts of each cell. Results Estimation of size of red and white blood cells. Scale diagrams of red and white blood cells. Record your results at the end of this worksheet under the section Results. You are required to record all estimates, show your working for any calculations and follow the rules for drawing scientifi c diagrams, a skill learnt in the Preliminary Course. Discussion and conclusion Answer all discussion questions. Re-read the aim and use your results to arrive at a valid conclusion. Investigation report: A microscopic investigation to estimate the size of red and white blood cells Aim: Materials: Safety: Method: 1. Place the mini-grid (or transparent ruler) on the microscope stage and view under the 10 objective. This gives a magnifi cation of 100 times (eyepiece objective = 10 10 = 100). 2. Estimate the field of view under low power. Use the grid or ruler to estimate the diameter of the fi eld of view in mm and μm (1 mm = 1000 μm) (see Fig. 2.4, steps 1 and 2). 3. Calculate the field of view under high power. Using the 40 objective, calculate the fi eld of view (¼ the size of your low power fi eld of view). 4. View a prepared slide of blood and estimate the size (length) of red and white blood cells. Estimate the size of a red blood cell. Count or estimate the approximate number of red blood cells that would fi t across the diameter (imaginary centre line) of the fi eld of view (using 400 magnifi cation). Using this number and the known diameter for the fi eld of view, calculate the size of each blood cell (see Fig. 2.5). Repeat this process three times and fi nd an average size for red blood cells. Estimate the size of white blood cells compared with the red cell. Use this estimate to calculate the size. Repeat with three different white blood cells and obtain an average. 5. Draw a scale diagram of each type of blood cell as follows: (a) Draw a your scale bar that represents 10 μm. (b) Using this scale, draw a red blood cell and a white blood cell. Label all parts of each cell. 3
Results 1. Field of view 40 = mm or μm Field of view 100 = mm or μm 2. Field of view 400 = mm or μm Blood cell type Estimate 1 Estimate 2 Estimate 3 Average 3. Scale diagram of a red blood cell 4. Scale diagram of white blood cell Conclusion 4
Discussion questions 1. Describe how you could distinguish between red cells and white cells under the microscope. 2. Which are more numerous, red cells or white cells? Explain, using evidence gathered in the investigation. 3. Compare the size of a red blood cell and a white blood cell. Assess the reliability of the measurements that you obtained. 4. Explain why it was necessary to repeat the estimation process three times and fi nd an average for each cell type 5. Assess the accuracy of your results for each cell type and justify. (Re-read the background information on measurement in science before attempting to answer this question.) 6. Discuss and report on the diffi culties you had in carrying out this investigation. 7. Describe how you could improve the accuracy and reliability of your investigation. 8. From this investigation and your knowledge of biology, prepare a table to outline the similarities and differences in structure between red blood cells and white blood cells. 5
Extension work 1. Describe the ratio that Anton Van Leeuwenhoek used to estimate the size of red blood cells (see the background information on pages 37 38 of the textbook). 2. Outline the method he would have had to use to arrive at this conclusion. 3. Using your knowledge of the actual size of a red blood cell, calculate the size of the grain of sand that he used for comparison with a red blood cell. Show your working. For this to be true, what assumptions do you need to make? 6
Answers Discussion questions 1. Describe how you could distinguish between red cells and white cells under the microscope. Colour: white blood cells have no pigmentation, whereas red blood cells are red (appear pale pink under the microscope). Nucleus: white blood cells have a nucleus that may be irregular in shape and stained purple, whereas red blood cells do not have a nucleus. Size and shape: white blood cells are larger and round or slightly irregular in shape; red blood cells are smaller and fl atter with a biconcave shape. 2. Which are more numerous, red cells or white cells? Explain, using evidence gathered in the investigation. Red cells are more numerous. Evidence: within the high power microscopic fi eld, there are many hundreds of red blood cells and only one or two white cells visible. 3. Compare the size of a red blood cell and a white blood cell. Assess the reliability of the measurements that you obtained. White blood cells are larger approximately one and a half times the size of red blood cells. The reliability of the results is increased by obtaining three sets of results for red cells and averaging this, then obtaining three sets for white cells and fi nding an average for each. These results were similar and so the results are fairly reliable. 4. Explain why it was necessary to repeat the estimation process three times and fi nd an average for each cell type. Repetition of the process gives more reliable results because several estimates give a greater percentage coverage of the fi eld of view. 5. Assess the accuracy of your results for each cell type and justify. (Re-read the background information on measurement in science before attempting to answer this question.) (Students should compare their results with those found in scientifi c literature and then comment on the accuracy. The degree of confi dence in the estimate also affects accuracy and students should mention this). A sample answer is given below. The accuracy using this method is not very high it is simply an estimate of the size of red blood cells, based on the number across the diameter of the fi eld. Our estimate of the size of a red blood cell was 10 μm, whereas scientifi c literature shows that it should be closer to 7.5 μm. Limitations in the accuracy of our measurements arose when: measuring the fi eld of view (use of a ruler was less accurate than use of a mini-grid smaller calibrations would have given a more accurate estimate of the diameter fi eld of view, so our less precise measurements led to some inaccuracy counting/estimating the number of cells across the diameter of the fi eld we are not very confi dent that we counted correctly as the cells are very small and we had some gaps across the diameter and had to estimate the number of cells that would fi ll them comparing the size and proportion of white blood cells with that of red blood cells. 6. Discuss and report on the diffi culties you had in carrying out this investigation. Diffi culties could include: how precisely the diameter of the fi eld of view could be measured (e.g. ruler, no mini-grids availalble) diffi culties trying to observe and count how many red blood cells fi t across the diameter cells are very small and we had some gaps across the diameter and had to estimate the number of cells that would fi ll them. 7. Describe how you could improve the accuracy and reliability of your investigation. Reliability can be increased further by comparing results with other students in the class, or even averaging the results of all students in the class. 7
Accuracy could be improved using more precise instruments of measurement, higher magnifi cation (smaller microscopic fi eld), measuring instrument with smaller calibrations or simple more experience in microscope work. It is important to note that results can be reliable without being accurate. This is because repeatedly getting the same result does not necessarily ensure that it is accurate (close to the correct measurement). Once accuracy has been determined (by comparing the measurements obtained in this practical with results in scientifi c journals), steps should be taken, using more precise instruments such as mini-grids and greater care when counting. Accuracy could be greatly improved if the technology used were upgraded for example, using an electron microscope and digital measuring equipment but this is not feasible at school level. 8. From this investigation and your knowledge of biology, prepare a table to describe the similarities and differences in structure between red blood cells and white blood cells. Cell type Density (per ml blood) Size (diameter) Where produced Structure Function Red blood cells 5 6 million 7 8 µm Bone marrow (destroyed in liver and spleen) Small, round, flattened biconcave, no nucleus, red (contain haemoglobin 250 million molecules in each RBC) Transport oxygen: 4 oxygen molecules combine with each haemoglobin to form oxyhaemoglobin White blood cells 4 000 11 000 ~ 12 µm Bone marrow (destroyed in liver and spleen) Larger than red blood cells, no pigmentation (i.e. no colour), contain a nucleus which may be unusual in shape Function as part of the immune system in defence against disease Platelets 400000 ~ 3 um Bone marrow Disc-shaped cell fragments, 50% smaller than red blood cells, non-pigmented Release a clotting enzyme, thromboplastin, to seal blood vessels and prevent excessive blood loss Extension work 9. Describe the ratio that Anton Van Leeuwenhoek used to estimate the size of red blood cells (see background information at the beginning of this investigation). 25,000 times smaller than a fi ne grain of sand, i.e. 1: 25 000. 10. Outline the method he would have had to use to arrive at this conclusion. To do this, he would have had to understand the magnifying power of the microscope and lenses that he was using: estimate the fi eld of view, count how many grains of sand fi t across the fi eld of view, then divide the size of the fi eld of view by the number of grains of sand to get the size of each grain of sand. 11. Using your knowledge of the actual size of a red blood cell, calculate the size of the grain of sand that he used for comparison with a red blood cell. Show your working. For this to be true, what assumptions does one need to make? Red blood cell = 7.5 μm. Grain of sand is 25 000 times larger, i.e. 7.5 25 = 187.5 μm. 8