Materials Included In Kit

Additional Materials Required

Prelab Preparation

Simulated blood solution: Weigh out 1.5 g of active yeast powder and 0.5 g of safranin O. Obtain 2 L of DI water in a large flask or beaker. Add the yeast and stir for a few minutes until large grains are no longer visible. At this time stir in the 0.5 g of safranin O until the solution is a uniform deep red color.

Safety Precautions

Simulated blood solution may stain skin and clothing. Have students wear chemical splash goggles, chemical-resistant gloves and a chemical-resistant apron. Remind students to wash their hands thoroughly with soap and water before leaving the laboratory. Please review current Safety Data Sheets for additional safety, handling and disposal information.

Disposal

Please consult your current Flinn Scientific Catalog/Reference Manual for general guidelines and specific procedures, and review all federal, state and local regulations that may apply, before proceeding. Each disposable hemocytometer is a single-use item—do not reuse. Hemocytometers may be disposed of in the regular trash after use. Extra simulated blood solution may be disposed of according to Flinn Suggested Disposal Method #26b.

Lab Hints

• Enough materials are provided in this kit for 10 groups of students. This laboratory activity can reasonably be completed in one 50-minute class period. The prelaboratory assignment can be completed before coming to lab, and the data compilation and calculations can be completed the day after the lab.
• Prepare the simulated blood fresh. The solution does not store well.
• Counts should be taken within a few minutes after students have added a sample to the hemocytometer. After about 10 minutes the yeast cells will begin to settle into the creases of the grid and will be more difficult to accurately count.
• Advise students not to turn the 100X objective lens (if the microscopes have them) into the viewing position—hemocytometers are thicker than conventional microscope slides and may scratch the objective lens.
• The dilution factor used in the above equation for real blood samples was not included in the simulated blood equation for simulated blood (see the Background section) since the simulated blood sample was prepared to the proper dilution and does not need to be diluted further.

Further Extensions

As a follow-up or alternate activity, real blood samples may be used. Follow all proper health and safety precautions for real blood products if performing this extension. The proper dilution is 1:200 blood to saline solution. To calculate erythrocytes per mL:

• # of cells in five squares x 5 x 200 (dilution factor) x 10⁴ (volume factor) = erythrocytes per mL
• Coverslips should be placed over the sample injection area of the slide after the sample is added when using real blood. As always when using real blood, students should only handle their own materials and blood. All materials used should be sterilized before disposal—an autoclave or pressure cooker works best for sterilization.
• If these items are not available, all items can be soaked for 24 hours in a 15% bleach solution and then disposed of in biohazard bags. A local hospital or blood bank may be willing to assist you in disposal of blood materials.

Answers to Prelab Questions

1. What is the primary function of red blood cells?

   To circulate the body carrying oxygen gas to cells from the lungs and waste materials away from cells.

2. What makes red blood cells unlike any other types of cells in the human body?

   Red blood cells are enucleated after maturity, meaning that they lack a nucleus. This is likely why RBC’s have a shorter lifespan when compared to other cells.

3. If an individual lacks A, B, ad Rh proteins on the surface of their red blood cells, what is their blood type?

   O negative

Sample Data

Answers to Questions

1. Calculate the number of red blood cells per mm³ using Equation 1.

   Sample A: 113 x 5 x 10⁴ = 5,650,000 cells in mm³ of simulated blood
   Sample B: 94 x 5 x 10⁴ = 4,700,000 cells in mm³ of simulated blood

2. Assume that sample A is from a woman and sample B is from a man. Are the number of cells for each sample within the normal ranges?

   5.65 million cells per mm³ is slightly higher than the average range for women, and may indicate polycythemia, however further testing would need to be performed by a medical professional for confirmation. A count of 4.7 million cells per mm³ falls into to the normal range for a man .

3. A woman has an RBC count of 3.9 million per mm3. Would this woman be considered anemic?

   Not necessarily. Anemia is a condition of low hemoglobin levels and is not always associated with low RBC counts. Anemia may arise from excessive bleeding, iron deficiencies, blood clots, or other conditions not associated with low RBC counts.

4. A medical professional draws blood from a vein in the arm, which appears blue when viewed through the skin. When the blood sample is obtained, it is red in color. Explain.

   Deoxygenated blood is a deep red color which appears to be blue through the skin because the dark color doesn't reflect light well. Once the blood is viewed without the obstruction of the skin, the characteristic “blood red” color can be observed.

References

Fankhauser, David B. PhD. Hemacytometer and Diluting Pipet Practice. Accessed March 2007, from University of Cincinnati Clermont College website: http://www.biology.clc.uc.edu/Fankhauser/labs/anatomy_&_physiology/A&P202/Blood/Blood_Counts_practice.htm

Scott, Ann Senisi; Fong, Elizabeth. Body Structures and Functions, 8th edition; Delmar Learning; Clifton Park, NY (1993).

Introduction

A hemocytometer is an instrument used to manually count cells under a compound microscope. Red blood cell counts are used to provide insight into medical conditions, such as anemia or polycythemia.

Concepts

• Blood
• Blood cell counting
• Blood typing

Background

Erythrocytes or red blood cells, also referred to as RBCs, are biconcave, disc-shaped cells that are unlike any other cells in the body. The unique shape of red blood cells is due to the fact that they are enucleated—the nucleus is squeezed out of the cell—after maturing in the bone marrow (see Figure 1). The lifespan of a red blood cell is usually about 3–4 months which is shorter than that of most other types of cells in the body. This is probably because they lack a nucleus, ribosomes, and mitochondria. Dead or damaged RBCs are filtered out of the blood by the liver and spleen where they are broken down. The iron portion of the heme is then recycled for use by maturing RBCs in the bone marrow.

The shape of erythrocytes increases their pliability and allows for easy movement through narrow capillaries as they exchange oxygen and waste materials with every cell in the body. RBCs contain a protein called hemoglobin that binds gases for transport between the lungs and body tissues. The name hemoglobin is derived from the composition of the protein, which consists of an iron-containing heme group and a globin protein. Each red blood cell contains millions of hemoglobin molecules. Oxygen binds to hemoglobin molecules to form a complex called oxyhemoglobin.

After oxygen is distributed to body cells, some of the carbon dioxide waste binds to the RBCs. Most of the carbon dioxide combines with plasma to form bicarbonate. A common misconception is that veins or deoxygenated blood are blue in color. Deoxygenated RBCs are in fact red in color. This deep red appears blue through the skin because the dark red color doesn't reflect light well. Both veins and arteries are a neutral beige color, and blood is never blue. Veins always carry blood to the heart, and arteries only carry blood from the heart. In most cases, veins transport deoxygenated blood and arteries transport oxygenated blood, but there are a few exceptions to this general rule. Blood that is seen in most injuries is venous blood. Arterial blood may be seen in major injuries and can be identified as it comes out in spurts synchronous with the heartbeat.

Blood typing (A, B, AB, O and Rh factor) is determined by the presence or absence of specific proteins on an individual’s red blood cells. A basic genetic principle is that an individual’s inherited genes determine which proteins are present on a person’s RBCs. In the ABO blood typing system, the blood proteins are referred to as A and B proteins. The presence or absence of the A and B proteins on the red blood cell’s surface determines an individual’s blood type. Individuals whose red blood cells contain protein A and lack protein B have type A blood. Those with protein B who lack protein A are called type B. Individuals with both protein A and protein B are called type AB, and individuals who lack both proteins have type O blood. The Rh factor is what is often referred to as positive or negative in reference to blood type. Any of the ABO blood types may or may not also have the Rh protein. If an individual has RBCs with both the A protein and the Rh protein present, the blood type is called A+. Blood types must be correctly matched in blood transfusions. If a person is given an incompatible blood type to their own, their body will not recognize the proteins on the cells, and the body will “tag” the cells as foreign material for termination by the immune system.

Normal RBC counts for women are between 4.2 and 5.4 million per mm³, and for men, 4.5 to 6.2 million per mm³. RBC counts lower than the normal range may indicate a medical condition, such as anemia. Specifically, anemia is a condition of low hemoglobin levels, usually due to a low red blood cell count, although this is not always the case. Anemia may arise from excessive bleeding, iron deficiencies, blood clots, radiation therapy or blood diseases, such as hemophilia, leukemia and sickle-cell anemia. RBC counts above normal range may indicate polycythemia, a condition of excess hemoglobin in the blood. Polycythemia may be due to chronic exposure to low oxygen levels (in people who live at high altitudes), “blood doping” (boosting the number of RBCs using drugs or special training to enhance athletic performance) or bone marrow diseases. If abnormal RBCs counts are found, further analysis or testing needs to be performed by a medical professional to find the specific cause or condition.

Red blood cells typically make up about 40% of the total volume of blood. Plasma is the liquid portion of the blood which contains many proteins, electrolytes, glucose, cholesterol, hormones, and metabolic waste products. Another important component of blood is white blood cells, or leukocytes. Generally, white blood cells are much larger in size and are found at a much lower concentration than red blood cells—3200 to 9800 cells per mm³. (The number of white blood cells tends to this higher end of the range when the body is fighting off an infection.)

A hemocytometer, also referred to as a hemacytometer, is an instrument used in medical settings to manually count cells. Although many types of cells and bacteria may be counted using a hemocytometer, originally it was designed to count red blood cells hence the name (hemo or hema = blood, cyto = cell). Hemocytometers have one counting grid per chamber that can be viewed in detail under the compound microscope. A 10-microliter (µL) blood sample is added to the injection site and the sample is pulled across the chamber by capillary action. Each grid on the hemocytometer contains nine large squares measuring 1 x 1 mm. The center square is divided into 25 smaller squares measureing, 0.2 mm x 0.2 mm, each of which is further subdivided into 16 0.05 mm x 0.05 mm squares (see Figure 2). Explanation of Equation 1:

(Sum of the five 0.2 mm x 0.2 mm squares counted) x 5 (there are 25 small squares, of which only 5 are counted—these 25 small squares = 1 mm²) x 10⁴ (the volume factor to calculate the number of cells per mm³.

Experiment Overview

The purpose of this activity is to use a hemocytometer to count simulated red blood cells in a simulated blood sample. Red blood cell concentrations will be counted under the microscope and RBCs per mm³ will be calculated using the same methods used in a real clinical setting.

Materials

Prelab Questions

1. What is the primary function of red blood cells?
2. What makes red blood cells unlike any other type of cells in the human body?
3. If an individual lacks the A, B and Rh proteins on the surface of their red blood cells, what is their blood type?

Safety Precautions

Simulated blood may stain skin and clothing. Wear chemical splash goggles, chemical-resistant gloves and a chemical-resistant apron. Wash hands thoroughly with soap and water before leaving the laboratory.

Procedure

1. Working in groups of two, determine which partner will count the first sample. Obtain 10 μL of simulated blood using a needle-tip pipet (see Figure 3).
   {10819_Procedure_Figure_3}
2. Open the hemocytometer wrapper, and add the 10 μL of simulated blood to the sample injection opening labeled “A” (see Figure 4).
   {10819_Procedure_Figure_4}
3. Focus the microscope on the center of the counting grid under the 4X scanning objective.
4. Switch to the 10X lens and fine focus the sample, and then switch to the 40X objective.
5. Count the number of red simulated blood cells in each of the 0.2 mm x 0.2 mm squares shaded and labeled 1–5 in Figure 5 and record the values in the data table. Count all the cells in all 16 small squares within the shaded squares. If there are cells in the crevasses outside the 0.2 mm x 0.2 mm square, do not count them. If there are clumps of cells visible, simply estimate the number of cells in each clump. Record all data for each square in the data table.
   {10819_Procedure_Figure_5}
6. Add up the total cells counted for all five squares and record the sum in the data table.
7. Have the other partner repeat steps 1–6 using the sample injection opening labeled “B.”
8. Dispose of the materials according to your teacher’s instructions and answer the Post-Lab Questions.

Student Worksheet PDF

10819_Student1.pdf