BISC110: Series 1 Lab 1 Boot Camp

==

In this laboratory, we will review the metric system as it applies to laboratory science, and learn how to use some basic laboratory measuring equipment by preparing a cupric chloride or cobalt chloride solution. You will also become familiar with the compound microscope by using it to examine the ciliated protozoan Tetrahymena pyriformis. Finally, you will be making dilutions of the stock solutions of cupric chloride and cobalt chloride to determine a concentration that is non-toxic to Tetrahymena.

Objectives: In this lab you will learn:

1. Metric measurements and conversions
2. Use of basic laboratory equipment
3. Preparation of solutions
4. Proper use of the microscope
5. Measurement of specimens using the micrometer
6. Serial dilutions

Series 1 Lab 1 Scientific Investigation Boot Camp

PART I: METRIC MEASUREMENTS

Measurements in pounds, miles and gallons are still commonly used in the United States, but the metric system, which was developed in France in the 1790’s, has several advantages over the English system and is more convenient for scientific use. The metric system uses decimals and a system of prefixes to define the measurements of a variety of parameters. Since it is based on powers of ten, calculations using the metric system are simpler than the English system.

The metric units you will commonly use in the lab are:

To describe objects that are larger or smaller than the base units, a system of prefixes is used. The prefixes and corresponding exponential values you will commonly use to change the size of the base units are:

The power of 10 in the exponent column in the table above indicates the placement of the decimal point for that measurement. For example, 103 is equivalent to 1.0 x 103 and can be converted to 1000 by moving the decimal point 3 places to the right corresponding to the exponent value equal to 3. Similarly, 2.5 x 103 is converted to 2500 by once again moving the decimal point 3 places to the right. If the exponent is negative, the decimal point must be moved to the left the correct number of places. For example, we can convert 2.5 x 10-2 to 0.025 by moving the decimal point 2 places to the left. The use of exponents of 10 to place the decimal point is called scientific notation. Scientific notation is used to represent very large or very small numbers in calculations and scientific writing.

It is important to become familiar with measurements expressed in metric units and to be able to convert between these units (from grams to milligrams, for example). Conversions are straightforward since the power of ten between each unit is known. Since there are 1000 milligrams in each gram, a sample that weighs 1 gram also weighs 1000 milligrams. To keep track of the decimal point, 1000 milligrams is best written in scientific notation as 1.0 x 103 mg.

Practice converting between the following units by writing your answers in decimal form and in scientific notation:

PART II: BASIC LABORATORY EQUIPMENT

In the first section of today’s lab, you reviewed the metric system as it is used to describe lengths, weights, volumes and temperatures. This section contains descriptions of some of the laboratory equipment available to make these measurements. With these tools, it is possible to make solutions of known concentrations and to accurately measure portions of these solutions. In Part III of this lab, you will use some of the equipment to prepare a solution needed for Parts IV and V of the lab. You will be using several types of containers and measurement devices in the laboratory this semester. The following pages describe some of the basic tools needed in the first series of laboratories. As the semester progresses, you will be introduced to more complex equipment. Please review the following descriptions before you use the equipment today, and direct any questions to your instructor.

A. DESCRIPTIONS OF LABORATORY EQUIPMENT

Beakers are used to prepare solutions and can range in size from 10mL to 4L. Any volume markings present on a beaker are only approximate and are not an accurate indication of volume. Beakers are not used to store solutions.

Erlenmeyer Flasks are used to prepare solutions and microbiological media. They range in size from 25mL to 6L. Volume markings on an Erlenmeyer flask are only approximate and are not an accurate measure of volume.

Test tubes will be provided to you throughout the semester for experimentation. Round bottom glass test tubes will be used for making dilutions and biochemical assays. These tubes are identified by the diameter of the tube, so a 13mm test tube has a diameter of 13mm. A 16mm tube has a diameter of 16mm and can hold a greater volume of liquid than the 13mm. Centrifuge tubes can be glass or plastic, are often pointed at the bottom, and identified by the maximum volume they can contain. They are used in a centrifuge to separate biologic materials.

Graduated Cylinders are used to measure volumes from 10mL to 2L. The gradations on the cylinder are an accurate measure of the volume of liquid contained in the cylinder. The liquid in the cylinder will form a meniscus as shown in the diagram below. To measure the proper volume, the bottom of the meniscus must rest on the desired volume mark on the cylinder.

Serological Pipettes accurately measure volumes from about 1.0mL to 10mL using the gradations on the pipettes. They may be made of glass or plastic. Glass pipettes are washed and reused, while plastic pipettes are disposable. The markings on these pipettes are often in descending order, so to deliver 6mL using a 10mL pipette, the liquid must be drawn to the 4 mark on the pipette. To use the serological pipettes a green or blue pipette pump is attached to the top of the pipette. Never pipette by mouth. The pointed end of the pipette is then submerged in the liquid, and the liquid drawn into the pipette by turning the wheel on the pump until the correct volume is measured. To dispense the liquid from the pipette, simply depress the plunger of the pump. Some serological pipettes will dispense all liquid from the pipette tip; others are designed to retain a small amount of liquid in the tip. Remember the glass pipettes are not disposable. They should be placed tips down in the plastic pipette canisters at each bench after use.

Micropipettes are used to measure volumes less than 1.0mL. They may be fixed volume or adjustable volume pipettes. You are provided with 3 adjustable volume pipettes: one for 200-1000 microliters (μL), one for 20-200 μL, and one for 1-20 μL. To use a micropipette, attach a plastic tip to the end, depress the plunger on the micropipette to the first stop, and insert the tip into the liquid you wish to measure. Slowly release the plunger taking care not to trap any air bubbles in the tip or splatter any liquid onto the pipette base. Withdraw the tip from the liquid and dispense the volume into the desired receptacle by depressing the plunger as far as you can. Be sure to keep the plunger depressed until it is removed from the liquid, or you will remove some of the material you have just measured. Used tips are discarded in the trash at your lab bench.

Pasteur Pipettes are named after Louis Pasteur. They are glass dropper pipettes that require the addition of a separate bulb at the top to create the suction. Pasteur pipettes are not used to measure volume but are used to transfer liquid from one place to another. For example, Pasteur pipettes are often used to remove all the liquid from a tube after centrifugation.

Top Load Balances are used to weigh solid materials greater than 1.0 gram accurately. Either a plastic weigh boat or weighing paper is placed on the balance, and the balance is set to zero (tared). The solid material is then placed in the weigh boat or on the weighing paper for measurement. When the proper amount of solid material has been added, it is transferred to a beaker or Erlenmeyer flask, solvent added, and mixed until the solid material is dissolved.

Magnetic Mixers are used in the laboratory to facilitate the mixing of materials. They are particularly useful when making solutions. The mixer has a magnetic core that can rotate at various speeds. If a beaker filled with materials to be mixed is placed on the mixer and a magnetic stir bar added into the beaker, the mixer could now be adjusted to mix the material at the proper rate. The mixing will continue until the mixer is turned off. A magnetic wand is then used to remove the stir bar from the solution before it is brought to final volume.

Vortex Mixers are used in the laboratory to mix materials contained in test tubes. The force of the mixing is adjustable, and the mixer can be set to run only when a tube is inserted into the mixer receptacle. The force of the mixer should be set so that the liquid in the tube forms a vortex as it mixes, and the tube containing the liquid should always be pointed away from you or your lab partner. If a tube is more than 2/3 full, a vortex mixer should not be used because the liquid will splatter out of the tube.

Centrifuges are instruments that use centrifugal force to separate biological materials. You will be using several types of centrifuges this semester. Clinical centrifuges are bench top models that accommodate various sizes of centrifuge tubes depending upon the type of holder installed. Refrigerated centrifuges are used when it is necessary to maintain the biological material at a constant cool temperature and can also accommodate several sizes of tubes. Microcentrifuges are used for very small plastic centrifuge tubes called microfuge tubes. All centrifuges require that the centrifuge tube containing the biological material be placed across from a tube of the same weight. We call this “balancing the centrifuge.” Tubes cannot be balanced by filling them to equal volumes, as these may not have the same weight. They must be balanced by weight, using a scale.

B. PRACTICE EXERCISES WITH PIPETTTES

1. Practice using the 10, 5, 2 or 1 milliliter pipettes by transferring the following volumes of deionized water into 13mm test tubes. Observe the volume differences. To insure the best accuracy, it is important that the pipette chosen to measure a specific volume has a total volume close to the volume to be measured. For example, the best pipette available to measure a volume of 1.7mL is a 2.0mL pipette.

Remember that many serological pipettes have the volume markings in descending order, so be sure to draw the liquid to the appropriate mark on the pipette so the correct volume is dispensed.

2. Adjustable micropipettes are very expensive, so please take great care in using them. To use a micropipette, attach a plastic tip to the end, depress the plunger on the micropipette to the first stop, and insert the tip into the liquid you wish to measure. Slowly release the plunger taking care not to trap any air bubbles in the tip or splatter any liquid onto the pipette base. Withdraw the tip from the liquid and dispense the volume into the desired receptacle by depressing the plunger as far as you can. Be sure to keep the plunger depressed until it is removed from the liquid, or you will remove some of the material you have just measured. Be sure never to hold a pipette upside down or sideways when liquid is in the tip. Also, if you feel resistance when changing the volume setting, stop immediately and ask your instructor for help. The following website [1] has more detailed information about micropipette use.

Practice using the micropipettes by completing the following exercise. Keep in mind that this is an exercise designed to allow you to practice and test your pipetting technique. When carrying out experiments, it is always important to choose the best pipette for the volume desired.

a. To test your micropipetting prowess and/or to calibrate your P1000, P 200, and P 20 micropipets, first label 6 microfuge tubes (1-6) and weigh them. Record the weights in the table below.

b. Following the table below, pipet the specified volumes into the pre-weighed microfuge tubes prepared above using either your P20, P200 or P1,000, as specified, ;then reweigh them. Record all weights.

c. Calculate the weight of the water in each tube in grams. (1000 microliters of water should weigh 1 gram at room temperature.)

d. If the water in any tube weighs more or less than 1 gram, your pipetting technique may need revision. Repeat steps 1-3 for the tube that is off the expected weight. If your water weight is significantly off after several repeated attempts, your pipet (or your technique) may need adjustment. Ask your instructor to watch your technique and/or recalibrate your pipet.

PART III: SOLUTIONS

When doing biological experiments, it is essential that the exact concentration of every solution be known. It is also important to know the pH of a solution since pH is often critical in biological reactions. Buffered solutions, which can absorb minor challenges to pH, are often used to maintain the pH of a solution. Different ways of describing the concentration of a solution as well as pH and buffers are reviewed below.

A. CLASSIFICATIONS OF SOLUTIONS

1. Percentage by weight (w/v) Solutions The number of grams in 100mL of solution is indicated by the percentage. For example, a 1% solution has one gram of solid in a final volume of 100mL solution. To make this type of solution properly, you should weigh 1.0g of the solid material and dissolve it in slightly less than 100mL of solvent. Once the solids have dissolved, you can bring the volume up to 100mL. If one liter of a 1% solution is needed, then 10g of solid would be dissolved in 1000mL of solution to maintain the 1% ratio of solid weight to solution volume.

2. Percentage by volume (v/v) Solutions In this case the percentage indicates the volume of the full strength solution in 100mL of dilute solution. For example, a 60% ethanol solution is made by mixing 60mL of 100% ethanol with 40mL of water. If only 10mL of a 60% ethanol solution is needed, then 6mL of 100% ethanol would be mixed with 4mL of water.

3. Molar Solutions A 1 molar (1M) solution is a solution in which 1 mole of a compound is dissolved in a final volume of 1 liter (1L). For example, the molecular weight of sodium chloride (NaCl) is 58.44, so one gram molecular weight (1 mole) is 58.44g. If you dissolve 58.44g of NaCl in a final volume of 1 liter, you have made a 1M NaCl solution. To make a 0.1M NaCl solution, you could weigh 5.844g of NaCl and dissolve it in a final volume of 1L water.

Sample Problem: Convert a 0.2M solution of NaCl into %(w/v) units. 0.2M = 0.2mol/L = (58.44g/mol)(0.2mol)/L = 11.7g/1000mL = 1.17g/100mL = 1.17%(w/v)

4. Buffered Solutions A buffered solution resists changes in pH. The pH of a solution is a measure of its acidity, and is defined as the negative log of the hydrogen ion concentration. The pH scale ranges from 0 to 14 where 0 is the most acidic, 14 is the most basic, and 7 is neutral. Since pH is a log scale, the difference between pH 5 and pH 6, for example, is a factor of 10. Buffers are used when biological samples need to be kept within a narrow range of pH to maintain activity. The enzymes involved in biochemical reactions often require a narrow pH range. This range is usually 7.2–7.4 for human and animal tissues. A phosphate buffer is commonly used in the biology laboratory, because it exhibits excellent buffering capacity in the neutral pH range. It is made up of a mixture of sodium monobasic phosphate (NaH2PO4) and sodium dibasic phosphate (Na2HPO4) dissolved in water.

B. SOLUTION PREPARATION FOR PARTS IV AND V

To prepare the solutions needed for Parts IV and V of this lab, the 4 students at one lab table will prepare two different solutions. One pair of students will prepare 100mL of a 0.11M solution of copper (II) chloride dihydrate (MW=170.5) and the other pair of students will prepare 100mL of a 0.26M solution of cobalt chloride hexahydrate (MW=237.9).

1. Calculate the correct weight of the material to yield 100mL of the proper molarity for the cupric or cobalt chloride solutions and record all calculations in your lab notebook. What would be the millimolar concentration of your solution? Have your lab instructor check your calculations before you weigh the material.

2. Put on gloves before handling chemicals in the solid state. Latex, vinyl or nitrile gloves are available on the bench at the front of the laboratory.

3. Using one of the top loading balances, weigh the correct amount of solid material and transfer it to a 250mL beaker.

4. Using a graduated cylinder, add 85mL of deionized water to the beaker. Add a magnetic stir bar and mix on a magnetic mixer until all the solid material has dissolved. Remove the magnetic stir bar.

5. Transfer the solution into an empty 100mL graduated cylinder and bring the volume to exactly 100mL with deionized water.

6. Transfer the solution to an empty storage bottle. Label the bottle with the name and concentration of the solution, your initials, your lab section and date.

PART IV: MICROSCOPY ==

Most cells measure about 1–100 micrometers (µm) in diameter. This size is smaller than can be detected by the unaided human eye, and so microscopes are needed to visualize cells and their component parts. The compound light microscope can magnify to about 1000 times the actual size of the specimen and can resolve details as fine as 0.2µm. In this part of today’s lab, you will learn to use the compound microscope by examining several types of cells.

A. CARE OF THE MICROSCOPES

Each bench space in the laboratory has in the cabinet below a compound microscope and a dissecting microscope. We will only be using the compound microscope today. Remember at all times that your microscope is a precision optical instrument and must be handled carefully. When removing the microscope from the cabinet, do not jar or drop it, always carry it upright with one hand below the base and the other hand on the arm of the microscope. Place the microscope at least 6 inches from the edge of the bench. When returning the microscope to the cabinet, check that:

1. the microscope light is turned off before the microscope is unplugged; 2. all lenses have been cleaned with lens paper, especially the oil immersion lens; 3. the lowest objective lens is near the stage and the stage itself is lowered; and 4. the microscope is covered.

B. PARTS OF THE MICROSCOPES

Figure 1 contains a diagram of a compound microscope and may help you locate some of the parts referred to in the following explanation. The compound microscope derives its name from the two sets of lenses it uses to magnify objects. These lenses are the objective lens, which can be found on the rotating nosepiece near the stage of the microscope, and the ocular lens, which is in the eyepiece. Your microscopes are equipped with several objective lenses, ranging from low to high magnification, including one oil immersion lens. The microscope magnifies by shining light from the light source through the iris diaphragm that limits the diameter of the light beam. The condenser lens focuses the light through the specimen that is on the stage. The stage is movable in order to view different parts of the specimen. The image we see is formed under the ocular lens by the objective lens and is a mirror image of the actual specimen.

C. REGULATION OF ILLUMINATION

1. The illumination intensity knob is located on the right side of the microscope just below the on/off switch. It has a setting range of 1¬-10 with 10 being the brightest level. This knob should ordinarily be set to 7.

2. Another way of adjusting illumination is by changing the position of the condenser lens. The condenser lens adjustment knob is located below the specimen stage and on the left side. It allows the user to move the condenser lens assembly up or down. As you move the condenser lens up, closer to the specimen, it concentrates (condenses) more light on your specimen. You will need to make this adjustment as you go up in magnification, so that you will have sufficient illumination.

3. The condenser aperture diaphragm is located below the specimen stage on the condenser lens assembly. It is an adjustable opening, which allows you to make fine adjustments in illumination. The lever, which adjusts the size of the aperture, faces the user. By sliding the lever to the left or right, you may adjust the illumination to the correct level for your specimen. Changing the size of this aperture also affects the amount of contrast in the image. Thus, adjusting the condenser aperture involves finding the brightness level, which gives you the best combination of illumination and contrast. This is the method used most often in adjusting illumination in the light microscope.

4. Another method of adjusting illumination is by using the field aperture diaphragm. This is mentioned here for the sake of completeness, as most light microscopes have an adjustable field aperture. However, the microscopes in the 110 lab lack this adjustability, which allows the user to obtain illumination that is uniformly bright and free from glare (Köhler illumination).

D. HOW TO LOCATE SPECIMENS USING A COMPOUND LIGHT MICROSCOPE

1. Place specimen slide on microscope stage and secure with clamping arm.

2. Using either the 4X or the10X objective lens, find the specimen and focus it using first the coarse and then the fine adjustment knobs. Whether you begin with the 4X or the 10X objective lens depends on the size of your specimen. Very small specimens, such as bacteria, may not be clearly visible using the 4X lens, so the 10x lens may be required for this step.

3. To increase the magnification of the specimen, rotate the nosepiece to the 40X objective lens and focus using the fine adjustment knob.

4. Immersion oil must be placed on the slide to use the 100X objective lens. After the specimen has been found using a lower magnification, move the objective lenses out of the way and place a small drop of immersion oil onto the slide. Rotate the nosepiece until the 100X (oil immersion) lens is selected. Once the 100X objective lens is in contact with the oil, focus the specimen using the fine adjustment knob only. Use of the course adjustment at this time could result in damage to the 100X objective lens or to the slide. Only lens paper should be used to remove oil from the 100X objective.

E. CALCULATION OF TOTAL MAGNIFICATION

Total magnification of the specimen is determined by multiplying the magnifying power of the ocular and objective lenses. For example, a 10X ocular and a 40X objective together give a total magnification of 400X (Table 1). This means that the specimen appears 400 times larger when viewed with a microscope than its actual size.