LAB 1:



LAB 3:

“Measurement Madness!”

Diffusion/Osmosis/Tonicity

I. Objectives:

Today’s laboratory will have two distinct sets of exercises. The first exercise will introduce you to several of the basic and most commonly used laboratory techniques in cellular and molecular biology. The second set of exercises will introduce you to the concepts of diffusion, osmosis, and tonicity and demonstrate how these natural phenomena can be visualized and studied in a laboratory setting. By the end of today’s lab, you should be able to:

❑ Accurately measure and dispense small volumes of liquid ranging from 1 µl to 5000 µl

❑ Perform calculations and unit conversions involving weight, moles, molecular weight, concentration, and volume

❑ Accurately articulate the difference between grams and moles of a substance

❑ Prepare a solution for sterilization in an autoclave

❑ Define the terms "diffusion," "osmosis," and "tonicity" and explain why they are important for the survival of cells

❑ Explain how the rate of diffusion of a substance is affected by temperature, concentration gradient, and molecular size and weight

❑ Define hypertonic, hypotonic, and isotonic solutions and describe the effects of placing living cells in each type of solution.

II. Safety Considerations

• Do not touch the agar plates used to observe diffusion. These plates contain either potassium permanganate or crystal violet, which are strong dyes that can permanently adhere to (stain) clothing.

• All of the chemical reagents used in this laboratory (NaCl, colored water, tryptone, yeast, etc.) are non-toxic and can be disposed of down the sink or in the trash. See specific lab clean-up instructions at the end of this chapter.

III. Introduction:

A: Measurement Madness!

Your success in the laboratory portion of this course, in future lab courses that utilize molecular and cellular biology techniques, and your future career (e.g. health care, forensics, biotechnology - should you decide on a career in science) will depend on your ability to make accurate calculations and measurements. In today’s lab, we will develop your hands-on measurement skills by introducing you to the tools that are commonly used to measure volumes and weights. Since these quantities are usually very small in cell/molecular work, we will focus specifically on the special tools that have been created to accurately measure very small volumes and weights in the lab.

Although performing calculations and making these kinds of measurements are basic skills, they are not necessarily easy to master, and even if you have handled some of this equipment previously, do not assume that you are an expert, or even necessarily proficient. Be ready to be careful and to learn and improve your skills even if you’ve done some of these things before! If you are unsure or just not confident at any point of this lab, it is your responsibility to ask the lab instructor or a TA for assistance.

1. MEASURING VOLUMES OF 1 mL OR LESS: care and use of micropipettors

MICROPIPETTORS ARE INDISPENSABLE TO MOLECULAR AND CELLULAR BIOLOGISTS BECAUSE THEY ALLOW THE ACCURATE MEASUREMENT OF VOLUMES OF 1 ML OR LESS. IF YOU HAVE NOT DONE SO ALREADY, IT IS VERY IMPORTANT THAT YOU KNOW THE FOLLOWING SIMPLE UNITS OF THE METRIC SYSTEM OF MEASUREMENT:

1 L or l (liter) = 1,000 mL (milliliters) = 1,000,000 µL (microliters)

For example, 650 mL = 0.65 L and 200 µL = 0.2 mL. You will need to practice making these kinds of conversions over and over again until they become second nature (and easy to do in your head). You will need to be able to do them quickly all the time in a cell/molecular biology laboratory setting, and we will start practicing today.

The type of micropipettor we will use is shown below. Each instrument is calibrated for a narrow range; e.g. 1-20 µL, 20-200 µL, and 200-1000 µL. We refer to each micropipettor by the highest volume it is able to administer: P20, P200 and P1000, according to the ranges above. Each micropipettor is labeled accordingly on the top of the plunger.

The volume is adjusted by turning the black wheel in the top-center of the handle. As the wheel turns, the reading in the front window changes. Be careful not to force the wheel beyond the volume of the micropipettor (e.g., a P20 should not be adjusted to above 20 µl) since this can strip the gears and severely damage the instrument. The volume is read in the window in the front of the instrument. Unfortunately, each of the three pipettors uses a different scale. The table on the next page should help you determine the volumes for each micropipettor. Refer to this table often as you are learning how to use the instruments, and after a while you won't need it anymore.

A micropipettor tip should always be placed on the end of the shaft prior to use. There are two sizes of tips for the pipettors that we will be using: one for the P1000 and one that fits both the P200 and P20. To place a tip on the shaft, open a box of tips and gently insert the narrow end of the shaft into a plastic tip. Then, after removing the tip from the box, you may also gently twist it on further with your fingers to ensure a snug fit. Do not pound the shaft into the plastic tip as this may bend the shaft of the micropipettor.

To measure a liquid: Gently press down on the plunger to the first stop position. (This is the position where you will first feel resistance to your pressing.) Then place the tip into the liquid to be measured (just below the level of the liquid’s surface) and gently and slowly release the pressure on the plunger. Then remove the pipette from the liquid and transfer the contents in the tip to the container you wish to fill. (The liquid will remain in the tip as you do this because it is being held in by the vacuum you created inside the micropipettor when you released the pressure on the plunger.)

To expel the liquid from the tip: Gently push the plunger down to the first stop position and then slightly past this position. It is possible to continue until you reach the second (final) stop position—but try to completely dispense the liquid by just passing the first stop. You may use either your thumb or the first finger to depress the plunger, whatever is most comfortable. If you are transferring the contents of the tip into a tube that already contains liquid, express the contents into the liquid, below the level of its surface. If you are transferring the contents of the tip into an empty tube, place the tip on one of the inside surfaces of the tube while expelling. (This will enable capillary action to help you release all the contents of the tip into the tube; if you do not do this, some liquid may remain in the tip.) As you expel, keep the pressure on the plunger. Do not release the pressure until you have removed the tip from the tube. (Otherwise you’ll just suck the liquid right back up into the tip!)

The best technique is always to watch the liquid to be absolutely sure that you got some in the pipette tip and that all of it was expelled. As you become used to using the micropipettors, you will get a good sense of how different volumes appear in a tip. This will help you avoid pipetting errors, particularly those that occur when you accidentally pick up the wrong pipettor. Some pipettors have an ejector on one side. After use, the tip can be popped off by pushing down on the ejector button at the top of the pipettor. If your pipettor does not have an ejector, you’ll have to remove the tip with your fingers.

Reading the windows on the micropipettors

|pipettor |Top value |Reading IN WINDOW at top value |sample reading |actual volume of sample reading |

|P1000 |1000 uL |1-0-0 |0-7-7 |770 µL |

|P200 |200 uL |2-0-0 |0-7-7 |77 µL |

|P20 |20 uL |2-0-0 |0-7-7 |7.7 µL |

Some do's and don'ts with micropipettors

Micropipettors are expensive instruments and should be handled with extreme care. Please, never 'mess around' or 'play' with these instruments. Each one costs about $285 and can be easily damaged by carelessness and inattention.

• Always use the appropriate pipettor for the volume you desire.

The P20 is used for volumes of 1 µL to 20 µL.

The P200 is used for volumes of 20 µL to 200 µL.

The P1000 is used for 200 µL to 1000 µL.

• Remember never to turn the wheel past the maximal volume for the pipettor. This breaks the instrument and it must then be sent in for repair (which costs about $50-$150).

• Always hold the instrument upright.

• NEVER hold the micropipettors with the tip above horizontal.

• Remember to depress and release the plunger slowly so that the liquid in the pipette tip does not splash into the shaft of the pipettor. If the shaft becomes contaminated, all downstream samples can become contaminated. If fluid enters the shaft, please call this to the attention of your instructor or a TA so that the instrument may be cleaned without damage. Please do not attempt to remove the contaminating fluid yourself.

• When placing a new tip on the end of the shaft, do so with care and attention.

2. measuring volumes GREATER than 1 ml: USING SEROLOGICAL PIPETTES

To accurately measure volumes larger than 1 mL, glass or plastic serological pipettes are used. These are available in a variety of volumes; we will be using 5 mL and 10 mL pipettes in this class. (The pipette size refers to the largest volume that it can deliver.) A pipette pump is used to get liquid into the pipette. You will be learning in the "To Do" section how the pumps are used.

3. MEASURING WEIGHTS OF 10 G OR LESS

To accurately measure the weight of a dry substance, particularly when the amount is small, plastic weigh boats or weigh paper are generally used. A small spatula is used to carefully transfer the powdered reagent to the weigh boat or paper and the substance is then weighed on a scale. Since the weight of the boat or paper needs to be subtracted from the total weight in order to get an accurate measurement, the weigh boat or paper is first placed on the scale and the scale is "zeroed" or "tared" (set to 0.00) before the substance is added for weighing.

When making solutions in the lab, it is critical that you understand the difference between weight (or mass), moles, molecular weight, and molarity. These terms are defined below. Make sure you commit them to memory.

1. Weight is a measurement of the gravitational force acting on an object. Near the surface of the Earth, the acceleration due to gravity is approximately constant; this means that an object's weight is roughly proportional to its mass. Therefore, because all our measurements will be used on planet Earth (there will be no field trips to Venus or Mars this semester!) we will use the terms weight and mass interchangeably in this class. Please remember, however, that weight and mass are not equivalent terms in physics.

2. A mole is defined as 6.02 ×1023 atoms or molecules of a substance. Note that if the molecule has a large molecular weight, one mole of that substance will weigh more than one mole of a molecule that has a lower molecular weight.

3. Molecular weight (also called formula weight) is defined as the weight of one mole of an atom or molecule. The most common unit of expression for molecular weight is g/mol. NaCl is a fairly small molecule, so 6.02 ×1023 molecules (one mole) of NaCl doesn’t weigh much (58.44 grams or about 2 ounces). On the other hand, one mole of human chromosome 1 (a molecule containing 247 million base-pairs of DNA) would weigh 160 billion grams, which is over 170,000 tons!

4. Molarity (M) is defined as the number of moles per liter of a solution of a substance. Obviously, the molarity of a solution can be manipulated by a researcher since it depends on how much of the substance is added to how much solvent (usually water). For example, 1 liter of 1 M solution of NaCl would contain 58.44 g of NaCl.

4. WORKING WITH SOLUTIONS

The concentrations of solutions in the lab are usually given in molarity (moles/L). However, they may also be expressed in grams/L or as a percent. By definition, 1 g/mL = 100%, so conversions can easily be made between these three methods of expressing solution concentration. For example, NaCl has a FW of 58.44. You have a 5 M solution of NaCl. What is its concentration in g/L? What percent solution is it?

(5 moles/L)(58.44 g/mole) = 294.65 g/L

(294.65 g/L)(100%/(1 g/mL)(1 L/1000 mL) = 29.465%

You will need to learn to do these kinds of conversions quickly and easily. In addition, you will find the following formula very helpful when working with solutions in the lab:

(Vi)(Ci) = (Vfi)(Cfi) where Vi = initial volume,

Ci = initial concentration

Vf = final volume

Cf= final concentration

Suppose, for example, that your stock solution of EDTA is at 2 M. You wish to make 50 mL of a 0.8 M solution of this reagent. How would you do it? What you really need to know is how much volume of the original solution you need to add to how much water to achieve the more dilute solution you are after.

Vi = (Vfi)(Cfi) = (50 mL)(0.8 M) = 20 mL

Ci 2 M

So you would need to add 20 mL of 2M EDTA to 30 mL of water to make 50 mL of 0.8 M EDTA solution.

B: Diffusion/Osmosis/Tonicity

Diffusion, osmosis, and tonicity are fundamental phenomena that govern cellular and organismal function.

1. DIFFUSION

Diffusion is the “random walk of particles” (paraphrased from Einstein, who first proved the molecular dynamics of the phenomenon) due to the fact that no substance can ever be brought to a temperature of absolute zero. Particles in solution (or in gas/air) are in constant motion and the direction of a particle at any given time is random. The net effect of diffusion is that over time, particles will tend to move from areas of high concentration (order) to areas of lower concentration (less order). This is a practical example of entropy, the Second Law of Thermodynamics. Summarized differently, things go down their concentration gradient (from high to lower concentration)…or, said another way, left to their own accord, molecules tend to spontaneously spread out and become less ordered over time. For example: Imagine that you are in a room with no cross-drafts and someone at the other end of the room strikes a match. Eventually you will smell the match, because the molecules that are perceived (by receptors in your nose) as having "odor" have diffused throughout the room.

When it comes to diffusion across membranes – such as into and out of membrane-bound cells – atoms or molecules such as ions and gasses tend to move from the side where they are highly concentrated to the side where they are less concentrated, if they can get across the membrane. In general, gasses and water are able to diffuse across cell membranes but charged atoms or molecules and large molecules (like proteins) cannot. They must enter and leave the cell through various processes that will be discussed at length in lecture, including endocytosis, exocytosis, and transport through transport proteins embedded in the membrane. There are many factors that can influence the rate of diffusion, including temperature, the difference in concentration across the area through which the substance can freely diffuse, molecular size, and solubility.

2. OSMOSIS

Osmosis is a special case of diffusion. Osmosis refers specifically to the movement of water across a membrane due to a concentration gradient. The net movement of water spontaneously tends to diminish the solute concentration differences across the membrane. Osmosis is easily observed when a wilted plant is provided water. When the plant is wilted, the cells have lost water and become limp. When water is added, it moves through the plant xylem to the cells and enters them, making them turgid. As a result, the plant "perks up" (assuming you haven't already killed it due to lack of water!).

3. TONICITY

Tonicity refers to the relative concentrations of non-penetrating (non-permeable) ions across a membrane. When compared with blood (which has a lot of dissolved salts and other membrane-impermeable substances), plain water is hypotonic (it has fewer non-permeable particles in it than blood). As a result, if you were to place blood cells into plain water, there would be a considerable concentration gradient, with a high concentration of dissolved substance inside the cells, and essentially none outside the cell. As a result of this concentration gradient, the tendency would be for the solute particles to cross the membrane. However, if the particles cannot cross the membrane (i.e. they are non-permeable), the only thing that can move to decrease the order (the concentration difference across the cell) is water. Indeed, water moves into blood cells when the cells are placed in a hypotonic solution - so much water, in fact, that the cells lyse (burst open)!

A hypertonic solution is one that has a higher concentration of non-permeable solutes than the solution on the other side of the membrane. If we were to place blood cells into a hypertonic solution, the cells would shrink due to water leaving the cells in order to decrease the concentration difference of solutes across the cell membrane.

An isotonic solution has the same concentration of dissolved solutes as that of the solution on the other side of the membrane. In an isotonic solution, blood cells will neither lyse nor shrink.

There are many interesting applications of osmosis and tonicity. For example, to check a human fetus for chromosomal abnormalities (such as Down Syndrome), a small amount of amniotic fluid is withdrawn from the sac that surrounds the embryo. This sac contains cells from the fetus that have sloughed off into the surrounding medium. The cells are then placed in a mildly hypotonic solution, which causes water to move into the cells by osmosis. Since animal cells lack cell walls, this causes the cells to swell and gently burst open, releasing the chromosomes into the surrounding solution. The chromosomes are then stained, ordered, and photographed to look for abnormalities, a process called karyotyping.

IV. Things to Do:

NOTE: Before proceeding with these activities, make sure you can define the following terms: solute, solvent, solution.

PART A. MEASUREMENT MADNESS!

1. Practice using the micropipettors. Although you are working in groups of 3, each person should practice with these precision instruments and make sure they feel comfortable with them and can operate them properly. Practice pipetting the following volumes. Use the colored liquid at your bench and pipette into a weigh boat so that you can see the size of the drop expelled. Pipette each of the volumes below three times (into the same weigh boat) and compare the sizes of the drops side-by-side.

a. Set a P1000 to measure 0.5mL (500 µl) of colored water. Place the appropriate tip on the P1000. Depress the plunger to the first stop. If you aren’t sure where the first stop is then continue to push the plunger in further until you cannot push it in any more. At this point you are past the 1st stop and are into the “ejection” region of the pipettor. Past the first stop is into the un-measured region – don’t go here unless you are ejecting your sample!

To withdraw the volume you set on the dial, you need to stop at the first stop! Read the previous sentence again! If you ever go past the first stop and then take up liquid, you will be beyond your desired volume and may have difficulty ejecting all that you have taken up. Once you and your lab partners are confident that you know where the first stop is on the plunger, depress the plunger to the first stop, submerge the tip (only) into the liquid and then slowly release the plunger to take up the liquid. Did the pipette tip bubble when you depressed the plunger? If so, you depressed the plunger after the tip was submerged. This would be a mistake if you were trying to not disturb your sample. You should always depress to the first stop PRIOR to submerging the tip. Do not remove the tip from the liquid until the plunger is completely released. It is advisable to watch the liquid go into the tip.

Once you have taken up the 0.5 ml of liquid, look at the tip. Are there any bubbles in the liquid (in the tip)? If so, you released the plunger too rapidly so some of the sample flew up high into the tip (and possibly contaminated/ruined the pipettor). Alternatively, bubbles will result in the tip if the tip comes above the surface of the sample before the plunger is fully released. So, don’t release the plunger too fast, and keep the tip below the surface the whole time the sample is being taken up! As soon as you have a good 0.5 ml sample, pipette it into the weigh boat. Repeat and place the second (and 3rd) samples near to the first sample.

Question (your answer should go in your Results section): Do all of the samples appear to be the same size?

b. Repeat the above procedure with the P200, the appropriate tip, pipetting 90 µL of the colored solution. Each person should repeat this at least 3 times. You can use the same weigh boat that you used the first time; just be sure to rinse out the weigh boat first.

c. Rinse out your weigh boat and dry it carefully. Using the scale, tare a weigh boat. Pipette 1 mL deionized (DI) water onto the weight boat. (The DI water comes from the lab tap with the white cap. If you can’t find it, ask your instructor or a TA.)

Questions (Record in your lab book Results section): How much does the 1mL drop weigh? Tare the weigh boat again and pipette 500 µl onto the weight boat. How much does this weigh? Now tare again and pipette 200 µl. How much does it weigh? How much does 100 µl weigh? How much does 7 µl weigh? Be sure to record all of these values in your lab notebook.

d. Now, PLOT the relationship between water volume and mass on graph paper and tape the graph into your lab notebook. Is the result a straight line (i.e., is the relationship linear)? If not, what do you see instead?

2. For volumes of 1-10 ml serological pipets are often used. P5000 pipets are available, but in many instances are less convenient than serological pipets. Examine the 5 and 10 ml pipets that are available in the lab. Question: What volume does each division represent?

3. Serological pipets require a device of some sort to draw up and dispense liquids (we do not allow mouth pipetting in any instance). We’ll have several of these available for you. Try out the pipet pump and pipet bulb with both 5 ml and 10 ml pipets. Before doing this, note whether the pipets you are using are marked TD or TC. TD pipets are designed “To Deliver”. These pipets are designed to leave a bit of liquid in the tip once the sample is dispensed. Therefore, do not dispense the last little bit that remains. TC pipets are “To Contain” and are designed to deliver the correct amount if all of the contents are “blown out” of the tip (using the device—not your mouth).

4. Practice drawing up and dispensing from the serological pipets using the colored liquid supplied or just use water.

5. Next, pipet 2.5 ml into a weigh boat, weigh it and record its weight. Question: Based on what you learned from plotting the smaller volumes, how much should it weigh? How close were you to that weight? Repeat this for 5, 7.5, 8.2 and 10 ml, recording the weights in your notebook. Question: How could you measure 12 ml with one of the 10 ml pipets supplied?

6. Examine the reagent bottle of NaCl. Note that the MW (sometimes also referred to as formula weight, FW) of NaCl is 58.44 g/mol. (This information should be clearly visible on the outside of the bottle.) Note also that the bottle is marked with the name of the manufacturer and a lot number. Record this information in your lab notebook.

7. You will now make 100 mL of a 1 M solution of NaCl. To do this, you will first need to calculate how much NaCl you will need to measure out in your weigh boat:

Perform the calculation that will give you the number of grams of NaCL that you need. Check the amount with the instructor before proceeding.

Now place a weigh boat on the scale and tare the scale. Then, using your spatula, add ______ g (from your calculation) of powdered NaCl to the weigh boat.

Fill up your graduated cylinder with about 90 mL of water. (DO NOT fill it all the way to 100 mL because the NaCl will increase the volume a little bit when added, and you want the FINAL volume of the solution to be exactly 100 mL.)

Add the water to your beaker and then add the powdered NaCl that you measured out. Then add a stir bar to the beaker and place the beaker onto one of the electronic mixers. Turn the mixer on so that the stir bar begins to move (it is metal and responds to a magnet in the mixer). It should not take long for the solution to become clear as all the NaCl dissolves into the water.

Pour the entire solution back into the graduated cylinder and add water to 100 mL. Then pour the solution back into the beaker. You now have 100 mL of a solution of NaCl that is exactly at 1 M (mol/L). Congratulations!

Question: By the way, did you notice how quickly the NaCl went into solution? What chemical properties of NaCl make it so soluble in water?

8. Working with your lab partners, you will now make 100 mL of LB broth. This is a nutrient-rich broth used to grow bacteria, and you will be using the broth you make today later in the semester(so be careful with your measurements!). Once you have made up your broth, you will pour it into the autoclave bottle (orange cap) for sterilization.

To make up your broth, add the following to an empty 250-mL glass beaker containing a stir bar:

1 g tryptone

0.5 g yeast extract

0.5 g NaCl

Using a graduated cylinder, collect 80 mL of tap water and add it to the beaker. Stir the powdered ingredients into the water until you have a clear solution. (It will be yellow in color.) Then pour the broth back into the graduated cylinder and add additional water to exactly the 100 mL mark.

Pool the broth from the graduated cylinder into a designated container supplied by your instructor. Your instructor will add a piece of autoclave tape to the lid. This tape looks white prior to autoclaving but will become striped during the autoclaving process. The tape therefore provides a visual mark indicating that the solution is sterile. An autoclave is a type of oven that sterilizes liquids and instruments by raising them to high temperature and pressure for a period of time. When solutions are autoclaved, they should be placed in a special autoclave tray so that if the glass breaks, the solution does not get all over the autoclave. The LB broth that you made will be autoclave (sterilized) for use later in the semester. The autoclave room is located in Humboldt Hall.

PART B. DIFFUSION/OSMOSIS/TONICITY

demonstration 1:

1. before class, a thin slice (~1/8" inch thick) of potato was placed in a Petri dish containing a strong salt (10% NaCl) solution. Enough salt solution was added to cover the potato slice. Another slice was also prepared and placed in a Petri dish with distilled water.

2. Using your fingers or the tweezers provided, pick up the potato slices and compare how they feel. Question: What is the difference? Record the results in your lab notebook.

3. Now interpret the results. Question: Does this experiment demonstrate osmosis, diffusion, tonicity or a combination of these? Explain.

DEMONSTRATION 2: Diffusion

4. Before class, six petri dishes of 1% agarose were prepared. The agarose was added to water, boiled, and then cooled, allowing it to solidify. After cooling, a few crystals of potassium permanganate (KMnO6; MW - 158 g/mol) were added to the center of three of the agarose plates and a few crystals of crystal violet (C25H30CIN3; MW = 408 g/mol) were added to the center of the other two.

5. One plate with potassium permanganate and one plate with crystal violet were then incubated at 37 o C (body temperature) overnight, one set of plates was incubated at room temperature overnight, and one set of plates was incubated in the lab refrigerator overnight. All the plates were incubated for exactly the same length of time and placed on the lab bench immediately before the lab period began.

6. Examine the plates. Questions: How does the MW of a substance influence its rate of diffusion through the agarose? How does temperature influence its rate of diffusion? Explain.

DEMONSTRATION 3: MOLES, WEIGHT, AND MOLECULAR WEIGHT

1. Both baggies (A and B) weigh exactly the same amount.

2. Questions: Assuming that the contents of these baggies represent molecules, which baggie (A or B) contains the most moles of the substance? Which substance (A or B) has the greater molecular weight (g/mol)?

3. Working with your lab partners, try to figure out a way to determine how many moles of rice grains are contained in the baggie of rice. Questions:

• What information do you need in order to make your calculation? How can you go about getting that information, using the equipment and materials available to you in the laboratory?

• This is a solvable problem – see if you can figure it out!

• Once you have figured out how to calculate the number of moles of rice grains in the baggie, calculate how much ONE mole of rice would weigh.

V. Lab Clean-Up

Before leaving the lab today:

• Place all used serological pipettes in the large plastic containers provided.

• Rinse your test tubes, spatula, stir bar, beaker and graduated cylinder, making sure they are free of colored water, salt solution, or powdered reagents.

• Wipe out weigh boats with a kimwipe and stack them the way you found them. (They can be reused by the next lab section.)

• Inform the instructor or TA if you are low or out of micropipettor tips or serological pipettes.

• Empty the contents of your solid waste beaker into the trash and rinse it out.

• Empty the contents of your liquid waste beaker into the sink and then rinse it out.

• Wash your hands.

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