Procedures for Conducting the Lab Activity

Overview:

Theory: Lysozyme (obtained from chicken egg white) is an enzyme/protein that protects chick embryos from bacterial invasion.  The enzyme attacks the bacterial cell wall and ruptures the bacterial cell (called lysing the cell).  Thus it is an enzyme that lyses, hence the term lysozyme.  The lysozyme protein is also found in humans (i.e. in tears, lysozyme attacks bacteria that invade the eye from air contact).  Lysozyme does not present a health hazard for students conducting the experiment!

Screening experiments are designed to systematically vary one condition (called a variable) at a time to find what works or what works the best.  In this experiment, the variable to be screened will be the salt (sodium chloride) concentration that produces the best lysozyme crystals for one lysozyme concentration at one pH (amount of acidity), at room temperature.  Changing the temperature affects the crystal form and size of the crystals.Also, vibration can be a factor in crystallization so the sample tube racks should be stored in a quiet place to eliminate this variable!

For this experiment, we need a solution (buffer solution) that resists changes in pH as pH is an unstable variable. This buffer solution is created by using distilled water, distilled white vinegar, and sodium acetate (note: the amount of molecular water present in the sodium acetate will affect the results.  The standard sodium acetate for this experiment is sodium acetate trihydrate).  The buffer solution is produced prior to conducting the experiment at the "stock station" for the "experiment stations" to use.

Normally, there are eight "experiment stations".  They will get the buffer solution from the "stock station" and dissolve a pre-measured amount of lysozyme protein in it.  At this point all eight "experiment stations" have identical experiment set ups.  Each "experiment station" will then dissolve a different pre-measured amount of salt (sodium chloride) according to the table below.  Each "experiment station" may also dissolve different combinations of food coloring into their samples to add color and facilitate the identification of the different salt concentrations.

Station #                 % salt (sodium chloride)                    color*

     1                                   2.0%                                        none
     2                                   2.5%                                         yellow
     3                                   3.0%                                         peach
     4                                   3.5%                                         green
     5                                   4.0%                                         rose
     6                                   4.5%                                         blue
     7                                   5.0%                                         purple
     8                                   5.5%                                         turquoise

· use combinations of standard food coloring
 

One 400 ml beaker
One 50 ml conical measuring tube (*)                    (*) a 50 ml graduated cylinder may
Two 15 ml conical measuring tubes (*)                        be substituted if desired
Two pipets
Distilled Water (one liter is more than enough)
Distilled white vinegar (at least 62.5 ml)
One tube containing 3.4 gm of sodium acetate
waste container

1. Using a 50 ml conical measuring tube and a 15 ml conical measuring tube, measure 187.5 ml of distilled water into the 400 ml beaker.
2. Add and dissolve the pre-measured 3.4 gm of sodium acetate.
3. Add 62.5 ml of distilled white vinegar (again using the conical measuring tubes) and mix thoroughly.

* This produces 250 ml of sodium acetate buffer of  0.1 Molar strength with a pH of about 4.3.

Each of the "experiment stations" should have the following:

One array rack (square plastic 'tubing' with 8 holes)
Eight small culture tubes fitted into the 8 holes of the array rack
One Erlenmeyer flask
One pipet (cut the length of the pipets at "experiment stations" to about 4 inches)
One powder funnel
One Gelatin capsule containing the pre-measured lysozyme (0.4 gm)
One snap top tube of sodium chloride distributed per station in the % assigned to
        that "experiment station" (note: the kit contains 3 tubes of each salt % in each
        envelope-enough for 3 experiments). Be careful to only use one snap tube for
        each experiment
One marking pen
One strip of sealing film

Optional setup items:
Pre-measured food coloring in 10 ml beaker

Prior to beginning the experiment, put 10 ml of the buffer solution from the "stock station" into the Erlenmeyer flask for each "experiment station".  Use the 15 ml conical measuring tube.  If too much is added to the tube, some can be removed using a pipet.  Put the excess into the waste container.  Do not put it back into the 400 ml beaker!
 

Now you are ready to begin the experiment!

1. Place a clean dry powder funnel in the Erlenmeyer flask.

2. Now add the lysozyme (0.4 gm) from the Gelatin capsule.  Carefully pull off the cap (sometimes it helps to turn or twist the cap first) and pour the lysozyme into the funnel.  Tap the capsule gently to get all of the lysozyme out.  Also, tap the funnel.  Now, recap the capsule.  Be careful not to create dust and do not inhale any of the lysozyme powder.

3. To dissolve the lysozyme, let it sit on the surface of the buffer solution (the less lysozyme on the glass, the better).  It may be necessary to 'tap' the flask once on a padded surface to start the dissolving process.  After the lysozyme has dissolved (Patience is a virtue!), roll or swirl the flask once to incorporate any lysozyme powder clinging to the glass.  It is important to do this gently to avoid getting any bubbles into the solution!

You now have a buffer solution at each station with a lysozyme level of 40 mg/ml.
 

4. To add the salt (sodium chloride), open the snap top tube and very slowly add the salt to the Erlenmeyer flask while swirling it gently.  Stop adding the salt if it begins to accumulate on the bottom of the flask. But, keep it swirling.  Once the accumulation has dissolved, slowly continue adding salt.  This is to keep the concentration from becoming too strong in a local area of the solution which could cause the solution to turn cloudy. (That may happen even if you are careful if your station is assigned one of the higher concentrations of salt.)

Each "experiment station" will now have a different ionic strength as a different amount of salt was added at each station.  At his point, those stations with the higher concentrations of salt may have some clouding of the solution due to some lysozyme precipitating out in a non-crystalline form (an amorphous precipitate).
 

Note: Save the Gelatin capsules and salt snap top tubes.  You may want to replace the chemicals yourself  for future experiment evolutions and these capsules and tubes will be very helpful for that purpose!
 

5. Add food coloring to your sample if you wish.  Our experience is that it is desirable to use color to mark the samples.  Do not use too much color as the protein crystals absorb the food coloring readily and too much color produces almost opaque crystals.  The crystals produced will be more remarkable if you use only enough color to   produce colored transparent crystals.  Pastel colors produce beautiful crystals! There are many dyes available other than food coloring that you may wish to experiment with.  We recommend the following as a starting point using McCormick food coloring available in most food stores:

% salt (sodium chloride)      Color               Number of Drops Required

    2.0%                                 none                none
    2.5%                                yellow              basic yellow color
    3.0%                                peach               1 red, 3 yellow
    3.5%                                green               basic color
    4.0%                                rose                 5 red, 1 blue
    4.5%                                blue                 basic color
    5.0%                                purple             1 red, 1 blue
    5.5%                                turquoise        5 blue, 1 green

Once you have mixed the color, use only one drop (or less) of the color in your Erlenmeyer flask to keep the crystals from appearing too opaque!!

A. 'Kit' Instructions: The concept of the procedure packed with 'the kit' is for each station to make their assigned  salt concentration lysozyme solution and then put about ½ of it into the 10 ml beaker so that the other stations can fill one of their culture tubes at each of the stations.

   To do it this way, have the students at each "experiment station" mark their culture tubes with the marking pen with each salt concentration (i.e. 2%, 2.5%, 3.0% etc.)

   Then with a pipet,  have each "experiment station" fill the small culture tube in their array rack that is numbered the same as the concentration of salt at their "experiment station".

   Since we want to avoid introducing any bubbles into the solution, first squeeze the bulb of the pipet then put the tip in the solution and release the bulb to fill the pipet.  Move the pipet to the culture tube to be filled and put the tip of the pipet into the tube near the bottom.  Slowly squeeze the bulb to expel the liquid, withdrawing the pipet as the tube fills.  Do not overfill!  Filling to about 2/3 of the culture tube is fine!

   Next,  the students from the various "experiment stations" rotate from one station to the next filling their appropriately labeled culture tubes at the corresponding "experiment station".  Be sure to use the pipet located at each station to prevent contamination of the solution at each station.

   Once each "experiment station" has all eight culture tubes filled with the appropriate salt concentrations, use a small piece of sealing film to cap off each culture tube with an air tight seal to prevent evaporation.
 

B.  Our Recommended Procedure:  Having the students from the various "experiment stations" go from station to station filling culture tubes creates a danger of contaminating the solutions at the stations and has proven to be difficult to monitor.  A second approach, which we also used at UAH when we certified our students to make the space station samples and that we have begun to use in our workshops is as follows:

Mark all eight culture tubes at each "experiment station" with the % salt that is being produced at that station.

Then, fill all eight culture tubes at each station with the % salt solution produced there.  Be careful to follow the procedure described above and repeated here to prevent getting any air bubbles in the culture tubes:

     Since we want to avoid introducing any bubbles into the solution, first squeeze the bulb of the pipet then put the tip in the solution and release the bulb to fill the pipet.  Move the pipet to the culture tube to be filled and put the tip of the pipet into the tube near the bottom.  Slowly squeeze the bulb to expel the liquid, withdrawing the pipet as the tube fills.  Do not overfill - filling about 2/3 of the culture tube is fine!

Next, cap off each of the tubes using a small piece of the sealing tape to prevent evaporation.

Now, you can have the students from the various "experiment stations" get a culture tube from each of the other stations so that each station has a complete set.  This procedure is much more efficient that the 'kit procedures'!

(Note:  This eliminates the requirement to use the small 10 ml beaker which you can use to mix food coloring if you wish!)

When you have completed the lab activity, store the culture tube racks in a quiet place at room temperature.

We have eight protein crystallization solution samples with the same concentration of the lysozyme protein (40mg/ml), the same pH (about 4.3), crystallizing at the same temperature (room temperature).  We eliminate evaporation as a factor by sealing the culture tubes. The only variable in each of the samples is the concentration of salt which varies in .5% increments from 2.0% to 5.5%.  The basic concept is that the salt (NaCl) will attract water away from the lysozyme protein concentrating the solution near the protein.  When the concentration of the solution in the vicinity of the protein increases to the right point, the lysozyme will precipitate out.  If the precipitation occurs slowly, the lysozyme may precipitate in the crystalline form.  From our possible experience with making crystals in the past, we might already know that when the crystallization process happens quickly, numerous small crystals form.  But, if the process occurs more slowly, fewer but larger crystals form.  So, in this experiment, we should expect that the higher % salt solutions will produce many small crystals (or even non-crystalline amorphous forms) quickly.  As the % salt is decreased, we should expect fewer but larger crystals.  And, at the low end (2.0%-3.0%), we might get no crystals at all even over a long period of time as long as there is no evaporation.

Comments:

1.  A large crystal in this experiment is .1 mm.  You will need a low power microscope to view the 'large' crystals.  The small crystals (high salt concentration samples) may appear simply as cloudy after crystallization has occurred.

2.  Once the experiment is completed, if you want to produce colored crystals in the lower % salt solutions, poke a small hole in the sealing tape covering the top of the culture tube.  A slow rate of evaporation will occur eventually producing crystals in those culture tubes.

3.   Salt concentration has little to do with the morphology (crystal shape/crystal system) of the crystal.  If the crystals are grown above 25 degrees C (77 degress F), the predominant form is orthorhombic (longer aspect ratio).  Below 20 degrees C (68 degrees F), the predominant form is tetragonal (shorter aspect ratio).  In between, the form is usually teragonal which sometimes interconverts to orthorombic.  The only way to tell for sure is to do an xray diffraction.
(Anna Holmes; Univ. of Alabama at Huntsville, 8/3/99)

Robert S. Smith, (bo_smith@bellsouth.net)
Director, Florida Protein Crystals in Space