Prep It

NOTE: All preparation instructions are intended to yield enough materials for one class of 36 students divided into groups of 3 per microscope, or a total of 12 microscopes. There will be extra plates for future use.

Materials Needed

• 80-100 Sterile culturing plates, (60mm diameter,15mm high)
• agar culturing medium - may be purchased or prepared by the teacher
• E. coli - HB101 or OP50 preferred; or K-12 from Carolina Biological
• LB broth for maintaining E. coli
• Saline medium - teacher prepared
• Microspatula
• Bunsen Burner
• Pasteur pipettes
• 28 Gauge (32 Gauge is better) nichrome wire, platinum wire preferred - you will need approximately 20 cm total (allowing for error.)
• Ethanol
• Eppendorf tubes
• Test tubes or squeeze bottles
• Indelible markers for labeling plates, tubes, etc.
• Sterile transfer pipettes
• Kimwipes or tissue
• C. elegans cultures - N2 wild type (and one or two mutant strains, if desired)
• Spreaders - may be made from Pasteur pipettes or from paper clips

Obtaining C. Elegans cultures
         
Wild type (N2):

• May be purchased from Carolina Biological for $9 per culture.
• Available from the Caenorhabditis Genome Center (CGC) free to educational institutions. Contact the CGC on the web at http://biosci.umn.edu/CGC/Strains/request.htm

Mutant Strains: http://biosci.umn.edu/CGC/Strains/request.htm

Contact the CGC on the web at the above address for available strains. You may order by email, mail, or fax.

Suggested Mutant Strains:

• lon - LONg - several varieties. Lons are longer and thinner than N2 wild type worms and are easy to distinguish from them.Their tracks are a larger amplitude.
• dpy - DumPY - smaller, "fatter" than N2. Locomotion easily distinguished from N2.
• unc - UNCoordinated. Several varieties, ranging from abnormal locomotion to paralysis.
• rol - ROLlers - "roll" to one side or other depending on strain; locomotion mutants.

Preparing Plates

Agar plates may be poured and stored in the original plastic sleeves in the refrigerator prior to the arrival of cultures. Cultures will be shipped on a starved plate which must be chunked onto seeded plates four days prior to the student lab.

NGM Agar recipe:

• 3 g NaCl
• 2.5 g Bacto-Peptone
• 17 g agar - 21 g agar
• 1 L distilled water

Autoclave 15 minutes at 120°C and cool in water bath to 55°C. Using sterile technique and swirling after each addition, add

• 1 mL cholesterol
• 1 mL 1 M CaCl2
• 1 mL 1 M Mg SO4
• 25 mL 1 M KH2PO4, pH 6.0

(NOTE: to make KH2PO4, pH 6.0: Add 136 g KH2PO4 to 900 mL distilled water, adjust pH to 6.0 with concentrated KOH, add water to 1 L, autoclave. This solution will remain stable at room temperature for approximately one year. It may be possible to have advanced or AP chemistry students prepare it for you.)

Pour 60mm plates about half full (approximately 8-10 mL per plate). Flame surface of plates with bubbles to eliminate them (bubbles will encourage worms to burrow and interfere with observation of their behavior) This recipe should make approximately 100 plates. Allow plates to cool overnight. The next day wipe any condensation off of the plate covers, mark the bottom of each plate with the date poured, and place plates in original plastic sleeves, closing top with tape. Store plates upside down in a vertical position in the refrigerator. They should be usable for up to two months.

S-BASAL saline solution (for washing worms):
Mix the following:

• 5.9 g NaCl
• 50 mL 1 M KH2PO4, pH 6.0
• 1 mL cholesterol (5 mg/mL in ethanol)
• water to 1 L

Autoclave 15 minutes at 120°C. Cool. Solution may be stored at room temperature for approximately one year.

Teacher Preparation Prior to the Lab

4 or more weeks ahead

• Decide which mutant strain or strains you wish to order, if any.
• Contact the CGC at http://biosci.umn.edu/CGC/Strains/request/htm
• Order N2 worms, and mutants, if desired.
• Begin to order necessary materials.

2 to 3 weeks ahead

• Prepare agar solution, pour 100 plates or order plates from Carolina Biological
• Prepare S-BASAL saline solution (Note: You will need 68 plates to complete both lab activities. If you prepare the agar solution according to the recipe included here, you will have enough to pour 100 plates. Plates may be kept in the refrigerator for 2-3 months.)
• Order E. coli solution from Carolina Biological (K-12 strain) or contact a local lab for HB101 or OP 50 strain. If bacteria is on a culture plate, inoculate sterile LB solution with a few colonies, allow to incubate at room temperature or at 37°C in an incubator overnight. Store E. coli solution in refrigerator.
• Prepare 13 picks: For one pick, cut approximately 1 cm of wire, insert into the end of a glass Pasteur pipette. Hold the end with the wire over a bunsen burner flame until the glass melts, fusing the wire into the end of the pick. The wire end may be flattened using a razor blade on a flat surface, or crimped flat using a pair of needle nosed pliers. This will create a mini-golf club shape that will facilitate picking the worms from one plate to another.
• Assemble all necessary materials (see list above.)

6 to 7 days ahead

• Using a sterile transfer pipette, drop 1-2 drops of E. coli solution onto each of 52 plates (this will yield enough plates for one class of 36 to complete Activity #1 and Activity #2). This is seeding the plates.
• Store plates on lab bench overnight. The next day return 48 plates to the plastic sleeves, label sleeve "Seeded", and refrigerate. Leave four seeded plates at room temperature for chunking (instructions follow.)

5 days ahead

• Chunking: Sterilize a microspatula by dipping it in ethanol and using a Bunsen burner or alcohol lamp to flame it. Carefully carve out a 0.5 cm square chunk of agar from the N2 culture plate and place it gently - worm side down - on the agar of a seeded plate. Cover the plate. Label the bottom of the plate N2 and date it. Repeat with three additional plates: you will have a total of 4 "stock plates". Store plates at room temperature. (Worms will emerge from their dauer state almost immediately in the presence of food. The next day there will be eggs and progeny.)
• If using a mutant strain for comparative purposes, repeat this chunking process with fresh plates, being sure to sterilize the spatula and to label the bottom of the plates with the name of the strain and the date.
• Leave 24 seeded plates on lab bench at room temperature. Place remaining seeded plates in plastic sleeves, being sure sleeve is labeled "seeded", and return to refrigerator for later use.

3 to 4 days ahead

• Pick 3 to 4 L-4's from the chunked N2 plates onto each of 12 seeded plates.
• Pick 3 to 4 L-4's from the chunked mutant plates onto each of 12 seeded plates.
• Leave plates at room temperature.

1 day before Activity #1

• Remove 12 seeded plates from refrigerator, store overnight at room temperature.
• Assemble the following lab materials for each of 12 lab groups:

Dissecting microscope
A plate of N2 worms
A plate of mutant worms
1 clean, seeded plate
A pick
Worm diagrams
Pencils, lab markers
Drawing paper if students do not have lab notebooks

• Decide how you are most comfortable with students using alcohol lamps or bunsen burners: if your students are capable of using these devices without extremely close supervision, you may choose to have a device for every two groups to use for sterilizing picks during the picking process. If you are not comfortable with this arrangement, an alternative is to set up a "Picking Station" which you will closely supervise. The Picking Station consists of 4 additional microscopes and two bunsen burners or alcohol lamps. A group of 2-3 students can work at each microscope and every two groups can share a flame device. (Instructions for sterilizing picks and picking worms follow in Do It!)

A note on timing: If Activity #2 is to be done approximately 4 days after Activity #1, the plates the students pick during Activity #1 should yield enough progeny for use in Activity #2. It the timing will be different the teacher will need to repeat the chunking procedure outlined above prior to Activity #2.

1 day ahead of Activity #2:

• Remove 24 unseeded plates from refrigerator, store overnight at room temperature.
• Assemble the following lab materials for each of 12 lab groups:

Disssecting microscope
1 plate of N2 worms
2 clean, unseeded plates
Test tube with S-Basal solution
2 Eppendorf tubes - 2 different colors - in rack
2 Kimwipes or tissues
1 sterile transfer pipette

IF YOU ARE NOT SETTING UP A SPREADING STATION*:

1 spreader
1 tube of 10% ethanol
1 tube of 20% ethanol
Bunsen burner or alcohol lamp

* You may choose to set up an Ethanol Plate Station in order to more closely supervise students using a flame source in the presence of ethanol. See Safety Concerns.