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Syllabus

• Agar slant tubes
• Aseptic technique
• Assays
• Assays Illustrated
• Colony morphology
• Gram stain
• Isolating colonies
• Making serial dilutions
• Culture media
• Preparing spread plates
• Relationship to oxygen
• Viewing Gram stains
• Viewing living bacteria

• Characterization writeup
• Lab responsibilities
• Project proposal guidelines
• Using Bergey's

Supplemental pdfs

• Inoculating sticks
• Microscopy procedures

Preparing and using agar slant tubes

Agar slants are used for storing pure cultures for a moderately long term. There is minimal risk of contamination or of losing the culture due to the medium drying out because the small volume of air inside the tube and narrow opening combine to limit water loss and exposure to outside air, including dust and other particles.

Preparing an agar slant

We prepare slants by preparing agar in a beaker, distributing it into tubes, sterilizing the capped tubes, and laying them at an angle to make a slanted surface as they cool.

1. Determine the volume of agar needed. We have found that 7 ml per 16 x 125 mm tube gives us a slanted surface with good surface area and a “butt” about 1 inch deep. Multiply the number of tubes needed by 7 ml and add 10% for waste.

NOTE: You may be able to tilt a rack so that tubes need not be laid out individually.

2. Place the desired number of tubes in a rack and obtain an equal number of screw caps.
3. Weigh out and mix the desired volume of agar in a beaker. The beaker should be close to double the volume of agar to prevent spilling.
4. Add a stir bar, place the beaker in a microwave, and heat until the agar begins to foam. Watch carefully so that the agar does not boil over.
5. Wearing gauntlet gloves for protection, carefully remove the beaker to a stir plate and stir to make a uniform mix. If the agar is not completely dissolved, heat some more.

***CAUTION*** Hot agar that appears inactive may be superheated and may boil over violently if disturbed. Handle with care, keeping the face away from the agar and bare skin protected.

6. While stirring to keep the agar mixed, use a large syringe (w/o needle) to distribute the required volume into each tube.
7. Place a cap on each tube but do not screw them down. That way the tubes are able to vent.
8. Sterilize on a liquid cycle (cycle 2 for our autoclave), remove the rack safely, then tilt the tubes to allow the agar to cure.

Inoculating an agar tube

Aseptically remove the cap and transfer material from a single isolated colony to the agar surface. To transfer material to a slant we hold the tube at an angle, loosen the cap so that it can be pulled straight off, and pick up some culture from a single colony. With the colony material on the loop, remove the cap using the little finger of the hand holding the loop, pass the mouth of the tube quickly through the flame (optional), then insert the loop or stick into the tube.

With the end of the loop or stick touching the agar surface near the bottom, move it back and forth slightly as you gradually pull it up toward the top of the slant. Remove it, again pass the mouth of the tube through a flame (optional), and replace the cap.

Different investigators and technicians will conduct the inoculation differently, as can be seen in videos on the web. Some do not flame the mouth of the tube, others use different ways of transferring material to the agar surface. In our lab you will have the option of using a loop to conduct the transfer (flame required) or using a sterile inoculating stick (no flame required).

            ***IMAGES TO BE ADDED LATER***

       
Fig. 1. Agar slant tubes (left) Slant tubes cooling at an angle after removal from an autoclave. (center) Single slant tube viewed from the side, prior to inoculation. (right) Isolates growing on slant tubes.