Ever wonder what is exactly growing on your teeth, incubated by your steamy 37 C mouth?  This exercise enables students to grow biofilms similar to plaque growth in culture tubes.

This exercise is slightly modified from one developed at Temple University School of Medicine by Dr. L. Packman from an idea provided by Dr. Ron Gibbons at the Forsyth Dental Clinic in Boston.  We thank Dr. Norman P. Willett for providing both the Streptococcus mutans GS5 strain and the idea for this addition to the web page.

 

Procedure:

•  Prepare Trypticase Soy Broth (without glucose) supplemented with 0.2% yeast extract and 5% sucrose.  Take a number of 20-mm culture tubes and insert  22 x 22-mm glass cover slips down their throats.  Into each of these tubes dispense 10-12 ml of the liquid media and autoclave.

•  Also sterilize 1-ml pipettes and a number of aluminum foil covered 50-ml beakers for collecting saliva.

•   In the sterile beakers, collect 1-ml aliquots of saliva from each student in the class.  Remember to use good technique to keep the beakers from contamination.

•   Let the beakers sit overnight in order to permit the air bubbles to dissipate in the saliva specimen.

• Inoculate each test tube with 1-ml of saliva.  Inoculate one tube with a pure culture of Streptococcus mutans GS5 to serve as a positive control.  An uninoculated tube may serve as the negative control.

• Incubate the test tubes for approximately 72 hours or longer at 37°C.

•  Gram Staining:  With a pair of sterile forceps, remove the cover slips and allow them to air dry.  DO NOT heat fix!  Heat fixing will caramelize media on the cover slips.

•  Gram stain the cover slips by immersing them in staining jars of the appropriate Gram Stain reagents.  After Gram staining, attach the cover slips to slides with a tiny drop of clear nail polish placed at each corner.  Observe under oil immersion.

•  Flow Through Gram Stain:  Follow procedures outlined in the On-Line Biofilm Laboratory Manual at www.personal.psu.edu/jel5/biofilms.  Observe under oil immersion.