Exercise Notes
Safety Notes

Safety Notes

Alternative autoclaving methods
Pressure cookers are one alternative, however, they can be dangerous and may not be large enough to autoclave a class load of reactors efficiently.

It is possible to "sterilize" media in a microwave (two  minutes is recommended).  However, that will not get rid of spores.  An alcohol rinse of the reactor would disinfect it.  The combination of both of these techniques could prove adequate for a high school lab setting  However,   a note of caution would be that these are not completely sterile, so it would be possible to have pathogenic microorganisms present.  Therefore, precautions must be taken in the handling of the samples taken from the reactor.

Gloves
Gloves should be selected consistent with the hazards involved and the activity to be conducted. Gloves must be worn when working with biohazards, toxic substances, hazardous chemicals and other physically hazardous agents. Temperature resistant gloves must be worn when handling hot material or dry ice.

Safety glasses
Splash shields or safety glasses with solid side shields should be required for anticipated splashes, sprays or splatters of infectious or other hazardous materials to the face.

Disposing of materials
Properly disposal of spent media, used slides, and  reactor vessels.  Items should be bleached and allowed to sit for a period of time and then washed or thrown away.  Use Biohazard Bags for contaminated waste.  It may be possible to arrange for the local hospital to autoclave the waste.

Exercise Notes

Harvesting and Dispersing Cells from Biofilms

Harvesting Cells: Alternative Method

For small surface areas and using automatic (digital) pipettes.

7. The procedure is as "Harvesting Cells: Standard Method" except that sterile 1.5 ml microcentrifuge tubes are used as dilution blanks. These are  prepared by the aseptic addition of 900 ml of sterile distilled water or PBS.
8. Scraping is as shown in the standard technique. The biofilm removed from the coupon is transferred with the stick and swirling into the microcentrifuge tube.
9. The scraped area of the coupon is rinsed with 100 ml aliquots of sterile distilled water of PBS and the volume of the microcentrifuge tube is brought up to 1 ml.

Dispersing Cells: TURBO MIX™Method

This technique employs a TurboMix^TM High Performance Attachment for the Vortex-Genie 2® (pictured at right), and is used in place of the Branson^TM or equivalent sonic cleaning water bath.  However, this device requires that the dilutions be made in 1.5 ml microcentrifuge tubes.

Also see www.mbcoct.com/sciind/90195.htm

Vortex Genie mixer with Turbo attachment

Instructions:

1. Place your microcentrifuge tube containing the harvested biofilm material into the TurboMix^TM attachment of the Vortex-Genie.
2. Lower the shield of the TurboMix and vortex for 2 minutes.
3. The cells should now be dispersed and ready for the next procedure.
4. The dilution tube should be vortex mixed before proceeding to dilution and plating to ensure that the dispersed cells are uniformly distributed.

---

Dr. John Lennox, Education Editor, Penn State Altoona

Educational Program Curricula and Teaching Resources
Supported in part by the Waksman Foundation for Microbiology
Developed in collaboration with Dr. John Lennox, Penn State University-Altoona
©1999-2006 Center for Biofilm Engineering, http://www.erc.montana.edu

NOTE: Links marked by the 3-arrow sign take you off the BiofilmsOnline.com web site. The Center for Biofilm Engineering is not responsible for content of sites marked with the 3-arrow sign.

Return to top

Return to Laboratory Exercises