2.2 Media, Reagent, and Supply Preparation

A. Objective

To familiarize the trainee with preparing, dispensing and sterilizing microbiological media, reagents and supplies.

1. To familiarize the trainee with media quality assurance procedures
2. To familiarize the trainee with safety concerns, such as autoclave safety and weighing of powders.

B. Assignment

1. The trainer will discuss and demonstrate the equipment used in media preparation such as balances, stirrers, dispensers, pH meters, and autoclave use.
2. The trainee will prepare several kinds of media and buffer solutions, representative of the types generally used. These include selective and non-selective enrichment broths, plating media and phosphate buffer. Examples include Lauryl sulfate tryptose broth, Brilliant green lactose broth, EC broth, Lactose broth, Tetrathionate broth, Plate count agar, Butterfields Phosphate diluent, Triple sugar iron agar, EMB agar, Hektoen enteric agar, Bismuth sulfite agar.
3. The trainee will prepare and sterilize supplies such as empty bottles and tubes, polyvinylchloride tubing, spoons etc. as dictated by individual lab use.
4. The trainer will discuss the function of the components that comprise the commonly used media.
5. The trainer will discuss storage and shelf life of prepared media

C. Questions

1. Which media should not be steam sterilized and why?
2. Where is agar derived from?  What are the special properties of agar that make it well suited as a solidifying agent in culture media?  Once melted, what temperature should agar be kept at to prevent it from solidifying?
3. In which media and buffer preparations is volume particularly critical?
4. Why is pH important in media and buffer preparation?
5. What are some safety concerns when autoclaving?

D. References for Media, Reagent, and Supply Preparation

1. Tomlinson, L. (Ed.). (1998). Bacteriological analytical manual (BAM) (8th ed., Rev. A, updated January 2001, Appendix 3-Media and Reagents). Washington DC: Merker, R. I, Ph.D., Office of Special Research Skills, Center for Food Safety and Applied Nutrition, U.S. Food & Drug Administration. Retrieve online at http://www.cfsan.fda.gov/~ebam/bam-mi.html
2. AOAC official methods of analysis. (current ed.) Arlington, VA: Association of Official Analytical Chemists.
3. U.S. Pharmacopeia/ National Formulary (current ed.).
4. Difco manual of dehydrated culture media and reagents for microbiology (current ed.). Detroit, MI: Difco Laboratories, Inc.
5. BBL Manual of Products and Laboratory Procedures (current ed.). Cockeysville, MD: BBL Division of Becton, Dickinson and Company.
6. Laboratory Quality Assurance Program.