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Training and Media Preparation

TRAINING SESSIONS An instructor will provide specialized training in the methods described below. Each team is assigned two training sessions, one on Monday and one on Wednesday. First session: Aseptic technique, culture methods, and Gram staining 1. Aseptically prepare three bacterial smears on a single marked slide from the three cultures on the agar plate provided 2. While the smears dry, prepare a dilution streak plate from a culture of Escherichia coli and Serratia marcescens D1 and incubate inverted at RT 3. Prepare a spread plate from the liquid sample provided and incubate inverted at 30 4. Prepare Gram stains from the dried smears (pt 1), to examined at the next session Second session: Microscopic observation of Gram stains and living bacteria 5. Examine and critique the streak and spread plates from session 1 6. Review use of the Nikon Labophot microscope, including dark field and phase contrast optics 7. Learn to use the oil immersion method, observe each Gram stain at 1000x, and record observations 8. Prepare and view wet mounts of living bacteria in dark field and in phase contrast, observing motility and endospores if possible Homework: Initial characterization of isolates From the respective folders in the Resources section on Owlspace, download the Gram negative and Gram positive contents sections reproduced from Bergey's Manual. For each culture that you Gram stained, narrow possible taxa to one or two Sections based upon the Gram stain result, cell morphology, and the relationship to oxygen that was noted on the plate. Use the Owlspace assessment "initial characterizations" (Tests & Quizzes) to report your findings within one week. MEDIA PREPARATION, STERILIZATION, AND CLEANUP Each student is to learn the following skills related to the culture and study of bacteria. Students with prior experience may demonstrate these skills at any time. Students with little or no relevant experience must consult with the instructor. properly prepare media including agar media and broth tubes load and start an autoclave using a liquid cycle; safely remove materials from an autoclave aseptically prepare agar plates using a laminar flow hood sterilize and properly clean contaminated glassware, including broth tubes, inserts, closures, and media bottles sterilize and dispose of contaminated agar plates

MEDIA AND SUPPLIES FOR MONDAY OCT 24 Teams are individually responsible for preparing media needed for the water analysis and characterization. Each team must prepare a minimum of 20 of each item listed under (1) and (2) to be ready to use the day the water samples are processed, with a few extras prepared in case of losses. Quantities of items 3-5 are described separately. All items must be sterilized in the autoclave. Broth media must be sterilized immediately after mixing and distributing into tubes. Agar media must be sterilized and plates poured all in the same day. If you plan to prepare media during a training session, please do not interrupt the instructor. Consult the instructor in advance if you need help with the media preparation. (1) Multiple tube fermentation (MTF) tubes for total coliforms (presumptive test) 18x150 mm tubes with 10 x 75 mm inserts for 10 ml water samples; fill with 15 ml 1.5x Lauryl Tryptose broth (including volume inside the insert) 13 x 100 mm tubes with 6 x 50 mm inserts for 1 ml samples; fill with 5 ml 1.2x broth 13 x 100 mm tubes for 0.1 ml samples with 6 x 50 mm inserts; fill with 6 ml 1x broth (2) Heterotrophic plate counts and spread plates for obtaining isolates Media bottle (Wheaton, 125 ml capacity) with 90 ml distilled water, cap loose R2A agar plates, dried ON in hood and stored upside down in sleeve on lab bench Tryptic soy agar plates, prepared and stored as above (3) Pipet tips, 100-1000 l (blue), one full box (4) Confirmed test (total coliforms) 13 x 100 mm tubes with 6 x 50 mm inserts, each with 5 ml brilliant green bile broth, total of 30/team MEDIA AND SUPPLIES FOR LATER In addition to the materials listed below, teams will need various types of assay media and reagents with which to characterize their isolates. Characterization will be discussed in a second presentation by the instructor. Assays, media, and assay procedures are described in the on-line manual. 5) Fecal coliform tests 13 x 100 mm tubes with 6 x 50 mm inserts, each with 5 ml EC medium, 30/team (6) Tryptic soy agar (TSA) plates for bacterial isolates Each team will need at least 2-3 sleeves (40-60 plates) for streaking out colonies rescued from R2A and TSA plates. (7) Agar slant tubes for bacterial isolates Teams will need at least one TSA or nutrient agar slant tube per pure isolate, to be prepared as needed. A team typically obtains at least two dozen unique isolates.

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