
Welcome page Timetable Background Local info Water issues Projects Characterization Sample article (pdf) How to use Bergey's Manuals Methods Rules Media/culture Aseptic technique Staining/interpretation Isolation strategies Assays/characterization Microscopy Cultural characteristics Media preparation and training Training sessions Media preparation Laboratory safety and conduct Experimental Biosciences Resources	Media and supplies needed Teams will be individually responsible for preparing media needed for the two projects. Please consult the Welcome Page for a detailed schedule of events. All materials must be prepared and sterilized before they are needed Listed quantities describe the minimum quantity needed; teams should prepare a few extras in order to be prepared for mistakes The laboratory will be available from 8 am to 6 pm daily, closed on weekend; the door locks automatically at 6 pm An autoclave cycle takes more than an hour; please check with others in the laboratory before starting a run just in case someone is also ready to use it We have a second autoclave on the third floor (east end) in addition to the autoclave in ABL 141; you are welcome to use the third floor autoclave if the one on the first floor is running or full In order to identify your team's supplies, label a few individual items with your team number in each batch; usse a marker – do not use labeling tape (e.g., if you are with team 3, label a few tubes as "T3") Sample processing for Project 1 Each team must prepare its own agar plates (minimum 20 of each type of plate). Prepare plates as outlined in the manual (Agar Plates, under the heading Media). Materials for preparing serial dilutions will be provided. 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 Sample processing for Project 2 Each team will need 20 of each type of multiple tube fermentation (MTF) tube for the presumptive test for total coliforms (use lauryl tryptose broth), and 20 of each type for the test for thermotolerant bacteria (use EC medium). 18x150 mm tubes with 10 x 75 mm inserts for 10 ml water samples; fill with 10 ml 2x concentrated broth 13 x 100 mm tubes with 6 x 50 mm inserts for 1 ml samples; fill with 5 ml 1.2x concentrated broth 13 x 100 mm tubes for 0.1 ml samples with 6 x 50 mm inserts; fill with 6 ml 1x broth Surface area in our shared laboratory will be in great demand. Try to consolidate each set of tubes into a single rack if possible. Durham tubes are self-filling thus they need not be filled manually. Broth tubes must be capped and sterilized within an hour or so of preparation and removed from the autoclave when the run is complete (about 1 hr 15 min). Media and supplies for later In addition to the materials listed below, teams will need assay media and reagents (described in the on-line manual) with which to characterize their isolates. Characterization will be discussed in a second presentation by the instructor. Confirmed test (total coliforms): 13 x 100 mm tubes with 6 x 50 mm inserts, each with 5 ml brilliant green bile broth, as needed to confirm each positive MTF tube 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 and for subsequent re-streaking in order to ensure pure cultures Agar slant tubes for bacterial isolates: Each team will need at least one TSA or nutrient agar slant tube per pure isolate, to be prepared as needed according to the guidelines in the manual Assay media: Assay media required to complete the characterization of an unknown will vary from one isolate to the next; teams should work together to estimate quantities needed in order to maximize efficiency and minimize waste.
	Created by David R. Caprette (caprette@rice.edu), Rice University 27 Dec 10 Updated 7 Oct 15 Copyright and Intended Use

Background

Media preparation
and training

Media and supplies needed

Teams will be individually responsible for preparing media needed for the two projects. Please consult the Welcome Page for a detailed schedule of events.

All materials must be prepared and sterilized before they are needed
Listed quantities describe the minimum quantity needed; teams should prepare a few extras in order to be prepared for mistakes
The laboratory will be available from 8 am to 6 pm daily, closed on weekend; the door locks automatically at 6 pm
An autoclave cycle takes more than an hour; please check with others in the laboratory before starting a run just in case someone is also ready to use it
We have a second autoclave on the third floor (east end) in addition to the autoclave in ABL 141; you are welcome to use the third floor autoclave if the one on the first floor is running or full
In order to identify your team's supplies, label a few individual items with your team number in each batch; usse a marker – do not use labeling tape (e.g., if you are with team 3, label a few tubes as "T3")

Sample processing for Project 1

Each team must prepare its own agar plates (minimum 20 of each type of plate). Prepare plates as outlined in the manual (Agar Plates, under the heading Media). Materials for preparing serial dilutions will be provided.

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

Sample processing for Project 2

Each team will need 20 of each type of multiple tube fermentation (MTF) tube for the presumptive test for total coliforms (use lauryl tryptose broth), and 20 of each type for the test for thermotolerant bacteria (use EC medium).

18x150 mm tubes with 10 x 75 mm inserts for 10 ml water samples; fill with 10 ml 2x concentrated broth
13 x 100 mm tubes with 6 x 50 mm inserts for 1 ml samples; fill with 5 ml 1.2x concentrated broth
13 x 100 mm tubes for 0.1 ml samples with 6 x 50 mm inserts; fill with 6 ml 1x broth

Surface area in our shared laboratory will be in great demand. Try to consolidate each set of tubes into a single rack if possible. Durham tubes are self-filling thus they need not be filled manually. Broth tubes must be capped and sterilized within an hour or so of preparation and removed from the autoclave when the run is complete (about 1 hr 15 min).

Media and supplies for later

In addition to the materials listed below, teams will need assay media and reagents (described in the on-line manual) with which to characterize their isolates. Characterization will be discussed in a second presentation by the instructor.

Confirmed test (total coliforms): 13 x 100 mm tubes with 6 x 50 mm inserts, each with 5 ml brilliant green bile broth, as needed to confirm each positive MTF tube
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 and for subsequent re-streaking in order to ensure pure cultures
Agar slant tubes for bacterial isolates: Each team will need at least one TSA or nutrient agar slant tube per pure isolate, to be prepared as needed according to the guidelines in the manual
Assay media: Assay media required to complete the characterization of an unknown will vary from one isolate to the next; teams should work together to estimate quantities needed in order to maximize efficiency and minimize waste.

Created by David R. Caprette (caprette@rice.edu), Rice University 27 Dec 10
Updated 7 Oct 15
Copyright and Intended Use

Welcome page

Timetable