Resistance 2012
Resistance 2012 kdorfman Thu, 01/17/2013 - 18:39Resistance 1
Resistance 1 kdorfman Wed, 01/11/2012 - 22:061/31/12 and 2/2/12
- 6 tubes of freshly growing E. coli BZB1011 in LB (one on each set of desks) from Peg's lab
- 6 tubes with 20 mL sterile LB (one on each set of desks)
- ~150 Sterile swabs (one beaker full per set of desks) (= 2 100-packs, sterilized)
- 12 Pipetteman and tips (1 per 2 students) (for 10 to 100 µL volumes)
- ~125 MacConkey plates (about 4 per student with extras available) @ 25 mL/plate = 3125 mL (@ 50 g/L = 62.5 g)
- 24 black markers
- shelf in an incubator
- shelf in a refrigerator
- camera to photograph plates (or students use iphones)
- scale for weighing 25 grams
- weigh boats
- vortexes
Have on hand for student use outside of class hours:
- Scales, weigh boats and vortexes
- 500 sterile capped tubes with 25 mL LB – need about 10/student
- pipetman and 100 µL tips – need about 10 tips per student
- BZB1011 for use as positive control – stored in refrigerator
- 500 MacConkey plates – need about 10 per student
- 500 sterile swabs – need about 10 per student
E. coli BZB1011
E. coli BZB1011 kdorfman Wed, 01/11/2012 - 22:17Get from Peg
Grow in LB
LB
LB kdorfman Mon, 01/16/2012 - 19:35Luria-Bertani broth (Miller)
LB broth (Fisher BP1426500) 500 g
25 g/L
= 20 g/800 mL (in a 1 L bottle)
Autoclave on liquid cycle 45 min
= 87.5 g/3.5L (in a 4 L flask)
Autoclave on liquid cycle 80 min
Aliquot 25 mL/tube 500 tubes (= 12.5 L)
MacConkey plates
MacConkey plates kdorfman Wed, 01/11/2012 - 22:17MacConkey Agar Fisher 212122 (Difco)
to distinguish Lac+ and Lac- bacterial strains.
50 g/L
Total plates needed =~625
in 2 L flasks
flasks | Total vol (L) | max # plates |
---|---|---|
1 | 1.75 | 70 |
2 | 3.5 | 140 |
3 | 5.25 | 210 |
4 | 7 | 280 |
5 | 8.75 | 350 |
6 | 10.5 | 420 |
7 | 12.25 | 490 |
8 | 14 | 540 |
- 2-L flasks, each with:
- 87.5 g MacConkey dry mix
- 1.75 L distilled water
- magnetic stir bar
- Stir to mix.
- Cover loosely with foil
- Pack Pour-Boy tubing in foil to go in same cycle if it's not already sterile
- Autoclave 55 min, liquid cycle
- Cool till handle-able on stir plate
- Dispense 25 mL per plate with Pour-Boy, stacking 10 tall.
in 4 L flasks
- 4 L flasks, each with
- 175 g MacConkey dry mix
- 3.5 L distilled water
- magnetic stir bar
- Stir to mix.
- Cover loosely with foil
- Pack Pour-Boy tubing in foil to go in same cycle if it's not already sterile
- Autoclave 75 min, liquid cycle
- Cool till handle-able on stir plate
- Dispense 25 mL per plate with Pour-Boy, stacking 10 tall
flasks | total vol (L) | max # plates |
---|---|---|
1 | 3.5 | 140 |
2 | 7 | 280 |
3 | 10.5 | 420 |
4 | 14 | 560 |
Mueller-Hinton agar
Mueller-Hinton agar kdorfman Fri, 01/20/2012 - 22:3990922 Mueller Hinton Broth 2 from Sigma
22 grams per liter, consisting of:
- Casein acid hydrolysate 17.5
- Beef extract 3.0
- Starch 1.5
Final pH 7.3 +/- 0.2 at 25°C
Add 17 g agar per L
Make 3.5 L in a 4L flask: (yields up to 140 plates)
77 g broth mix
stir till dissolved. Leave stir bar in
59.5 g agar
autoclave 80 minutes
QSB antibiotics
QSB antibiotics kdorfman Thu, 01/12/2012 - 14:40From Peg's list (weight indicates how much should be on one disk.)
Calculate concentration from volume needed per disk (25 uL)
Make 35 mL of each (because the filter syringe only holds that much)
See recipes in stock solutions.
Make 35 mL each, make 48 0.7 mL aliquots
Antibiotic | ug/disk | mg/mL | category | solubility | Fisher # |
---|---|---|---|---|---|
Ampicillin | 10 | 0.4 | beta-lactam | 50 mg/mL in 1 M NH4OH | |
Erythromycin | 15 | 0.6 | macrolide | 50 mg/mL in EtOH | ICN19452901 1g $33.68 |
tetracycline | 30 | 1.2 | tetracycline | 10 mg/mL in water | |
cipro | 5 | 0.2 | quinolone | 30 mg/mL in water | |
Kanamycin | 30 | 1.2 | Aminoglycoside | 50 mg/mL in water |
cipro solubility http://www.inspiralis.com/go/antibiotics.php 30 mg/mL in water
ampicillin solubility 50MG/ML IN 1M AMMONIUM HYDROXIDE http://www.sigmaaldrich.com/catalog/DataSheetPage.do?brandKey=SIGMA&sym… ampicillin sodium salt is soluble 50 mg/mL in water http://www.bioxys.com/i_gentaur/ampicillin_sodium.htm
erythromycin solubility 50MG/ML IN ETHANOL http://www.sigmaaldrich.com/catalog/DataSheetPage.do?brandKey=SIGMA&sym…
Kanamycin 50 mg/mL in water
Tetracycline 10 mg/mL
The HCl forms are more water soluble
Not using Spring 2012
Antibiotic | ug/disk | mg/mL | category | Fisher # |
---|---|---|---|---|
Amikacin | 30 | BP2643250 250 mg $28 | ||
Cefazolin | 30 | NC9863037 1g/10mL $17 | ||
gentamicin | 10 | ICN1676045 10mg/mL 10 mL $29 | ||
ticarcillin | 7 | 50-121-7983 1 g $45 | ||
trimethoprim | 5 | ICN19552701 1 g $34 | ||
tobramycin | 10 | 50-146-785 100 mg $56 |
Media Tally
Media Tally kdorfman Tue, 02/28/2012 - 14:22Medium | started with (approx) | left over | actually used |
---|---|---|---|
LB 25 mL in 50-mL tube | 500 | 489 | 11 |
LB 20 mL in 125 mL flask | 14 | 14 | 0 |
MacConkey plates | 650 | 162 | 488 |
MH plates | 560 | 325 | 235 |
Soy-Trypticase plates | 32 | 0 | 32 |
Antibiotic | started with | left over | actually used |
---|---|---|---|
amp | 48 | 26 | 22 |
cipro | 48 | 27 | 21 |
erythro | 48 | 34 | 14 |
Kan | 48 | 31 | 17 |
Tetra | 48 | 30 | 18 |
resistance supplies
resistance supplies kdorfman Thu, 01/19/2012 - 15:54Fisher cotton-tipped applicators 23-400-101 (come in 100-count autoclave bags.)
Fisher 43-400-102 plain wood applicators for colony picking
Fisher 11-312A solid glass beads 3mm for spreading liquid on plates
Krackeler 10-231039 Blank discs 1/4" dia. 6 vials of 50 $23.76