Antibiotic Resistance

Resistance 2012

Resistance 1

1/31/12 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

Get from Peg

Grow in LB

LB

Luria-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 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

90922 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

AttachmentSize
sigma-mueller-hinton.pdf2.18 MB

QSB antibiotics

From 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&symb... 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&symb...

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

AttachmentSize
kanamycin_specs.pdf19.11 KB

Media Tally

Medium 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

Fisher 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

Resistance 2013

AttachmentSize
antibiotic_resistance_week_1_prep.docx73.18 KB
antibiotic_resistance_week_2_prep.docx66.6 KB
antibiotic_resistance_week_3_prep.docx78.76 KB
welcome_to_the_resistome_lab_manual.pdf1.07 MB

Antibiotics 2013

(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
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&symb... ampicillin sodium salt is soluble 50 mg/mL in water http://www.bioxys.com/i_gentaur/ampicillin_sodium.htm

Kanamycin 50 mg/mL in water

Tetracycline 10 mg/mL

The HCl forms are more water soluble

Plate Tally 2013

type poured remaining used
LB 222 145 77
McC 1006 280 726
M-H 1150

Resistance 1 - 2013

Jan 29 & 31, 2013

Resistance 2 2013

  • 20 MacConkey plates per student

  • sterile swabs

  • gloves

  • spray ethanol

  • markers

Resistance 3 2013

  • 26 Mueller Hinton plates

  • sterile swabs

  • 26 1-mL aliquots physiological saline

    0.9% NaCl (=0.9 g/100 mL = 9 g/L)

  • sterile sticks

  • parafilm

  • markers

  • gloves

  • micropipettors & tips

  • vortex

  • antibiotic solutions in freezer

  • sterile disks

  • sterile forceps

  • rulers

  • microtube racks

Resistance 2015

Change antibody concentrations so 10 uL contains the correct number of mg.

Antibiotic ug/disk mg/mL category solubility Fisher #
Ampicillin 10 1 beta-lactam 50 mg/mL in 1 M NH4OH
Erythro 15 1.5 macrolide 50 mg/mL in EtOH
tetracycline 30 3 tetracycline 10 mg/mL in water
cipro 5 0.5 quinolone 30 mg/mL in water
Kanamycin 30 3 Aminoglycoside 50 mg/mL in water
Doxycyline * 30 tetracycline 50 mg/mL in water

*One group wants 1.2 mg/mL for project