Practical Session 1: Bacteria Collection

This training session has the aim that you register at least 2 Bacteria manually and 3 Bacteria with a Batch Registration.

In addition the aim is to create a General Protocol how to make a LB-Medium and a LB-Agar with and without an Antibiotics supplement

Please try to follow this list of tasks.

Create in a Test Project within your own user space:

Bacteria Collection (BACTERIA)

Chemical Collection (CHEMICAL)

Antibiotic Collection (CHEMICAL)

Solution Buffer Collection (SOLUTION_BUFFER)

Media Collection (MEDIA)

Protocol Collection (GENERAL_PROTOCOL)
Add 2 Bacteria to your Bacteria Collection
Add 3 Bacteria via the Batch Registration
Create all necessary Chemical, Antibiotics for a standard LB-Medium and LB-Agar
Create 2 Media in the Media Collection, one for the LB-Medium and one for the LB-Agar
Create a stock soultion for the Antibiotic
Create a Protocol how to create the LB-Medium
Create a Protocol how to create the LB-Agar
Create a Protocol how to add an Antibiotic to the LB-Medium and LB-Agar
View the Hierachy Graph of one of the created Protocols
View the Hierachy Graph of one of the created Media

Resources

Bacteria

Manually

Escherichia coli str. K-12 substr. MG1655
Escherichia coli O157:H7 str. Sakai

Batch Registration

Pseudomonas fluorescens SBW25 Rainey et al., 1996
Pseudomonas aeruginosa PAO1 Stover et al., 2000
Pseudomonas syringae pv. tomato DC3000 Buell et al., 2003

Chemicals

Yeast extract

Tryptone/Peptone

Sodium chloride

CAS No. 7647-14-5
Specification: Sodium chloride

Agar-Agar

Carbenicillin disodium salt

CAS No. 4800-94-6
H317-H334 (may cause an allergic skin reaction, may cause allergy or asthma symptoms or breathing difficulties if inhaled)
P272 P280 P302+P352 P333+P313 P342+P311 (contaminated work clothing should not be allowed out of the workplace, wear protective gloves/eye protection, IF ON SKIN: Wash with plenty of water, if skin irritation or rash occurs: Get medical advice/attention, if experiencing respiratory symptoms: Call a POISON CENTER/doctor)
Specification: Carbenicillin disodium salt
storage: +4°C
Stock solution: 50 mg/ml in 50 % ethanol / 50 % water.
Working concentration: 20-60 µg/ml
β-lactam antibiotic, semi synthetic penicillin.

Ethanol

CAS No. 64-17-5
H225-H319 (highly flammable liquid and vapour, causes serious eye irritation)
P210 P233 P305+P351+P338 (keep away from heat, hot surfaces, sparks, open flames and other ignition sources. No smoking, keep container tightly closed, IF IN EYES: Rinse cautiously with water for several minutes. Remove contact lenses, if present and easy to do. Continue rinsing)
Specification: Ethanol

LB-Medium protocol

5g / 1L Yeast Extract
5g / 1L NaCl
10g / 1L Tryptone
Weigh the indicated powder given above using weighing boats and place powders into an appropriate size beaker, add the corresponding amount of dH2O and mix using a magnetic stirrer.
Dispense the resulting 1000ml mixture into a 1000ml flask
Autoclave to make the culture media sterile
Remove flasks from autoclave, label with production date and store at room temperature or +4°C until required
(Optional) add Antibiotic

LB-Agar protocol

5g / 1L Yeast Extract
5g / 1L NaCl
10g / 1L Tryptone
15g / 1L Agar-Agar
Weigh the indicated powder given above using weighing boats and place powders into an appropriate size beaker, add the corresponding amount of dH2O and mix using a magnetic stirrer (all will dissolve apart from agar).
Dispense the resulting 1000mL mixture into a 1000mL flask
Autoclave to make the culture media sterile
Remove the flasks from the autoclave and allow the media to cool to about 55°C (until they can be held by hand)
(Optional) add Antibiotic (under the safety fume hood)
Promptly and smoothly pour LB-Agar medium +/- antibiotic (approx. 15-20mL) into the base of Petri dishes, cover with Petri lid and allow to cool and completely solidify (under the safety fume hood)
Label Petri dishes with manufacture date and antibiotic and store at +4°C until they are used if not used immediately

Note: To avoid contamination, plates should be discarded if not used after 1 month.

Antibiotic stock solution

Preparing 1000x Carbenicillin disodium salt stock solution:

Clean work surface and work space
Using the electronic balance, carefully weigh out 500mg Carbenicillin disodium salt into an appropriate size weighing boat using a spatula
Carefully tip the powder into a sterile 15mL falcon tube
Using a 10mL measuring cylinder, measure out 2.5mL of 100% Ethanol and add 2.5mL of sterile distilled or deionised water
Pour 4mL into the 15mL falcon tube containing the Carbenicillin disodium salt
Use the remaining 1mL to rinse the weighing boat and pouring that into the 15mL falcon tube to make 5mL total and mix gently by inversion
Using a sterile 20mL syringe, aspirate the solution from the falcon tube
Affix a sterile 0.22 µm micro filter to the end of the syringe and dispense thee solution into a new sterile 15mL falcon tube
Pipette 1000µL of the filtered 50mg/mL Carbenicillin disodium salt solution into 5x sterile 1.5mL microcentrifuge tubes
Close the tubes, label and store them at -20°C if they are not to be used immediately

This is a 1000x stock of 50µg/µL Carbenicillin disodium salt which is the normal working concentration in liquid or solid media. The stock can be diluted before use to reach the desired concentration.

Note: Carbenicillin disodium salt does degrade with time and there can be differences in potency and stability between batches. It is recommended when starting with a new batch to titrate the concentration to ensure 100% of non-resistant bacteria are killed. If the antibiotic properties start to drop off, ideally make a new batch from scratch or titrate to an increased effective concentration.

LB-Medium / LB-Agar + Antibiotic protocol

For a final concentration of 50µg/mL add 1µl per mL LB-Medium or LB-Agar from a 50mg/ml stock solution (1000x)

Next >>> Practical Session 2: DNA Extraction

26. Practical Session 1: Bacteria Collection