Antibiotic Stock & Working Solutions

A Fundamental Technique in Microbiology.

What are Antibiotic Stock and Working Solutions?

In microbiology and molecular biology, antibiotics are critical for selecting specific bacteria (e.g., those that have successfully taken up a plasmid). To use them, scientists prepare two types of solutions: stock solutions and working solutions.

A Stock Solution is a highly concentrated solution of the antibiotic that is prepared for long-term storage. It's convenient because you only need to weigh out the antibiotic powder once to make many future solutions.

A Working Solution is a much more dilute solution prepared from the stock solution. It is the final concentration that will be used in an experiment, for example, added to a petri dish.

Example: Preparing and using antibiotic solutions is a fundamental and daily task in any lab that works with bacteria for genetic engineering.

Why Use a Two-Step Process?

Using a stock solution to make a working solution is standard practice for several reasons:

Accuracy: It is very difficult to accurately weigh the tiny amount of antibiotic powder needed for a single experiment. It's much more accurate to weigh a larger amount for a stock solution and then accurately dilute it.

Convenience & Speed: Once the stock is made, preparing a working solution is a quick and simple dilution step.

Stability: Many antibiotics are more stable when stored at high concentrations and in specific solvents (like ethanol or DMSO), often frozen. The working solution is typically made fresh before use.

Example:It's like making orange juice from a frozen concentrate. The concentrate is the 'stock solution' that's easy to store, and the final juice is the 'working solution'.

Key Formulas: Mass and Dilution

Two simple formulas are all you need for these preparations:

1. Preparing the Stock Solution (from powder):

This is based on the definition of concentration: Mass = Concentration × Volume. You decide the concentration and volume of your stock and calculate the mass of powder to weigh.

2. Preparing the Working Solution (from stock):

This is a standard dilution calculation using the formula: C₁V₁ = C₂V₂, where '1' refers to the stock solution and '2' refers to the final working solution.

Example:For C₁V₁ = C₂V₂, C₁ is the stock concentration, V₁ is the volume of stock to use, C₂ is the final working concentration, and V₂ is the final volume you want to make.

Real-World Application: Preparing Ampicillin Plates

A common task is preparing LB agar plates with the antibiotic ampicillin for selecting E. coli bacteria.

Goal: Prepare 1 liter (1000 mL) of LB agar with a final 'working' concentration of 100 µg/mL ampicillin.

Step 1 (Make Stock): The researcher first prepares a 'stock' solution, for example, at 100 mg/mL. This is 1000 times more concentrated than the working solution (100 mg/mL = 100,000 µg/mL).

Step 2 (Dilute into Media): The researcher then uses the C₁V₁=C₂V₂ formula to calculate how much stock to add to the 1 liter of agar.

Calculation: (100 mg/mL) * V₁ = (100 µg/mL) * (1000 mL). First, make units consistent: (100,000 µg/mL) * V₁ = (100 µg/mL) * (1000 mL). V₁ = 100,000 / 100,000 = 1 mL.

Result: The researcher adds just 1 mL of the concentrated stock solution to the 1 L of molten agar before pouring the plates.

Example:This 1000x stock concentration is very common in labs and is often referred to as a '1000x stock'.

Key Summary

A **stock solution** is a concentrated solution used for storage. A **working solution** is the dilute solution used in an experiment.
Preparing a stock requires a mass calculation: **Mass = Concentration × Volume**.
Preparing a working solution from a stock requires a dilution calculation: **C₁V₁ = C₂V₂**.
This two-step process ensures accuracy, convenience, and stability.

Practice Problems

You want to make a 10 mL stock solution of kanamycin at a concentration of 50 mg/mL. How much kanamycin powder do you need to weigh out?

Use the formula: Mass = Concentration × Volume.

Solution: Mass = (50 mg/mL) * (10 mL) = 500 mg, or 0.5 grams of kanamycin powder.

You have a 100 mg/mL ampicillin stock solution. You need to make 200 mL of LB agar media with a final ampicillin concentration of 100 µg/mL. How much of your stock solution should you add?

Use the dilution formula C₁V₁ = C₂V₂. First, make sure the concentration units are the same.

Solution: C₁ = 100 mg/mL = 100,000 µg/mL. C₂ = 100 µg/mL. V₂ = 200 mL. (100,000 µg/mL) * V₁ = (100 µg/mL) * (200 mL). => 100,000 * V₁ = 20,000. => V₁ = 20,000 / 100,000 = 0.2 mL, or 200 µL.

Frequently Asked Questions

Why do you add the antibiotic after autoclaving the media?

Many antibiotics, including ampicillin, are heat-sensitive and would be destroyed by the high temperature and pressure of an autoclave. The antibiotic stock is filter-sterilized separately and is always added to the agar media after it has cooled down to a manageable temperature (around 50-55°C).

How should I store my antibiotic stock solutions?

Storage conditions depend on the specific antibiotic. Most are stored in a dark container (e.g., wrapped in foil) at -20°C. Some are dissolved in water, while others require solvents like ethanol or DMSO for stability. It's critical to look up the correct solvent and storage for each specific antibiotic.

What does '1000x' or '100x' mean for a stock solution?

This is common lab shorthand. A '1000x' stock solution is 1000 times more concentrated than the final working concentration. To use it, you would perform a 1 to 1000 dilution (e.g., adding 1 mL of stock to 999 mL of media to make a final volume of 1 L).

Antibiotic Stock

Antibiotic Stock & Working Solution Calculator

Step 1: Prepare Stock Solution (C₁)

Step 2: Prepare Working Solution (C₂)

How It Works