How do you make research antibiotics last longer? This is a common question in the lab, and researchers could follow easy practices to extend the shelf life of antibiotics.

To make research antibiotics last longer, verify the storage conditions, store them in a dark and dry place, prepare aliquots to prevent multiple freezing and thawing cycles, and test your antibiotic efficacy over time with a disk diffusion assay.

Antibiotics are such a universal reagent in the lab, and ensuring their efficacy over time is critical for your research. In this article we will not only explain how to increase the shelf life of your research antibiotics, but we will explain why and any additional details in each section.


Article Contents

Tip 1: Verify the form of the antibiotic (powder or liquid)

Tip 2: Prepare research antibiotic stock solutions following the manufacturer’s indications

Tip 3: Verify the storage conditions

Freezing

Light

Contamination

Tip 4: Reduce freezing and thawing cycles

Tip 5: Test the antibiotic efficacy using the disk diffusion assay

GoldBio Antibiotics

Useful sources

Keywords:

References



Tip 1: Verify the form of the antibiotic (powder or liquid)

Antibiotics can come in powder or liquid forms. Powder antibiotics last longer than liquid form because they have more extended stability periods. The stability of an antibiotic means it keeps its antibacterial ability over time without degradation.

For instance, powdered forms of amoxicillin can last around two to three years if properly stored.

But when it’s mixed in water for a solution, it can expire after 14 days at room temperature!

Many biotech manufacturers often provide powder antibiotics and give instructions about how the powder should be made into an antibiotic stock solution.

The process of adding a diluent (commonly water) to a powdered antibiotic to prepare a solution or suspension is called reconstitution.

The drawback of having powder antibiotics is that the reconstitution process needs more care because some powder antibiotics are very hygroscopic (the phenomenon of holding water molecules).

Therefore, powder particles adhere to the internal and external walls of the container leading to waste and affects the final concentration in the reconstitution process. Here, the invitation is to prepare powdered antibiotics very carefully in closed places without air currents to avoid any waste.

Review the label or product description when ordering your research antibiotics to know if the antibiotic is in powder or liquid.

Descriptions or labels may not always be explicit. For powdered antibiotics, the units as mg (milligrams) or g (grams) are on the label (figure 1A) will signal a powder antibiotic.

Liquid antibiotics will have the units as mL (milliliters) (figure 1B).

Labelling of GoldBio antibiotics for powdered (A) and liquid (B) forms.

Figure1. Illustrated example of labeled antibiotic bottles for powdered (A) and liquid (B) forms.


For powdered antibiotics, pack sizes range between 5 g up to 250 g. In the case of liquid antibiotics, they come in concentrations around 50 to 100 mg/mL. Also, you can find solutions in pack sizes ranging between 10 mL up to 100 mL.

Commercial antibiotics come as sulfates (e.g., gentamicin sulfate) or disodium reagents (e.g., carbenicillin disodium) because sulfate and disodium molecules facilitate the dissolution of antibiotics in water.


Tips:

  • For powdered antibiotics. If you have several bottles of the same antibiotic, do not prepare them all simultaneously. Carefully prepare stock solutions (50 to 100 mg/mL) from one of the bottles and store the rest until needed. Keep them out of light and under freezing conditions as the manufacturer suggests (generally at -20°C for desiccated antibiotics). Also, make sure the container is tightly closed.
  • For antibiotics in solution. If the antibiotic comes in a liquid form, take an aliquot with clean pipette tips. This aliquot will work as a stock solution (from 50 to 100 mg/mL) to prepare further working solutions. Proceed to store in freezing conditions of -20°C for up to one year.


Tip 2: Prepare research antibiotic stock solutions following the manufacturer’s indications

Most antibiotic solutions are completely dissolved in water. It is important to filter them using a prepared 0.22 µm syringe filter before storing it at -20°C for around one year.

You may find our video that demonstrates how to prepare sterile filtered antibiotics helpful.

At GoldBio, we have protocols to prepare stock solutions for most of the antibiotics. We present a selected list of protocols in table 1, which you can access just by clicking on the antibiotic name.

Table 1. Selected and commonly used antibiotic stock solution protocols.

Antibiotic

Description

GoldBio Catalog

Ampicillin

Ampicillin Stock Solution - 100 mg/ml

A-301


Amoxicillin

Amoxicillin Sodium stock solution - 25 mg/ml

A-551


Bialaphos

Bialaphos Stock Solution - 10 mg/ml

B0178


Carbenicillin

Carbenicillin Stock Solution - 50-100 mg/ml

C-103


Cefotaxime

Cefotaxime Stock Solution - 100 mg/ml

C-104


Chloramphenicol

Chloramphenicol Stock Solution - 25-50 mg/ml

C-105


Erythromycin

Erythromycin stock solution - 50 mg/ml

E-350


Gentamicin

Gentamicin Stock Solution - 50 mg/ml

G-400


Hygromycin

Hygromycin Stock Solution - 50-100 mg/ml

H-271


Kanamycin

Kanamycin Stock Solution - 50 mg/ml

K-120


Nourseothricin

Nourseothricin Stock solution - 200 mg/ml

N-500


Spectinomycin

Spectinomycin Stock Solution - 50-100 mg/ml

S-140


Streptomycin

Streptomycin Stock Solution for molecular biology applications - 50 mg/ml

S-150


Tetracycline

Tetracycline Stock Solution - 5-10 mg/ml

T-101


Trimethoprim

Trimethoprim stock solution - 25 mg/ml

T-350



You may also find our stock solution and solution dilution calculators helpful as well.

1) Stock Solution calculator:

Link: https://www.goldbio.com/stock-solution-calculator

This calculator allows you to estimate how much volume you'll get from a product pack size or weighed mass of powder.


2) Solution Dilution calculator:

Link: https://www.goldbio.com/solution-dilution-calculator

The solution dilution calculator lets you calculate final volume needed for a solution.


Tip 3: Verify the storage conditions

Storage conditions such as light and freezing are critical to extend the shelf life of the antibiotics. Storage conditions can be different depending on the type of antibiotic.

Furthermore, it is important to occasionally verify that your antibiotics are not contaminated.


Freezing

Most antibiotics that come in powder or solution are stored at -20 °C. However, some stock solutions must be kept at -80°C to preserve them due to their stability.

For instance, ampicillin solutions degrade 13% after one week at -20°C. For this reason, it is recommended to store ampicillin stock solutions at -80 °C up to three months. Note – for better energy savings, -70 °C is acceptable and has stability for up to three months.

Tips:

  • Store ampicillin stock solutions at -80 °C up to three months. In the case of amoxicillin, stocks may be stored in small aliquots at -70°C for up to 3 months. Other stock solutions of antibiotics such as Bialaphos, Carbenicillin, Cefotaxime, Chloramphenicol, Gentamicin, Kanamycin, Spectinomycin, Streptomycin, Tetracycline may be stored at -20 °C up to one year.
  • Refer to your Antibiotic Stock preparation protocols to verify the storage conditions of your antibiotic.

Light

Light degrades antibiotics over time in a process called photolysis. Photolysis is a process where photons break down a molecule.

Diagram showing the breakdown of molecules mediated by light through the photolysis process.

Figure 2. Diagram showing the breakdown of molecules mediated by light through the photolysis process.


Ellepola & Rubasinghege (2022) studied the photodegradation of amoxicillin and found that sunlight produces different subproducts of amoxicillin like amoxicillin penicilloic acid, amoxicillin penilloic acid and amoxicillin diketopiperazine (figure 3).

However, the toxicological effects of these subproducts have not been evaluated yet.

Reactions of amoxicillin degradation by natural light through photolysis.

Figure 3. Reactions of amoxicillin degradation by natural light through photolysis.


The level of photodegradation depends on several parameters (Kumar et al., 2017):

  • concentration of the antibiotic
  • light intensity
  • time of irradiation
  • pH of the solution
  • oxygen level in the environment
  • temperature
  • surface area exposed to light
  • nature of the light (UV/sunlight)
  • type and structure of the antibiotic


Tips:

  • To avoid photodegradation of your antibiotics, keep them under dark conditions, and use a tightly closed container to prevent the entrance of oxygen and humidity, which may accelerate the photodegradation.
  • Prepare working solutions from your stock solutions and use them at the bench to avoid continuous exposure to light and degradation.


Contamination

Cross-contamination may occur while handling your antibiotics.

Cross-contamination is the presence of microorganisms in the stock or working solutions that may interfere with the microbiological analysis.

Once in a while, you should perform cultures of small aliquots of your stock and working solutions and evaluate if there is any growing colony. If nothing grows on your plate, the solution is free of microorganisms.


Tips:

  • A quick way to visualize contamination is through turbidity. You can see if the vials where working and stock solutions are stored have a foggy appearance. If contamination is not evident enough, proceed with agar plates as I explain in the next tip.
  • Prepare an agar plate of commonly used media like LB broth culture and malt agar media to screen bacteria or yeast growth. Use sterilized pipette tips to take small aliquots (about 100 μL) and place two or three drops on the agar plate. Then, with the help of a sterilized inoculation loop or autoclaved glass beads, distribute the drops over the entire plate. Seal the plates and place them in an incubator at 24 °C overnight. On the next day, review under a stereoscope for the presence of colonies. Perform all these steps under a clean flow chamber.
  • Use our GoldBio culture media for bacterial/yeast screening like LB Broth, Malt agar, or our Bacteria Screening Medium.



Tip 4: Reduce freezing and thawing cycles

Freezing and thawing cycles (freeze-thaw cycles) refer to repeated moments where your reagent will be frozen and then thawed or partially thawed.

Leaving reagents on the bench too long, even holding the freezer door open for a long period can potentially lead to repeated freezing and thawing.

Of course, lab work necessitates using your antibiotics at different periods throughout the experiment.

The temperature changes caused by freezing and thawing cycles reduce the stability of the antibiotics and make them more prone to degradation by light, oxygen, and contamination.

An aliquot can be made from the stock solutions to prepare a working solution.

An aliquot is a fraction of the reagent that is dissolved in a liquid solvent, in this case water. From the powdered or liquid reagent, you can make a stock solution. A stock solution of an antibiotic is a solution prepared to an X concentration.

From a stock solution you prepare a working solution. A working solution is a solution usually in a lower concentration than the stock solutions and is ready to use for your experiments.

Many aliquots of working solutions can be prepared in different concentrations. These concentrations depend on the experiment's needs.

For this reason, you can spend the complete working solution in an experiment avoiding freeze-thawing cycles.

For instance, if the final antibiotic concentration in your experiment is 2 mg/mL, your stock solution should be at least four or five times higher.

Most stock solutions range between 50 mg/mL and 100 mg/mL.

Tips

  • Evaluate the presence of contamination (a bacterial screen) in both working and stock solutions using the agar plate cultivation. This will avoid having to start your experiment from the very beginning and ultimately save you time.


Tip 5: Test the antibiotic efficacy using the disk diffusion assay

Testing the effectiveness, or efficacy of your antibiotics is critical to achieve good results in your experiments.

This can be done using the disk diffusion assay. This test compares the antibiotic's ability to prevent bacterial growth to a common set of standards created by the CLSI (Clinical Laboratory Standards Institute), in order to assure its functionality at a certain concentration.

As a control, use a strain sensitive to the antibiotic to determine its effectiveness, and a resistant strain to your antibiotic.


Tips:

  • If the results of your disk diffusion assay show your antibiotic is not effective, you will need to purchase new antibiotics. For instance, having a low inhibition zone (less than 50% compared to the positive control) for the strain sensitive signals that your antibiotic has low efficacy
  • To perform the disk diffusion assay, you can follow our Disk Diffusion Assay Protocol or get more information and tips in our GoldBio video.



GoldBio Antibiotics

GoldBio carries more than 150 quality research antibiotics, including a variety of aminoglycosides, glycopeptides, cephalosporins, and penicillins.

Below is a list of commonly used GoldBio antibiotics to help you succeed in your experiments.

Amoxicillin

Ampicillin

Bialaphos

Carbenicillin

Cefotaxime

Chloramphenicol

Erythromycin

Gentamicin

Hygromycin

Kanamycin

Spectinomycin

Streptomycin

Tetracycline

Trimethoprim

Complete list of GoldBio antibiotics




Useful Resources

Antibiotics & Selection



Keywords:

Antibiotics, antibiotics shelf life, antibiotic storage, light sensitive antibiotic, photodegradation.



References

Ellepola, N., & Rubasinghege, G. (2022). Heterogeneous Photocatalysis of Amoxicillin under Natural Conditions and High-Intensity Light: Fate, Transformation, and Mineralogical Impacts. Environments, 9(7), 77. https://doi.org/10.3390/environments9070077

Kumar, A. (2017). A Review on the Factors Affecting the Photocatalytic Degradation of Hazardous Materials. Material Science & Engineering International Journal, 1(3), 106–114. https://doi.org/10.15406/mseij.2017.01.00018

Lin, J. S., Pan, H. Y., Liu, S. M., & Lai, H. T. (2010). Effects of light and microbial activity on the degradation of two fluoroquinolone antibiotics in pond water and sediment. Journal of Environmental Science and Health - Part B Pesticides, Food Contaminants, and Agricultural Wastes, 45(5), 456–465. https://doi.org/10.1080/03601231003800222

Singh, G. D., & Gupta, K. C. (2014). Photo and UV degradation of Ciprofloxacin Antibiotic. International Journal of Current Microbiology and Applied Sciences, 3(6), 641–648.

Timm, A., Borowska, E., Majewsky, M., Merel, S., Zwiener, C., Bräse, S., & Horn, H. (2019). Photolysis of four β‑lactam antibiotics under simulated environmental conditions: Degradation, transformation products and antibacterial activity. Science of the Total Environment, 651, 1605–1612. https://doi.org/10.1016/j.scitotenv.2018.09.248.

Yang, X., Chen, Z., Zhao, W., Liu, C., Qian, X., Zhang, M., & Wei, G. (2020). Recent advances in photodegradation of antibiotic residues in water. Chemical Engineering Journal, January.