Tips & Tricks to Reduce Bioburden

Next-Gen sequencing has enabled massive growth in the microbiomics field, allowing scientists to study microbiomes at an incredible depth. While the high sensitivity of these tools means increased quality and quantity of data, it also means that the data collected includes organisms that were not original to the sample. Bacterial or nucleic acid contamination introduced throughout the workflow, such as during sample collection, extraction, or library preparation, are also reflected in microbiome sequencing data.

This contamination, or bioburden, should be a major consideration in developing microbiomics workflows as it can skew profiling results and interpretation. To ensure accurate representation of microbiome samples and to minimize foreign contamination, the following tips and tricks can be used to reduce bioburden in microbiome workflows.

Work In Clean Environments

The first step in ensuring successful, uncontaminated microbiome results is working in the proper environments, such as an enclosed workstation or a clean room. Bacteria can persist on a variety of surfaces, and trace amounts of DNA can be present at any time and in any place. Keep in mind that you are working in a laboratory environment where amplicons, specifically amplicons targeted for your microbiomics analyses, exist. Ensure that you have a separate workstation for each application and clean the surfaces adequately. A clean work environment is essential to minimize bioburden.

Tips for Keeping Your Lab Clean

Place an adhesive entry mat (sticky mat) at the entry of your clean room or wear shoe covers upon entering the clean room
Use a disposable or dedicated lab coat when in the clean room
Wear a protective face mask or procedure mask for facial concealment
Wear a bouffant cap (hairnet)
Wear sterile gloves
Work in a HEPA filtered room
Use designated sterile pipette tips for the extraction, and if possible, use filtered tips
To clean your workbench effectively, apply 10% bleach on the countertop, and let it sit for approximately 15 minutes before removing. Additionally, clean the rotor, lid, and internal walls of the microcentrifuge as well as pipette tips with 10% bleach, followed by 70% ethanol. Ethanol is used to ensure neutralization of the bleach to prevent downstream interference.
If at any time you must leave this clean environment and return, reapply fresh clean-room attire, such as bouffant cap, protective face mask, etc. Lab coats should never leave the clean room environment.

Choose Low Bioburden Reagents

In addition to working in proper environments, utilizing certified low bioburden DNA extraction kits and reagents is also important for reducing bioburden in microbiome studies. Taking extra care to prevent the introduction of contaminating DNA and microbes during extraction and library preparation is also key. Using low bioburden reagents and careful practices will help prevent the introduction of contaminants that may skew the microbial profiles of your samples.

Top Tricks for Ensuring Low Bioburden Reagents

Use clean manufacturing procedures when making reagents. All ZymoBIOMICS reagents are formulated with clean manufacturing procedures (e.g. HEPA filtered environment) and are filtered before aliquoting.
If using kits, use certified low bioburden kits such as the ZymoBIOMICS DNA Kits. These kits have been designed for users who work with high-sensitivity, low-biomass samples to ensure minimal contaminating bacterial background DNA. Low bioburden is determined and quantified by 16S rRNA amplification using qPCR.
For purification and subsequent analyses, use fresh reagents that are opened in a clean room or an enclosed workstation.
Ensure beads used for mechanical lysis are contaminant free. ZR BashingBead tubes used in the ZymoBIOMICS DNA Kits are autoclaved while 0.1 mm and 0.5 mm beads are baked at high temperatures (250˚C for 5 hours) to degrade any contaminating bacterial DNA.
Take caution to ensure there is no cross-contamination between samples during handling.
Ensure the elution buffer or DNase/RNase free water is fresh and free of contaminants. ZymoBIOMICS DNase/RNase-Free Water is DEPC-treated (diethyl pyrocarbonate), incubated overnight, and then inactivated by autoclaving at 121˚C. The water is then aliquoted into polypropylene bottles, capped loosely, and then autoclaved at 121˚C to eliminate any contaminating DNA that may have been introduced during the aliquoting process. After this treatment, the bottle is capped tightly to prevent further contamination.

Best DNA Extraction and qPCR Handling Practices

In addition to working in the proper environments, we recommend a few measures to minimize potential contamination during extraction and qPCR set up or similar processes:

Keep the ice chest or bucket used for stabilizing reagents in the clean room and use a second ice chest to transfer ice to the chest in the clean room.
Use DNase/RNase-Free Water. For ease and reassurance, ZymoBIOMICS DNase/RNase-Free Water (D4302-5), which is certified low bioburden, is available for purchase.
Use new filtered pipette tips for each prep and, if possible, use new pipette tips even when reloading sample to the same column.
Use new pipette tips for elution of DNA. It is recommended to pipette the elution buffer of DNase/RNase free water directly on to the column matrix, so the pipette tip may come into close contact with the column matrix.
Maintain calibration of pipettes. Pipettes must be calibrated for accurate quantification of low amounts of DNA present within a sample.
Change pipette tips frequently to reduce contamination of reagents.
When a bottle, container, tube, etc. is open, use the cap to shield the mouth of the container. By doing so, users can avoid any contaminants falling into the reagent.
Aliquots of reagent should be made to encompass one (or at most a couple) of experimental set-ups. By doing so, users can minimize both the frequency of freezing and thawing of reagents and the reuse of previously contaminated reagents (if contamination is present).
Between different preparations, clean used tools as necessary with 10% bleach, followed by 70% ethanol.
Initially, a master mix should be made and aliquoted for the assay. Remember to flush out the pipette tip to ensure accurate quantification when applying the sample to each well. DNA can remain within the pipette tip, and efficiency in pipetting is required when working with low amounts of DNA.
Take caution not to lean or hover over reagents or tools. Tilt PCR plates or similar items at an angle to be able to see into the plate while avoiding potential contaminations that could be caused by hovering.
Lastly, don’t forget your no template controls (NTC)!

Properly cleaning lab spaces and tools, utilizing low bioburden reagents, and using good handling practices are very important for microbiome workflows. Following these tips can help prevent the introduction of contaminants and ensure accurate microbial profiling.