Many infections in humans, including medical device infections, chronic wounds, staph skin infections, and endocarditis, involve biofilms (1). Troublingly, due to their protective matrix, many biofilms can evade the immune system and may have an inadequate response to antibiotic treatment. This can lead to chronic infections and antimicrobial resistance. Therefore, the development of effective prevention and treatment strategies requires thorough investigation of biofilms. In this blog post, we highlight and discuss two major problems that plague biofilm investigations and their potential solutions.
A numbers problem – Inoculum Size
Reliable investigation of biofilms requires proper assessment of the number of bacteria, or inoculum. A known quantity of bacteria allows investigators to accurately determine the effectiveness of antibiotic treatments and dose-response relationships. However, accurate determination of inoculum size can be difficult because bacteria can range in size between 1-10 µm (Figure 1), but many automated cell counters are limited to a minimal detection size of ~5 µm. Additionally, bacteria replicate quickly, with many strains being capable of doubling their number within 20 minutes. This replication speed can make accurate determination of inoculum size difficult if there is a lag between counting and experimentation.
Figure 1.
Figure 1. Scale showing the varying sizes of common microorganisms.
Considering the limitations of most automated cell counters, many microbiology labs employ other methods of enumeration, including direct microscopic count, culture turbidity, and plate count. Some of the pros and cons of these methods are outlined in Table 1 below.
Table 1.
Although these common bacteria counting methods each have pros and cons, none of these methods provide a reliable way to assess bacterial aggregation. Under physiological conditions, adherence between bacterial cells is important for colonization and survival. However, in experimental conditions, aggregates can contribute to inaccurate counts, greatly distorting results. Though methods of aggregate discrimination, such as flow cytometry, sedimentation, and atomic force microscopy exist, these methods are extremely time-consuming and expensive and may fall outside of the expertise of many microbiology labs.
To improve the speed and accuracy of bacterial biofilm investigations, Cell Microsystems recently began offering the CASY cell counter and analyzer. Unlike optical-based cell counters, CASY measures the electrical pulse that is generated between two electrodes as a cell passes through a defined pore. This signal directly correlates with cell volume, giving CASY a broad detection range of 0.7-100 µm, making it ideal for counting bacteria. Furthermore, CASY greatly limits the need for dilution and ensures that up to 50,000 bacteria can be enumerated in 45 seconds or less. In addition to counting individual bacterium, CASY’s aggregation discrimination is more accurate than visual methods that rely on morphological differences because CASY uses a mathematical correction to determine sample aggregation (Figure 2). Together, these features substantially improve the ease, speed, and accuracy of counting bacteria.
Figure 2.
Figure 2. Examples of volume-based aggregation correction.
Nature & Nurture – Physiological Relevance of Biofilms
In addition to counting accuracy, a second major problem biofilm investigators face is the challenge of building physiologically relevant biofilm models. In nature, and specifically in the human body, biofilms rarely exist in dry static environments. Bacteria in humans are continuously exposed to fluid movement, from saliva in the mouth to blood and interstitial fluid throughout the body. Biofilms grown in static plates or flasks can be useful for examining early-stage biofilm formation and genetic screening. However, the static nature of these systems has several drawbacks, including inadequate nutrient exchange, limited biofilm maturity, and perhaps most importantly, a lack of physiological relevance (2). These drawbacks can have a severe negative impact on the accuracy of antibiotic testing and other biofilm research investigations.
An obvious solution to this problem is to grow adherent biofilms under liquid flow. This has led many microbiologists to try their hand at fluid engineering by constructing “do-it-yourself” shear flow systems or relying on commercial shear flow systems that were not designed for biofilm growth and analysis. Although DIY systems can be effective, the construction and use of these systems are often less than ideal and lack standardization, as highlighted in a previous blog post. Furthermore, although a limited number of commercial shear flow systems are available, these systems are ripe for contamination issues and suffer from very low-throughput, making them difficult to use for antibiotic discovery and development.
BioFlux shear flow systems overcome the problems of DIY and other commercial shear flow systems by providing a contamination-free, high-throughput shear flow system. BioFlux leverages pneumatic pumping through microfluidic channels embedded into a well-plate format to ensure that nothing in the disposable plate contacts the system, making BioFlux ideal for physiologically relevant biofilm investigations (Figure 3). Furthermore, changing the flow rate is as simple as entering a number into the software, making BioFlux a convenient system to both grow and assay biofilms.
Figure 3.
Figure 3. Image of a BioFlux plate and the contactless pneumatic pump interface on a BioFlux 1000z microscope stage.
Enhance your biofilm investigations
Here, we have highlighted two common issues in many biofilm investigations and their potential solutions. Leveraging these tools can improve biofilm workflows, accuracy, and relevance. These benefits have the potential to accelerate antimicrobial discovery, enhance biofilm engineering, and improve experimental standardization. Together, the features of CASY and BioFlux provide substantial benefits to biofilm investigators.
References
Dr. Anson Blanks completed his BS in exercise physiology at East Carolina University and his MS in clinical exercise science at Appalachian State University. After working as a clinical exercise physiologist in cardiopulmonary rehabilitation, Dr. Blanks decided to pursue a career in scientific research. He attended Virginia Commonwealth University, where he completed his Ph.D. in Rehabilitation and Movement Science. After spending several years as a research and development scientist in biotechnology industry, Dr. Blanks is now a scientific marketing manager for Cell Microsystems in Durham, NC.
In the ongoing battle against antibiotic-resistant bacteria, or “superbugs,” scientists are constantly seeking innovative tools to understand the mechanisms of resistance and develop effective treatments. One such groundbreaking technology is shear flow systems, which have emerged as game-changers in microbiological research. By simulating the dynamic conditions of bacteria’s natural environments, such as the human body or food processing, shear flow systems provide unique insights into bacterial behavior and resistance mechanisms that traditional static methods simply cannot achieve. In this blog post, we’ll explore the benefits of using shear flow systems to combat antibiotic resistance and develop next-generation therapies.
Antibiotic resistance is a complex phenomenon influenced by a variety of factors, including genetic mutations, horizontal gene transfer, and the formation of biofilms. Traditional laboratory methods often fail to capture the dynamic nature of these processes, leading to incomplete or misleading results. Shear flow systems address this limitation by providing a more realistic environment for bacterial growth and interaction. Here’s how:
1. Mimicking Real-World Conditions
In the human body, bacteria are rarely in a static state. They are constantly exposed to fluid flow, such as blood circulation or urine flow, which influences their behavior and resistance mechanisms. Shear flow systems can replicate these conditions, allowing researchers to study how bacteria respond to antibiotics under realistic physiological conditions. This is critical for understanding how resistance develops and persists in vivo. This benefit was recently highlighted in a webinar presented by Dr. Katharina Richter, a microbiology researcher from the University of Adelaide in Australia. Using a high-throughput BioFlux Shear Flow System, Dr. Richter and her team were able to test 3 different methods of superbug treatments under physiological conditions.
2. Studying Biofilm Formation and Resistance
Biofilms—structured communities of bacteria encased in a protective matrix—are a major contributor to antibiotic resistance. Biofilms are notoriously difficult to treat because they shield bacteria from antibiotics and the immune system. Shear flow systems enable researchers to study biofilm formation in real time, observing how bacteria adhere to surfaces, form microcolonies, and develop resistance under flow conditions. For example, using a BioFlux, Dr. Richter was able to leverage high-resolution imaging to obtain unprecedented insight into the biofilm killing and prevention efficacy of a copper and diethyldithiocarbamate (DDC) combination nanoparticles¹. The group is currently investigating the use of a Cu-DDC infused gel that can be injected into wounds that are at high risk for chronic infection, such as hernia.
The study of biofilm communities presents unique challenges in experimental design and analysis. While mixed bacterial populations offer fascinating insights into microbial interactions and community dynamics, accurately characterizing and investigating these complex systems has previously required sophisticated and tedious technical approaches. Here, we will discuss advances in technology that have helped to simplify and streamline polymicrobial investigations.
Throughout the last decade, a trend for growing and assaying biofilms under shear flow has gained traction among microbiologists. The rationale for investigating biofilms under flow is multi-dimensional. A recent Cell Microsystems survey asked why microbiologists are using shear flow found that the top responses were:
Of the scientists surveyed, 40% indicated that they use a BioFlux Shear Flow System. This system provides unique advantages over other methods to induce shear stress, such as slide-based parallel plate flow chambers and benchtop rockers.
BioFlux uses microfluidic channels embedded onto the bottom of standard-sized 6, 24, or 48-well plates to create isolated and independent flow chambers where user-defined wall shear stress is initiated and maintained by digitally controlled air pressure. This not only simplifies the setup and experimental processes but also ensures that experiments are contained and do not contaminate the shear flow system.
Although shear flow has been thoroughly demonstrated to improve the biological relevance of biofilm investigations1, an often overlooked aspect of biofilm physiology is the significant role that multicellular aggregates play in biofilm formation2. When investigating Pseudomonas aeruginosa, it has been demonstrated that multicellular aggregates lead to faster biofilm formation and enhanced resistance to antimicrobials and immune responses3. In addition, multispecies microbial aggregates have been shown to dominate single cells in human saliva4. Together, these data show that aggregates are important areas of investigation because they are more likely to represent physiological biofilm formation and growth.
