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Tech Talk: Enhancing Microbiology Investigations

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.

microbe sizes

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.

Microbe counting method table

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.

CASY aggregates

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. 

BioFlux 1000Z interface and microscope

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 

  1. Gondil, V.S., Subhadra, B. Biofilms and their role on diseases. BMC Microbiol 23, 203 (2023). https://doi.org/10.1186/s12866-023-02954-2
  2. Thomen P, Robert J, Monmeyran A, Bitbol AF, Douarche C, Henry N. Bacterial biofilm under flow: First a physical struggle to stay, then a matter of breathing. PLoS One 12(4):e0175197. (2017). doi:10.1371/journal.pone.0175197

Dr. Anson Blanks completed his BS in exercise physiology at East Carolina University in 2003. Dr. Blanks then attended Appalachian State University, where he earned his Master of Science in clinical exercise science in 2009. After working as a clinical exercise physiologist in a cardiopulmonary rehabilitation center in Washington, DC, 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 in 2018. 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.

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