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Biosafety in Microbiology Labs: Principles and Biosafety Levels

Introduction

Microbiology is the study of tiny, microscopic organisms called microbes.[1] For studies like learning the shape and size of organisms, microscopic study is the preferable approach. However, when it comes to genetic analysis, molecular functions, and metabolic studies, the microbes are required to be cultured in labs in their preferable environment.

To ensure a particular strain of microbe is maintained in the cultures, strict aseptic or sterile conditions are required to be maintained in the labs. Moreover, people working in these labs also have a high likelihood of contracting diseases and infections caused by cultured microbes. 

Therefore, standard practices exist to ensure a safe work environment for people and the containment of the microbe inside the labs. These practices came into existence when scientists noted that there was a history of people contracting diseases, such as typhoid, tetanus, and cholera, that were associated with laboratories. 

Some of these reports include the[2]:

  • Sulkin and Pike (1949) report: Here, 21 of the 222 reported viral infections were considered to be serious and a third of the infection was attributed to poor handling of infected tissues.
  • Hanson et al (1967) report which links Arboviruses to 428 laboratory-associated infections and the commonest infection source in labs was infectious aerosols.
  • Skinhoj (1974) report on a survey of Danish clinical chemistry labs, where around 1000 lab personnel contracted hepatitis in a year. 

These studies emphasized the importance of learning and implementing biosafety measures in microbiology labs, especially while working with life-threatening microbes, such as viruses.

Principles of Biosafety

Several safe methods are available today that allow for better containment of microbes in labs, maintain their favorable environment, and prevent lab personnel from becoming infected with these infectious agents. This complete process is known as the containment of microbes and it’s divided into two[2]:

  • Primary containment: Here, personnel and the immediate laboratory environment are safeguarded from infectious agents. Primary containment is done by utilizing good microbiological techniques, proper safety equipment, and vaccinating working professionals in the labs.
  • Secondary containment: It’s the process of protecting the environment outside of the lab from the spread of infectious agents. This is done by combining highly-advanced designs of facilities with best operational practices.

Elements of Microbial Containment

The containment of microbes is composed of three elements: safety equipment, best laboratory practices and techniques, and facility design.

1. Safety Equipment

It’s also called the primary barrier which includes biosafety cabinets and a variety of other enclosed containers. The biosafety cabinet is beneficial to contain infectious aerosols, which are generated while performing microbiological procedures. 

The biosafety cabinets are categorized into three groups: Class I, Class II, and Class III biosafety cabinets. 

Class I and class II biosafety cabinets are open-fronted biological safety cabinets, thus they only offer partial containment. Class III biosafety cabinets provide maximum protection level to both laboratory personnel and the environment.

Other than biosafety cabinets, some other equipment, such as gloves, lab coats, shoe covers, lab gowns, face shields, respirators, boots, and safety glasses provide an extra level of protection to lab personnel.[2]

2. Best laboratory practices and techniques

Standard laboratory training and practices are essential for the containment of microbial organisms in labs. Lab personnel should be aware of the potential hazards of infected materials and agents they are required to work with. They must be taught standard practices and microbiological techniques for handling and maintaining such materials.

The lab director has an important role to play when it comes to choosing appropriate training for lab personnel. Moreover, when the safety measures and training are not sufficient to contain the spread of infectious agents, they are required to select additional practices to minimize/eliminate hazards associated with infectious materials/agents.

Additionally, a biosafety operation manual should be available in all labs explaining the hazards associated with each infectious agent and the procedures to minimize the risks. 

A lab expert or scientist trained in using laboratory techniques, aware of lab safety measures, and hazards associated with working with an infectious agent must direct such lab activities.[2]

3. Facility design

Appropriate facility design and engineering need to supplement lab personnel’s efforts, techniques, and best safety practices for proper containment and safety of people, inside or outside the labs. It serves as an additional barrier against the spread of infectious agents.

Based on the level of containment, the facility design is of three types[2]:

  • The basic laboratory

It includes the space created for working with viable infectious agents or materials that are not infectious or potentially have low levels of hazard, with biosafety level 1 and level 2 facilities.

For example, the locations of animal rooms and waste staging areas, which are known to be sources of general contamination, should not be adjacent to patient care areas, but they can be in the same building or complex.

Moreover, it is important to separate the public areas and offices needed by non-laboratory staff from the laboratories.

  • The containment laboratory

The containment lab contains some specialized engineering features to deal with potential hazards and protect lab personnel, the environment, and the community. It’s described as a biosafety level 3 facility.

Its access controls and specialized ventilation system are the distinguishing features of the containment lab.

The lab settings can be the building itself, a single module within it, or a complex of modules. And regardless of the situation, the laboratory is always separated from public areas by a controlled access zone.

  • The maximum containment laboratory

This lab has special engineering and containment systems. It permits the safe handling of infectious agents that can be extremely dangerous to lab workers or can cause epidemic diseases. This level of facility is described as biosafety level 4.

The lab should be built in isolated areas within the building or a separate building. It has secondary barriers to protect the environment from hazardous materials. 

The barrier can be airlocks or liquid disinfectant barriers, sealed entrances to the laboratory, a treatment system to eliminate contaminants from exhaust air, and a clothes-change room and a shower room adjacent to the laboratory ventilation system.

Four Biosafety Levels

Biosafety levels are divided into four levels based on their combination of safety equipment, laboratory techniques, best practices, and appropriate laboratory facilities to perform operations, control hazards by infectious agents, and run lab activities safely.

The combination of elements, designed and fit into these four levels decides the extent or degree of protection the labs provide to lab personnel, the environment, and the community.

The following is a brief overview of all four protection levels in ascending order.

Biosafety Level 1

The standard practices, equipment, and facilities included in this level suit undergraduate, secondary education training, or any other teaching labs or facilities where experiments are performed on viable microbial strains that don’t infect any healthy adult human. 

A few examples of these types of strains include Naegleria gruberi, Bacillus subtilis, and infectious canine hepatitis virus[2].

Even so, some opportunistic organisms can infect young, elderly, immunodeficient, or immunosuppressed individuals.

Standard microbiological practices:

  • When experiments are in progress, the laboratory director restricts or limits access as needed.
  • Decontamination is performed for all contaminated liquids and solids before disposal.
  • Once a day, work surfaces are decontaminated after a spill of any viable material.
  • To prevent contamination or soiling of street clothes, laboratory gowns, coats, or uniforms should be worn.
  • Prohibition of eating, drinking, smoking, storing food, and applying cosmetics inside the lab.
  • Pipetting is done with mechanical devices, and mouth pipetting is not permitted.

Special practices:

  • The decontaminated materials are placed in a leak-proof container before being taken away from the laboratory for decontamination.

Containment equipment:

  • Biosafety level 1 agents do not generally require special containment equipment.

Laboratory facilities:

  • The lab is designed to be easy to clean.
  • Each laboratory is equipped with a sink for handwashing.
  • A benchtop should be resistant to water, alkali, organic solvents, acid, and moderate heat.

Biosafety Level 2

The biosafety level 2 practices and equipment are suitable for diagnostic, clinical, and facilities that work with a wide range of indigenous moderate-risk infectious agents. 

These agents are generally associated with some human diseases with different degrees of severity. A few examples of the microbes in this category include salmonellae, Hepatitis B virus, and Toxoplasma spp[2].

Standard microbiological practices:

  • The standard microbiological practices of Biosafety Level 2 are similar to Biosafety Level 1.

Special practices:

  • The decontaminated materials are placed in a leak-proof container before being taken away from the laboratory for decontamination.
  • The lab director creates policies that ensure that only those who have been informed of potential hazards and meet entry requirements (e.g., immunizations) are allowed into the lab or animal rooms.
  • Rodent and insect control programs are in place.
  • Infectious material spills and accidents are immediately reported to the lab director. An appropriate medical evaluation, surveillance, and treatment are provided and records are maintained.

Containment equipment:

Lab personnel is required to use biosafety cabinets and personal protective equipment to perform lab procedures, especially when:

  • Working with a high concentration of infectious agents. These agents are required to be centrifuged in an open lab if using centrifuge safety cups and are only opened in biosafety cabinets.
  • Using a procedure with high potential to create aerosols.

Laboratory facilities:

The lab facilities are similar to Biosafety Level 1, except:

  • An autoclave is available for decontaminating infectious laboratory wastes.

Biosafety Level 3

This level of biosafety is applicable to research, teaching, diagnostic, clinical, and production facilities that work with indigenous or exotic agents, and where aerosols may pose a threat of infection and can have lethal consequences. 

Examples of agents for which biosafety level 3 is required include St. Louis encephalitis virus, Mycobacterium tuberculosis, and Coxiella burnetii[2].

Standard microbiological practices:

  • The standard microbiological practices of Biosafety Level 3 are similar to Biosafety Level 1.

Special practices:

In addition to the practices mentioned in Biosafety Level 2, it includes:

  • Lab doors should be closed while performing the experiments.
  • High-efficiency particulate air (HEPA) filters and liquid disinfectant traps protect vacuum lines.
  • It is prohibited to keep animals and plants in laboratories that are not related to the research conducted.
  • A biosafety manual is prepared or adopted.

Containment equipment:

  • All classes of biosafety cabinets, physical and personal protective equipment should be present while performing activities having the potential to cause exposure to infectious aerosols. 
  • These include masks, gloves, special protective clothing, respirators, centrifuge safety cups, containment caging for animals, and sealed centrifuge rotors.

Laboratory facilities:

In addition to the facilities mentioned in Biosafety level 1, it also includes:

  • Within the building, the laboratory is separated from areas where the flow of traffic is unrestricted.
  • A sink, which can be operated with an elbow, foot, or automatic, should be placed near the lab exit door to wash hands.
  • Access doors to the laboratory or containment module are self-closing.
  • Class I and Class II Biological safety cabinets with HEPA-filtered exhaust air are vented to the outdoors or through the building’s exhaust system.

Biosafety Level 4

The level of safety procedures and equipment are applicable when labs work with agents that can cause life-threatening diseases, such as the Lassa fever virus[2].

Standard microbiological practices:

  • It includes all standard practices as mentioned in biosafety level 1.

Special practices:

In addition to practices mentioned in Biosafety Level 3:

  • While removing viable biological material from a class III biosafety cabinet to the outside lab, it should be placed in a sealed primary container followed by a sealed secondary container and removed through a disinfectant dunk tank, fumigation chamber, or an airlock designed for this purpose.
  • Facility or individual laboratory room access is restricted to those whose presence is necessary for program or support purposes.
  • Lab personnel is only permitted to enter and exit the facility through the clothing change and shower rooms (each time, a shower is required).
  • The laboratory keeps a report of workplace accidents, employee absences, and potential illnesses associated with the lab.

Containment equipment:

  • A Class III biosafety cabinet is required to perform the procedures. But Class I and Class II biosafety cabinets are also used in combination with one-piece positive-pressure personnel suits ventilated by a life support system. 

Laboratory facilities:

In addition to facilities mentioned in biosafety level 3:

  • Maximum containment facilities are either separate buildings or delineated and isolated areas inside buildings.
  • Facility interiors, such as light fixtures, air ducts, and utility pipes are designed to minimize dust accumulation on horizontal surfaces.
  • Central vacuum systems do not serve areas outside the facility.
  • Before being released from the maximum containment facility, liquid effluents from laboratory sinks, biological safety cabinets, floors, and autoclave chambers are decontaminated by heat treatment.
  •  The facility may include a specially designed suiting area.

To learn all about the facilities, equipment, and special practices of microbiology labs, refer to the materials mentioned in the reference section.

Conclusion

Microbiology laboratories dealing with infectious agents that may pose a threat to laboratory personnel require biosafety measures. These measures include information on standard microbiological practices and procedures, safety equipment, and biosafety facilities.

However, not all microbes possess the same level of threat to humans after their exposure. Therefore, according to the level of danger posed to the public by an infectious agent, four types of biosafety levels are created for the safety of lab personnel, the community, and the environment. According to the levels of their biosafety, the labs are ordered as: Biosafety Level 1, Biosafety Level 2, Biosafety Level 3, and Biosafety Level 4.

Considering the history of people contracting diseases, safety measures are a must in microbial labs. And following these rules, will not only help the people working in labs, but also the people around them, and the whole community.

References:

  1. Biosafety Practices and Procedures for the Microbiology Laboratory. Retrieved from https://milnepublishing.geneseo.edu/suny-microbiology-lab/chapter/biosafety-practices-and-procedures-for-the-microbiology-laboratory/
  2. National Research Council (US) Committee on Hazardous Biological Substances in the Laboratory. Biosafety In The Laboratory: Prudent Practices for the Handling and Disposal of Infectious Materials. Washington (DC): National Academies Press (US); 1989. Appendix A, Biosafety in Microbiological and Biomedical Laboratories. Available from: https://www.ncbi.nlm.nih.gov/books/NBK218631/

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Introduction

In behavioral neuroscience, the Open Field Test (OFT) remains one of the most widely used assays to evaluate rodent models of affect, cognition, and motivation. It provides a non-invasive framework for examining how animals respond to novelty, stress, and pharmacological or environmental manipulations. Among the test’s core metrics, the percentage of time spent in the center zone offers a uniquely normalized and sensitive measure of an animal’s emotional reactivity and willingness to engage with a potentially risky environment.

This metric is calculated as the proportion of time spent in the central area of the arena—typically the inner 25%—relative to the entire session duration. By normalizing this value, researchers gain a behaviorally informative variable that is resilient to fluctuations in session length or overall movement levels. This makes it especially valuable in comparative analyses, longitudinal monitoring, and cross-model validation.

Unlike raw center duration, which can be affected by trial design inconsistencies, the percentage-based measure enables clearer comparisons across animals, treatments, and conditions. It plays a key role in identifying trait anxiety, avoidance behavior, risk-taking tendencies, and environmental adaptation, making it indispensable in both basic and translational research contexts.

Whereas simple center duration provides absolute time, the percentage-based metric introduces greater interpretability and reproducibility, especially when comparing different animal models, treatment conditions, or experimental setups. It is particularly effective for quantifying avoidance behaviors, risk assessment strategies, and trait anxiety profiles in both acute and longitudinal designs.

What Does Percentage of Time in the Centre Measure?

This metric reflects the relative amount of time an animal chooses to spend in the open, exposed portion of the arena—typically defined as the inner 25% of a square or circular enclosure. Because rodents innately prefer the periphery (thigmotaxis), time in the center is inversely associated with anxiety-like behavior. As such, this percentage is considered a sensitive, normalized index of:

  • Exploratory drive vs. risk aversion: High center time reflects an animal’s willingness to engage with uncertain or exposed environments, often indicative of lower anxiety and a stronger intrinsic drive to explore. These animals are more likely to exhibit flexible, information-gathering behaviors. On the other hand, animals that spend little time in the center display a strong bias toward the safety of the perimeter, indicative of a defensive behavioral state or trait-level risk aversion. This dichotomy helps distinguish adaptive exploration from fear-driven avoidance.

  • Emotional reactivity: Fluctuations in center time percentage serve as a sensitive behavioral proxy for changes in emotional state. In stress-prone or trauma-exposed animals, decreased center engagement may reflect hypervigilance or fear generalization, while a sudden increase might indicate emotional blunting or impaired threat appraisal. The metric is also responsive to acute stressors, environmental perturbations, or pharmacological interventions that impact affective regulation.

  • Behavioral confidence and adaptation: Repeated exposure to the same environment typically leads to reduced novelty-induced anxiety and increased behavioral flexibility. A rising trend in center time percentage across trials suggests successful habituation, reduced threat perception, and greater confidence in navigating open spaces. Conversely, a stable or declining trend may indicate behavioral rigidity or chronic stress effects.

  • Pharmacological or genetic modulation: The percentage of time in the center is widely used to evaluate the effects of pharmacological treatments and genetic modifications that influence anxiety-related circuits. Anxiolytic agents—including benzodiazepines, SSRIs, and cannabinoid agonists—reliably increase center occupancy, providing a robust behavioral endpoint in preclinical drug trials. Similarly, genetic models targeting serotonin receptors, GABAergic tone, or HPA axis function often show distinct patterns of center preference, offering translational insights into psychiatric vulnerability and resilience.

Critically, because this metric is normalized by session duration, it accommodates variability in activity levels or testing conditions. This makes it especially suitable for comparing across individuals, treatment groups, or timepoints in longitudinal studies.

A high percentage of center time indicates reduced anxiety, increased novelty-seeking, or pharmacological modulation (e.g., anxiolysis). Conversely, a low percentage suggests emotional inhibition, behavioral avoidance, or contextual hypervigilance. reduced anxiety, increased novelty-seeking, or pharmacological modulation (e.g., anxiolysis). Conversely, a low percentage suggests emotional inhibition, behavioral avoidance, or contextual hypervigilance.

Behavioral Significance and Neuroscientific Context

1. Emotional State and Trait Anxiety

The percentage of center time is one of the most direct, unconditioned readouts of anxiety-like behavior in rodents. It is frequently reduced in models of PTSD, chronic stress, or early-life adversity, where animals exhibit persistent avoidance of the center due to heightened emotional reactivity. This metric can also distinguish between acute anxiety responses and enduring trait anxiety, especially in longitudinal or developmental studies. Its normalized nature makes it ideal for comparing across cohorts with variable locomotor profiles, helping researchers detect true affective changes rather than activity-based confounds.

2. Exploration Strategies and Cognitive Engagement

Rodents that spend more time in the center zone typically exhibit broader and more flexible exploration strategies. This behavior reflects not only reduced anxiety but also cognitive engagement and environmental curiosity. High center percentage is associated with robust spatial learning, attentional scanning, and memory encoding functions, supported by coordinated activation in the prefrontal cortex, hippocampus, and basal forebrain. In contrast, reduced center engagement may signal spatial rigidity, attentional narrowing, or cognitive withdrawal, particularly in models of neurodegeneration or aging.

3. Pharmacological Responsiveness

The open field test remains one of the most widely accepted platforms for testing anxiolytic and psychotropic drugs. The percentage of center time reliably increases following administration of anxiolytic agents such as benzodiazepines, SSRIs, and GABA-A receptor agonists. This metric serves as a sensitive and reproducible endpoint in preclinical dose-finding studies, mechanistic pharmacology, and compound screening pipelines. It also aids in differentiating true anxiolytic effects from sedation or motor suppression by integrating with other behavioral parameters like distance traveled and entry count (Prut & Belzung, 2003).

4. Sex Differences and Hormonal Modulation

Sex-based differences in emotional regulation often manifest in open field behavior, with female rodents generally exhibiting higher variability in center zone metrics due to hormonal cycling. For example, estrogen has been shown to facilitate exploratory behavior and increase center occupancy, while progesterone and stress-induced corticosterone often reduce it. Studies involving gonadectomy, hormone replacement, or sex-specific genetic knockouts use this metric to quantify the impact of endocrine factors on anxiety and exploratory behavior. As such, it remains a vital tool for dissecting sex-dependent neurobehavioral dynamics.
 The percentage of center time is one of the most direct, unconditioned readouts of anxiety-like behavior in rodents. It is frequently reduced in models of PTSD, chronic stress, or early-life adversity. Because it is normalized, this metric is especially helpful for distinguishing between genuine avoidance and low general activity.

Methodological Considerations

  • Zone Definition: Accurately defining the center zone is critical for reliable and reproducible data. In most open field arenas, the center zone constitutes approximately 25% of the total area, centrally located and evenly distanced from the walls. Software-based segmentation tools enhance precision and ensure consistency across trials and experiments. Deviations in zone parameters—whether due to arena geometry or tracking inconsistencies—can result in skewed data, especially when calculating percentages.

     

  • Trial Duration: Trials typically last between 5 to 10 minutes. The percentage of time in the center must be normalized to total trial duration to maintain comparability across animals and experimental groups. Longer trials may lead to fatigue, boredom, or habituation effects that artificially reduce exploratory behavior, while overly short trials may not capture full behavioral repertoires or response to novel stimuli.

     

  • Handling and Habituation: Variability in pre-test handling can introduce confounds, particularly through stress-induced hypoactivity or hyperactivity. Standardized handling routines—including gentle, consistent human interaction in the days leading up to testing—reduce variability. Habituation to the testing room and apparatus prior to data collection helps animals engage in more representative exploratory behavior, minimizing novelty-induced freezing or erratic movement.

     

  • Tracking Accuracy: High-resolution tracking systems should be validated for accurate, real-time detection of full-body center entries and sustained occupancy. The system should distinguish between full zone occupancy and transient overlaps or partial body entries that do not reflect true exploratory behavior. Poor tracking fidelity or lag can produce significant measurement error in percentage calculations.

     

  • Environmental Control: Uniformity in environmental conditions is essential. Lighting should be evenly diffused to avoid shadow bias, and noise should be minimized to prevent stress-induced variability. The arena must be cleaned between trials using odor-neutral solutions to eliminate scent trails or pheromone cues that may affect zone preference. Any variation in these conditions can introduce systematic bias in center zone behavior. Use consistent definitions of the center zone (commonly 25% of total area) to allow valid comparisons. Software-based segmentation enhances spatial precision.

Interpretation with Complementary Metrics

Temporal Dynamics of Center Occupancy

Evaluating how center time evolves across the duration of a session—divided into early, middle, and late thirds—provides insight into behavioral transitions and adaptive responses. Animals may begin by avoiding the center, only to gradually increase center time as they habituate to the environment. Conversely, persistently low center time across the session can signal prolonged anxiety, fear generalization, or a trait-like avoidance phenotype.

Cross-Paradigm Correlation

To validate the significance of center time percentage, it should be examined alongside results from other anxiety-related tests such as the Elevated Plus Maze, Light-Dark Box, or Novelty Suppressed Feeding. Concordance across paradigms supports the reliability of center time as a trait marker, while discordance may indicate task-specific reactivity or behavioral dissociation.

Behavioral Microstructure Analysis

When paired with high-resolution scoring of behavioral events such as rearing, grooming, defecation, or immobility, center time offers a richer view of the animal’s internal state. For example, an animal that spends substantial time in the center while grooming may be coping with mild stress, while another that remains immobile in the periphery may be experiencing more severe anxiety. Microstructure analysis aids in decoding the complexity behind spatial behavior.

Inter-individual Variability and Subgroup Classification

Animals naturally vary in their exploratory style. By analyzing percentage of center time across subjects, researchers can identify behavioral subgroups—such as consistently bold individuals who frequently explore the center versus cautious animals that remain along the periphery. These classifications can be used to examine predictors of drug response, resilience to stress, or vulnerability to neuropsychiatric disorders.

Machine Learning-Based Behavioral Clustering

In studies with large cohorts or multiple behavioral variables, machine learning techniques such as hierarchical clustering or principal component analysis can incorporate center time percentage to discover novel phenotypic groupings. These data-driven approaches help uncover latent dimensions of behavior that may not be visible through univariate analyses alone.

Total Distance Traveled

Total locomotion helps contextualize center time. Low percentage values in animals with minimal movement may reflect sedation or fatigue, while similar values in high-mobility subjects suggest deliberate avoidance. This metric helps distinguish emotional versus motor causes of low center engagement.

Number of Center Entries

This measure indicates how often the animal initiates exploration of the center zone. When combined with percentage of time, it differentiates between frequent but brief visits (indicative of anxiety or impulsivity) versus fewer but sustained center engagements (suggesting comfort and behavioral confidence).

Latency to First Center Entry

The delay before the first center entry reflects initial threat appraisal. Longer latencies may be associated with heightened fear or low motivation, while shorter latencies are typically linked to exploratory drive or low anxiety.

Thigmotaxis Time

Time spent hugging the walls offers a spatial counterbalance to center metrics. High thigmotaxis and low center time jointly support an interpretation of strong avoidance behavior. This inverse relationship helps triangulate affective and motivational states.

Applications in Translational Research

  • Drug Discovery: The percentage of center time is a key behavioral endpoint in the development and screening of anxiolytic, antidepressant, and antipsychotic medications. Its sensitivity to pharmacological modulation makes it particularly valuable in dose-response assessments and in distinguishing therapeutic effects from sedative or locomotor confounds. Repeated trials can also help assess drug tolerance and chronic efficacy over time.
  • Genetic and Neurodevelopmental Modeling: In transgenic and knockout models, altered center percentage provides a behavioral signature of neurodevelopmental abnormalities. This is particularly relevant in the study of autism spectrum disorders, ADHD, fragile X syndrome, and schizophrenia, where subjects often exhibit heightened anxiety, reduced flexibility, or altered environmental engagement.
  • Hormonal and Sex-Based Research: The metric is highly responsive to hormonal fluctuations, including estrous cycle phases, gonadectomy, and hormone replacement therapies. It supports investigations into sex differences in stress reactivity and the behavioral consequences of endocrine disorders or interventions.
  • Environmental Enrichment and Deprivation: Housing conditions significantly influence anxiety-like behavior and exploratory motivation. Animals raised in enriched environments typically show increased center time, indicative of reduced stress and greater behavioral plasticity. Conversely, socially isolated or stimulus-deprived animals often show strong center avoidance.
  • Behavioral Biomarker Development: As a robust and reproducible readout, center time percentage can serve as a behavioral biomarker in longitudinal and interventional studies. It is increasingly used to identify early signs of affective dysregulation or to track the efficacy of neuromodulatory treatments such as optogenetics, chemogenetics, or deep brain stimulation.
  • Personalized Preclinical Models: This measure supports behavioral stratification, allowing researchers to identify high-anxiety or low-anxiety phenotypes before treatment. This enables within-group comparisons and enhances statistical power by accounting for pre-existing behavioral variation. Used to screen anxiolytic agents and distinguish between compounds with sedative vs. anxiolytic profiles.

Enhancing Research Outcomes with Percentage-Based Analysis

By expressing center zone activity as a proportion of total trial time, researchers gain a metric that is resistant to session variability and more readily comparable across time, treatment, and model conditions. This normalized measure enhances reproducibility and statistical power, particularly in multi-cohort or cross-laboratory designs.

For experimental designs aimed at assessing anxiety, exploratory strategy, or affective state, the percentage of time spent in the center offers one of the most robust and interpretable measures available in the Open Field Test.

Explore high-resolution tracking solutions and open field platforms at
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References

  • Prut, L., & Belzung, C. (2003). The open field as a paradigm to measure the effects of drugs on anxiety-like behaviors: a review. European Journal of Pharmacology, 463(1–3), 3–33.
  • Seibenhener, M. L., & Wooten, M. C. (2015). Use of the open field maze to measure locomotor and anxiety-like behavior in mice. Journal of Visualized Experiments, (96), e52434.
  • Crawley, J. N. (2007). What’s Wrong With My Mouse? Behavioral Phenotyping of Transgenic and Knockout Mice. Wiley-Liss.
  • Carola, V., D’Olimpio, F., Brunamonti, E., Mangia, F., & Renzi, P. (2002). Evaluation of the elevated plus-maze and open-field tests for the assessment of anxiety-related behavior in inbred mice. Behavioral Brain Research, 134(1–2), 49–57.

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