The purpose of this three-article dissertation was to expand knowledge and theory regarding digital tool use in biology laboratory courses, such as microbiology, which requires specific laboratory skill development through the perception of instructors and students. Article 1 establishes the broad digital literacy and fluency problem in education by providing definitions and the context behind digital literacy fluency and its impact on acquisition of knowledge in digital learning environments. The study provided a picture of the lack of knowledge about the use of digital tools in education and practical problems around appropriate implementation, infrastructure, and preparedness. Article 2 presents results of a literature research study about the foundational, pandemic-induced, and current digital tool use in biology and microbiology lab courses. Recommendations for improvement in digital tool implementation, pedagogical approach, and appropriate selection to meet learning outcomes were provided. Article 3 describes a 3-layered study to build a new instrument to understand minority-student perceptions of identity and digital literacy and technology barriers on student success in STEM courses. The study identified challenges and benefits of digital tool use in virtual microbiology lab courses, unique challenges of underrepresented populations, and the need to develop an instrument to capture the context of this unique population.