Ateneo - UPO

The course aims to provide the basics of bacterial culturing, such as preparation of culture media, with emphasis on the differences between media: generic, selective, differential, broth and agarized; the various sterilization techniques necessary for the preparation of material used in microbiology will be covered; techniques for isolation and transplantation of bacterial strains from mixed culture with the aim of obtaining pure strains; techniques for enrichment and freezing of pure strains for the establishment of a “collection” of microorganisms (identification by MALDI-TOF); DNA extraction and purification techniques from pure strains; DNA amplification techniques, such as PCR (Polymerase Chain Reaction) and NGS techniques; preservation of strains by lyophilization; assessment of cell viability, Gram staining, assessment of bacterial density in a generic matrix (dilutions, measurement of optical density using spectrophotometer, preparation of bacterial inocula, bacterial cell counts).

The educational material will be provided in the form of slides and operational procedures, as well as multimedia and bibliographic resources available on websites such as PubMed.

The course aims to provide basic theoretical skills related to microbiology and its practical applications, as well as the use of both basic and advanced equipment found in microbiology laboratories. Students will acquire knowledge for the characterization, isolation, transfer, enrichment, and freezing of microorganisms (pure strains) present in various biological matrices: environmental and/or clinical. Methods for freeze-drying bacterial strains for long-term preservation will be illustrated. Additionally, students will be provided with information about basic methodologies and principles of safety and sterility to be used in a microbiology laboratory, ensuring the safety of both the operator and the sample. The course will cover analytical methods for the main matrices, including extraction, amplification, purification, and sequencing (kits for bacterial DNA extraction, PCR, principles of NGS). Practical principles for assessing cell viability, Gram staining, and evaluating bacterial density in a general matrix will also be addressed (dilutions, measurement of optical density using a spectrophotometer, principles of operation, preparation of bacterial inocula, and cell counting).

Basic knowledge of microbiology and biology in general is required. In particular, knowledge of the morphology and physiology of bacterial and eukaryotic cells (yeasts) is needed. Basic understanding of biological macromolecules, organic chemistry, general chemistry, physics, and mathematics is also essential. Additionally, students must possess adequate language skills and scientific proficiency, as well as appropriate behavior and seriousness necessary for conducting activities in the laboratory.

Lectures in the laboratory and practical application during exercises will be supported by slide presentations, multimedia materials, and bibliographic research, including reading, discussion, and presentation of ongoing scientific articles.

Students with physical disabilities, Learning Disabilities or Special Education Needs can request specific services and tools via the Staff Sviluppo e Coordinamento Carriere e Servizi alle Studentesse e agli Studenti, consulting the University webpage: https://www.uniupo.it/en/services/services-students-physical-or-learning-disabilities Students with disabilities, learning disabilities or special education needs, once they have contacted the University Staff, can refer to the tutor in charge of the course to define the examination modalities, concerning academic aspects.

The exam is a written test based on multiple choice questions and descriptive response questions with the aim of verifying the knowledge and understanding of the points of the entire program treated. Students will have 1 hour to complete the assignment, composed of multiple choice questions and questions on specific topics with descriptive answer. The final mark will be the sum of the two partial marks. The final score will result from the partial scores obtained in the single questions and it is expressed in thirtieth (the passing mark is 18). The consultation of any material is not allowed during the exam.

The course aims to provide the fundamentals for culturing bacterial strains from various matrices, such as environmental samples like the rhizosphere or food and/or clinical matrices. Methodologies for preparing both agar and broth culture media will be addressed, with particular attention to general and more specific (selective and differential) media. The main sterilization and sanitation techniques for plasticware, surfaces, and materials used will be covered, which are necessary for safe operation (both for the operator and the sample) and to prevent contamination during standard laboratory procedures.
Different techniques for isolating and transferring bacterial strains from mixed cultures to obtain pure strains, represented by a single bacterial population, will be illustrated. With knowledge of appropriate enrichment and freezing techniques for pure strains, it will be possible to build a “collection” of microorganisms that can be frozen and preserved for long periods without undergoing phenotypic and/or genotypic changes, allowing their use for experimental research studies when needed. The identification of the strains, aimed at their collection, will also rely on mass spectrometry using MALDI-TOF with specific dedicated software.
Furthermore, techniques for extracting and purifying DNA from pre-treated pure strains will be examined, and methods for amplifying DNA using specific kits that utilize thermocyclers, such as PCR (Polymerase Chain Reaction) for amplifying target regions of the bacterial 16S rDNA gene, will be illustrated. Techniques for purifying extracted DNA and sequencing using next-generation sequencing (NGS) methods will also be covered, as well as strain preservation through freeze-drying based on sublimation.
The course will provide the fundamentals for assessing cell viability, plating counts, and Gram staining, as well as evaluating bacterial density in a general matrix (principles of dilutions, measurement of optical density using a spectrophotometer, preparation of bacterial inocula, and counting bacterial cells). Information on the use of laboratory equipment will be provided, such as biosafety cabinets (DPC), personal protective equipment (PPE), thermostatic ovens, incubators, spectrophotometers, mass spectrometers, electrophoresis cells for DNA fragment separation in agarose gel, thermocyclers, various types of centrifuges, and vortex mixers. Guidelines for serious and responsible behavior in a microbiological laboratory (BSL1, 2, and 3) will also be provided.

Acquisition of mastery in the basic theoretical knowledge related to microbiology and its practical applications. Acquisition of foundational knowledge of the methods that can be applied for manipulating biological matrices in a microbiological context, along with the adoption of suitable and safety-oriented behavior for conducting activities within a scientific laboratory. Development of both theoretical and practical skills regarding the main methods employed in a microbiology laboratory. Familiarization with the use of basic and complex laboratory equipment. Acquisition of appropriate scientific language aimed at understanding specific and complex topics. Development of serious and responsible behavior within a microbiological laboratory (BSL1, 2, and 3).
Acquisition of skills in identification, classification, in-depth analysis, and interpretation of scientific data, methodologies, protocols, and safety issues, as well as the ability to apply appropriate techniques for the isolation, identification, and manipulation of microorganisms. Furthermore, students will develop a certain degree of critical judgment regarding the ability to analyze issues related to the handling of pathogens in a microbiology laboratory, as well as independence in evaluating experimental data.
Regarding communication skills, the course will provide the foundation for using appropriate language in this field. The ability to relate and collaborate with colleagues, produce scientific material (reports and/or presentations) using appropriate scientific language in both oral and written forms will also be assessed. The capacity for independent learning, in-depth study, and self-updating through reading texts and scientific articles will be considered, along with practical autonomy and the ability to utilize educational materials for critical and reasoned study.