B9153 - TECNICHE DI BATTERIOLOGIA E VIROLOGIA (CE)

Learning outcomes

By the end of the course, students will have acquired comprehensive knowledge of the manual and automated techniques used in the diagnosis of bacterial and viral infections. They will also have gained a solid understanding of the new and innovative molecular techniques used in diagnostic practice.

Course contents

1. Biological Risk and Safety in the Microbiology Laboratory

• Definition and main characteristics of microorganisms.

• Classification of microorganisms into four infectious risk groups.

• Factors determining the level of biological risk.

• Principles of laboratory containment and safety.

• Personal and collective protective equipment.

• Management of accidental exposure and prevention of biological risk.

• Sentinel microorganisms and their use in microbiological monitoring.

2. Organisation of the Diagnostic Workflow and the Pre-analytical Phase

• Objectives of the clinical microbiology laboratory.

• Stages of the microbiological diagnostic workflow.

• Turnaround time and the main factors affecting reporting times.

• Appropriateness of diagnostic requests.

• Collection and identification of biological specimens.

• Selection of suitable containers and transport systems.

• Specimen storage and transportation.

• Specimen contamination and the main causes of non-conformity.

• Collection and transport media for bacteriological and virological specimens.

• Specimen acceptance and rejection criteria.

3. Microscopy and Microbiological Staining

• Principles of microscopy applied to microbiology.

• Basic and acidic dyes.

• Simple, differential and special staining methods.

• Gram staining: principles, procedure and interpretation.

• Ziehl-Neelsen staining for acid-fast microorganisms.

• Staining methods for capsules, spores, flagella and fungi.

• Evaluation of bacterial morphology and cellular arrangement.

• Main technical and interpretative errors.

4. Culture Media and Inoculation Techniques

• Composition and characteristics of microbiological culture media.

• Classification of culture media according to composition, physical state and function.

• General-purpose, enriched, selective and differential media.

• Principal culture media used in diagnostic microbiology.

• Stages and characteristics of bacterial growth.

• Factors affecting microbial growth: nutrients, temperature, pH, salinity and oxygen availability.

• Culture of aerobic and anaerobic microorganisms.

• Selection of culture media according to the specimen and suspected microorganism.

• Plate inoculation techniques.

• Streak-plate technique for obtaining isolated colonies.

• Quantitative culture and determination of colony-forming units.

• Applications to urine cultures, faecal specimens and positive blood cultures.

5. Identification Using Biochemical Tests

• Metabolic and enzymatic principles of bacterial identification.

• Catalase, coagulase and oxidase tests.

• Urease, citrate and indole tests.

• Assessment of haemolysis on blood agar.

• CAMP test.

• Bacitracin and optochin susceptibility tests.

• Miniaturised and multiparametric biochemical systems.

• Enterotube: inoculation, incubation and interpretation of results.

6. Antimicrobial Resistance and Susceptibility Testing

• Definition of drug resistance and antimicrobial resistance.

• Main classes of antibacterial agents and their cellular targets.

• Mechanisms of intrinsic and acquired resistance.

• Enzymatic inactivation, target modification, reduced permeability and efflux.

• Genetic transfer of resistance through transformation, conjugation and transduction.

• Principles of antimicrobial susceptibility testing.

• Kirby-Bauer disk-diffusion method.

• Determination of the minimum inhibitory concentration.

• Broth-dilution methods and gradient-diffusion tests.

• Clinical interpretation of susceptibility results.

• Automated systems, including VITEK 2 and MicroScan.

• Principles of genotypic investigation of antiviral drug resistance, with particular reference to HIV.

7. Identification by MALDI-TOF Mass Spectrometry

• General principles of mass spectrometry.

• MALDI ionisation and time-of-flight analysis.

• Specimen preparation and application of the matrix.

• Production and interpretation of protein mass spectra.

• Database comparison and microorganism identification.

• Applications in clinical microbiology.

• Advantages, limitations and complementary identification approaches.

8. Advanced Techniques for Investigating Antimicrobial Resistance

• Importance of identifying specific resistance mechanisms.

• Phenotypic detection of extended-spectrum beta-lactamases.

• Double-disk synergy test.

• Phenotypic detection of carbapenemases.

• Modified Hodge test.

• EDTA test for metallo-beta-lactamases.

• Boronic acid test for KPC carbapenemases.

• Molecular methods for detecting antimicrobial resistance genes.

• Multiplex PCR platforms and integrated systems, including Allplex, GeneXpert and FilmArray.

9. Blood Culture Diagnostic Workflow

• Definition of bacteraemia, sepsis and endocarditis.

• Clinical indications for blood cultures.

• Pre-analytical considerations: timing of collection, number of sets, blood volume and skin disinfection.

• Aerobic and anaerobic blood-culture bottles.

• Transportation and storage of inoculated bottles.

• Automated incubation and detection of microbial growth.

• Management of positive blood-culture bottles.

• Gram staining and prompt communication of preliminary results.

• Subculture, microorganism identification and antimicrobial susceptibility testing.

• Post-analytical procedures and integration with the laboratory information system.

10. Principles of Virological Diagnosis and Serological Investigations

• Direct and indirect virological diagnosis.

• Types of specimens used for viral investigations.

• Detection of viral genomes, antigens and proteins.

• Molecular techniques, antigen detection, cell culture and electron microscopy.

• Diagnostic significance of IgM, IgG and IgA.

• Principles of antigen-antibody reactions.

• Direct and indirect agglutination.

• Haemagglutination.

• Immunochromatographic tests.

• Complement-fixation tests.

• Immunodiffusion and Western blotting.

11. ELISA and Immunofluorescence Techniques

• Principles of enzyme immunoassays.

• Direct, indirect, sandwich and competitive ELISA.

• Operational stages: immobilisation, specimen addition, incubation, washing and detection.

• Chromogenic reactions and interpretation of results.

• Applications to antigen and antibody detection.

• Direct and indirect immunofluorescence.

• Fluorochromes, specimen preparation and interpretation of fluorescent signals.

• Principal advantages, limitations and potential sources of error.

12. Molecular Biology Techniques

• Extraction and purification of DNA and RNA.

• Main extraction methods: phenol-chloroform, silica columns and magnetic beads.

• Role of DNases and RNases.

• Assessment of nucleic acid quantity, purity and integrity.

• Interpretation of A260/A280 and A260/A230 absorbance ratios.

• Principles of the polymerase chain reaction.

• End-point PCR and gel electrophoresis.

• Real-time PCR and reverse-transcription PCR.

• Nested PCR, multiplex PCR, hot-start PCR and touchdown PCR.

• Applications of molecular methods in diagnostic microbiology.

13. Sequencing and Next-Generation Sequencing

• Principles of nucleic acid sequencing.

• Sanger sequencing.

• Reaction preparation and capillary electrophoresis.

• Chromatogram reading and interpretation.

• General principles of next-generation sequencing.

• Ion Torrent, Illumina and Oxford Nanopore platforms.

• Library preparation, adapters, indexes, amplification and enrichment.

• Sequencing chemistries and quality parameters.

• 16S rRNA gene sequencing for bacterial identification and characterisation.

• Selection of 16S rRNA gene variable regions.

• Illumina 16S sequencing workflow.

• Applications of sequencing in clinical microbiology and in the investigation of microbial communities and antimicrobial resistance.

Readings/Bibliography

The teaching materials presented during the lessons and made available by the lecturer constitute the primary reference for examination preparation.

Recommended Textbooks

• Murray P.R., Rosenthal K.S., Pfaller M.A. Medical Microbiology. 10th ed. Elsevier, 2025.

• Tille P.M. Bailey & Scott’s Diagnostic Microbiology. 16th ed. Elsevier, 2026.

• Carroll K.C., Pfaller M.A., Karlowsky J.A., Landry M.L., McAdam A.J., Patel R., Pritt B.S., editors. Manual of Clinical Microbiology. 13th ed. ASM Press, 2023.

Reference Documents and Resources

• World Health Organization. Laboratory Biosafety Manual. 4th ed. WHO, 2020.

• European Committee on Antimicrobial Susceptibility Testing. Clinical Breakpoint Tables for the Interpretation of MICs and Zone Diameters. Current version available on the EUCAST website.

• Clinical and Laboratory Standards Institute. Performance Standards for Antimicrobial Susceptibility Testing. CLSI M100, 36th ed., 2026.

• Additional guidelines, diagnostic protocols and technical documents indicated by the lecturer during the course.

The recommended textbooks are intended as supplementary resources and do not replace the presentations and topics covered during the lessons.

Teaching methods

Teaching is delivered through lectures supported by presentations, images, interpretative diagrams and examples from clinical microbiology laboratory practice.

The different methods are presented within the complete diagnostic workflow, from biological specimen management to the production and interpretation of laboratory results. The lessons include discussion of practical cases, analytical results and the main pre-analytical, analytical and post-analytical issues.

Active student participation is encouraged through questions, reasoned discussion of diagnostic procedures and connections with the professional activities of biomedical laboratory technicians.

Assessment methods

Learning outcomes will be assessed through an oral examination designed to evaluate:

• knowledge of the principles underlying the methods presented during the course;

• the ability to describe the microbiological diagnostic workflow correctly;

• understanding of the applications and limitations of the different techniques;

• the ability to interpret the main laboratory results critically.

Teaching tools

The following resources are used to support teaching activities:

• multimedia presentations prepared by the lecturer and made available to students in PDF format;

• microscopic images, diagrams and photographs of the main laboratory procedures;

• examples of analytical results, antimicrobial susceptibility tests, MALDI-TOF spectra, amplification curves and sequencing data;

• videos and animations explaining laboratory methods and workflows;

• discussion of practical cases derived from clinical microbiology laboratory practice;

• guided consultation of current guidelines, diagnostic protocols, databases and technical documents;

• the University’s online learning platform for sharing presentations, announcements and supplementary materials.

Office hours

See the website of Massimiliano Guerra