Lab Skills in Microbiology

Syllabus

• Biosafety
  Students will learn the general good laboratory practices (GLPs) in a Microbiology Laboratory, the risk groups of microorganisms and the corresponding biosafety levels and also the rules that have to be followed
• Microorganism isolation from environmental samples
  Students will learn the process of isolating microorganisms from the field (e.g. soil, fruit surfaces, aquatic environments, etc.). After isolation, they will become familiar with the preservation of their samples and the safe storage of microorganisms. Introduction to the different categories of microorganisms, Observation of distinctive characteristics (size, movement, morphology).
• Sterilization / aseptic methods – Microorganism growth and preservation Students will learn how to work under aseptic conditions and how to cultivate pure cultures. Inoculation in liquid nutrient media (with a negative control) to verify aseptic conditions. The “streaking” method on solid media will aim on isolation of a single colony. Introduction to sterilization methods (wet and dry sterilization, with chemicals, etc.). Introduction to the microorganism preservation (Low temperatures, freeze drying, drying).
• Quantification of microbial population with colony forming units (cfu) method
  Students will use the samples they isolated in the previous exercise and will quantify the total microbial population by plating their sample on agar media in petri dishes after subsequent decimal dilutions in order to form individual colonies. They will later find the exact number of microbes of their initial sample and express the population as colony forming units (cfu). Moreover, isolates will be plated on general and selective agar media (TSA, LB-Agar, YGC-Agar for yeasts, PDA, Sudan black staining, Nile blue and Nile red staining, culture for anaerobes, etc.) will be used for the isolation of microorganisms with different properties.
• Gram Staining
  Students will learn the most popular method for staining to differentiate bacteria. They will use two bacterial cultures (Gram+ and Gram-), carry out the staining stages (smearing on a slide, fixing, staining and preparation for observation) and finally observe their samples under the optical microscope
• Microorganism identification
  Students will learn about the methodology of microorganism classification (using classical microbiology and biochemical tests) and modern identification methodology (16S or 26S rRNA gene sequencing, MLST, FAMEs) and then the students will carry out the identification of the microbial isolates through the sequencing of the 16S or 26S rRNA gene or ITS DNA regions. They will then create a phylogenetic tree with the closest relatives.
• Determination of antibacterial activity
  Students will learn about antibacterial activity determination such as measuring the Minimum Inhibitory Concentration (MIC) and the Minimum Bactericidal Concentration (MBC) and then they will evaluate the antibacterial activity of honey using the Agar Well Diffusion Assay method, comparing it with the corresponding activity of antibiotics and artificial honey
• Microalgae cultivation
  Students will learn about microalgae, their environmental role as well as the specificities of their energy requirements. Microalgae are cultivated in photobioreactors and observed under a microscope.
• Phage cultivation
  Students will learn the basic difference between viruses and phages, their cultivation requirements, the use of cell cultures, the cell lines used as well as the detection of cytopathic effect (CPE). Students, through the use of bacteriophage and bacterial culture will detect the presence of phage, observe the lysis of bacteria and quantify the initial viral titer of the sample with the technique of calculating Plaque-Forming Units (PFUs).
• Protists and their ecological role
  Introduction to protists (ecological role, diversity and diversity, functions). Observation of cultures of protists with bacteria and protists with fungi where protists are the predators and bacteria or fungi the prey.
• Archaea and their ecological role
  The role of archaea in the environment and health, aerobic (ammonia oxidizers) and anaerobic archaea (methanogenic archaea from the gut microbiota of humans and termites or from biogas plants), cultivation of archaea (focusing on ammonia oxidizers) in liquid cultures and monitoring of ammonia oxidation

Student Performance Evaluation

Assessment is based on laboratory reports (30%) and the written exam of the course during the examination period (70%).

The written exam includes:
• open-ended questions
• multiple choice
• correct or incorrect sentence
• matching two categories of sentences
• targeted questions that require short and precise answers

The total number of correct answers corresponds to 100 points, which are reduced to a grade of 10. Based on this reduction, each student’s grade in the theoretical examination of the course is calculated. This grade corresponds to 80% of the total course grade.

Suggested Bibliography

• BROCK Biology of Microorganisms, M. Madigan, J. Martinko, J. Parker, 2018
• Prescott Microbiology (Willey, Sandman, Wood) Broken Hill Publishers 2023
• JAWETZ, MEDICAL MICROBIOLOGY,2023
• Microbial Biotechnology-A Laboratory Manual for Bacterial Systems, Surajit Das, Hirak Ranjan Dash, Springer India, 2015

Teaching Material / E-class

https://eclass.uth.gr/courses/BIO_U_200/