1. AGRICULTURAL AND ENVIRONMENTAL MICROBIOLOGY Department of Microbiology, Nutrition and Dietetics Course syllabus – laboratory exercises Lecturer: Prof. Vojtěch Rada Office No.: 221 rada@af.czu.cz

2. Course syllabus – laboratory exercises Microscopy of bacteria: Negative staining, simple staining Microscopy of bacteria: Gram staining Yeast study: Methylene blue staining Mould study: Zygomycetes (Mucor, Rhizopus), Ascomycetes (Eupenicillium) and Deuteromycetes (Penicillium, Aspergillus. Asexual reproduction (conidiospores and sporangiospores) Actinomycetes and antibiotics

3. Course syllabus – laboratory exercises Identification of bacteria (staphylococci) Microbiology of drinking water Microbiology of milk: fermented milk products, starter cultures. Carbon cycle: amylolytic bacteria Nitrogen cycle: Nitrogen fixing bacteria (Azotobacter, Rhizobium)

4. LABORATORY SAFETY Do not drink, eat and smoke Protective clothing Aseptic technique Bacteriological loop, needle Bunsen burner Bacteriological stains

5. Brightfield microscopy low-power objectives (4-20x) high-dry objectives (40-60x) immersion objectives (90-100x) Resolution limit (0.4 μm) Magnification (1500x) Oil immersion technique.

6. PROTOZA100 μm MOULDS AND YEASTS 5 – 10 μm BACTERIA - COCCI1 μm - RODS 1 x 2 – 4 μm VIRUSES0,1 μm

7. Methylene blue staining This method distinguish live (colourless) and dead (coloured) cell. Saccharomyces cerevisiae – yeast; baker, beer production

8. Methylene blue staining A drop of water is placed in the centre of a slide. Two loopful of yeast are transferred to slide One loopful of methylene blue is added Examine with dry objectives

9. YEASTS budding

10. Bacteria

11. COCCI staphylococci diplococci streptococci pediococci, tetrades sarcina

12. Axis of division diplococci staphylococci sarcinas tetrades streptokoky

13. RODS regular - nonsporing

14. RODS- regular endospore-forming Clostridium Bacillus plektridium

15. RODS - curved spirilla spirochaeta vibrio

16. VIBRIO SPIROCHAETA

17. RODS- irregular mycobacteria bifidobacteria

18. Actinomycetes

19. Negative Staining (Background staining) This method consist of mixing the microorganisms in a small amount of nigrosine and spreading the mixture over the surface of a slide.

20. Negative Staining Drops of water and nigrosine are placed in the centre of a microscopic slide. Remove a small amount of material from between your teeth with a sterile straight toohpick. Spread the mixture of water, nigrosine and sample over the slide. Allow the slide to air-dry and examine with an oil immersion objective