AGRICULTURAL AND ENVIRONMENTAL MICROBIOLOGY Department of Microbiology, Nutrition and Dietetics Course syllabus – laboratory exercises Lecturer: Prof.

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

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

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)

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

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.

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

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

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

YEASTS budding

Bacteria

COCCI staphylococci diplococci streptococci pediococci, tetrades sarcina

Axis of division diplococci staphylococci sarcinas tetrades streptokoky

RODS regular - nonsporing

RODS- regular endospore-forming Clostridium Bacillus plektridium

RODS - curved spirilla spirochaeta vibrio

VIBRIO SPIROCHAETA

RODS- irregular mycobacteria bifidobacteria

Actinomycetes

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.

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