AGRICULTURAL AND ENVIRONMENTAL MICROBIOLOGYDepartment of Microbiology, Nutrition and Dietetics

• Course syllabus – laboratory exercises• Lecturer: Prof. Vojtěch Rada• Office No.: 221• rada@af.czu.cz

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.

PROTOZA 100 μm

MOULDS AND YEASTS 5 – 10 μm

BACTERIA - COCCI 1 μm

- RODS 1 x 2 – 4 μm

VIRUSES 0,1 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

COCCICOCCI

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