AGRICULTURAL AND ENVIRONMENTAL MICROBIOLOGY
Department of Microbiology, Nutrition and Dietetics
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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
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Do not drink, eat and smoke
Protective clothing
Aseptic technique
Bacteriological loop, needle
Bunsen burner
Bacteriological stains
Brightfield microscopy
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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
VIRUSES
1 x 2 – 4 μm
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
COCCI
pediococci, tetrades
diplococci
streptococci
sarcina
staphylococci
Axis of division
diplococci
streptokoky
tetrades
sarcinas
staphylococci
RODS regular nonsporing
RODS- regular endospore-forming
plektridium
Clostridium
Bacillus
RODS - curved
vibrio
spirilla
spirochaeta
SPIROCHAETA
VIBRIO
RODS- irregular
bifidobacteria
mycobacteria
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