Microbiology

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					     AGRICULTURAL AND ENVIRONMENTAL MICROBIOLOGY
                       Department of Microbiology and Biotechnology
                                     Faculty of Agronomy
Lecturer: Ass.Prof. Vojtěch Rada, CSc.




Course syllabus – laboratory exercises:




   1) Microscopy and bacteria


   Brightfield microscopy, objectives (low-power objectives, high-dry objectives and
   immersion objectives), resolution limit, magnification, oil immersion technique.


   Microscopy of bacteria: negative staining (bacterial flora of oral cavity), bacterial
   morphology (cocci, rods, spore formation), smear preparation, simple staining (Bacillus
   subtilis), Gram staining (Micrococcus luteus and Escherichia coli).




   2) The Fungi and antibiotics


   Yeasts and Moulds,
   Yeasts study: Methylene blue staining (vital staining, Saccharomyces cerevisiae),
   observation of nucleus, vacuole and budding.
   Moulds study: Zygomycetes (Mucor, Rhizopus), Ascomycetes (Eupenicillium) and
   Deuteromycetes (Penicillium, Aspergillus), fungi with septate and nonseptate hyphae.
   sexual reproduction (zygospores and ascospores), asexual reproduction (conidiospores
   and sporangiospores)


   Antibiotics: Microbial sources of antibiotics (bacteria, fungi), narrow-spectrum and broad
   spectrum antibiotics, cidal and static antibiotics, determining the level of antimicrobial
   activity (dilution susceptibility tests, disk diffusion tests, minimal inhibition concentration
   (MIC), minimal lethal concentration).



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3) Culture methods


Microbiological media:
Liquid, semisolid and solid media, nutritional needs of bacteria (water, carbon, energy,
nitrogen, minerals, growth factors, hydrogen ion concentration), composition of
bacteriological media (synthetic and nonsythetic media), special media (selective and
differential media), media preparation and sterilization.


Bacterial population counts:
Quantitative plating method - standard plate count (SPC), Most probable method,
Enumeration of total viable bacteria in raw cow milk.




4) Microbiology of fermented milk products. Soil microbiology




Starter organisms (lactic acid bacteria, miscellaneous bacteria, yeasts, moulds),
mesophilic, thermophilic and therapeutic bacteria, microscopy of selected dairy products
(buttermilk, yoghurt, acidophilus milk, bifighurt, kefir and kefir-like products.).


Soil bacteria: Azotobacter, Rhizobium, Clostridium




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




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Brightfield microscopy


Objectives
- low-power objectives (4-20x)
- high-dry objectives (40-60x)
-   immersion objectives (90-100x)



Resolution limit (0.4 μm)

Magnification (1500x)

Oil immersion technique.




                                     4
Microscopy of bacteria: negative staining (bacterial flora of oral cavity), bacterial
morphology (cocci, rods, spore formation), smear preparation, simple staining (Bacillus
subtilis), Gram staining (Micrococcus luteus and Escherichia coli).




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1) Microscopy and bacteria


Brightfield microscopy, objectives (low-power
objectives, high-dry objectives and immersion
objectives), resolution limit, magnification, oil
immersion technique.


Microscopy of bacteria: negative staining
(bacterial flora of oral cavity), bacterial
morphology (cocci, rods, spore formation), smear
preparation, simple staining (Bacillus subtilis),
Gram staining (Micrococcus luteus and
Escherichia coli).




                                                    6
                     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.


Microflora of oral cavity
1) Drops of water and nigrosine are placed in the centre
  of a microscopic slide.
2) Remove a small amount of material from between
  your teeth with a sterile straight toohpick.
3) Spread the mixture of water, nigrosine and sample
  over the slide.
4) Allow the slide to air-dry and examine with an oil
  immersion objective




                                                           7
Simple Staining


 1) Smear preparation:
     a) A drop of water is placed in the centre of a
        slide
     b) One loopfuls of organisms is transferred to
        the centre of slide
     c) Spread the organisms over the slide
     d) The smear is allowed to dry
     e) Slide is passed through flame several times
        to heat-kill and fix organisms
 2) A bacterial stain is stained with crystal violet
    (fuchsin, methylene blue) 1 min
 3) Stain is briefly washed off slide with water
    Allow the slide to air-dry and examine with an
    oil immersion objective




                                                       8
                    Gram Staining
1884 Christian Gram
Staining technique that separates bacteria into two
groups:
  - Gram-positive bacteria
  - Gram-negative bacteria


Based on the ability to retain crystal violet during
decolorization with alcohol




                                                       9
                  Gram Staining


1) Smear preparation.
2) Stain with crystal violet 1 min.
3) Add Lugol solution 1 min.
4) Decolorize with alcohol 10-15 seconds.
5) Wash with water.
6) Stain with fuchsin 2 min
7) Allow the slide to air-d
8) y and examine with an oil immersion objective




                                                   10
Grampositive bacteria
Steptococcus

Staphylococcus


Lactobacillus


Bacillus


Clostridium


Gramnegative bacteria


Escherichia


Salmonella


Vibrio


Treponema;


                        11
2) The Fungi and antibiotics


Yeasts and Moulds,
Yeasts study: Methylene blue staining (vital
staining, Saccharomyces cerevisiae), observation of
nucleus, vacuole and budding.
Moulds study: Zygomycetes (Mucor, Rhizopus),
Ascomycetes (Eupenicillium) and Deuteromycetes
(Penicillium, Aspergillus), fungi with septate and
nonseptate hyphae. sexual reproduction
(zygospores and ascospores), asexual reproduction
(conidiospores and sporangiospores)


Antibiotics: Microbial sources of antibiotics
(bacteria, fungi), narrow-spectrum and broad
spectrum antibiotics, cidal and static antibiotics,
determining the level of antimicrobial activity
(dilution susceptibility tests, disk diffusion tests,
minimal inhibition concentration (MIC), minimal
lethal concentration).

                                                        12
3) The Fungi and antibiotics


Yeasts and Moulds,
Yeasts study: Methylene blue staining (vital
staining, Saccharomyces cerevisiae), observation of
nucleus, vacuole and budding.
Moulds study: Zygomycetes (Mucor, Rhizopus),
Ascomycetes (Eupenicillium) and Deuteromycetes
(Penicillium, Aspergillus), fungi with septate and
nonseptate hyphae. sexual reproduction
(zygospores and ascospores), asexual reproduction
(conidiospores and sporangiospores)




                                                     13
The Fungi: Yeast and Molds



Taxonomy

Kingdom: Mycota (Fungi)

 Division: Eumycota (True fungi)

     Subdivision: Zygomycotina

         Genus: Mucor, Rhizopus

     Subdivision: Ascomycotina

         Genus: Aspergillus, Penicillium,

                 Saccharomyces

     Subdivision: Deuteromycotina

                 (Fungi imperfecti)

         Genus: Candida, Monilia




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                           Yeast study


Methylene blue staining
This method distinguish live (colourless) and dead (coloured) cell.


  1) A drop of water is placed in the centre of a slide.
  2) Two loopful of yeast are transferred to slide
  3) One loopful of methylene blue is added
  4) Examine with dry objectives




                                                  Budding




Live cell                                   Dead cell




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Observation of molds

 5) A drop of lactophenol is placed in the centre of a slide.
 6) One loopful of molds are transferred to slide
 7) Add cover slide
 8) Examine with dry objectives. Look for sporangiophjores
    or conidiophores




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Antimicrobial Susceptibility Testing

Antimicrobial agents:

- Antibiotics (Secondary metabolites of specific

microorganisms: bacteria especially actinomycetes,

molds)

- Chemotherapeutics: Sulphonamides

Antibiotic Susceptibility Testinig

  - Dillution methods

  - Disk diffusion method




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                   DISK DIFFUSION TESTS


Microorganisms tested:




Escherichia coli                 Micrococcus luteus




Procedure:
    1) Pipe 1 ml of sterile water to Petri dishes
    2) Add 1 loop of bacterial culture
    3) Mix well
    4) Pour with nutrient agar
    5) Allow to cool
    6) Place disks on the medium




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Culture methods


Microbiological media:
- Liquid, semisolid and solid media
- Nutritional needs of bacteria
- Composition of bacteriological media
- Media preparation and sterilization


Cultivation, isolation and identification of bacteria


Bacterial population counts:
Quantitative plating method - standard plate count
(SPC), Most probable method, Enumeration of
total viable bacteria in raw cow milk.




                                                    19
                   Microbiological media


Media Consistency
- Liquid consistency (nutrient broth, glucose broth, litmus
 milk)
- Solid media (gelatine, agar, silica gel)
- Semisolid media (bacterial motility)


Nutritional Needs of Bacteria
  - Water (tap water, distilled water)
  - Carbon (autotrophs, heterotrophs)
  - Energy (phototrophs, chemotrophs)
  - Nitrogen (NH3, amino acids, peptides, peptone)
  - Minerals (Ca, P, Fe….)
  - Growth factors (amino acids, vitamins, yeast extract)
  - Hydrogen ion concentration (pH 6.8)


  SPECIAL MEDIA
  - Selective media
  - Differential media
Media Preparation (Self-made media, dehydrated media)


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IDENTIFICATION OF UNKNOWN BACTERIA


1st Condition: pure culture (cultivation condition)


Identification tests
Morphological Study (negative staining, Gram
staining)
Cultural Characteristics (colony characteristics –
shape and colour)
Physiological Characteristics (relation to oxygen,
incubation temperature)
Biochemical tests (Enzymes, fermentation tests)
Molecular Characteristics (Analyses of DNA, RNA)




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         IDENTIFICATION OF SKIN BACTERIA


Genus             Colony     Catalase Morphology Gram
                  colour
Staphylococcus    White         +    cocci        +
Micrococcus       Yellow,       +    cocci        +
                  orange
Propionibacterium variable      +    rods         +
Corynebacterium   White,        +    rods         +
                  grey
Brevibacterium    White,        +    rods         +
                  yellow




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              IDENTIFICATION OF BACTERIA


Genus             Colony Catalase Morphology Gram
                  colour
Lactobacillus     White      -   Rods           +
Micrococcus       Yellow,    +   Cocci          +
                  orange
Bacillus          variable   +   Rods,          +
                                 sporeforming
Streptococcus     White      -   Cocci, long    +
                                 chains
Escherichia       White,     +   Rods           -
Bifidobacterium   White      -   Irregular      +
                                 rods
Enterococcus      White      -   Cocci, short   +
                                 chain




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STAPHYtest 24 – procedure


  - Create a suspension of pure culture (Staphylococcus
    sp.) in 5 ml of saline
  - - Pipe 0.1 ml into each out of 24 wells (three rows for
    each strain)
  - Add few drops of oil in F, G, H wells
  - Incubate at 37oC for 48 h
  - Read color reactions




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-
QUANTITATIVE PLATING METHOD, STANDARD PLATE
METHOD
Total Bacterial Counts


SAMPLE     10 ml         1 ml   1 ml   1 ml




MOST PROBABLE METHOD
E. COLI




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26
 CULTURE MEDIA


Media consistency
 - Liquid media (nutrient broth, glucose broth,
     lithmus milk)
 -   Solid media (agar, gelatin, silica gel)
 -   Semisolid media




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        MICROBIOLOGY OF MILK PRODUCTS.
Organisms used
  - Lactic acid bacteria – mesophilic, thermophilic
  - Miscellaneous bacteria – Acetobacter,
    Brevibacterium
  - Yeast – Candida, Kluyveromyces
  - Moulds – Moulds – Penicillium


Starter cultures
      7) Buttermilk: Lactococcus lactis, Leuconostoc
         mesenteroides (20oC, 24h)
      8) Yoghurt : Lactobacillus delbrueckii ssp.
         bulgaricus, Streptococcus thermophilus (45oC,
         4h)
      9) Acidophilus milk: Lactobacillus acidophilus
         (37oC, 16h)




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Observation of Bacteria in Fermented Milk Products


 1) Spread one loopful of sample over the slide.
 2) Allow the slide to air-dry.
 3) Fix with ether-alcohole for 1 min.
 4) Add a drop of 1%NaOH for 10 sec.
 5) Wash with water.
 6) Stain with methylene blue for 2 min.
 7) Wash with water.
 8) Allow the slide to air-dry and examine with an
    oil immersion objective.




Yoghurt        Acidophilus milk     Buttermilk




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                        Water Microbiology
  - Surface water
  - Swimming pool water
  - Bottled natural water
  - Drinking water
Testing water for sewage contamination
Indicator organisms: E. coli, enterococci
Enteric diseases – Cholera, typhoid fewer, bacillary dysentery.
Other diseases – Pseudomonas aeruginosa, Legionella


Coliform bacilli: Escherichia, Citrobacter, Enterobacter, Hafnia,
Klebsiella, Serratia (37oC, ß-galactosidase)
Fecal coli: E. coli (44oC, indol-positive)
Enterococci: Enterococcus (10oC and 45oC, 40% bile, 6% NaCl,
aesculin-positive).


                           Coliform test
  - MPN method
  - Membrane filter method
                         Total viable counts
  - 37oC/24 h
   - 20-22oC/72 h




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           WATER MICROBIOLOGY


          Detection of coliform bacteria:


Presumptive tests
- Fermentation of lactose
- Gas production


Confirmed test – isolation of pure culture
- Gram staining
- Detection of ß-glucuronidase
- Indole production
- Differential media (Endo, TBX)




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Kefir   Bifidobacteria




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   MICROBIOLOGICAL STANDARDS FOR DRINKING
                            WATER


Total plate counts
< 100 cfu/ml at 22oC
< 10 cfu/ml at 37oC


E. coli, coliforms
Absence from 100 ml


Enterococci (faecal streptococci)
Absence from 100 ml


Nitrates (NO3-)
< 50 mg/l (adults)
< 15 mg/l (infants)




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               BOTTLED NATURAL WATERS


Total viable counts
< 100 cfu/ml at 22oC
< 20 cfu/ml at 37oC


Pseudomonas aeruginosa
Absence from 250 ml


Sulphite-reducing clostridia
Absence from 50 ml




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     Methods for analysis of the intestinal microflora

Culture methods
  - Non-selective culture methods
  - Selective culture methods

Culture-independent techniques
  - Direct microscopic analysis
  - Enzyme/metabolite analysis
  - Molecular methods – PCR, DGGE,
                          FISH

Fluorescence in situ hybridization (FISH)

FISH is a staining Metod in which fluorescent DNA probes
are hybridized to the ribosomal RNA’s in intact cells. The
technique allows the immediate identification of individual
cells under the microscope.




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               SOIL MICROBIOLOGY




The carbon cycle
- Cellulolytic and Amylolytic bacteria (Clostridium)




The Nitrogen cycle
- N2 fixation, Azotobacter, Rhizobium




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          Observation of amylolytic bacteria
                     (Clostridium sp.)
1) Tranfer a drop of fermented potato from test tube to
  slide
2) Add a drop of Lugol solution (containing iodine)
3) Put cover slide
4) Observe using dry objectives (10x, 45x)




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               NITROGEN CYCLE




              NITROGEN FIXATION

- Free-living nitrogen-fixing bacteria: Azotobacter,
  Clostridium

- Symbiotic nitrogen fixation : Rhizobium




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                       Capsule stain
                  Azotobacter chroococcum


1) Drops of water and nigrosine are placed in the centre
of a microscopic slide.
2) Remove a small amount of material from colony of
Azotobacter chroococcum growing on Aschby agar
3) Spread the mixture of water, nigrosine and sample
over the slide.
4) Allow the slide to air-dry
5) Stain with crystal violet
6) Wash gently with saline solution, dry
7).Examine with an oil immersion objective




                                                       39
Simple Staining (for bifidobacteria)


  4) Smear preparation:
      a) A drop of water is placed in the centre of a
         slide
      b) One loopfuls of organisms is transferred to
         the centre of slide
      c) Spread the organisms over the slide
      d) The smear is allowed to dry
      e) Slide is passed through flame several times
         to heat-kill and fix organisms
  5) A bacterial stain is stained with methylene blue
    2 min
  6) Stain is briefly washed off slide with water
    Allow the slide to air-dry and examine with an
    oil immersion objective




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Probiotic : life bacteria or other beneficial
(microorganisms).


Prebiotics : food or ingredients which support
probiotic bacteria in the gut.


Synbiotics : combination of probiotics and prebiotics.




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