Foundation in Microbiology

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Foundation in Microbiology

• The Tools of the Laboratory
• The methods for studying Microorganisms
• Chapter 3

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Microbiology

• The study of organisms too small to be seen
  without magnification
• bacteria
• viruses
• fungi
• protozoa
• helminths (worms)
• Algae (al'je) "seaweeds"

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Microbe are involved in

• nutrient production energy flow
• decomposition
• production of foods, drugs vaccines
• Bioremediation (the introduction of microbes into
  the environment to restore stability or to clean
  up pollutants)
• causing disease.

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Impact of pathogens

• Nearly 2,000 different microbes cause diseases
• 10 B infections/year worldwide
• 13 M deaths from infections/year worldwide

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Taxonomy - system for organizing, classifying
naming living things

• Domain - Archaea, Bacteria Eukarya
• Kingdom - 5
• Phylum or Division
• Class
• Order
• Family
• Genus
• species

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Naming micoorganisms

• Binomial (scientific) nomenclature
• Gives each microbe 2 names
• Genus - noun, always capitalized
• Species (spe'sh z, spe's z) - adjective,
  lowercase
• Both italicized or underlined
• Staphylococcus aureus (S. aureus)
• Bacillus subtilis (B. subtilis)
• Escherichia coli (E. coli)

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The 5 Is of culturing microbes

• Inoculation introduction of a sample into a
  container of media which provide growth
  requirements
• Incubation under conditions that allow growth
  (temperature, pH, gas)
• Isolation separating one species from another
  (separate colonies on solid media)
• Inspection macroscopic observation (color, size,
  and texture), microscopic.
• Identification type of microorganism to level of
  species (genetic analysis, DNA)

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Isolation

• If an individual bacterial cell is separated from
  other cells has space on a nutrient surface, it
  will grow into a mound of cells- a colony
• A colony consists of one species

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Isolation technique
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Media-providing nutrients in the laboratory

• Physical states of media
• - Liquid media
• - Semisolid media 0.3-0.5 agar.
• - Solid media 1-5 agar.

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Media

• Most commonly used
• nutrient broth liquid medium containing beef
  extract peptone
• nutrient agar solid media containing beef
  extract, peptone agar
• agar is a complex polysaccharide isolated from
  red algae
• solid at room temp, liquefies at boiling (100oC),
  does not resolidify until it cools to 42oC
• provides framework to hold moisture nutrients
• not digestible for most microbes

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Types of media

• synthetic contains pure organic inorganic
  compounds in an exact chemical formula
• complex or nonsynthetic contains at least one
  ingredient that is not chemically definable ( e.g
  blood agar, MacConkey agar.)
• general purpose media- grows a broad range of
  microbes, usually nonsynthetic (nutrient agar)
• enriched media- contains complex organic
  substances such as blood, serum, hemoglobin or
  special growth factors required by fastidious
  microbes (blood agar)

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Enriched media
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• selective media- contains one or more agents that
  inhibit growth of some microbes and encourage
  growth of the desired microbes (MacConkey, MSA)
• differential media allows growth of several
  types of microbes and displays visible
  differences among desired and undesired microbes
  (blood agar, MacConkey)

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selective differential media
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Selective media
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Miscellaneous media

• reducing medium contains a substance that
  absorbs oxygen or slows penetration of oxygen
  into medium used for growing anaerobic bacteria
  (Sodium thioglycollate)
• carbohydrate fermentation medium- contains sugars
  that can be fermented, converted to acids, and a
  pH indicator to show the reaction basis for
  identifying bacteria and fungi

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The Microscopy
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Transmission Electron Micrograph
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Scanning Electron Micrograph
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Terms

• Staining means coloring the microorganisms with
  dyes to make some structures more visible.
• Smear consists of a thin film made from a liquid
  suspension of cells on a slide and air-drying it.
• Air-Dry
• Fixation means killing and attaching the
  microorganisms to the slide by heat or alcohol.

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Specimen preparation

• Fresh living preparations (wet mounts hanging
  drop mounts ) allow examination of
  characteristics of live cells motility, shape,
  arrangement
• Fixed, stained smear are made by drying
  heating a film of specimen. This smear is stained
  using dyes to permit visualization of cells or
  cell parts.

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Staining

• Most microorganisms appear almost colorless when
  viewed through a standard light microscope.
• cationic dyes - basic, with positive charges on
  the chromophore (colored positive ion)
• anionic dyes - acidic, with negative charges on
  the chromophore (colored negative ion)
• surfaces of microbes are negatively charged and
  attract basic dyes positive staining. (Crystal
  violet, methylene blue, malachite green, and
  safranin)
• negative staining microbe repels dye it
  stains the background (eosin, nigrosin, acid
  fuchsin, and india ink.) no heat fixation (less
  distortion) p.g. 81

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Staining

• Simple stains is an aqueous or alcohol solution
  of a single basic dye. (all cells appear the same
  color under the microscope)
• Differential stains use a primary stain and a
  counterstain (two dyes) to distinguish cell types
  or parts. examples Gram stain, acid-fast stain
  and endospore stain
• Special stains used to emphasize certain cell
  parts that are not revealed by conventional
  staining methods (capsule and flagellar stains)

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Types of differential stain

• Gram stain classifies the bacteria into two
  large groups (G and G- bacteria). Four reagents
  are used
• 1- Crystal violet (purple) because it impacts
  its color to all cells its referred as primary
  stain.
• 2- Iodine (mordant)
• 3- Alcohol (decolorizing) removes the color from
  some species (G-) but not from others (G)
• 4- safranin (red) has a counterstain color to
  the primary stain, and called (counterstain).

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The results

• Gram-positive bacteria retain the primary dye
  and remain purple,
• Gram-negative bacteria dont retain the primary
  dye and thus appear red or pink from counterstain.

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Types of differential stain

• Acid-fast stain in this stain the primary dye
  binds only to bacteria with waxy materials in
  their cell wall, three reagents are used
• 1- Carblfuchsin (red dye) soluble in the lipids
• 2- Acid-alcohol (decolorizing) removes red dye
  from bacteria which are not acid-fast.
• 3- Methylene blue (blue, counterstain) affects
  only non acid-fast cells which appear blue.

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The results

• Acid-fast cells retain carbolfuchsin after
  decolorizing and appear red.
• Non acid-fast cells take up the methylene blue
  and appear blue.

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Special stains

• Special stains spore, capsule, and flagella.
• Spore staining a spore is special resistance,
  dormant structure formed within a cell that
  protects a bacterium from adverse environmental
  conditions. (malachite green)
• Flagella staining a mordant is used to build up
  the diameters of flagella to become visible.
  (carbolfuchsin)
• Capsule staining demonstrates the presence of
  capsule. Positive stain may be used, but most
  commonly to be used is a negative stain, the
  capsules appear as halos around bacterial cells
  and stand out against dark background.

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Types of stains
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