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Blueboom/Microbiology

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Medical Microbiology

Study of microscopic organisms includes

  • Bacteriology Bacteria simplest smallest single celled free living organisms
  • Virology viruses - non cellular parasitic, not living organisms
  • Mycology fungi - microscopic (molds and yeast), macroscopic (mushrooms and puff balls), unicellular and multicellular
  • Protozoology - protozoa, single celled
  • Phycology/Algology - algae - simple aquatic organisms ie seaweeds

Koch's postulates

  1. The same microbe is always associated witha specific disease
  2. this microbe can be recovered and grown in pure culture
  3. The pure culture must cause disease in an experimental animal
  4. The original microbe must be recovered from t he experimental disease

Examples of Problems:

  • Mycobacterium leprae - requres animal host
  • Neisseria gonorrhoeae - only human host
  • Opportunistic pathogens - immunocompromised host

Use of Molecular Postulates

  • Criteria a for determining cause of infections using molecular techniques
  1. Virulence factor: gene or gene product must be found in pathogenic stains but not in non-pathogenic stains
  2. When a virulence factor is introduced into a non-pathogenic strain it should become a pathogenic strain
  3. Genes for virulence must be expressied during the course of disease
  4. Antibodies specific for the virulence gene products should be protective


Classifications of Microogranisms

Organisms:

  • Bacteria, Archaeabacteria and Eukaryotes.
  • Infectious Agents: Viruses, viroids, prions

Bacterial and fungal classification using taxonomy

  • Kingdom, Phylum, Class, Order, Family, Genus, Species
  • Order has suffix of ales, Family has suffix of aceae
  • Biovar - variant strain that differs physiologically or biochemically
  • Serovar - sub division of species
  • Strain - a genetic varient or subtype

Viral Taxonomy

  • Order suffix virales
  • Family suffix viridae
  • Subfamily suffix virinae
  • Genus suffix virus
  • Species individual virus

Bacterial Morphology

Atypical bacteria

Fungi

Viruses

Atypical Virus like agents

Micobial gowth and multiplication

  • Physical and chemical requirements are required to grow and multiply
  • Physical requirements such as temperature osmotic pressure and pH
  • Chemical requirements Carbon Nitrogen Sulfur phosphorus trace elements oxygen organic growth factors
  • Survival of bacteria in a hostile environment requires that there must be a continous macromolecular synthesis and genome replication. -> increase in cell mass and in bacterial genome necessitiates that the cell mst divide to maintain its physical size and the size of its genome
  • Therefore one bacterial cell divides into 2 daughter cells by asexual process known as binary fission
  • The time required for a single cell to divide into 2 daughter cells is termed as doubling time
  • doubling time is deined as formation of new daughter bacterial cells from parent cell. its growth to propersize due to replication fo bacterial macromolecules and eventual division into two cells

Sample doubling times :

  • Bacillus stearothermophilus 11min
  • Escherichia coli 20 min
  • Staphylococcus aureus 28min
  • Lactovacillus acidophilus 60-80 min
  • mycobacterium tuberculosis 360 minutes
  • treponema pallidum (causative agent of syphyllis) 1980 minutes

growth curve

in a closed system where nutrients and space are finite no removal of waste products

lag phase

newly inoculated cells need adjustment to the new environment (nutrients temp ph) 2) no cell divisiion 3) very few bacterial cells population is sparse/dilute

exponential (log) phase

1) cell adjustment is complete metabolism and replication at full speed growth at geometric / logarithmic rate cells reach maximum rate of cell division (this continues as long as nutrients and environment is favorable)

stationary phase

1) population reaches maximum numbers ate of cell inhibition (death) = rate of multiplication factors: nutrient depletion, ph changes, accumulation of waste, reduced O2 (can induce endospores of bacillus and clostridium species)

death

1) decline in growth rate 2) death in geometric proportions causes : depletion of nutrients, O2, excretion of toxic waste products, increased density of cells (limited space) factors same as stationary phase+ release of lytic enzymes

measurement of viable bacteria

serial dilution: dilute concentrated samble 10 folds and meansure enumerated bacteria in each plate count

plate count use for enumeration of viable bacteria (CFU colony forming unit) . A fraction of each dilution is placed on agar plate and counted. Assume each colony is formed by one bacterium and total # of viable bacteria = total number of colonies. Total viable count= # of colonies x sample size (.1) and the dilution factor.

Filtration; for water testing and highly diluted samples Turbitidy for blood cultures

Physical factors affecting growth

-temperature- whether a bacteria multiplies slowly or luxuriantly at certain temperatures 3 groups

  1. minimum - lowest temp at which bacteria can survive and permit microbial growth and metabolism
  2. maximum - highest temp at which bacteria can survive and permit microbial growth and metabolism
  3. optimum - narrow range of temp that promotes the fastest growth of a bacterial species
Min Max Optimum
Ecoli 15-20 45 37


Psychrophile

psychrophilic opt temp~ 4 celcius capable of growth at 0 degrees celcius not involved in human infections found in snow fields polar ice deep ocean examples: psydomonas, flavobacterium, alcaligenes, achromobacte species

Facultative psychrophile

grow slowly in cold condition but optimial above 20c staph aureus c. monocytogenes contamination of food/dairy


Mesophile

opt temp 20-40c capable of growth 10-50 group containing human pathogens (30-37) example: e. coli

Thermophile

optimum temp 60 cel. capable of growth 45-85 celcius incapable of growth at usual body temp not involved in human infections found volcano, direct exposure to sun

Osmotic Pressure

AKA Water activity

  • all bacteria require water for growth and reproductions . It is an essential solvent involved in all biochemical reactions and indirectly maintain osmotic pressure
  • increase of salts leads to a decrease in the rate of growth , cell swells , replication halts
  • aw = index of free available water= atmospheric measure , relative humidity = rh
  • rh= 1.00 aw
  • therefore 90% rh = .9 Aw
  • absorption and solution factors reduce availabilty of free water (decreased Aw)

pure distilled water (Aw =1) Saturated solution of NaCl (Aw=.8) Seawater NaCla = 3%

  • Most bacteria have an active metabolism at Aw > .0
  • Foods that have more free water will spoil more quickly as opposed to foods which have lower Aw (which would have less water0
  • Lower Aw -> slow growth
  • Below aw of .09 -> Bacteria unable to grow
  • Exceptions to this
    • Xerotolerant: lower Aw
    • Fungi can grow at Aw of .6
    • Yeasts ~ .6
    • Salt tolerant bacteria, Halophiles (high solute, low Aw)


Effects of pH

Growth rates are influenced by pH values

Oxygen Requirements

  • limting factor in bacterial growth because O2 has limited solubiity in water
  • Enzymes are needed to reduce oxygen into water as well as ROI byproducts
  1. Catalase 4H2O2 -> 3H2O + 2O2
  2. Peroxidase H2O2 + NADH + H+ -> 2H2O + NAD+
  3. Superoxide dismutase 2O2- + 2H+ -> H2O2 + 2

Based on oxygen requirements

Obigate aerobes

  • Totally dependant on O2
  • Need at least 1atm
  • have catalase and SO dismutase

Microaerophiles

Grow in presence of O2 but only tolerate 4% of I2

  • possess enzymes but growth will be inhibited if toxic products accumulate

Obligate anaerobes

Grow only in absense of O2

  • Do not possess detoxifying enzymes
  • Low levels of O2 -> LETHAL

Examples

  1. Clostridum species
  2. Bacteroides species

Capnophiles

  • grow better in high CO2 but tolerate O2

Faculative anaerobe

  • can grow in with / without O2 present
  • grow best under aerobic conditions
  • use aerobic respiration when O2 is present

example: Enterobacteriacea

Bacterial Enzymes related to O2

Microorganism Type Catalase SO Dismutase
Aerobe + +
Facultative anaerobe + +
Microaerophile - +
Obligate Anaerobe - -

Growth Location

Growth location based on Gaseous requirement
[  ]  [  ]  [  ]  [  ]  [  ]
 --    --    --    --    --
|  |  |..|  |  |  |  |  |..|
|  |  |..|  |  |  |  |  |..|
|  |  |  |  |..|  |  |  |..|
|  |  |  |  |..|  |  |  |..|
|  |  |  |  |  |  |  |  |..|
|  |  |  |  |  |  |..|  |..|
|  |  |  |  |  |  |..|  |..|
 --    --    --    --    --
 1     2     3     4     5
  1. uninoculated
  2. obligate aerobe
  3. microaerophile
  4. obligate anerobe
  5. facultative anerobe


Culture media

Developed to isolate and grow bacteria

  • Types :
  • Chemically defined media
  • Complex media
    • chemicals + complex organic compoinds added to facilitate bacterial growth
  • Anaerobic growth media
    • known as Reduced medium
    • reducing agents added which remove any free oxygen from medium
    • adds reducing medium (thioglycolate cystine or ascorbate to remove O2
  • Selective media
    • contain nutrients and inhibitiong agents for unwanted bacteria
    • addition of specific C or energy source, adjust pH, increase osmotic pressure, adjust O2 tension
    • examples Salmonella-Shigella agar, mannitol salt agar
  • Enrichment media
    • special nutrients are added to make it 'tasty' for bacteria
  • All purpose media
    • nutrient agar and broth support growth of most microbes
  • Enriched media
    • Also contains additional nutrition ie: blood agar
  • Transport medium
    • Used to preserve microbes for transit following initial procurement from patient
  • Differential Medium
    • more than one type of organism can grow but speration is based on growth pattern
    • examples:
      1. MacConkey agar - enterobacteriaceae
      2. Eosin-methylene blue (EMB) E coli Enterbacter aerogenes
      3. Blood Agar - differential for streptococci only

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This page was last modified on 16 March 2008, at 20:11.
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