PROPAGATION by xiaoyounan

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									 MICROBIAL
PROPAGATION
PROPAGATION
               REPRODUCE
               MULTIPLY




FOR MICROBES, AN INCREASE OF CELL NUMBER IS

              GROWTH
Microbial Growth
   I. The requirements for growth
     – A. Physical requirements
     – B. Chemical requirements
   II. Population growth
I. The requirements for growth
    A.   Physical requirements
     – 1. Temperature
        » psychrophiles Cold loving. 0 - 15oC.
        » mesophiles Moderate temperature
          loving. 10 - 45oC
        » thermophiles Heat loving. 40 - 70oC.
        » extreme thermophiles 65 - 110oC.
        a minimum, optimum, and
    Have
    maximum temperature of growth
I. The requirements for growth
     A. Physical requirements
      – 2. pH
          » Optimum for most bacteria, is pH 6 to
            pH 8
          » Optimum for yeast is about pH 5
          » Acidophiles grow at very low pH.
          » Alkalinophiles grow at high pH.
I. The requirements for growth
  A.Physical requirements
   – 3.Osmotic Pressure
      » Most cells live in isotonic solutions
      » Hypertonic solutions dehydrate cells
         Used for food preservation

      » halophile Salt loving
         Extreme or obligate halophiles require
           high salt (archaea)
         Facultative halophiles can grow in high
           salt
I. The requirements for growth
     B. Chemical Requirements
      – 1. Carbon
          » autotroph Gets carbon from CO2.
          » heterotroph Gets carbon from organic
            source
      – 2. Nitrogen, sulfur, & phosphorous
          » Nitrogen for proteins, DNA, RNA, ATP
          » Sulfur for some amino acids & vitamins
          » Phosphorous for nucleic acids, ATP,
            phospholipids
I. The requirements for growth
     B. Chemical requirements
      – 3. Oxygen
          » Both useful and harmful
             Useful in respiration

             Harmful because is a strong oxidizing
              agent
I. The requirements for growth, cont.
 Classificationof organisms base on their
  response to oxygen
     » obligate aerobe Requires oxygen.
     » facultative anaerobe Grows with or without
       oxygen.
     » obligate anaerobe Grows only in absence of
       oxygen.
     » aerotolerant anaerobe Grows in presence of
       oxygen, but does not use it.
     » microaerophile Grows in low oxygen
       concentration.
OBLIGATE AEROBES
OBLIGATE ANAEROBES
FACULTATIVE AEROBES
FACULTATIVE ANAEROBES
MICROAEROPHILES
Why is oxygen is harmful?
     All organisms produce superoxide ( O2-)
     It is a strong oxidizing agent
     It pulls electrons off other molecules
     Superoxide is toxic to cells
   Superoxide    must be neutralized
Superoxide dismutase (SOD)
     O2- + O2- + 2 H+ -------> H2O2 + O2

   Hydrogen    peroxide is also toxic to cells and it
      must be neutralized
Catalase
         2 H2O2 --------> 2 H2O + O2
     Obligate Anaerobes lack:
      – Superoxide dismutase ( SOD )
      – Catalase
I. The requirements for growth
     SOD                      CATALASE
       – Obligate aerobes       – Obligate aerobes
       – Facultative            – Facultative
         anaerobes                anaerobes
       – Aerotolerant           – (not aerotolerant
         anaerobes                anaerobes)
       – (Not obligate          – (Not obligate
         anaerobes)               anaerobes)
       – (Usually not in        – (Usually not in
         microaerophiles)         microaerophiles)
Special Culture Techniques for
Anaerobic Microbes
  a.   Reducing Media
  b.   Anaerobic Container
  c.   Agar Stab
  d.   Agar Shake
Special Culture Techniques for
Microaerophilic Microbes
 Grow best under reduced O2 levels and
                 increased CO2 levels
 Normal Atmosphere 21 %             O2
                      0.3 to .03 % CO2
Growing Microaerophilic Microbes



                          16 %   O2
                          4%     CO2




             Candle Jar
Growing Microaerophilic Microbes




        CO2 Generating Packet
I. The requirements for growth
     B. Chemical requirements
      – 4. Trace elements: potassium, magnesium,
         calcium, iron, copper, molybdenum, and
         zinc. Are needed as cofactors for enzymes
I. The requirements for growth
     B. Chemical requirements, etc.
      – 5. Organic growth factors may be needed
          » Vitamins
          » Amino acids
          » Purines
          » Pyrimidines
Culture Media

 1. Chemically Defined
   – the exact chemical composition is known
   – used to grow fastidious organisms
 2. Complex Media
   – exact chemical composition is not known
   – most bacteria and fungi are grown with this
Selective Media

   Inhibits the growth of some bacteria while
    selecting for the growth of others
   Example:
     – Brilliant Green Agar
        » dyes inhibit the growth of Gram (+)
          bacteria
        » selects for Gram (-) bacteria
        » Most G.I. Tract infections are caused by
          Gram (-) bacteria
Selective Media
  EMB    (Eosin Methylene Blue)
   – dyes inhibit Gram (+) bacteria
   – selects for Gram (-) bacteria
   – G.I. Tract infections caused by Gram (-)
     bacteria
Differential Media

  Differentiates between different organisms
   growing on the same plate
  Example:
    – Blood Agar Plates (TSA with 5% sheep
      blood)
       » used to differentiate different types of
         Streptococci
Alpha Hemolytic Streptococci




     Incomplete lysis of RBC’s
Beta Hemolytic Streptococci




     Complete lysis of RBC’s
Gamma Hemolytic Streptococci




         No lysis of RBC’s
Selective and Differential Media
  MacConkey’s    Agar
   – used to identify Salmonella
  MacConkey’s Agar
   – Bile salts and crystal violet (inhibits Gram
     (+) bacteria)
   – lactose
   – pH Indicator

  Many Gram (-) enteric non-pathogenic bacteria can
  ferment lactose, Salmonella can not
I. Population growth

  Binary   Fission

  Generation   Time (Doubling Time)
   – time required for a cell to divide
   – most about 1 Hr. To 3 Hrs.
       » E. coli - 20 minutes
       » Mycobacterium tuberculosis - 24 Hrs.
I. Population growth
II. Population growth, cont.
   Steps   of binary fission:
    – 1. Cell elongates, and DNA replicates.
    – 2. Cell wall and plasma membrane increase in
      size and form septum.
    – 3. Cross wall forms around DNA.
    – 4. Cells separate.
II. Population Growth, cont.
   Generation time varies
    with:
     – Organism
     – Available nutrients
     – Temperature
     – pH, etc.
   Can be short (10 min)
    or long (hours)
II. Population growth, cont.
   Case:
     – 1 cell
     – Generation time 30 min
     – In 10 hrs have 1,048,576 cells
     – 10 hrs = 20 generations
   Get large numbers of cells quickly
   Plot arithmetically or logarithmically
II. Population growth, cont.
   Phases   of growth:
    – 1. Lag phase
       » Cells are adjusting to environment.
       » Cells are synthesizing needed
         macromolecules.
    – 2. Log phase or exponential phase
       » Cells are undergoing binary fission.
II. Population growth, cont.
    – 3.Stationary phase
       » Growth rate slows down.
       » Some cells die.
       » Due to depletion of nutrients and/or
         accumulation of waste
    – 4. Death phase
       » Cells die.
Fig. 6-9
Fig. 6-7
Fig. 6-19
Fig. 6-6
Fig. 6-10

								
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