Microbial Growth

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Chapter 3

• Microbial Growth

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Microbial Growth means
increase in number of cells, not cell size. It is
very confusing with reproduction (??) in other
forms of life (e. g. animals).
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Growth requirements
Physical
1 Temperature
2 pH
3 Osmotic pressure (water)
4 Light
Chemical
1 Carbon
5 Inorganic element
2 Nitrogen
6 Oxygen
3 Sulfur
7 growth factors
4 Phosphorus
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Physical Requirements for Growth 1 temperature

• Temperature
• Minimum growth temperature
• Optimum growth temperature
• Maximum growth temperature

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Arctic/antarctic
mammal/soils
Spring/sea volcano
compost
Sea/refrige
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Question Is it always safe if foods are stored
in refrigerator for long time?
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Figure 6.2
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Physical Requirements for Growth 2 pH

• pH
• Most bacteria grow between pH 6.5 and 7.5
• Molds and yeasts grow between pH 5 and 6
• Acidophiles grow in acidic environments

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Physical Requirements for Growth 3 osmotic
pressure

• Osmotic Pressure (water availability)
• Hypertonic (??) environments, increase salt or
  sugar, cause plasmolysis(????)
• Extreme or obligate (??) halophiles require high
  osmotic pressure
• Facultative (??) halophiles tolerate high osmotic
  pressure

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Plasmolysis
Cell in normal osmotic pressure environment
Cell in hypertonic environment
Figure 6.4
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Physical Requirements for Growth 4 Light

• Light (radiation)
• Necessary for phototrophic bacteria (using light
  as energy source)
• Radiation in different wavelength has different
  effects (ultraviolet, x-ray, and gamma-ray kills
  bacteria while green light induces development of
  life cycle, e. g. mushrooms)

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Chemical Requirements for Growth 1 carbon

• Carbon
• Structural organic molecules, energy source
  (recall the chemical constituents of cells)
• Chemoheterotrophs (?????) use organic carbon
  sources
• Autotrophs (???) use CO2

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Chemical Requirements for Growth

• Nitrogen
• In amino acids, proteins
• Most bacteria decompose proteins
• Some bacteria use NH4 or NO3?
• A few bacteria use N2 in nitrogen fixation
• Sulfur
• In amino acids, thiamine (???Vb 1), biotin (???
  VH)
• Most bacteria decompose proteins
• Some bacteria use SO42? or H2S

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Chemical Requirements for Growth

• Phosphorus
• A. In DNA, RNA, ATP, and membranes
• B. PO43? is a source of phosphorus
• Trace elements
• A. Inorganic elements required in small amounts
• B. Usually as enzyme cofactors

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Chemical Requirements for Growth

1. Oxygen (O2)

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Why O2 is toxic to some bacteria

• Singlet oxygen O2 boosted to a higher-energy
  state
• Superoxide free radicals (???????) O2?
• Peroxide anion (???????) O22?
• Hydroxyl radical ??(OH?)

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Chemical Requirements for Growth

• Organic Growth Factors
• Organic compounds obtained from the environment
• Vitamins, amino acids, purines (??), pyrimidines
  (??)

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Culture Media

• Culture Medium Nutrients prepared for microbial
  growth
• Sterile No living microbes
• Inoculum Introduction of microbes into medium
• Culture Microbes growing in/on culture medium

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Agar

• Complex polysaccharide
• Used as solidifying agent for culture media in
  Petri plates (??), slants (??), and deeps (????)
• Generally not metabolized by microbes
• Liquefies at 100C
• Solidifies 40C

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Culture Media

• Chemically defined media exact chemical
  composition is known
• Complex media Extracts and digests of yeasts,
  meat, or plants, e. g.
• Nutrient broth (????)
• Nutrient agar (????)

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Examples of Culture Media
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Anaerobic Culture Methods

• Reduced media
• Contain chemicals (thioglycollate ?????or cystine
  (???) or ascorbate (????) to remove O2
• Heated to drive off O2

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Anaerobic Culture Methods
?

• Anaerobic jar

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Anaerobic Culture Methods

• Anaerobic chamber

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Some bacteria require high CO2

• Candle jar O2-packet

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Selective Media
Differential Media

• Enhance the growth of certain wanted organisms
  but suppress unwanted microbes.

• Make it easy to distinguish colonies of different
  microbes.

Figure 6.9b, c
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Enrichment Media

• Encourages growth of desired microbe
• Assume a soil sample contains a few
  phenol-degrading bacteria and thousands of other
  bacteria
• Inoculate phenol-containing culture medium with
  the soil and incubate
• Transfer 1 ml to another flask of the phenol
  medium and incubate
• Transfer 1 ml to another flask of the phenol
  medium and incubate
• Only phenol-metabolizing bacteria will be growing

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All-purposed (rich) medium

• An all-purposed (rich) medium is rich in a wide
  variety of nutrients (including many growth
  factors) and will, therefore, support the growth
  of a wide range of bacteria.

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Minimal Medium

• A Minimal medium supplies only the minimal
  nutritional requirements of a particular organism.

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Summary of Commonly-Used Constituents of Media

• Substance Function Composition
  Source
• AGAR Solidifying agent Impure
  polysaccharide marine algae
• PEPTONES nutrient Animal/Plant
  proteins cow,soy
• EXTRACTS nutrient
  Animal/Bacteria paste cow,yeast
• BODY FLUIDS hormones Blood animals
  
• BUFFERS pH K2PO4NaHPO3CaCO3 -
  
• REDUCTANTS e- source thioglycolate
  -
• SELECTIVES bacteriostat Antibiotics,
  sodium azide varies
• INDICATORS pH bromothymol
  blue,phenol red -
• WATER hydration H2O (DI tap) -
  

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A few words

• A pure culture contains only one species or
  strain
• A colony is a population of cells arising from a
  single cell or spore or from a group of attached
  cells
• A colony is often called a colony-forming unit
  (CFU)

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Streak Plate (????)
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Medium sterilization
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Sterilization all living cells, viable spores,
viruses are killed or removed from object or
habitat though
Irradiation destroys/distorts nucleic acids
X-rays microwaves. UV commonly used on object
surfaces Filtration physical removal from liquid
or gas. Sterilize solns that are denatured by
heat, i.e., antibiotics, injectable drugs, amino
acids, vitamins, etc Gas formaldehyde,
glutaraldehyde, ethylene oxide. Toxic chemicals
(require gas chamber) used for large items Heat
important, widely used. Endospores (theromoduric)
destruction guarantees sterility
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Treatment
Temp
Effectiveness
Vaporizes organic material on non-flammable
surfaces but may destroy many substances in the
process
Incineration
gt500o
30 minutes boiling kills microbial pathogens
vegetative forms of bacteria BUT may not kill
endospores
Boiling
100o
100o
Intermittent Boiling
3x30 min intervals, followed by periods of
cooling kills endospores
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Treatment
Temp
Effectiveness
Autoclave/pressure cooker (steam under pressure)
121o/15mins _at_ 15lbs pressure
Kills all forms of life including endospores.
Sterilization requires maintenance at effective
temp for full time period
Dry heat (hot air oven)
160o/2hrs
Materials that must remain dry
Dry Heat (hot air oven)
170o/1hr
Same as above NB Increasing temp by 10o
shortens sterilizing time 50
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Treatment
Temp
Effectiveness
Pasteurization (batch method)
63o/30mins
Kills most vegetative bacterial cells including
pathogens, i.e., streptococci, staphylococci
Mycobacterium tuberculosis
Pasteurization (flash method)
72o/15secs
Similar to batch method For milk conducive to
industry fewer undesirable effects on quality
taste
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Some more words
Sterilants Chemical agent used in chemical
sterilization
Disinfectants agents (chemical) used in
disinfection only on inanimate objects
Disinfection killing, inhibition or removal of
microbes that may cause disease
Sanitization related to disinfection. Microbial
popln reduced to levels considered safe by public
health standards
Antisepsis prevention of infection or sepsis
Antiseptics chemical agents applied to tissue to
prevent infection by killing or inhibiting
pathogen growth (not toxic)
Germicide kills pathogens non pathogens (not
endospores)
Bactericide, Fungicide, Algicide or Viricide
disinfectant/antiseptic effective against
specific microbial group
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Autoclave
to sterilize medium
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Filtration apparatus
to sterilize medium
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How Bacterial Cells Divide
Binary fission(????) 1 cell divides into 2 new
cells Growth rate rate of cell to reproduction
Generation(???) Time required for a complete
fission cycle i.e., 1 parent cell 2 new
daughter cells 1st Generation 2 cells
2nd 4 cells 3rd 8
cells
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Exponential growth
Log growth
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Assume that cells are at the exponential growth
stage. Then we can calculate cell numbers at ant
time of the stage.
N0 is the number of cells at time zero. Nt is the
number of cells at time t. n is the generations
G is the time needed to complete one generation.
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Growth stage in a batch culture
stationary
decline
log

lag
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LAG PHASE 1. Newly inoculated cells,
adjustment (can be diauxic) 2. NO cell
division taking place 3. Population is sparse
or dilute LOG PHASE 1. Population growth at
geometric/logarithmic rate 2. Cells reach
maximum rate of cell division (while nutrients
and environment are favorable) STATIONARY PHASE
1. Population reaches maximum numbers, rate of
cell inhibition (death) Rate of
multiplication DEATH PHASE 1. Decline in
growth rate (reverse Log phase) 2. Death in
geometric fashion
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Continuous cultures
Cells in continuous culture vassal can be kept at
log phase.
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The elemental composition of biomass
The elemental composition of biomass is
surprisingly constant across the variety of
commercially utilised strains of bacteria and
fungi.   For Example
C/N
mole ratio Escherichia coli CH1.77O0.49N0.24
? Saccharomyces
cerevisiae CH1.83O0.56N0.17
? Pseudomonas C12B CH2.00O0.52N0.23
?   Average CH1.79O0.50N0.20
? This average elemental composition can be
utilised if no other value is available.   However
, values for many other organisms are available
in literature.
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The end