Cultivation, Reproduction and Growth of Bacteria

1
Cultivation, Reproduction and Growth of Bacteria

• Chap 5

2
Nutritional Considerations

• Energy source
• Carbon source
• Nitrogen, sulfur and phosphorus sources
• Metallic elements
• Vitamins
• Water

3
Nutritional Types of Organisms

• Autotrophs and Heterotrophs

4
Autotrophs

• Make their own food using simple carbon sources
• Photoautotroph gtgt Energy from sunlight and carbon
  from CO2
• Chemoautotroph gtgt Inorganic compounds as carbon
  and energy source

5
Heterotrophs

• Not able to synthesize food from inorganic
  compounds, need carbs, fatty acids or alcohols
• Photoheterotroph gtgt Sunlight as energy source,
  preformed organic molecules for carbon source
• Chemoheterotroph gtgt Use preformed organic
  molecules for both carbon and energy source

6
Types of Media

• Complex and Synthetic and Modifications of Them

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Types of Media

• Complex
• Most commonly used
• Exact make up is unknown
• Beef extract, yeast extract, tryptone, peptone,
  salt
• Synthetic
• To determine growth requirements
• Glucose, salt, magnesium, potassium, etc.
• Nutrient agar

8
Types of Media

• Differential gtgt allows identification based on
  specific properties
• Blood agar - hemolysis
• Eosin Methylene Blue (EMB)

E. coli produces metallic green colonies
http//www.rlc.dcccd.edu/mathsci/reynolds/micro/la
b_manual/throat.html http//www2.austin.cc.tx.us/m
icrobugz/html/eosin_methylene_blue_agar.html
9
Types of Media

• Selective Media gtgt contains ingredients to
  inhibit the growth of some while allowing the
  growth of others
• Sodium Chloride Agar
• Antibiotic Containing Agar

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Types of Media

• Enriched gtgt for fastidious organisms
• Blood agar
• Chocolate agar
• Haemophilus influenzae and Neisseria species

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Types of Media

• Characterization
• Gelatin looking at liquefaction patterns

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Even with all of this, less than 1 of all known
bacteria can be cultured in the lab
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Chlamydia

• Obligate intracellular bacterial parasite
• Must be grown inside cells

Reticulate bodies
14
Physical Growth Conditions

• Temperature, Oxygen, pH, Pressure

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Temperature

• Psychrophiles - low temperature range
• less than15C
• Mesophiles - mid temperature range
• 10-45C gtgt human pathogens
• Thermophiles - high temperature range
• around 60C
• Hyperthermophiles - extremely high temperatures
• above 80C
• Psychrotrophic bacteria mesophiles growing at
  the low temperature range certain Campylobacter
  sps growing at fridge temperature

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Growth Rates in Response to Temperature
37C
Fig 5.8 pg 147
Note the overlapping ranges and how quickly the
curves decline outside of the optimal range
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Oxygen

• Aerobic bacteria - Bacillus
• Anaerobic bacteria Clostridium
• sulfur
• Microaerophilic Treponema palladium
• Facultative anaerobe Bacillus
• prefers oxygen, but can grow without

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Effect of Oxygen on Bacterial Growth
Fig 5.10 pg150
Thioglycollate broth binds free oxygen, so the
only available is at the top where it enters from
the environment
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Acidity/Alkalinity

• Optimal pH for growth
• Human body between 7.2 7.4
• Prokaryotic cytoplasm 7.0
• Acid-tolerant bacteria gtgt acidophiles
• Lactobacillus
• Streptococcus
• Alkaline-tolerant bacteria gtgt alkalophilic
• Vibrio cholerae as low as 2.0

20
Osmotic and Hydrostatic Pressure

• Bacteria are under constant pressure from the
  inside out
• Hypertonic to their environment gtgt This is why
  prokaryotes have cell walls!
• Halophiles gtgt up to 30 environmental salt
• Marine organisms live at 16,000 psi, but die at
  the surface, 14.7 psi gtgt barophiles

21
Reproduction of Bacteria
22
Types of Reproduction

• Binary fission
• Budding
• Fragmentation
• More common in fungi

23
Binary Fission
Fig 5.2 pg 139
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Incomplete Binary Fission

• Tetrads
• Cell arrangement that is a consequence of
  binary fission with incomplete separation of
  cells, occurring in two planes, producing a
  square consisting of four cocci, one at each
  corner

25
Growth

• Increase in cell size
• Happens but irrelevant
• Increase in cell number
• Colony gtgt A visible mass of microorganisms of one
  type arising from one parental cell

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Generation Time

• Interval of time between cell divisions
• Reproductive potential of E. coli 18-20min
• Reproductive potential of S. aureus 30min
• Reproductive potential of M. tuberculosis 18 hr

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Bacterial Growth Curves
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Rapid Generation Times
1cell to 2 million cells in 7 hours! Only a
build up of waste or depletion of food will
stop growth
Fig 5.3 pg 140
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Bacterial Growth Curve

• Lag phase gtgt preparing for division
• Logarithmic phase gtgtreproduction
• Disease symptoms develop
• Vulnerability to antibiotics highest
• Stationary phase gtgt equilibrium
• Decline phase gtgt cell death

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Growth Curve
Living Dead
Fig 5.4 pg 142
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Quantification of Bacteria

• Direct Cell Count, Plate Count, Membrane Filter,
  Turbidity, Dry Weight

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Direct Cell Count

• Counts cells per ml
• Very common method
• Count bacteria in vaccines and other cultures

33
Hemocytometer
Add trypan blue to show the dead cells
34
Plate Count

• Counts Colony Forming Units (CFU) per ml
• Count bacteria in milk, water, etc
• Standard Plate count must have between 30 and 300
  colonies per plate

35
Turbidity

• Measures optical density (OD)
• gtgt cells per ml
• Light absorption compared to a standard
• Estimate cell numbers in heavy suspensions
• Faster than direct count

36
Membrane Filter

• Counts CFU per ml
• Count bacteria in very dilute samples of milk,
  water
• Common in industry

37
Dry Weight

• Counts mg of dry bacteria per ml
• Remove the water and weigh
• Count bacteria in very heavy suspensions