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1
MICROBIAL GROWTH
I.
Growth:
Increase in cell # & cell mass
Binary fission:
Individual cell enlarges & divides  2 progeny of equal size
Asexual reproduction:
Does not involve genetic recombination
II.
The Growth Curve
Used to study changes in total population number
A.
Lag phase:
Cells don't divide
Metabolically active adapting to new medium:
Synthesizing:
ATP
Cofactors
Ribosomes
Enzymes
B.
Log phase:
Cells grow and divide at their maximal rate
Rate of growth is constant
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Cell # increases exponentially
Exponential growth:
1
1st generation
2
2nd generation
4
3rd generation
8
4th generation
Generation time:
Time required to double the # of cells in a population during the
log phase of growth
Depends on:
Species
Nutrients
O2
pH
Temperature
Cell # increases by 2n where n = # of generations
Large populations develop quickly:
During log phase population uniform in:
Chemical composition
Physiological characteristics
Log phase cultures used in:
biochemical studies
physiological studies
C.
D.
Stationary phase:
# of cells dying = # of cells dividing
Cell # remains constant:
Population reaches maximum density (109 cells/ml)
Caused by:
Accumulation of:
acidic wastes
toxic products
Depletion of:
O2
nutrients
Death phase:
Cells die faster than new cells are produced
Caused by:
Depletion of nutrients
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Accumulation of wastes
Decrease in pH
Usually logarithmic:
Constant # of cells dies every hour
III.
MEASUREMENT OF GROWTH
A.
Total cell count:
Measures # of cells in a given volume of sample
Place on slide & count:
Petroff-Hausser counting chamber:
Special slide with grids:
holds given volume
Advantages of total cell count:
Quick
Inexpensive
Requires small volume of sample
Gives information about:
Cell size
Cell morphology
Disadvantages:
Hard to count small cells
Doesn't distinguish between living & dead cells
B.
Viable cell count
more accurate
Counts only living cells (cells able to divide & produce colonies)
Colony - asexual progeny of a single cell growing on solid medium
Use:
Spread plates:
Spread O.1 ml sample of bacteria on agar plate:
Incubate until colonies appear.
Every cell is a completely separate colony
Pour plates:
Sample mixed with liquid agar medium:
Poured into sterile Petri dishes
Cells stuck in agar are colonies
Sample should be 25 - 250 colonies/plate:
Thick suspensions must be diluted
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Plate 1 ml sample of each dilution
Incubate:
Count colonies
Total # of colonies equals the # of viable organisms in the
diluted sample
Multiply plate count by dilution factor:
Reciprocal of dilution:
If dilution is 10-6 dilution factor would be 106
Find # of cells present in 1 ml of the original undiluted
sample
Plate counts - spread plates & pour plates used to do viable cell counts
Results expressed as colony forming units (CFU) since it is not absolutely
certain that each colony arose from an individual cell
Viable cell counts very sensitive:
Any viable cell  colony
Allow:
Identification of organisms
Isolation of pure cultures
Widely used to count bacteria in:
Soil
Food
Water
Cosmetics
IV.
The Influence of Environmental Factors on Growth:
Growth greatly influenced by the chemical and physical nature of surroundings:
A.
Solutes and water activity:
Microorganisms:
Affected by changes in the osmotic concentration of their
surroundings
Rigid cell wall maintains:
Shape
Integrity
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B.
Compatible solutes:
Solutes compatible with growth and metabolism at high concentration:
Choline
Betaine
Proline
Glutamic acid
Other Amino acids
Used to increase internal osmotic concentration of cell:
Keeps cell membrane firmly pressed against cell wall
If placed in hypotonic environment:
Plasmolysis occurs
H2O leaves cell
Cell membrane pulls away from cell wall:
Cell is dehydrated
C.
Water Activity:
Quantitative expression of the degree of water available to a
microorganism:
1% relative humidity of a solution:
Equals the ratio of the solution's vapor pressure (Psoln) to that of pure H2O
(Pwater):
Aw = Psoln
Pwater
Most microorganisms only grow well at aw = 0.98 (seawater):
Large quantities of :
Sugar
Salt
are effective in preserving foods
Osmotolerant organisms:
Maintain high internal solute conc.:
Retain H2O
Grow over a wide range of water activity:
Staphylococcus aureus
Saccharomyces rouxii
Dunaliella viridis
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Halophiles:
Extremely specialized:
Found only in:
Dead Sea
Great Salt Lake
Other highly salty habitats
Require high levels of NaCl (2.8 -6.2 M)
Have modified:
Proteins
Membranes
Ribosomes
D.
Acidity or alkalinity:
Measured on log scale
Expressed in terms of pH:
pH 7 is neutral:
Number of H+ = Number of OHpH above 7 is alkaline:
More OH- ions than H+
pH below 7 is acidic:
More OH- ions than H+
Each species has definite pH growth range & pH growth optimum
Most organisms:
Grow best at pH of about 7:
Will not grow if medium is:
Too acid
Too alkaline (basic)
No matter what the optimum environmental for growth:
The pH in cytoplasm of all organisms is maintained at @
7.0:
H+ & OH- ions actively pumped out of cell
Acidophiles:
Growth optima between pH 1.0 & 5.5:
Important in production of fermented foods:
Fermented milk products
Pickles
Vinegar
Wine
Beer
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Examples of acidophiles:
Thiobacillus
Yeasts
Molds
Alkalophiles:
Grow best between pH 8.5 & 11.5
Example of Alkalophiles:
Vibrio cholerae
NEUTROPHILES:
Growth optima between 5.5 & 8.5
Note: In general pH of media should be at about 7:
Add NaOH if too acid
Add HCl if too basic
During growth pH of medium tends to change:
Becomes more acid as metabolic waste products accumulate:
E.
Buffer:
Added to keep pH constant
Can absorb large numbers of H+ & OH- ions
Must be non-toxic to microorganisms
Proteins & PO4 often used as buffers
V.
Temperature:
A.
Generalities
Important component of organism's environment:
Rate of chemical reactions depends on temperature:
Increased temperature increases the rate of enzymatic reactions:
Rate doubles for every 10o increase in temperature
Affects:
Rate of growth
Metabolism
Morphology
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Pigment Production
After certain temp growth slows:
Enzymes & other proteins denature
DNA denatures
Microorganisms as a group grow over a wide temperature range:
Growth range for a particular species spans about 30o:
Stenothermal:
Narrow temp range:
Neisseria gonorrhoea
B.
Eurythermal:
Wide temp range:
Streptococcus faecalis
Cardinal Temperatures:
Temperatures at which an organism can grow
Specific for each group:
Each group has characteristic temp dependence
Three cardinal temperatures for each group:
1.
Maximum temperature:
Temperature above which an organism will not
grow
C.
2.
Minimum temperature:
Temperature below which the organism will not
grow
3.
Optimum temperature:
Temperature at which a particular organism grows
fastest:
Psychrophiles:
Organisms with temperature optima at 15oC or lower:
Maximum around 20oC:
Grow well at 0oC:
Enzyme systems:
Function well at low temp.
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Protein synthesizing mechanisms:
Function well at low temp.
Cell membranes have:
High levels of unsaturated fatty acids:
Function well at low temperatures:
But begin to leak cellular components at 25 - 30oC
Found in:
Arctic & Antarctic habitats:
Snow fields
Glaciers
Oceans (average temp = 5oC)
Examples:
Pseudomonas
Flavobacterium
Achromobacterium
Alcaligenes
D.
Facultative Psychrophiles or Psychrotrophs:
Growth optima between 20 & 30oC & maxima at about 35oC:
Can grow at 0oC
Major factors in the spoilage of refrigerated foods
E.
Mesophiles:
Organisms with growth optima between 20-45oC:
Maximum is 45oC or lower:
Includes most organisms:
Saprophytes
Pathogens
F.
Most human pathogens have temperature optima of around 37oC (body
temperature)
Thermophiles:
Grow at temperatures above 55oC:
Grow minimum is about 45oC
Optimum usually between 55 & 60oC
Some maxima are above 100oC
Heat stable:
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Enzyme systems
Function well at high temperatures
Protein synthesizing mechanisms:
Function well at high temperatures
Cell membranes have high levels of saturated fatty acids:
Have higher melting points then those of mesophiles:
Remain intact at high temperatures
Found in:
Hot springs
Areas of volcanic activity
Compost
Hay stacks
Hot water lines
Mostly bacteria but also includes a few algae
VI.
Oxygen Requirements:
A.
Obligate Aerobes:
Require oxygen
Cannot generate energy by fermentation
Require O2 for the synthesis of unsaturated fatty acids & sterols
B.
Facultative Anaerobes:
Grow best when O2 is present but are able to grow in its absence.
C.
Microaerophiles:
Damaged by normal 20% O2 in the atmosphere but require between 2-10%
O2
D.
Obligate Anaerobes:
Die when exposed to O2:
Poisoned by O2
Examples:
Clostridium
Bacteroides
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Special techniques are used protect obligate anaerobes from O2
Gaspak
Thioglycolate medium
E.
F.
Aerotolerant Anaerobes:
Ignore O2:
Grow equally well whether it is present or not:
But do not use O2 as the terminal electron acceptor during electron
transport
Oxygen:
Toxic in high conc. even for aerobic organisms:
Toxic oxygen products produced during respiration and electron transport:
H2O2
Superoxide radical (O2-)
Hydroxyl radical (OH )
Obligate aerobes & facultative anaerobes have enzymes which destroy
toxic oxygen products:
Obligate aerobes:
Catalase
Superoxide dismutase
Aerotolerant anaerobes:
Catalase
No superoxide dismutase
Obligate anaerobes
No catalase
No superoxide dismutase
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