Chapter 6
Microbial Growth
Lab 2 Goals and Objectives :
Lecture: Chapter 6 (Microbial Growth)
Learn aseptic technique and pure culture isolation
• Exercise 9: Aseptic Technique
• Exercise 10: Pure Culture Technique
• Turn in cultures from home for incubation
• Finish microscope worksheet if necessary
• Microbiology is the study of life and organisms that are
too small to be seen with the naked eye.
• Microbial growth - an increase in the number of cells
in a population , not increase in size.
Reproduction of Prokaryotes - Binary fission
Bacterial growth in culture
• Bacteria multiply by binary fission
• The population grows in geometric progression
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12
2 4
3 8
4 16
5 32
6 64
7 128
8 256
9 518
10 1036
11 2064
12 4128
13 8256
………
-Lag phase: initial period of little to no cell division as bacteria
acclimate to new media
-Log phase: period of exponential growth with a constant generation
time
-Stationary phase: cell growth is equal to cell death
-Death phase: cell death exceeds cell growth
Number of generation
Generation time
• Generation time - the time required for a cell to divide, to undergo
one round of binary fission
– If 100 cells growing for 5 hours produced 1,720,320 cells:
• E. coli has a generation time of 20 min
• Common bacterial generation times range 1-3 hrs
Growing microorganisms
1. Culture Medium: Nutrients prepared for microbial growth
• Sterile: No living microbes
2. Inoculation: Introduction of microbes into medium
Inoculum: The material used in an inoculation
3. Culture: Microbes growing in/on culture medium
Culture Media
• Nutrient broth- liquid form
• Nutrient agar- solid form
– Agar - complex polysaccharide
• Used as solidifying agent for culture media in Petri plates,
slants, and deeps
• Generally not metabolized by microbes
• Liquefies at 100°C
• Solidifies ~40°C
Culture Media
• Complex Media:
Extracts and digests of yeasts, meat, or plants
• Chemically Defined Media:
Exact chemical composition is known
• Differential Media:
– Make it easy to distinguish colonies of different microbes.
• Enrichment Media
Encourages growth of desired microbe
• Reducing media for anaerobic culture,
Contain chemicals (thioglycollate or oxyrase) that combine O2
Heated to drive off O2
Figure 6.9b
Quantifying Microbial Growth
• Direct Measurements
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Plate Counts
Filtration
Most Probable Number (MPN)
Direct Microscopic Count
• Indirect Estimations
– Turbidity
– Metabolic Activity
– Dry Weight
Microbial Growth
• The requirements for microbial growth
• Physical
– Temperature
– pH
– Osmotic pressure
• Chemical
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Oxygen
Carbon
Nitrogen
Sulfur
Phosphorus
Trace elements
Organic factors
Physical Requirements -Temperature
– Minimum growth temperature
• the lowest temperature at which the species will grow
– Optimum growth temperature
• species grow best
– Maximum growth temperature
• the highest temperature at which growth is possible.
Physical Requirements - 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
Physical Requirements - Osmotic Pressure
• Osmotic pressure is the hydrostatic pressure produced by a
difference in concentration between solutions on the two sides of a
surface such as a semipermeable membrane.
• An isotonic environment for a cell is created when the solution outside of the
cell is isotonic (having equal accent )with the cytoplasm of the cell.
• Hypotonic environment - the solution outside of the cell is hypotonic in
comparison to the cytoplasm of the cell.
– cause cell lyses for some cells and nothing to bacteria, because of cell wall .
• Hypertonic environments - hypertonic solution has more dissolved solute
than the cytoplasm of the cell ( increase salt or sugar)
– cause plasmolysis ( shrink the cells )
– Facultative halophiles tolerate high osmotic
pressure
– Extreme or obligate halophiles require
high osmotic pressure
Figure 4.18 - Overview
Chemical Requirements
• Carbon
– Structural organic molecules, energy source
– Autotrophs use CO2
– Chemoheterotrophs use organic carbon sources
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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, biotin
– Most bacteria decompose proteins
– Some bacteria use SO42 or H2S
Phosphorus
– In DNA, RNA, ATP, and membranes
– PO43 is a source of phosphorus
• Inorganic elements required in small amounts- potassium, magnesium and
calcium
• Trace Elements - iron, copper, molybdenum and zinc - Usually as enzyme
cofactors
Chemical Requirements
• Organic Growth Factors
– Essential organic compounds an organism is unable to
synthesize
– Organic compounds obtained from the environment
– Vitamins, amino acids, purines, pyrimidines
Chemical Requirements -
Oxygen (O2)
Toxic Forms of Oxygen
• Singlet oxygen: O2 boosted to a higher-energy state
• Superoxide free radicals: O2
• Peroxide anion: O22
• Hydroxyl radical (OH)
Figure 6.6 A jar for cultivating anaerobic bacteria on Petri plates.
Lid with
O-ring gasket
Clamp with
clamp screw
Envelope containing
sodium bicarbonate and
sodium borohydride
Anaerobic indicator
(methylene blue)
Petri plates
Palladium
catalyst pellets
Biofilms
• Microbial communities
• Form slime or hydrogels
– Bacteria attracted by chemicals via quorum sensing
Clumps of bacteria
adhering to surface
Migrating
clump of
bacteria
Surface
Water currents
Obtaining Pure Cultures
• 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)
• The streak plate method is used to isolate pure cultures
• Exercise 9: Aseptic Technique
• Each person make 3 inoculations:
1. Broth to broth - Escherichia coli - 37°C
2. Slant to slant - E. coli - 30°C
3. Plate to slant - Serratia marcescens - 30°C (changed)
• Exercise 10: Pure Culture Technique
– Each person make 2 streak plates: Quadrant Streak Method B,
page 82.
– Incubate at 25°C
• Turn in cultures from home for incubation
• Finish microscope worksheet if necessary
Streak Plate
Flame the loop,
cool it
Flame the loop,
cool it
Flame the loop,
cool it
Figure 6.10a, b
• Exercise 9: Aseptic Technique
• Each person make 3 inoculations:
1. Broth to broth - E. coli - 37°C
2. Slant to slant - E. coli - 37°C
3. Plate to slant - S. marcescens 30°C (changed)
• Exercise 10: Pure Culture
Technique
– Each person make 2 streak
plates
– Quadrant Streak Method B,
page 85.
– Incubate plates at 25°C
Each pair will need:
1 broth culture Escherichia coli,
1 slant culture Escherichia coli
1 plate culture Serratia marcescens
Each person will need:
1 Nutrient Broth/BHI tubes,
2 Nutrient Agar/BHIA slants
Each pair will need:
1 mixed culture (which contains:
Escherichia coli, Serratia
marcescens and Micrococcus
luteus)
Each person will need:
2 Nutrient Agar/BHIA plates
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