CHAPTER 6

MICROBIAL
GROWTH
 The Requirements for Growth
Physical Requirements
Boiling Water Hyperthermophiles
• Temperature
- Minimum growth > Optimum growth > Maximum growth
• Temperature
- Psychrophiles - extremely cold temperatures
Grow from 0°C – 20°C
Examples: Pseudomonas, Flavobacterium, Alcaligenes Thermophiles

- Mesophiles - moderate temperatures

Body Temperature
Optimum growth temperature of 25°C to 40°C
Mesophiles
Example: E. coli, Salmonella, Clostridium botulinum
- Thermophiles - extreme heat
Optimum growth temperature of 50°C to 60°C Psychotrophs
Example: Bacillus stearothermophilus
Freezing
- Hyperthermophiles Psychrophiles
Optimum growth temperature of 80°C or higher
Example: Pyrolobus fumarii
Psychrotrophic – cause food spoilage
Grow between 0°C and 20°C to 30°C
Example: Listeria monocytogenes
 The Requirements for Growth
Potential of Hydrogen (pH)
• Most bacteria grow best in a narrow pH range near neutrality, between
pH 6.5 and 7.5.
• Very few bacteria grow at an acidic pH below about pH 4.
• Molds and yeast grow between pH 5 and pH 6.

- Acidophiles – grow in acidic environment

Examples: genus Ferroplasma
- Neutrophiles – grow optimally at a pH within one or two pH units
of the neutral pH of 7
Example: : E. coli, Salmonella
- Alkaliphiles – grow best at pH between 8.0 and 10.5
Example: Vibrio cholerae
 The Requirements for Growth
Osmotic Pressure
• A microbial cell is in a solution whose concentration of solutes is higher
than in the cell– the environment is hypertonic to the cell.
• Hypertonic environments (higher in solutes than inside the cell) cause
plasmolysis due to high osmotic pressure.

- Extreme halophiles – adapted so well to high salt concentration

Termed as obligate halophiles
- Facultative halophiles – tolerate high osmotic pressure

• If the osmotic pressure is unusually low – the environment is

hypotonic
 The Requirements for Growth

Chemical Requirements

Carbon
• One of the most important requirements for microbial
growth.
• Structural backbone of living matter.
• Chemoheterotrophs use organic molecules as energy
• Autotrophs use CO2
• Proteins, DNA, carbohydrates, and other molecules that
distinguish living matter are all composed of carbon
compounds
 The Requirements for Growth
Chemical Requirements
Nitrogen
•Component of proteins, DNA, and ATP
•Most bacteria decompose protein material for the nitrogen source
•A few bacteria use N2 in nitrogen fixation
•The nitrogen fixed in the symbiosis is used by both the plant and the bacterium
 The Requirements for Growth
Chemical Requirements
Sulfur
•Used to synthesize sulfur-containing amino acids and vitamins such as thiamine and biotin.
• Important natural sources of sulfur include the sulfate ion (SO42-), hydrogen sulfide, and the sulfur-containing amino
acids.
 The Requirements for Growth
Chemical Requirements

Phosphorous
•Essential for the synthesis of nucleic acids and the phospholipids of cell membranes.
•Used in DNA, RNA, and ATP
 The Requirements for Growth
Chemical Requirements
Trace Elements
•Inorganic elements required in small amounts.
•Usually as enzyme cofactors
•Include iron, copper, and zinc
 The Requirements for Growth
Chemical Requirements
Oxygen
• Different microbes have different tolerances to oxygen
• There was no free oxygen in the early Earth atmosphere.
 The Requirements for Growth
Chemical Requirements
Organic Growth Factors
• Organic compounds obtained from the environment
• Vitamins, amino acids, purines, and pyrimidines
 The Requirements for Growth
Biofilms
•Microbial communities
•Form slime or hydrogels that adhere to surfaces
- Bacteria communicate cell-to-cell via quorum sensing
- Bacteria secrete an inducer (signaling chemical) to attract
other bacterial cells
• A biofilm usually begins to form when a free-swimming
(planktonic) bacterium attaches to a surface.
• Bacteria that use quorum sensing pro- duce and secrete a
signaling chemical called an inducer.
•Share nutrients
•Shelter bacteria from harmful environmental factors; more
resistant to treatments
 The Requirements for Growth
Culture Media
•Culture medium: nutrients prepared for microbial growth
•Sterile: no living microbes
•Inoculum: introduction of microbes into a medium
•Culture: microbes growing in or on a culture medium
Agar
•Complex polysaccharide
•Used as a solidifying agent for culture media in Petri plates,
slants, and deeps
•Generally not metabolized by microbes
•Liquefies at 100°C; Solidifies at ~40*C
 The Requirements for Growth
Culture Media
Chemically Defined Media

• A chemically defined medium is one whose exact chemical composition

is known.
• Complex media: extracts and digests of yeasts, meat, or plants: chemical
composition varies batch to batch
• Nutrient broth
• Nutrient agar
 The Requirements for Growth
Anaerobic Growth Media and Methods
Reducing Media
•Used for the cultivation of anaerobic bacteria
•Contain chemicals (sodium thioglycolate) that combine O, to deplete it
•Heated to drive off 02
 The Requirements for Growth
Special Culture Techniques
Biosafety Levels
• BSL-1: no special precautions; basic teaching labs
• BSL-2: lab coat, gloves, eye protection
• BSL-3: biosafety cabinets to prevent airborne transmission
• BSL-4: sealed, negative pressure; "hot zone”
• Exhaust air is filtered twice through HEPA filters
 The Requirements for Growth
Special Culture Techniques
Selective Media
• Suppress unwanted microbes and encourage desired microbes 16
- Contain inhibitors to suppress growth 18

EMB agar is selective for gram-negative bacteria. The dye methylene blue in the medium inhibits the growth of
gram-positive bacteria; small amounts of this dye effectively inhibit the growth ..of most gram-positive bacteria
 The Requirements for Growth
Special Culture Techniques
Differential Media
• Allow distinguishing of colonies of different microbes on the same plate
• Some media have both selective and differential characteristics
 The Requirements for Growth

Special Culture Techniques

Enrichment Culture

• Encourages the growth of a desired microbe by

increasing very small numbers of a desired
organism to detectable levels.
• Usually a liquid
 The Requirements for Growth

Obtaining Pure Culture

Enrichment Culture

• 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) 20
• The streak plate method is used to isolate pure cultures
 The Requirements for Growth

Preserving Bacterial Cultures

• Two common methods of preserving microbial cultures for long periods are deep-freezing and lyophilization.

- Deep-freezing is a process in which a pure culture of microbes is placed in a suspending liquid and quick-
frozen at temperatures ranging from -50°C to -95°C.
- Lyophilization (freeze-drying), a suspension of microbes is quickly frozen at temperatures ranging from -
54°C to -72°C, and the water is removed by a high vacuum (sublimation).
 The Requirements for Growth

The Growth of Bacterial Cultures

Bacterial Division
• Increase in number of cells, not cell size
• Binary fission
• Budding
 The Requirements for Growth

The Growth of Bacterial Cultures

Generation Time

• Time required for a cell to divide

- 620 minutes to 24 hours
• Binary fission doubles the number of cells each generation
• Total number of cells = 2 ^ number of generations
• Growth curves are represented logarithmically
 The Requirements for Growth

The Growth of Bacterial Cultures

Generation Time
 Understanding the Bacterial Growth Curve

Bacterial populations follow a sequential series of growth phases:

Phases of Growth
1. The Lag Phase
2. The Log Phase
3. Stationary Phase
4. Death phase
 Understanding the Bacterial Growth Curve
Direct Measurement Of Microbial Growth
- The growth of microbial populations can be measured in a number of ways. Some
methods measure cell numbers; other methods measure the population’s total mass,
which is often directly proportional to cell numbers.
• Plate Counts
- The most frequently used method of measuring bacterial populations.
• Serial Dilutions
- A stepwise series of dilutions that are performed to reduce the
concentration of a substance in a solution to a more usable concentration.
 Understanding the Bacterial Growth Curve
Direct Measurement Of Microbial Growth
• Pour Plates and Spread Plates
- A plate count is done by either the pour plate method
or the spread plate method.
• Filtration
- Bacteria are retained on the surface of a membrane
filter and then transferred to a culture medium to
grow and subsequently be counted.
• The Most Probable Number (MPN) Method
- Can be used for microbes that will grow in a liquid
medium; it is a statistical estimation.
• Direct Microscopic Count
- The microbes in a measured volume of a bacterial
suspension are counted with the use of a specially
designed slide.
 Understanding the Bacterial Growth Curve
Estimating Bacterial Numbers by Indirect Methods
• Turbidity
- A spectrophotometer is used to determine turbidity by
measuring the amount of light that passes through a
suspension of cells.
• Metabolic Activity
- This method assumes that the amount of a certain
metabolic product, such as acid, CO2, ATP, or DNA, is
in direct proportion to the number of bacteria present
• Dry Weight
-For filamentous bacteria and molds, the usual
measuring methods are less satisfactory.
Flavobacterium
Alcaligenes
E. coli
Salmonella
Clostridium botulinum
Bacillus stearothermophilus
Pyrolobus fumarii
Listeria monocytogenes
Ferroplasma
Vibrio5. The Requirements for Growth cholerae
Biofilms
Binary Fission
Lag Phase
Log pahse
Stationary & Death phase
END