Lecture #10: Factors that Affect Bacterial Growth

Measuring Growth

• Total Cell Counts

• Viable Counts

• Turbidometric Counts

Environmental Factors

–Temperature

–pH

–Water availability

–Oxygen

Direct Measurements

Total Cell Counts

• Viable Counts

Indirect Measurements

Turbidometric Methods

Using a

Counting

Chamber

• Cover slip is placed on

top and cell suspension

to be counted is applied

underneath.

• The grid is viewed under

the microscope and the

organisms in the

specimen are counted.

• The concentration in the

sample is calculated from

the area counted and

known depth of the

sample

Considerations when Using Direct

Cell Counts

• Direct method of cell population determination.

• Counts both viable (live) and non-viable (dead)

cells. Dyes such as methylene blue can be used for

differentiation.

• Hard to count with motile cells. Sometimes CHCl3

is added to kill the sample to kill them and make

counting easier.

Viable Count

• Spread-plate method

  • Surface colonies

• Pour-plate method

  • Spread colonies

Sources of Error in Plate Counting

Unsuitable culture conditions

• inappropriate medium

• growth environment (oxygen, temperature, etc)

• source of “the great plate count anomaly.”

Procedural Errors

• inaccurate transfers/dilutions

• inadequate mixing of culture

• Cell clumping (counting colonies, not cells)

A Few Environmental Factors that

Affect Microbial Growth

• Temperature

• pH

• Water availability

• Oxygen concentrations

Cardinal Temperatures

Minimum

Maximum

Optional

Temperature Classes of Organisms

• psychrophile

• mesophile

• thermophile

• hyperthermophile

• hyperthermophile

Growth in Cold Environments

Psychrophilic organisms

have optimal growth

temperatures below 15ºC,

maximum growth

temperatures below 20ºC,

and minimum growth

temps of 0ºC or lower.

Growth in Cold Environments

Psychrotolerant organisms

can grow at 0ºC [or <5ºC by

some definitions], but have

optimal growth

temperatures of 20-40ºC.

They are found in

temperate climates

Molecular Adaptations to Psychrophily

• Enzymes active in the cold

– tend to have more α-helicies and less β-sheets.

– tend to have more polar side-chains (helps

flexibility).

– have fewer weak interactions.

• More unsaturated lipids in the membrane

• Cryoprotective molecules reduce dehydration

and ice-crystal formation

Microbial Growth at High Temperatures

• Thermophiles have growth optima over 45ºC

• Hyperthermophiles have

  growth optima over 80ºC

Thermophiles

Three Generalizations:

• Prokaryotes have higher

temp. growth abilities than

eukaryotes.

• The archaeal branch

contains the most

thermophilic species.

• Non-phototrophs can

exceed phototrophs in

thermophilic growth

abilities

Molecular Adaptations to Thermophily

• Amino acid substitutions at key places in enzymes to

increase stability at high temperatures (higher

temperature optima).

• More ionic bonds and denser hydrophobic protein

cores.

• Cytoplasmic solutes (e.g., diglycerol phosphate) may

help stabilize proteins.

• High saturation rates of fatty acids in membranes of

Bacteria

• Use of lipid monolayers in Archaea

Thermophily and Biotechnology

• Industrially important enzymes from

thermophiles

– can be used to increase reaction efficiencies.

– are typically more stable.

– examples:

• thermostable polymerases (e.g., Taq) in molecular

biology

• thermostable xylanase in lignin extraction from pulp

(paper industry)

Acidophiles

Picrophilus oshimae

• pH optimum 0.7

• lyses above pH 4

• grows in volcanic

soils.

Alkaliphiles

Bacillus firmus

• up to pH 11

• uses Na+ gradient to

drive transport and

locomotion

Archaeal strains

many are halophilic

Obligate aerobes

require O2 for growth; they use O2 as a final electron acceptor in

aerobic respiration.

Obligate (strict) anaerobes

do not need or use O2 as a nutrient. In fact, O2 is a toxic

substance, which either kills or inhibits their growth. These organisms are

fermentative or respire anaerobically.

Facultative anaerobes (or facultative aerobes)

are organisms that can switch

between aerobic and anaerobic types of metabolism. Under anaerobic conditions

(no O2) they grow by fermentation or anaerobic respiration, but in the presence of

O2 they switch to aerobic respiration

Microaerophiles

equire some oxygen because they are obligately aerobic, yet

atmospheric levels of oxygen are toxic. These organisms grow only under

conditions where the oxygen is lower than that of air

Aerotolerant anaerobes

are bacteria with an exclusively anaerobic (fermentative)

type of metabolism but they are insensitive to the presence of O2. They live by

fermentation alone whether or not O2 is present in their environment.