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Microbial growth

Chapter 6

BIO 220

Requirements for growth

• Physical

– Temperature

– pH

– Osmotic pressure

• Chemical

Temperature

• Psychrophiles (cold-loving microbes)

• Mesophiles (moderate temperature)

• Thermophiles (heat-loving)

• Each type of bacteria grows within a limited

range (min and max only @ 30 °C apart)

Growth rates – temperature

Fig. 6.1

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Psychrophiles vs. Psychrotrophs

• One group can grow at 0 °C but has an optimal

of about 15 °C

– Found mostly in ocean depths or polar regions

• Another group can grow at O °C but has an

optimal temperature of about 20 – 30 °C

– Common in low temperature food spoilage

– Pseudomonas, Listeria monocytogenes

Food spoilage

Fig. 6.2

How does amount of food affect chance of

spoilage?

Fig. 6.3

Mesophiles

• Optimal growth temperature of 25- 40 °C

• Those microbes that live in the bodies of

animals have an optimal growth temperature

close to that of their hosts

• Many pathogens have an optimal temperature

of 37 °C

• Most common spoilage and disease organism

• S. aureus, S. pyogenes, S. pneumoniae, E. coli

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Thermophiles

• Many have an optimal temperature of 50 – 60

°C and are unable to grow below 45 °C

• If endospores are formed, they are likely to

survive heat treatment

• Some members of Archaea have an optimal

growth temperature of 80 °C or higher and are

known as hyperthermophiles

pH

• Most bacteria grow at a relatively narrow pH

range @ 7

• Yeasts and molds have an optimal pH that is

more acidic

• Acidophiles are tolerant of acidity

Osmotic pressure

• Microbes obtain most of their nutrients from

their aqueous surroundings, making water a

requirement for growth

Fig. 6.4

Extreme halophiles

• Require a high salt environment for growth

• Obligate vs. facultative halophiles

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Chemical requirements for growth

• Water

• Carbon

• Nitrogen

• Sulfur

• Phosphorous

• Potassium, magnesium, calcium and trace elements

• Organic growth factors

Oxygen

• Obligate aerobes – require O2 to live

– Mycobacterium tuberculosis

• Facultative anaerobes – use O2 when present,

but can still grow (at a slower rate) when O2 is

absent

– Escherichia coli, Salmonella, Shigella

• Obligate anaerobes – unable to use O2 for ATP

production

– Clostridium botulinum

Toxic forms of oxygen

• Singlet oxygen

• Superoxide radicals

– Superoxide dismutase neutralizes these anions

• Hydrogen peroxide (peroxide ions)

– Catalase and peroxidase break down H2O2

• Hydroxyl radicals

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What are biofilms?

• Biofilms are complex microbial aggregates consisting of one type or a variety of microbes encased in an extracellular matrix, a hydrogel which is composed of a complex polymer containing many times its dry weight in water

• Biofilms are the bacterial slime layers that line drains, can be found in the kitchen, and even line internal body surfaces

Biofilm formation

• Biofilm deposition on a solid surface begins when a free-swimming (planktonic) bacterium lands on and attaches to the surface

• The biofilm grows as the original microbes reproduce and recruit other microbes

• Instead of growing in a monolayer, microbes will often form pillar-like structures

• Water flow past these structures allows for nutrient acquisition and waste removal

Biofilms

Fig. 6.5

Why make a biofilm?

• The bacteria and other colonists are protected

from environmental challenges by the

extracellular matrix

• Quorum sensing allows communication

between microbes

• Each member of the biofilm performs a

designated metabolic function which benefits

all

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Biofilms can be beneficial

• Root biofilms help plants harvest nutrients

and moisture from the soil

• Biofilms can also help remove organic wastes

from sewage

• Bioremediation

Biofilms can cause problems

• Form on indwelling medical devices such as catheters, artificial heart valves, and prosthetic joints

• Can promote cavity formation

• Can promote eye infections

• Disease

– Otitis media, cystic fibrosis, Legionnaire’s disease, bacterial endocarditis, corneal infections associated with contact lens use

Culture media

• This is the nutrient material that is required for microbial growth in a laboratory

• Some microbes can grow on almost any nutrient medium, other microbes are more fussy (fastidious)

• Inoculum – microbes that are introduced into a culture medium to promote growth

• A culture refers to microbes that grow and multiply on or in a culture medium

Culture media

• Must meet the requirements for microbial

growth

• Should contain sufficient moisture

• Appropriate pH

• Suitable amount of oxygen

• Must be sterile (initially)

• Must be incubated at the appropriate

temperature

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Culture media cont.

• In order to produce a solid medium, a

solidifying agent like agar is used

• Advantages of agar

– Few microbes can degrade it

– Liquefies around 100 °C

• Agar media can be used in

– Petri plates

– Slants & deeps

Chemically defined media

• Exact chemical composition is known

• In order to support microbial growth, the

medium must provide an energy source,

sources of carbon, nitrogen, sulfur,

phosphorus, and any other organic molecules

the microbe is unable to synthesize

Fastidious organisms require

many growth factors in their

chemically defined media.

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Complex media

• Most heterotrophic bacteria and fungi are grown on complex media

• Chemical composition varies

• Energy, carbon, nitrogen, and sulfur requirements are primarily provided by proteins or peptones

• Vitamins and other organic growth factors are provided by meat or yeast extracts

• Nutrient agar vs. nutrient broth

Anaerobic growth media and methods

• Reducing media must be used in order to

deplete the oxygen in the culture medium

– Sodium thioglycolate

Figs. 6.6, 6.7

Selective media

• Designed to suppress the growth of unwanted

bacteria and encourage the growth of the

desired microbes

• Sabouraud Dextrose Agar – medium has a low

pH (pH=5.6), fungi can grow in these

conditions but most bacteria can not

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Differential media

• Distinguishes between different types of

microbes that grow on media

• Blood agar allows us to distinguish between

organisms that can lyse red blood cells

– α vs. β vs. γ hemolysis

Media can be selective & differential

• Levine eosin methylene blue agar

– Selective for Enterobacteriaceae and related gram

(-) enterics

– Differential for lactose fermentation

• Mannitol salt agar

– Selective for salt-tolerant skin microbes

– Differential for mannitol fermentation

Mannitol salt agar

Fig. 6.10

Enrichment culture

• The enrichment medium for this type of

culture is usually liquid and provides nutrients

and environmental conditions that favor the

growth of a particular microbe but not others

• Supports growth of fastidious organisms

• Designed to increase very small numbers of

the desired microbe to detectable levels

• i.e. Blood agar, brain heart infusion

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Obtaining pure cultures

• Theoretically, microbial colonies arise from

individual cells or spores that come into

contact with the medium

• So, these colonies are composed of cells that

are genetically identical (clones)

• Over time, as mutation and genetic

recombination occurs the cultures will

eventually become axenic

Streak plate method

• Used to get pure cultures

Fig. 6.11

Preservation of bacterial cultures

• Refrigeration

• Deep – freezing

– Culture quick-frozen at a temperature between

-50 °C and -95 °C

• Lyophilization (freeze-drying)

– Cultures frozen between -54 °C and -72 °C and

then dehydrated by sublimation

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Bacteria normally divide by binary fission

Fig. 6.12

Generation time

• This is the time required for a cell to divide

and its population to double

• Generation time is dependent on the microbe,

temperature, and other factors as well

Fig. 6.13 Fig. 6.14

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Fig. 6.15

Bacterial growth curveLag Phase

• Cells are not dividing, but are preparing to

divide in the medium.

• Increased metabolic activity characterized by

the synthesis of enzymes and various

molecules.

Log Phase

• This is a period of cell division resulting in an

exponential increase in growth.

• Most active metabolically.

• Rate of division is constant.

• Nutrients are still in excess and the cells are

maximizing the utilization of the nutrients.

• Production of new cells is greater than the

number of dying cells.

Stationary Phase

• This represents a period of equilibrium, when

the number of cell deaths is balanced by the

production of new cells.

• Nutrients are now limiting and wastes

continue to accumulate.

• Cell walls may start to weaken.

• Carbon dioxide may increase, pH decrease,

and oxygen decrease.

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Death Phase

• Number of cell deaths exceeds new cell

production.

• Death occurs at a constant and maximal rate.

This exponential decline is the reverse of the

Log Phase.

• Continues until the population is diminished

to a tiny fraction of the cells in the previous

stage or until the population dies out.

Estimation of microbial growth

• Direct measurement

– Plate counts

– Pour plates/spread plates

– Filtration

– MPN

– Microscopic count

Measurement of microbial growth

• Plate counts (CFUs)

• Serial dilutions

Fig. 6.16

Pour plates and spread plates

Fig. 6.17

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Filtration

Fig. 6.18

Most probable number (MPN)

Fig. 6.19

Direct microscopic count

Fig. 6.20

Estimation of microbial growth

• Indirect measurement

– Turbidity

– Metabolic activity

– Dry weight

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Turbidity

Fig. 6.21

Other indirect assessments

• Metabolic activity

– Measure amount of some metabolic product

• Dry weight

– Remove organism from growth medium, filter, dry

in desiccator, weigh