Characteristics and study of prokaryotic growth

How do we grow bacteria in the laboratory?

What is required for growth?

How do we measure bacterial growth?

What do bacteria do while they are growing?

How do bacteria grow in the environment?

What is a pure culture?

All of the organisms are descended from a single cell(and therefore are identical)

Bacteria do not grow that way naturally

Most bacteria cannot be cultured that way

So why do it?

Historically (19th century):

Koch’s postulates –applied to prove that a particular organismcaused disease-isolate organism from diseased animal-establish a pure culture of the organism (and prove it)- infect another animal with cultured bacteria

does this animal get sick?are the same microbes isolated from the secondanimal?

Koch proved that B.anthracis causes anthrax and thatM. tuberculosis causes tuberculosis this way

Koch also developed new methods for bacterial growth

What is required for pure culture?

Sterile apparatusAseptic techniqueAppropriate media

solid media: separable colonies

Bacteria “grow” (i.e, divide) by binary fission

With each generation the population doubles

“generation time,” or doubling time, varies withbacterium and growing conditions

Can be as little as 20 minutes

Thus bacterial contamination can be rapidly established

What sorts of factors affect bacterial growth?temperature

How can they grow at such a wide range of temperatures?

Implicationssubstances that function at extreme temperaturesfor prevention of contaminationfor pathogenesis(why do we develop fevers?)

Oxygen requirements

Availability varies in the environment and on/in our bodies

pH

Bacteria maintain an internal neutral pH, but may survivein a wide range of acidic or alkaline environments

How?H. pylori- produce urease; generates ammonia andraises pH of immediate environment

Other bacteria use proton pumpsacidophiles- outalkalophiles- in

Salt tolerance

Synthesize or pump solutes inside the cell (and drawwater)

Osmotolerant organism- can grow in dry environments(like skin)

Halophiles- require high salt conditions

High solutes concentrations are historically used topreserve foods

What do bacteria require to grow?

NutrientsEnergy sourceSome organisms require growth factors (fastidious)

Which is easiest to grow in the laboratory?

Some media are richer than others

Some are used to look at mixed cultures of microorganisms

Selective- allow some organisms to grow but not others

Differential- bacteria have different characteristics

Some are both!

Adjusting atmospheric requirements

CO2 levels (capnophiles)

Candle jars (microaerophiles)

Anaerobe jars/chambers (guess)reducing agents

Why would you use selective enrichment medium?

Measuring bacterial growth

Considerations

RapidityViable vs. total bacteriaLarge numbers or small numbers of bacteria

Growth in progress?

Direct counting

RapidCan count organisms that can’t grow in medium

Can’t distinguish live from dead cellsMotile organisms are hard to count!

Special instruments needed

Viable counts

Of living (and growing) bacteriarequires some skill and time

Can be performed on special mediaNeed fairly concentrated culture

Dilute samplesmembrane filtrationMPN (most probable number)- statistical estimate,NOT direct count

Measuring cell mass

Most common method- turbidity (spectrophotometry)

Cells grown in liquid mediumCan measure “progress” of cell growth

Changes (in turbidity) correlate with increase in cell numbers

Growth can be measured either by turbidity or bycounting to determine growth curve

Metabolic activities change in late log/stationary phase

Metabolites are often of research/commercial interestBacteria can be grown to enhance production

How do bacteria actually grow in nature?

Are nutrients always available, and in constant amounts?

They may elaborate protective structures when necessaryglycocalyxendosporesbiofilm (protects communities of microbes)

Interactions may be required (one organism produces substances required by another