Growth of bacteria in culture

Culture systems based on having:1. a pure culture2. suitable nutrient medium3. satisfactory growth conditions4. sterile technique to maintain pure culture

Different micro-organisms have different nutrient requirements

• Heterotroph- organism that uses complex organic carbon compounds as a source of carbon and energy

• Photoautotroph- uses light as source of energy and CO2 as carbon source

• Chemoautotroph- uses oxidation of simple inorganic compounds for energy and fixes CO2 as a source of carbon

Chemoautotrophic bacteria appear purple,

Heterotrophic E.coli

Phototrophic cyanobacteria

Bacteria can be grown on solid media or liquid media:

• agar in Petri plates• agar in glass bottles or deep

tubes (less dehydrating and anaerobic conditions)

• flat sided bottles allowing growth as a monolayer

• Liquid culture in a culture flask

Dynamics of growth usually studied in liquid culture• Many cells can be grown as a homogeneous single celled

suspension e.g. many bacteria, yeast, some animal cells….

Need to be able to monitor the growth of organisms with time as

1. Want to make sure growth is occurring2. Want to monitor for contamination3. Want to follow stage of growth as some products produced

during growth, others at the end of growth

Batch culture• Cells inoculated into a sterile vessel containing a fixed amount

of nutrients. Growth in the vessel normally follows the predicable “growth curve”

• No nutrients are added or removed (a closed system)• Cells grow by binary fission, as cell divides population

increases exponentially

A. Lag- • No increase in cell number, cells

getting ready to grow and divide• length related to how the cells used

as inoculum were previously treated

B. Log/exponential- • Cells growing at maximum rate for

these conditions. • Growth is balanced i.e. nothing is

limitingC. Stationary- • Cell growth begins to be limited by

lack of particular nutrients or accumulation of inhibitory compounds

• No. Cells dividing are equal to cells dying, a plateau is reached.

D. Death Phase- cells dying faster than dividing

Measuring growth

1. Direct counts

e.g. using a haemocytometer

2. electronic

3. culture based

e.g. dilution and spread plating

4. using a spectrophotometer

Direct counts using a Hemocytometer

Direct counts using a Hemocytometer

•Sample is viewed on special slide with fixed volume

•Sample may be stained first to increase visability or to discern whether cells are viable

•A count of number of cells in a primary square is made

•This is repeated for several other squares

•An average cells per primary square is calculated

•As volume counted is known cells per ml can be calculated

Electronic counters

e.g. Coulter counter The device counts because whenever a particle goes through the hole, the electronic system detects a sudden and momentary increase in resistance (a partial interruption of current flow) and a green vertical line appears on the screen, as shown at the top.

Issues with coulter counter • Does not distinguish between live and

dead• Clumping is a problem• Interference is an issue • expenseOther electronic devises include Flow

cytometers that incorporate fluorescence and lasers to distinguish and count cells. These are very expensive and require dedicated technologists

Culture based methods1. Pour plate- cells are mixed with molten

agar and poured into petri plate, allowed to grow and colonies counted

2. Spread plate- cells are spread on the surface of an agar plate allowed to grow and colonies counted

Both methods assume one cell gives rise to one colony

Colorimetric/spectrophotometric methods: Turbidity is a measure of the # of microbes:

• measured by light scattering or absorbance in a spec

• Cells must be single cells in suspension• Clumping makes the relationship non linear• Can make a standard curve for specific

organism of cell number versus absorbance so can correlate absorbance with number of cells