Microbial Growth Mikro 2010

8/12/2019 Microbial Growth Mikro 2010

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MICROBIAL GROWTH


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DEFINITION OF GROWTH

Bacterial growth is defined not bygrowth in size, but by an increase in

number. Fungal growth occurs at the hyphal

tip by the fusion of characteristic

membrane-bound vesicles derivedfrom the Golgi.


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Bacteria grow bybinary fission.

What is microbial growth?

Increase in cell numbers

How do bacteria reproduce ?

Binary fission Budding

Fragmentation

Bacterial Growth


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What occurs during binary fission? DNA duplication

DNA replication Separation of DNA and cytoplasmic contents Cross wall formation

Generation time = doubling time time required for a cell to divide or apopulation to double

1 to 2 or 100 to 200 or 1 million to 2 million

Most common bacteria have a generation time30-60 min under optimum conditions. Most common pathogens in the body, about

5-10 hours.


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Bacterial Growth Curve


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LAG PHASE : During this phase,bacteria are growing in size, but theyare not undergoing binary fission.Hence, there is no increase in cellnumber. The bacteria are adapting tothe new environment and are

synthesizing cellular components suchas ribosomes, enzymes, and otherproteins.

Bacterial growth phases :


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LOG PHASE : This phase is alsoreferred to as the exponential phasebecause there is a logarithmicincrease in cell number. This

exponential growth is expressed asthe bacterias generation time.During this phase, the conditions are

optimal for growth and binary fissionoccurs.


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STATIONARY PHASE : There is nonet increase or decrease in cell

number in this stage. In other words,cell growth (division) equals celldeath. The birth rate decreases due

to limited nutrients, lack of space,and the build up of secondarymetabolic products (e.g. toxins). Theinsufficient supply of nutrients also

causes some bacteria to form sporesduring this phase.


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DEATH PHASE : This phase ischaracterized by an exponentialdeath of cells. When the media runs

out of nutrients and there are toomany toxins, cells begin to die at afaster rate.


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Fungal Growth curve


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During an initial LAG phase the rate ofgrowth or cell division is very slow.

Growth or cell division then starts toaccelerate into the EXPONENTIAL

phase. This exponential phaserepresents the period when the fungusis growing or multiplying most rapidly.This phase will continue until one or

more nutrients become limiting,oxygen becomes depleted and/ormetabolic by-products accumulate totoxic levels.


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Growth will start to DECELERATE(DECLINE).

This may be followed by aSTATIONARY phase, during whichthere is no discernible change in cell

concentration or biomass.

Finally, we may observe a phase ofCELL DEATH and LYSIS - whichresults in a decrease in cell numberand/or biomass.


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Measurement of growth

1. Total Cell count

Petroff-Hausser chamber slide -- needs large

conc. (107

cells/ml minimum) Coulter Counter (for larger microbes; fungi,

yeasts, protozoa, etc.) -- uses electricalcharge difference in passing through small hole.

Not so useful with bacteria, get errors due toclumping, debris, etc.


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Petroff Hausser


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2. Viable count This is typically carried out by CFU (colony

forming units) assay: carry out dilution series

plate known volumes on plates

count only plates with 30-300 colonies (beststatistical accuracy)

extrapolate to undiluted cell conc.

CFU may or may not be same as number of cells

Method is accurate, but requires time forincubation.


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Two ways to carry outviable count :

Spread plate : bacteria are spread on thesurface of agar using some sterile spreadingdevice.

Advantages : if properly carried out, allcolonies should be easily counted.

Disadvantages : takes some time, cellswith low tolerance to oxygen won't grow.If "spreaders" are present, may overgrow

plate surface.


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Pour plate : bacteria are mixed with meltedagar and cooled; colonies grow throughout the

agar. Advantages : almost fail-proof technique,colonies well separated. Can allow growth oforganisms with lower oxygen tolerance in

agar. Disadvantages : colonies variable size,harder to see similarity in colonymorphology between those on surface and in

agar. Counting may be more difficult. Heatmay kill some cells before agar cools andgels.


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Plate count


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Plate method


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Filtration


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MPN method


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3. Optical techniques

Often, can estimate cell numbers accuratelyby measuring visible turbidity.

This only works above cell densities of 107 inpure cultures. With less than 107 cells/ml,cannot detect bacteria.


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Eyeball method. This is not a precisemeasurement, but shoud allow estimation withinan order of magnitude

no turbidity means less than 107cells/ml

slight turbidity = 10

7

-10

8

cells/ml high turbidity = 108-109cells/ml

very high turbidity = greater than 109cells/ml (cultures rarely get as high as 1010

cells/ml)


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Absorbance method Use a spectrophotometer to accurately

measure absorbance, usually at wavelengthsaround 400-600 nm.

Accurate measure of cells whenconcentration not too high. Easy and quickto measure (can sample in less than aminute)


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Turbidity


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Microbial Growth Mikro 2010

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