MICROBIAL GROWTH

DEFINITION OF GROWTH Bacterial growth is defined not by growth in size, but by an increase in number. Fungal growth occurs at the hyphal tip by the fusion of characteristic membrane-bound vesicles derived from the Golgi.

Bacterial Growth
Bacteria grow by binary fission. What is microbial growth? Increase in cell numbers How do bacteria reproduce ? Binary fission Budding Fragmentation

 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 a population to double 1 to 2 or 100 to 200 or 1 million to 2 million Most common bacteria have a generation time 30-60 min under optimum conditions. Most common pathogens in the body, about 5-10 hours.

Bacterial Growth Curve

Bacterial growth phases :

LAG PHASE : During this phase, bacteria are growing in size, but they are not undergoing binary fission. Hence, there is no increase in cell number. The bacteria are adapting to the new environment and are synthesizing cellular components such as ribosomes, enzymes, and other proteins.

 LOG PHASE : This phase is also referred to as the exponential phase because there is a logarithmic increase in cell number. This exponential growth is expressed as the bacterias generation time. During this phase, the conditions are optimal for growth and binary fission occurs.

 STATIONARY PHASE : There is no net increase or decrease in cell number in this stage. In other words, cell growth (division) equals cell death. The birth rate decreases due to limited nutrients, lack of space, and the build up of secondary metabolic products (e.g. toxins). The insufficient supply of nutrients also causes some bacteria to form spores during this phase.

 DEATH PHASE : This phase is characterized by an exponential death of cells. When the media runs out of nutrients and there are too many toxins, cells begin to die at a faster rate.

Fungal Growth curve

 During an initial LAG phase the rate of growth or cell division is very slow. Growth or cell division then starts to accelerate into the EXPONENTIAL phase. This exponential phase represents the period when the fungus is growing or multiplying most rapidly. This phase will continue until one or more nutrients become limiting, oxygen becomes depleted and/or metabolic by-products accumulate to toxic levels.

 Growth will start to DECELERATE (DECLINE). This may be followed by a STATIONARY phase, during which there is no discernible change in cell concentration or biomass.

Finally, we may observe a phase of CELL DEATH and LYSIS - which results in a decrease in cell number and/or biomass.

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 electrical charge difference in passing through small hole. Not so useful with bacteria, get errors due to clumping, debris, etc.

Petroff Hausser

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 (best statistical accuracy) extrapolate to undiluted cell conc. CFU may or may not be same as number of cells Method is accurate, but requires time for incubation.

Two ways to carry out viable count :

Spread plate : bacteria are spread on the surface of agar using some sterile spreading device. Advantages : if properly carried out, all colonies should be easily counted. Disadvantages : takes some time, cells with low tolerance to oxygen won't grow. If "spreaders" are present, may overgrow plate surface.

 Pour plate : bacteria are mixed with melted agar and cooled; colonies grow throughout the agar. Advantages : almost fail-proof technique, colonies well separated. Can allow growth of organisms with lower oxygen tolerance in agar. Disadvantages : colonies variable size, harder to see similarity in colony morphology between those on surface and in agar. Counting may be more difficult. Heat may kill some cells before agar cools and gels.

Plate count

Plate method

Filtration

21

MPN method

3. Optical techniques
Often, can estimate cell numbers accurately by measuring visible turbidity. This only works above cell densities of 107 in pure cultures. With less than 107 cells/ml, cannot detect bacteria.

 Eyeball method. This is not a precise measurement, but shoud allow estimation within an order of magnitude no turbidity means less than 107 cells/ml slight turbidity = 107-108 cells/ml high turbidity = 108-109 cells/ml very high turbidity = greater than 109 cells/ml (cultures rarely get as high as 1010 cells/ml)

 Absorbance method Use a spectrophotometer to accurately measure absorbance, usually at wavelengths around 400-600 nm. Accurate measure of cells when concentration not too high. Easy and quick to measure (can sample in less than a minute)

Turbidity

See you