Microbiology

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Bacterial population growth curve

Lag: slow increase in cell number. Bacteria adjusting to environment: switching on genes, producing digestive enzymes to secrete to digest the medium extracellularly, DNA replication

Log: nutrients in plentiful supply, waste products are at low concentrations, rate of cell division is at maximum, growth is exponential.

Stationary: carrying capacity reached, equilibrium established. Limiting factors (nutrient depletion and waste accumulation) prevent a further increase in population.

Death: more cells dying than being produced, due to nutrient shortage and accumulation of waste.

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Optimum conditions for growth

Nutrients: source of carbon, nitrogen and phosphorous. A respiratory substrate usually glucose. Vitamins and minerals to act as coenzymes. Water, as all metabolic reactions occur in solution. If any one of these is depleted, it will become a limiting factor to growth rate.

Temperature: for enzymes. Too high- denaturation of enzymes causes cell death. Too low- enzyme catalysed reactions too slow to sustain life. Mesophiles have optimum between 20-40C. Thermophiles have optimum above 40C. Psychrophiles have optimum below 20C.

pH for enzymes. Microbes in general can tolerate a wider range of pHs than plant or animal cells.

OxygenObligate aerobes require oxygen for growth at all times. Obligate anaerobes find oxygen toxic as it inhibits their respiration so cannot grow in its presence. Facultative anaerobes grow best in the presence of oxygen, but can survive without it, albeit population growth rate is slow.

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Measuring population growth

Total count: living and dead cells. Haemocytometry. Numbers in the population can be overestimated.

Viable count: only living cells. Dilution plating: growing bacteria to form distinct colonies that can be counted - based on assumption that one cell gives rise to one colony. Population can be underestimated because of clumping of cells - several cells form one colony.

total viable cell count = number of colonies x dilution factor

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Metabolites

Primary metabolites are required for normal microbe metabolism, e.g. enxymes. Continous fermentation used where microbes kept in exponential phases to maximise production

Secondary metabolites are not required for normal metabolism e.g. antibiotics, produced in overcrowded conditions to kill competing microbes. To maximise production, batch fermentation is used, allowed to reach stationary phases, where antibitiotics produced.

Penicillium notatum fungus produces penicillin as a secondary metabolite when glucose has become depleted. Produced commercially in a batch culture fermenter: a closed stainless steel tank that resists the corrosive effect of acidic fermentation productions and withstands pressure.

Pencillin prevents the formation of cross links between peptidoglycan units during cell division. This weakens cell walls and leaves the bacteria susceptible to osmotic lysis.

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Penicillin production process

1. Vessel sterilised using high pressure steam to kill any microbes, preventing contamination.

2. Sterile nutrient medium added.

3. Pure Penicillium notatum culture added.

4. Fungus provided with suitable conditions for growth:

- Sterile air intoduced to provide oxygen for aerobic respiartion. Prevents contamination from airborne microbes.

- Culture heated to optimum temperature for growth, mainting by cooling water jacket that removes excess heat from fungal respiration.

- sparging ring/paddles thoroughly mixes the culture and nutrients by forced aeration.

- once glucose depleted, fungus enters stationary and penicillin is produced.

- culture fluid drained and filtered to remove the mycelium and the filtrate is purified to extract the penicllin.

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