Growing bacteria in the lab Bacteria can be grown if given the right conditions, nutrients and water. There are a number of physical factors that effect bacterial growth: – Temperature – pH – Oxygen availability
Physical Factors for bacterial growth Temperature Psychrophiles grow optimally below 15°C Thermophiles multiply best around 60°C Hyperthermophiles are Archaea that grow optimally above 80°C Mesophiles thrive at the medium temperature range of 10° to 45°C, including pathogens that thrive in the human body
Most bacteria are happiest at 37 o C and wont grow much about 42 o C Some can produce spores and survive at very high temperatures e.g. Bacillus Spores can resist sterilisation and can be used as an indicator
pH The majority of species grow optimally at neutral (~7.0) pH Acidophiles are acid-tolerant prokaryotes For example, those used to turn milk into buttermilk, sour cream, and yogurt
Nutrients These are supplied in a nutrient media Carbon – usually in an organic form eg glucose Nitrogen – organic or inorganic Other growth factors – vitamins and minerals The usual source of energy used is glucose
Factors affecting bacterial growth All of these factors can affect bacterial growth: Extend lag phase Decrease exponential (log) phase Premature stationary/death phase
The perfect growth curve Can be used to calculate growth rate Can be used to calculate generation time Need linear part of growth curve Calculate growth rate first Calculate generation time once you know the growth rate
Generation times Linear part Growth rate (k) = log 10 X t – log 10 X 0 T Generation time = 1/(k)
arithmetic semilog arithmetic semi-log Rate of growth Choose two points on linear part of graph Higher value is X t Lower value is X 0 Measure time interval between them (T) Log the X t and X 0 values and put into following formula: T Gives gen/hr (k) Calculate generation time: gen time = 1/k Gives answer in hr per gen convert to min/gen by multiplying the answer by 60
Sterile equipment Equipment and media must be sterilised Autoclave – 121˚C for 15mins – Under pressure Bunsen for inoculating loops Heat labile plastics are irradiated Must be protected from contamination after sterilisation
Methods for measurement of cell numbers Direct microscopic counts are possible using special slides known as a haemocytometer. Dead cells cannot be distinguished from living ones. Only dense suspensions can be counted (>10 7 cells per ml), but samples can be concentrated by centrifugation or filtration to increase sensitivity.
Indirect viable cell counts, also called plate counts, involve plating out (spreading) a sample of a culture on a nutrient agar surface. – The sample or cell suspension can be diluted in a nontoxic diluent (e.g. water or saline) before plating. – If plated on a suitable medium, each viable unit grows and forms a colony. – Each colony that can be counted is called a colony forming unit (cfu) and the number of cfu's is related to the viable number of bacteria in the sample.
Estimating size of viable bacterial population 500 µl 4.5 ml 10 -1 10 -2 10 -3 10 -4 10 -5 10 -6 10 -7 10 -8
10 -4 10 -5 10 -6 10 -7 10 -8 1 ml Gently swirl plate to mix
Flame the neck of the bottle Make sure you work close to the bunsen burner Pouring an agar plate
Motile bacteria Some bacteria are motile: These tend to be Gram negative rods Flagellar make bacteria motile These can be stained Unstained bacteria can be visualised swimming using light microscopy 45
Motility testing The hanging drop method: Bacteria are suspended in a drop of liquid They can be seen by light microscopy Motile bacteria swim in straight line Non-motile bacteria vibrate a bit (Brownian motion) 46
Using bacteria commercially A lot of commercial activities use microbiology directly in their processes – Food manufacturing (think yogurt, cheese, tofu and brewing) – Drug production – Waster water treatment, bioremediation etc It is truly applied biology!
Streak plate to obtain a pure culture Many processes require pure cultures to start with. How do you do that in the lab? Streak Plates!
The microorganisms are grown in very large vessels called fermenters
The large stainless steel cavity is filled with a sterile nutrient solution, which is then inoculated with a pure culture of the carefully selected fungus or bacterium. Paddles rotate the mixture so that the suspension is mixed well. As the nutrients are used up, more can be added. Probes monitor the mixture and changes in pH, oxygen concentration and temperature are all computer controlled.
A water jacket surrounding the fermenter contains fast flowing cold water to cool the fermenter since fermentation is a heat generating process. Most of the air, including carbon dioxide and other gases produced by cell metabolism, leave the fermenter by an exhaust pipe. There are two main types of culture used in industrial processes: batch cultures and continuous cultures.
Batch CultureContinuous Culture cells are grown in a fixed volume of liquid medium in a closed vessel nutrients are added and cells harvested at a constant rate No microorganisms, fluid or nutrients are added or removed from the culture during the incubation period Volume of suspension is kept constant Used for producing secondary metabolites, such as penicillin and other antibiotics, which are relatively unstable and not essential for the growth of the culture Fermenter does not have to be emptied, cleaned and refilled very often Secondary metabolites can be extracted economically only when they reach a high concentration in the culture Production is almost continuous Continuous cultivation needs sophisticated equipment to maintain constant conditions. Highly trained staff need to operate the equipment. Therefore this process can be expensive
Penicillin Production Penicillium notatum (fungus) grown in batch culture It produces the antibiotic after the growth (log) phase when glucose is depleted (limiting) Can you think why it does that? Made to reduce competition for its food The fungal mycelium by filtration The residual liquid is then processed to purify the antibiotic.
For more info on penicillin production: http://penicillin.wikispaces.com/General+bioprocess+flow http://penicillin.wikispaces.com/General+bioprocess+flow