19 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:TemperaturepHOxygen availability
20 Physical Factors for bacterial growth TemperaturePsychrophiles grow optimally below 15°CThermophiles multiply best around 60°CHyperthermophiles are Archaea that grow optimally above 80°CMesophiles thrive at the medium temperature range of 10° to 45°C, including pathogens that thrive in the human body
21 Most bacteria are happiest at 37oC and wont grow much about 42oC Some can produce spores and survive at very high temperatures e.g. Bacillus Spores can resist sterilisation and can be used as an indicator
26 pH The majority of species grow optimally at neutral (~7.0) pH Acidophiles are acid-tolerant prokaryotesFor example, those used to turn milk into buttermilk, sour cream, and yogurt
27 These are supplied in a nutrient media NutrientsThese are supplied in a nutrient mediaCarbon – usually in an organic form eg glucoseNitrogen – organic or inorganicOther growth factors – vitamins and mineralsThe usual source of energy used is glucose
28 Factors affecting bacterial growth All of these factors can affect bacterial growth:Extend lag phaseDecrease exponential (log) phasePremature stationary/death phase
30 The ‘perfect’ growth curve Can be used to calculate growth rateCan be used to calculate generation timeNeed linear part of growth curveCalculate growth rate firstCalculate generation time once you know the growth rate
31 Generation times Linear part Growth rate (k) = log10Xt – log10X0 T Generation time = 1/(k)
32 arithmeticsemi-logRate of growthChoose two points on linear part of graphHigher value is XtLower value is X0Measure time interval between them (T)Log the Xt and X0 values and put into following formula:TGives gen/hr (k)Calculate generation time: gen time = 1/kGives answer in hr per gen convert to min/gen by multiplying the answer by 60arithmeticsemilog
37 Sterile equipment Equipment and media must be sterilised Autoclave 121˚C for 15minsUnder pressureBunsen for inoculating loopsHeat labile plastics are irradiatedMust be protected from contamination after sterilisation
38 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 (>107 cells per ml), but samples can be concentrated by centrifugation or filtration to increase sensitivity.
40 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.
41 Estimating size of viable bacterial population 4.5 ml10-110-210-310-410-510-610-710-8
42 1 ml10-410-510-610-710-8Gently swirl plate to mix
44 Make sure you work close to the bunsen burner Pouring an agar plateMake sure you work close to the bunsen burnerFlame the neck of the bottle
45 Motile bacteria Some bacteria are motile: These tend to be Gram negative rodsFlagellar make bacteria motileThese can be stainedUnstained bacteria can be visualised swimming using light microscopy
46 Motility testing The hanging drop method: Bacteria are suspended in a drop of liquidThey can be seen by light microscopyMotile bacteria swim in straight lineNon-motile bacteria ‘vibrate’ a bit (Brownian motion)
48 Using bacteria commercially A lot of commercial activities use microbiology directly in their processesFood manufacturing (think yogurt, cheese, tofu and brewing)Drug productionWaster water treatment, bioremediation etcIt is truly applied biology!
49 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!
50 The microorganisms are grown in very large vessels called fermenters
52 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.
53 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.
54 Batch CultureContinuous Culturecells are grown in a fixed volume of liquid medium in a closed vesselnutrients are added and cells harvested at a constant rateNo microorganisms, fluid or nutrients are added or removed from the culture during the incubation periodVolume of suspension is kept constantUsed for producing secondary metabolites, such as penicillin and other antibiotics, which are relatively unstable and not essential for the growth of the cultureFermenter does not have to be emptied, cleaned and refilled very oftenSecondary metabolites can be extracted economically only when they reach a high concentration in the cultureProduction is almost continuousContinuous cultivation needs sophisticated equipment to maintain constant conditions. Highly trained staff need to operate the equipment. Therefore this process can be expensive
55 Penicillin Production Penicillium notatum (fungus) grown in batch cultureIt 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 foodThe fungal mycelium by filtrationThe residual liquid is then processed to purify the antibiotic.
56 For more info on penicillin production: http://penicillin. wikispaces