Lecture 6: Microbial Growth and Control

PowerPoint® Lecture Presentations CHAPTER 5 Microbial Growth and Its Control © 2018 Pearson Education, Inc. Binary Fission Microbial growth 3 Microorganisms and Concepts Staphylococcus aureus • lag, exponential, stationary and death phases • measuring bacterial growth • cell count vs. viab...

PowerPoint® Lecture Presentations CHAPTER 5 Microbial Growth and Its Control © 2018 Pearson Education, Inc. Binary Fission Microbial growth 3 Microorganisms and Concepts Staphylococcus aureus • lag, exponential, stationary and death phases • measuring bacterial growth • cell count vs. viable count • dilution series • continuous culture 1. GROWTH; chemical & physical factors. 2. Industrial, health, and research considerations. 3. For example: imagine you are hired by Pfizer to help develop a new antibiotic from a strain of Bacillus. You have one 10,000 gallon tank available, costs $1000/day to run. You must decide how much inoculum to put in tank, when to harvest bacteria for maximum profit, minimum cost. How will you decide? What numbers do you need? 4.Another example. You are hired as a research assistant in a laboratory. You need to grow bacteria to a concentration of 5 x 108 cells/ml, then harvest these cells for use in an experiment. When should you inoculate? How can you know when you have reached the desired cell concentration? Batch vs. Continuous culture methods Batch method: put small inoculum of pure culture into sterile medium, let grow. Common lab procedure, but not typical of many real environments. Continuous culture: use chemostat or turbidostat. Trickle fresh medium into culture at slow but steady rate, displace = volume of culture as overflow. Cells remain in exponential (but suboptimal) state, growing at known rate. Good simulation for study of many natural environments. Reproductive Strategies • the reproductive strategies of eukaryotic microbes – asexual and sexual, haploid or diploid • Bacteria and Archaea – haploid only, asexual - binary fission, budding, filamentous – all must replicate and segregate the genome prior to division http://www.youtube.com/watch?v=gEwzDydciWc&feature=relate d -bacterial growth is exponential 1.Why lag phase? - cells ran out of food - made adaptations necessary for dormancy and protection. - regenerate pools of essential nutrients before growth - new enzyme synthesis and time for pathways to function. 2.Why exponential phase? -cells in optimum growth state, divide repeatedly by binary fission at maximal rate. Note: useful to calculate doubling time; can vary from 20 min to several days 3.Why stationary phase? - exhaustion of some critical nutrient, or accumulation of waste products (e.g. acid buildup from fermentation). 4.Why death phase? -accumulation of wastes, exposure to oxygen, loss of cell's ability to detoxify toxins, etc. Note: in practice, try to prolong stationary phase, reduce death phase. - transfer to slants (tubes), store capped in refrigerator once grown. Nf=( No) 2n or where n = number of generations, Nf = final conc. of cells (e.g. 109/ml), No = initial conc. of cells (e.g. 103/ml), and .301 is factor to convert log2 to log10 Example: measure culture at 9 a.m.: No = 10,000 cells/ml measure culture at 3 p.m.: Nf = 100,000 cells/ml calculate n = (5 - 4)/.3 = 1/.3 = 3.33 generations total time = 6 hours = 360 minutes 360 minutes/3.33 generations = 108 min/generation Conclude: generation time = 108 minutes Eg.: Staphyloccocus aureus has contaminated an egg sandwich. How many cells (Nf) will be in the sandwich after it sits in a warm car for 4 hr. Assume that No is 10 ( number of cells deposited in the sandwich while it was being prepared). To derive n we need to divide 4 hrs (240 min) by the generation time. We will assume from previous studies that it is 20 minutes. Apply: Nf=( No) 2n Nf =? No =10 n= 240 minutes total time elapsed / 20 = 12 generations Nf = 10 x 212 = 40,960 cells in the sandwich Measurement of growth 1.Total Cell count Coulter Counter uses electrical charge difference in passing through small hole. -errors due to clumping, debris, etc. or Petroff-Hauser chamber 2.Viable count This is typically carried out by CFU (colony forming units) assay: 1.dilution series 2.plate 3.count only plates with 30-300 colonies (best statistical accuracy) 4.extrapolate to undiluted cell conc. CFU may or may not be same as number of cells -Method is accurate, but requires time for incubation. Counting Bacteria • Viable counts – Counts only cells able to reproduce • Form colonies – Requires time to form colonies (overnight) Two ways to carry out viable count: 1.Spread plate: Advantages: if properly carried out, all colonies should be easily counted. Disadvantages: takes some time, not always reliable in inexperienced hands, cells with low tolerance to oxygen won't grow. If "spreaders" are present, may overgrow plate surface. 2. Pour plate: bacteria are mixed with melted agar and cooled; colonies grow throughout the 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. Spread-plate vs Pour-plate Dilution plate method Serial dilutions of the sample. Most representative value is Between 30 and 300. • Dilution in liquid culture – Reduces number of cells in each tube – Spread liquid on plate to see single colonies Optical techniques. Often, can estimate cell numbers accurately by measuring visible turbidity. Light scattered is proportional to number of cells. 1. Eyeball method. This is not a precise measurement, but should 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) 2. Counting Bacteria • Petroff-Hauser chamber – Counts cells directly – Gives accurate number – Can’t tell if cells are alive or dead • Use stain to distinguish living cells 3.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) 28 Continuous culture: Chemostat Microbial population density is controlled by the concentration of the nutrient in the reservoir. Microbial growth rate is controlled by the flow rate. Therefore, growth rate and growth yield can be controlled independently. This cannot be done in batch culture. Microbial populations can be Maintained in exponential phase. 1.Psychrophiles -- optimum temp. typically 15 deg C or lower. Note: some organisms are psychrotolerant -- optimum temperature is 20-40 deg, but can grow as low as 0 deg. These are not considered psychrophiles. 2.Mesophiles -- optima from 20-45 deg, minimum around 15-20 deg. 3.Thermophiles -- optima 55 deg or higher. Some (hyperthermophiles) have optima of 80 deg or higher (mostlyArchaea in this group). Found in hot springs, deep-sea hydrothermal vents, other locations. 33 Effects of oxygen on growth 1.Obligate aerobes -- grow only when oxygen is present 2.Facultative anaerobes -- grow with or without oxygen, grow better in oxygen (respire) 3.Aerotolerant anaerobes -- ignore oxygen, grow equally well with or without 4.Obligate anaerobes -- die in presence of oxygen 5.Microaerophiles -- won't grow at normal atmospheric oxygen (20%), but require some oxygen for growth (2-10%) Biofilms • most microbes grow attached to surfaces (sessile) rather than free floating (planktonic) • these attached microbes are members of complex, slime enclosed communities called a biofilm • biofilms are ubiquitous in nature in water • can be formed on any conditioned surface 36 Where to find biofilms • Biofilms are ubiquitous in nature in water – Seen as layers of slime on rocks or other objects, or at water-air interface • Biofilms also form on man-made objects exposed to water • Pipes, boats, submerged objects • Can form in the human body and on teeth – Concern for medical devices such as catheters, hip and knee replacements – Pseudomonas aeruginosa commonly forms a biofilm in the lungs of CF patients – Implicated in several medical and dental conditions, e.g. chronic wounds, kidney stones, periodontal disease P. Aeruginosa and aliginate (white) in sputum of CF patient. Cell to Cell Communication Within the Microbial Populations • bacterial cells in biofilms communicate in a density-dependent manner called quorum sensing • produce small proteins that increase in concentration as microbes replicate and convert a microbe to a competent state – DNA uptake occurs, bacteriocins are released Quorum Sensing • acylhomoserine lactone (AHL) is an autoinducer molecule produced by many gram-negative organisms – diffuses across plasma membrane – once inside the cell it induces expression of target genes that regulate a variety of functions – many microbes produce effect 39 40 Can you think of any other factors which may affect microbial growth?