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Chapter 6: Microbial Growth 1. Requirements for Growth 2. Culturing Microorganisms 3. Patterns of Microbial Growth 4. Measuring Microbial Growth 1. Requirements for Growth Chapter Reading – pp. 166-174 Factors that affect Microbial Growth Microbial growth depends on physical factors… • temperature • pH • osmotic pressure …and chemical factors • availability of a useable carbon source • useable sources of nitrogen, sulfur & phosphorus • availability of trace elemental nutrients (Fe, Mg…) • presence (or absence) of oxygen gas (O2) Temperature & Microbial Growth Growth rate Thermophiles -10 Mesophiles Hyperthermophiles Psychrophiles 0 10 20 30 40 50 60 70 Temperature (°C) 80 90 100 110 120 Microorganisms can be grouped based on the temperature range in which they can grow… • each has an optimal temp. & minimum, maximum growth temps. pH • most microorganisms grow best at pH levels near neutral (6.5-7.5) • few microorganisms grow at the more extreme pH levels (below 4.0, above 10.0) • microbial growth tends to acidify the growth medium, inhibiting further growth Osmotic Pressure Hypertonic solutions can draw water out of cells via osmosis: • causes membrane to detach from cell wall (plasmolysis) • caused by high salt, sugar… • inhibits bacterial growth Oxygen (O2) As we’ve learned, oxygen can promote growth (via respiration in aerobes) or inhibit growth (of obligate anaerobes). Why is oxygen so toxic to some organisms? • O2 can undergo changes resulting in the formation of very toxic reactive oxygen species (ROS): superoxide radical (most toxic!): singlet oxygen: O2- O2 (“energized” O2) hydroxyl radical: peroxide anion: OH O22- • aerobic organisms, unlike obligate anaerobes, have enzymes to eliminate these dangerous radicals e.g. superoxide dismutase (SOD), catalase, peroxidase Oxygen & Microbial Growth Oxygen concentration High Low Obligate aerobes Loosefitting cap Obligate anaerobes Facultative anaerobes Aerotolerant anaerobes • thioglycollate medium produces an O2 gradient • a given bacterial species will grow only in the regions it can tolerate (e.g., anaerobes at bottom) Chemical Factors for Growth Source of Carbon • autotrophs simply need access to CO2 to grow • heterotrophs require an organic carbon source • proteins, carbohydrates, lipids **The carbon source a given organism can use depends depends on its metabolic abilities (i.e., its enzymes!)** Trace Elemental Nutrients • all organisms need trace (small) amounts of many so-called “mineral” elements: iron (Fe), zinc (Zn), magnesium (Mg), calcium (Ca)… • most are essential cofactors for various enzymes Nitrogen, Sulfur & Phosphorus • all organisms need access to nitrogen, sulfur & phosphorus to make proteins, nucleic acids, vitamins • some organisms require organic sources of these elements, others are more flexible: • e.g., nitrogen fixers are unique in being able to obtain nitrogen from the atmosphere (N2), most other organisms need Nitrogen in other forms *One can effectively promote or inhibit the growth of a microorganism of interest (or concern) by controlling its physical & chemical environment!* Biofilms It is estimated that the majority of bacteria in nature live in biofilms, and that most bacterial diseases are due to bacteria in a biofilm. SO WHAT’S A BIOFILM? • a gelatinous extracellular matrix (ECM) consisting primarily of polysaccharides in the glycocalyces of the bacteria in the biofilm • forms on hard surfaces (rocks, teeth, prosthetics…) • multiple bacterial species live synergistically in a biofilm • when sufficient bacterial numbers are present, a signaling process called quorum sensing induces biofilm **bacteria in biofilm are MUCH harder to get rid of than isolated bacteria** Biofilm Formation Free-swimming 1 microbes are vulnerable to environmental stresses. Water flow Chemical structure of one type of quorum-sensing molecule Water channel Escaping microbes Bacteria Matrix Some microbes 2 land on a surface, such as a tooth, and attach. Quorum The cells begin 3 4 sensing producing an triggers cells to extracellular matrix change their and secrete quorumbiochemistry sensing molecules. and shape. New cells arrive, 5 possibly including new species, and water channels form in the biofilm. Some microbes 6 escape from the biofilm to resume a free-living existence and, perhaps, to form a new biofilm on another surface. 2. Culturing Microorganisms Chapter Reading – pp. 174-182 Culture Medium The culturing of microorganisms requires an appropriate growth medium: • material containing all nutrients required for the desired organism to grow • can be liquid or solid (i.e., solid agar) • must initially be sterile (i.e., no live organisms) • media can be sterilized by heat or by filtration • growth should only occur following inoculation of the medium with the desired organism Defined vs Complex Medium Defined medium has a precisely known chemical composition • used for assessing metabolic characteristics Complex medium is rich in nutrients though chemical composition is not known • used to sustain rapid growth Selective & Differential Media Selective media promote the growth of desired organism(s), suppress growth of others: • include something in the growth medium that desired organism can tolerate, most other organisms cannot (e.g., antibiotic, low pH, high salt) • use defined media that sustain growth of desired organism, not others (e.g., lactose as carbon source) On differential media, microorganisms can be distinguished based on appearance • e.g., contain substances that change color due to pH change, production of particular by-product A B Selective medium • compare A (non-selective) with B (selective) C D Differential medium • C illustrates differential growth • D is differential & selective Culturing Obligate Anaerobes • special chambers are used to remove and exclude any oxygen (O2) that would otherwise kill such organisms How to Obtain a Pure Culture “quadrant streak” or “streak plate” will yield isolated colonies if done correctly • inoculate an isolated colony (derived from a single original cell) into liquid medium to obtain a pure culture Plating Bacteria 2 basic methods: 1) mix 1 ml of culture with molten agar 1 2 (not too hot, ~45-50o C.) & pour in plate • colonies grow IN as well as ON agar • some cells may be harmed by higher temp. 2) spread small volume of culture (0.1 ml) on solid agar surface • best method! **Each colony starts with 1 CFU!** 3. Patterns of Microbial Growth Chapter Reading – pp. 182-185 Bacterial Growth • most bacteria divide by binary fission ( a few by budding) • increase in cell numbers is exponential 1 bacterium can become 1 billion in just 30 generations!!! Arithmetic vs Exponential Growth 70 70 arithmetic 60 60 Species A 40 30 20 10 Species B 50 Number of cells 50 Number of cells exponential • real living organisms reproduce exponentially 40 30 20 10 0 2 1 Time (hours) 0 2 1 Time (hours) Rates of Microbial Growth The rate of microbial growth depends on the generation time: • the time for a microbial cell to divide • depends on the type of microorganism • also depends on the growth medium ***can be as short as 20 minutes (E. coli) or >24 hr*** • a practical measure is the the time it takes a microbial population to double in size (doubling time) • i.e., when every cell divides once! Microbial Growth Patterns Microorganisms cannot undergo unlimited growth, eventually the chemical and physical environment in which they’re growing will no longer be able to sustain such numbers: • sources of carbon, nitrogen, etc, get used up • waste products accumulate, pH may change Therefore, microbial growth tends to follow a characteristic pattern: Lag phase > Log phase > Stationary phase > Death phase Phases of Microbial Growth log phase growth is “linear” (straight line) on a logarithmic plot Lag phase: cells adjust to medium before dividing Log phase: exponential growth (unrestricted) Stationary phase: growth = death (wastes, lack of nutrients) Death phase: poor environment results in death > growth 4. Measuring Microbial Growth Chapter Reading – pp. 185-190 How to Measure Microbial Growth? There are a number of methods used to count microorganisms and thus determine the growth rate. The method used depends on several things: • the organism being analyzed • how quickly one needs the result • the degree of accuracy needed • the nature of the sample being tested Counting by Serial Dilution 1 ml original culture 9 ml broth + 1 ml original culture 1.0 ml 1:10 dilution 0.1 ml of each 0.1 ml transferred to a plate 1.0 ml 1:100 dilution 1.0 ml 1:1000 dilution 0.1 ml 0.1 ml 1.0 ml 1:10,000 dilution 1:100,000 dilution 0.1 ml result takes ~24 hr Incubation period Too numerous to count (TNTC) TNTC * 65 colonies 6 colonies 0 colonies **ea colony starts w/1 CFU** Counting by Filtration Direct Microscopic Counts • place sample of culture “counting chamber” slide • count cells within grid and calculate the cell density • the volume covering each grid or square is known so the number of cells per unit volume is easily determined • gives immediate and relatively accurate results! Spectrophotometry One of the quickest, most convenient methods to determine cell density is with a spectrophotometer. • measures how much light is transmitted through a liquid culture sample • more light blocked = greater cell density (i.e., turbidity) • % transmittance can be used to calculate cell density **Less precise, but gives immediate results!** Key Terms for Chapter 6 • psychrophile, mesophile, thermophile, hyperthermophile • superoxide dismutase, catalase, peroxidase • defined, complex, selective & differential media • binary fission, exponential growth, generation time • Lag, Log, Stationary and Death phases • biofilm, extracellular matrix, quorum sensing • serial dilution, spectrophotometry Relevant Chapter Questions MC: 1-5, 7, 9-12, 15 FB: 1-7, 9-11