Chapter 6: Microbial Growth 1. Requirements for Growth 2. Culturing Microorganisms 3. Patterns of Microbial Growth
1. Requirements for Growth
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 (O 2 )
Temperature & Microbial Growth Microorganisms can be grouped based on the temperature range in which they can grow each has an optimal temp. & minimum, maximum growth temps.
Temperature is the easiest way to control Microbial Growth If you want microbial growth (e.g., in the lab), then you incubate them at the optimal temperature. If you don t (e.g., food), you can incubate at temperatures above the range of tolerance (cook!), and/or store at temperatures below the range of tolerance (freeze) or close to it (refrigerate).
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) 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 microbial growth tends to acidify the growth medium, inhibiting further growth
Oxygen (O 2 ) 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? O 2 is a very reactive molecule which can result in the formation of very destructive free radicals (molecules or atoms with an unpaired electron) e.g. superoxide (O 2- ), peroxide anion (O 2 2- ) aerobic organisms, unlike obligate anaerobes, have enzymes to eliminate these dangerous radicals e.g. superoxide dismutase (SOD), catalase, peroxidase
Oxygen & Microbial Growth in thioglycollate medium (binds O 2 ), the top is O 2 -rich, the middle O 2 -poor, & the bottom lacks O 2 a given bacterial species will grow only in the regions it can tolerate (e.g., anaerobes at bottom)
most are essential cofactors for various enzymes Chemical Factors for Growth Source of Carbon autotrophs simply need access to CO 2 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)
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 (N 2 ), 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!*
2. Culturing Microorganisms
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
Plating Bacteria 2 basic methods: 1) mix 1 ml of culture with molten agar (not too hot, ~45-50 o C.) & pour in plate 1 2 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 cell!**
How to Obtain a Pure Culture inoculate an isolated colony (derived from a single original cell) into liquid medium to obtain a pure culture streak as shown to obtain isolated colonies
3. Patterns of Microbial Growth
Bacterial Cell Division Most bacteria divide by binary fission ( a few by budding)
1 bacterium can become 1 billion in just 30 generations!!! Microbial Growth is Exponential Increase in numbers by the same factor (i.e., x 2) each generation also referred to as logarithmic growth differs from arithmetic growth, increase by the same amount each generation (e.g., + 2)
How Fast do Microorganisms Grow? 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 Lag phase: cells adjust to medium before dividing Log phase: exponential growth log phase growth is linear (straight line) on a logarithmic plot Stationary phase: growth = death (wastes, lack of nutrients) Death phase: poor environment results in death > growth
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 result takes ~24 hr * ea colony starts w/1 cell Dilute culture sample in series & plate each dilution: (# of colonies x dilution factor) / sample vol. = cell density count a plate with a practical # of colonies (~30-300*)
Counting by Filtration Specific volume of a test solution (e.g., a water sample) is filtered to trap microbes Grow filter on agar plate, # of colonies = # of microbes in test sample e.g., 200 colonies from 100 ml of filtered test sample = 200 cells/100 ml or 2/ml **Not all cells can grow on culture medium!
Direct Microscopic Counts place sample on counting chamber slide known volume covering each grid or square is used to calculate cell density gives immediate results!
Cell Density via Spectrophotometry One of the quickest, most convenient methods is to measure the optical density (OD) of a culture. a spectrophotometer is used to measure how much light is absorbed by a liquid culture sample more light blocked or absorbed = greater cell density (i.e., turbidity) Less precise, but gives immediate results! the absorbance or OD value is used to calculate cell density (e.g., 1 OD unit = 10 8 cells/ml)
Key Terms for Chapter 6 psychrophile, psychrotroph, mesophile, thermophile & hyperthermophile superoxide & peroxide ions superoxide dismutase, catalase, peroxidase defined, complex, selective & differential media logarithmic (exponential) growth, generation time Lag, Log, Stationary and Death phases serial dilution, spectrophotometry Relevant Chapter Questions rvw: 1-6, 9-13 MC: 1-8