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Bacterial Growth & Growth Conditions Bacteria grow only if their environment is suitable and assures optimum growth conditions; growth conditions which differ between different bacterial species have to be carefully examined to be able to allow bacterial growth in an artificial lab environment - it is not surprising that there are many different recipes of bacterial growth media and growth conditions listed for the many different bacterial species Generally, the growth of bacteria is dependent on the existence of water in their environment from which they absorb the dissolved growth-supporting nutrients and factors; optimum growth of bacteria requires that certain essential requirements for growth are full-filled Besides elementary nutrient requirements which includes the 10 macroelements C, O, H, N, S, P, K, Ca, Mg and Fe, bacteria also need so-called micro- or trace-elements, such as Mn, Mo, Zn, Co, Cu, Ni, V, B, Cl, Na, Se, Si and W, for optimum growth - the macro-elements are usually part of the nutrients which bacteria find in form of salts or carbon sources in their unique environments, while the trace elements are usually amply supplied as impurities of common slats and soil minerals Critical parameters for optimum bacterial growth are: 1. Supply of suitable and retrievable nutrients - the nutrients have to present in the environment and in a (usually dissolved) form in order to allow passive or active take up by the bacterial cell (--> see bacterial transport processes) 2. Presence of a source of carbon or other forms of energy - No life form exists which does not take up some form of energy to survive - Microorganisms which receive the primary energy from sunlight and perform photosynthesis require the gas carbon dioxide (CO2) as carbon source; these C-autotrophic organisms reduce CO2 to build up more complex carbon molecules, most importantly glucose and fructose (see Image below) - these organisms, e.g. algae, diatoms, and cycanobacteria, have to be irradiated

with light under supply of carbon dioxide in order to promote growth - A vast majority of microorganisms, the C-heterotrophs, retrieve their macronutrients and carbon for build-up of cell material from organic nutrients, e.g. cellulose, starch, sucrose, glucose, from other organisms - examples of this type of organisms are the protists, fungi, and most bacteria - A third type of microorganisms are chemotrophs; they retrieve their nutrients from simple organic compounds - The fourth type are the so called lithotrophs; these forms of life derive their energy from inorganic molecules, such as H2, NH3, H2S and S.

3. Existence of water - as mentioned earlier, bacterial growth is strictly dependent on

water, which is easily explained by the volume increase of the bacterial cell during binary fission and that water assures the dissolution of the nutrients necessary for bacterial growth 4. Existence of the appropriate temperature - microorganisms have quite different expectations regarding the temperature in their environment in order to show optimum growth - regarding the optimum temperature or temperature range, following types of bacteria have been classified: 1. Mesophilic bacteria - show maximum growth at temperatures between T = 20oC 42oC - most soil and water-prone bacteria - e.g. enterobacteriaceae (Klebsiella, E.coli) & Clostridia species 2. Thermotolerant bacteria - are organisms which are able to show growth up to 50oC - e.g. Methylococcus capsulatus 3. Thermophilic bacteria - are bacteria which show maximum growth at temperatures between T = 40oC - 70oC - many soil and hot spring bacteria - e.g. Bacillus stearothermophilus, Thermoactinomyces vulgaris, Clostridium thermocellum, Thermotoga elfii 4. Extreme thermophilic bacteria - are organisms which temperature optimum for growth is higher than 70oC - many of these species belong to the domain Archaea bacteria - they are very common in hot geyser springs, volcanic hot springs - e.g. Thermus aquaticus, Caldicellulosiraptor saccharolyticus, Sulfolobus solfataricus, diverse Bacillus and Clostridium species - some even show growth at temperatures higher than 80oC (Sulfolobus acidocaldarius) and even 100oC (Pyrodictium occultum, an anaerobic sulfurreducing bacterium)

5. Psychrophile (or kryophile) bacteria - are organisms which temperature optimum for growth is lower than 20oC - many of these species are marine organisms which includes luminescent bacteria and iron bacteria, e.g. Gallionella 5. Appropriate pH of the environment - the pH, which means the concentration of free protons (H+) in a given medium, plays an enormously important role in the optimum growth of bacteria - most microorganisms grow best when the pH is around 7 (= neutral pH), i.e. when the free proton concentration is 1 x 10-7 M - many bacteria which produce pH-lowering acids during metabolism, e.g. enterobacteria, are not acid-tolerant; they inhibit their own growth over time - many bacteria prefer to grow at higher, means more alkaline (or basic) pH values - prominent examples of alkalophile bacteria are: Nitrifying bacteria, Rhizobia bacteria, Actinomycetes - only some bacteria are able to grow at very low (= acidic) pH values; these so called acido-tolerant and acidophilic bacteria manage to grow at pH values around 5 - prominent examples of acid-tolerant bacteria are: Lactobacilli, Acetobacter, Sarcina ventriculi - a prominent example of an acidophilic bacterium is Thiobacillus; also many biomassdecaying soil fungi prefer acidic environments and grow best at pH 5 In order to avoid the acidification of the growth medium and eventual killing of the bacteria in the growth medium due to the toxic effects of the generated acids as metabolic end products, buffering substances, (or so called buffers) are usually added to the growth medium. Prominent buffers added to growth media are inorganic phosphates, e.g. K2HPO4/Na2PO4, or (in the case of stronger acid formation) calcium carbonate or sodium bicarbonate. Another important buffering system (which plays a crucial role in natural aqueous environments) is the carbon dioxide (CO2)-bicarbonate system which involves the huge amount of atmospheric CO2 (see equation below): CO2 CO2 CO2 + H2O H2CO3 H+ + HCO3(gaseous) (water) (Carbonic Acid) (Carbonate)

6. Optimum levels of oxygen (or the absence thereof) & Aeration - many organisms, the obligate aerobic forms of life, are dependent on molecular oxygen (O2), which has an important function as primary electron acceptor during aerobic respiration - during aerobic respiration oxygen is reduced to water (see cellular respiration section) - even though cells can derive oxygen from water and carbon dioxide, it is mostly the molecular oxygen contained in air or dissolved in water which cells take up via diffusible processes; since under normal STP conditions there is not a lot of oxygen gas dissolved in liquid growth media, and is quickly consumed by the growing bacteria in the deeper regions of the growth vessels (or bio-reactor), oxygen gas has to be continuously supplied to the medium in form of blown in air in a process called aeration, to allow continued growth of obligate anaerobic bacteria 1 liter H2O (at STP) only contains 6.2 ml (or 0.28 mmol) O2 --> this is only enough to oxidize about 8.3 mg glucose - regarding their dependence and requirement for molecular oxygen, three types of microorganisms can be classified: 1. Obligate aerobic organisms - are organisms which are strictly dependent on the presence of oxygen - they are only able to generate cellular energy (in form of ATP) via cellular respiration including some form of electron transport chains Pseudomonas sp. 2. Obligate anaerobic organisms - are organisms which are only able to metabolize and survive in environments that are characterized by the absence of oxygen (= anaerobic environments) - the presence of oxygen is toxic to these organisms, due to the lack of protective enzymes, e.g. catalase (CAT) and superoxide

dismutase (SOD) - includes many human and dental pathogens, such as: Porphyromonas gingivalis Bacteroides forsythus Clostridium perfringens 3. Facultative anaerobic organisms - these are organisms that are able to grow either in the presence or absence of molecular oxygen (O2) - two different types are characterized: a. aerotolerant lactic acid bacteria - are able to grow in the presence of oxygen but don't use it as terminal electron acceptor (no electron transport chain) - retrieve energy through fermentation (= substrate chain phosphorylation) b. enterobacteriaceae type - can switch between anaerobic (= fermentative) and aerobic respiration depending on the absence or presence of oxygen E.coli, Citrobacter, Salmonella, Enterobacter 7. Presence of sulfur (S) and nitrogen (N) - both chemical elements are essential for the build up of proteins, catalytic centers of enzymes, nucleotides and other molecules - sulfur and nitrogen exist in the cell primarily in reduced form as sulfhydryl (SH-) or amino-groups - most microorganisms are able to take up these two elements in their oxidized versions in form of sulfate and nitrate, which are then reduced by the cell - the most common form of bio-available nitrogen is in form of ammonium salts, e.g. NH4Cl, but some microorganisms require amino acids as nitrogen source - in the lab, the latter are supplied to bacteria in form of tryptone or peptone digests - an important sulfur-containing amino acid is cysteine - only some bacteria, the so called nitrogen fixers or diazotrophic bacteria, are able to directly reduce atmospheric (molecular) nitrogen (= N2) to ammonia and amino acids - they achieve this ecologically important feat with the help of a unique enzyme called nitrogenase

8. Presence of absence of carbon dioxide (CO2) - is especially important for the growth of auto/phototrophic microorganisms, which utilize this factor during carbon fixation for the build-up of glucose - some heterotrophic bacteria, e.g. parasitic bacteria growing in blood, tissues and the GI tract, also require carbon dioxide for optimum growth - CO2 it is supplied to the growth media usually in form of sodium bicarbonate (Na2CO3) or by growing the organism in a carbon dioxide atmosphere in a closed vessel 9. Presence of trace elements and supplemental factors - many bacteria only show optimum growth in the presence of ample or trace amounts of so-called trace elements, e.g. nickel, iron, copper, molybdenium, manganese, (many of which are co-factors of important metabolic enzymes) - most bacteria only will grow when they are supplied with amounts of so-called supplemental compounds (or supplines) or growth factors; supplines are molecules which bacteria need as their basic molecular constituents and which bacteria cannot build up by themselves; 3 main types of supplines are known: 1. Amino acids 2. Purines & Pyrimidines 3. Vitamins - many of them are co-substrates or prosthetic groups of enzymes - e.g. biotin, nicotinic acid, folic acid (see Table below)

In order to be able to grow a certain microorganism in a controlled lab environment, microbiologists prepare defined simple or complex synthetic growth media; the Table I below shows the recipe of such a bacterial growth medium which contains several macro- and micro-nutrients Table I: Recipe of a typical synthetic bacterial growth medium (NFb Medium) (this medium supports the growth of the nitrogen-fixing bacteria Herbaspirillum and Azospirillum) Malate..................... ....................... 5 g K2HPO4.......................................... 0.5 g MgSO4.7H2O.................................. 0.2 g NaCl........................... ..................... 0.1 g

CaCl2 x 7H2O................................. 1.0 g FeSO4.7H2O.................................. 0.1 g Bromothymol blue....... .................. 0.5% 0.2 M KOH................... .................. 2 ml Vitamin solution............................. 1 ml Micronutrient solution.................... 2 ml ________________________________ adjust pH to 6.5 ad 1 l Dissolved nutrients in a medium or natural environment, e.g. salts, sugars, are usually taken up by microorganisms into their cell(s) by 5 different transport mechanisms, which are: 1. Simple diffusion - spontaneous molecular transport along a concentration gradient - mostly gases, e.g. oxygen, carbon dioxide 2. Facilitated diffusion - a type of transport enabled with the help of carrier and antiporter proteins - energy-independent transport along a concentration gradient - e.g. mostly transport of sugars, acids and amino acids 3. Active transport - energy (ATP)-dependent transport enabled with the help of transport proteins - allows molecular transport against a concentration gradient 4. Endocytosis - energy (ATP)-dependent process for bulk transport of larger molecules and liquids - coordinated invaginations of the cell membrane and formation of transport vesicles - e.g. proteins, lipid droplets, whole cells 5. Siderophores - the iron needed as important co-factor in the cytochromes of the electron transport chains of microorganisms is often taken up by cell secreted unique low molecular weight molecules called siderophores (see Image below) - they are usually hydroxamates, or phenolates-catecholates - bacteria secrete siderophores when little free iron in the environment is available

- siderophores complex with free ferric iron and allows efficient uptake into the cell usually with the help of ABC transporter proteins Different siderophores

Bacteria can be grown on simple/synthetic or complex, liquid or solid nutrient media - a synthetic or defined nutrient media is a solution which is comprised of defined amounts chemical ingrediences and compounds - microbiologists try to find the minimum growth requirements for an identified microorganism and try to develop so-called minimum media - a minimum medium is a solution which contains just enough of the chemicals and compounds to allow good growth of the specific microorganism - many photolithotrophic autotrophic microorganisms, e.g. cyanobacteria, are able to grow on relatively simple, minimum media - often need only CO2 - given in form of carbonate - as carbon source and and an ammonium salt, e.g. NH4Cl, as a nitrogen source

- some heterotrophic bacteria, such as enterobacteria, can also easily grown on minimum media with glucose as carbon source - a nutrient solution which contains defined amounts of chemically not defined extracts, e.g. extracts from yeast, brewing malt, peptone, tryptone or meat, are referred to as complex media (see Table below) - they are often the choice or only option if the exact growth conditions and requirements of a given microorganism are not known - commonly used complex media in microbiology are: 1. Tryptic soy broth 2. Nutrient broth 3. Peptone yeast extract broth 4. Luria Bertani (LB) broth 5. MacConkey agar - e.g. for some microorganisms such as Leuconostoc mesenteroides and many extreme thermophilic bacteria, the growth media may contain more than 40 different chemical components! Table: Recipes of complex media

- if a solid medium is needed for surface cultivation and isolation of bacteria, liquid media are usually solidified by adding defined amounts - usually 1.0 - 2.0% - of a red algae-derived polysaccharide called agar-agar (or simply agar) to the media - the agar-containing broth or media is usually poured into a sterile plastic dish to create a typical agar plate for further use of bacterial streaking or spreading (see section below) - agar is a sulfated polymer comprised primarily of D-galactose, 3,6-anhydro-galactose and D-glucuronic acid; it hardens at temperatures below 40 42oC and does not melt again until the temperature rises to about 80 - 90oC - in order for certain heterotrophic bacteria to grow in or on the

surface of agar plates, a specific carbon source is added to the medium, for example, in order to select for bacteria capable of breaking down cellulose in order to retrieve the glucose monomers of the cellulose polysaccharide, cellulose is added to medium before pouring the plates; - bacteria capable of breaking down the cellulose in the agar plates with the help of special enzymes called cellulases, will form typical cleared zones (so-called "halos") on the agar plates - to select for fat-degrading bacteria on agar plates, certain triglycerides, such as tributyrine, are added to the medium; - bacteria which produce triglyceride degrading enzymes called lipases and secrete these into the surrounding medium, will form halos on the turbid agar plates after a certain incubation time (see Image below); - prominent lipase-positive and halo-forming bacteria are: Pseudomonas aeruginosa Staphylococcus aureus Bacillus subtilis - the bacteria Salmonella sp. and E.coli will grow on these plates but do not form halos since they do not secrete the lipase enzyme into the medium. Tributytrin Agar Plate (to test for bacterial lipase activity)

2. Isolation of Pure Bacterial Cultures In order to be able to adequately study and characterize a certain microorganism, microbiologists need to separate and isolate this microorganism from the many other microorganisms with which it usually shares its natural environment or habitat; proper and professional execution of bacterial isolation techniques are at the center of routine activities in any professional microbiology laboratory - bacterial isolation and development of pure bacterial cultures which was pioneered and developed by the great German microbiologist Robert Koch and his associates at the end of the 19th century - lead to the successful isolation and identification of many important human pathogens, such as Bacillus anthracis and Vibrio cholerae The task of the microbiologist is to isolate the microorganism under investigation from the mixed culture of the sample and to create a pure culture "A pure culture is defined as a population of cells which arose from a single cell by repeated cell division..."

Today, microbiologists routinely rely on following ways and techniques to establish pure microorganism cultures: 1. The spread plate technique (see Figure below) - a mixture of cells or of a cell culture is spread out on the Agar surface of a Petri dish with the help of a flame-sterilized glass rod-made cell spreader (see Image below) - after incubation in an incubator over a defined time period usually between 12 and 48 hours - the surface-dispersed cells grow into distinctive and visible cell clusters called colonies - because the number of colonies on the agar surface should equal the number of viable cells in the sample/cell suspension, this method can be used to: 1. count the bacterial population 2. isolate pure colonies from a mixed bacterial culture

2. The streak plate technique (see Figure below) - a flame-sterilized platinum loop is used to apply a loop-full of a bacterial suspension to the edge of a agar-medium-filled Petri dish and streaked out over the agar surface following a distinctive zick-zack pattern (see Figure below) - the bacterial cells which are streaked along the agar surface will grow into distinctive colonies after incubation in a humified incubator - this technique allows - like the spread plate method - the isolation of pure bacterial colonies

3. The pour plate method - an original bacterial or fungal cell suspension is diluted several time in the chosen growth media (= serial dilution) to reduce the cell density to a level which allows the separation of individual colonies after plating on agar-growth media-filled Petri dishes (see Image below) - after the serial dilution of the sample, small volumes of the diluted samples are mixed with liquid agar - after cooling of the agar to about 45oC, the mixtures are poured into a sterile Petri dish and are allowed to solidify - each cell fixed in place within the solidified agar plate will eventually grow into a defined colony which can be picked for further isolation and propagation of pure cultures The Pour Plate Technique

The culture dishes or Petri dishes used in all three microbiological plating methods are named after the German inventor Julius Richard Petri, an assistant of R. Koch, who - in 1887 developed and introduced the use of these practical round dishes for convenient and sterility-assuring routine culturing of microorganisms 3. Bacterial Shapes, Colony Morphology & Growth Bacteria come in a huge diversity of cell shapes and morphologies (see Images below), which to the vast extent explains their unique colony morphologies on culture dishes

Colony development of microorganisms, i.e. bacteria or fungi, on culture plates helps the microbiologist to identify and further study individual bacterial or fungal species - it often complements the use of a microscope and of biochemical and/or molecular biological tests Especially the unique size, color and shape (= morphology) of the microorganisms growing as a distinctive colony on the agar surface of the culture dishes is of tremendous analytical value and helps to identify the bacterial or fungal species Bacterial colonies growing on agar surface in defined growth media show a huge variety of different shapes and texture (see Image below)

- one has to keep in mind that these shapes and patterns vary with nutrient availability and hardness of the agar surface - e.g. the right panel of the image below shows the aesthetic snowflake and fractal-forming colonies of Bacillus subtilis it forms when growing on nutrient-poormedium - also, the most rapid cell growth usually occurs at the colony edge, while often cell autolysis and cell death happens in the colony center due to mal-supply of nutrients, gases, etc. Bacterial Colony Morphology

3. Bacterial Growth & Growth Curves One of the hallmark features of life is growth, i.e. the increase in cell mass and number over time; especially bacteria are known to have the fastest growth rates of biological organisms - microorganisms reproduce and increase the cell number by relatively rapid processes, most importantly via binary fission (bacteria)

or budding (e.g. yeasts) Growth is defined as the irreversible increase of living substance over time, mostly in form of body enlargement (in case of multicellular organisms) or in number (in case of single-celled organisms) The careful and scientific study of microorganism growth is an important part of microbiological lab routines; the growth of a population of microorganisms, e.g. a pure bacterial culture in growth medium, is usually studied by analyzing the growth curve of the microorganism under investigation - for this the microorganism is cultivated in its growth medium usually in as a batch culture - batch ( or closed) culture means, no new nutrients or supplements are added to the culture while it is growing - since under batch growth conditions no fresh medium is added to the cells, nutrient concentrations decline over time and concentrations of metabolic waste products, e.g. organic acids, increase - determination of the bacterial number or mass usually starts with a homogenous bacterial suspension (liquid culture) and the microbiologist determines the: 1. Bacterial concentration in cell count / ml or 2. Bacterial density in mg / ml A typical bacterial growth curve (see Figure below) under batch conditions shows 4 different phases, each with unique events: 1. Lag Phase - no immediate increase in cell number and mass occurs - cells adapt to new medium and factors - evtl. new enzymes and metabolic pathways are induced - temperature adaptation - length of lag phase dependent on different factors: e.g. age of cell culture, type of medium, temperature, presence or absence of important co-factors or supplements

2. Exponential or Log Phase - microorganisms are dividing and growing at the maximum possible rate - rate of growth is constant and cellular constituents, e.g. ribosomes, are manufactured at constant rates ("balanced growth") - bacteria are dividing and doubling in number at constant rates - the cell count N of a cell population with a number of N0 cells (at the beginning of the inoculation) and after n cell divisions can be calculated using following formula: N(n) = N0 2n Number of cell divisions n: n = lgN - lgN0 / lg2 Division rate or mean growth rate constant k: k = n / t = lgN - lgN0 / lg2 (t - t0) --> number of generations per unit of time Mean generation time or Doubling time g: g = 1/ k --> time it takes for a population to double in size --> can be graphically determined from a semi-logarithmic plot of the growth data (see right panel of Figure below) --> vary greatly amongst different microorganisms and in different growth environments, e.g. media - the chemical and physiological properties of the culture are uniform in this phase --> ergo: exponential phase cultures are used in biochemical and physiological studies! 3. Stationary Phase - population growth ceases and growth curve becomes horizontal - bacterial population density reaches maximum number: - usually 109 cells / ml - several factors account for this phenomenon (observed under batch growth conditions): 1. Carbon source (e.g. glucose, amino acids) becomes depleted ("Starvation effect") 2. Limitation of oxygen availability (--> aerobic bacteria)

3. Accumulation of toxic waste products, e.g. acids 4. Energy-consuming production of starvation proteins - e.g. increased peptidoglucan cross-linking - e.g. DNA-protecting proteins (Dps), chaperons, stress-response proteins 4. Death Phase - decline in number of viable cells due to build-up of toxic metabolic end products - cells loose ability to reproduce and die off; some produce endospores - death rate may be exponential Phases of a typical bacterial growth curve

Microorganisms differ greatly regarding their generation times (see Table below), an

observation which is helpful for identification of a newly isolated organism

4. Measurement of Bacterial Growth

Microbiologists rely on many different methods to determine bacterial numbers in a laboratory setting for final calculation of growth rates and generation times - no single method introduced below is always best or ideal; the final choice of the bacterial growth technique depends on the experimental question and situation Following bacterial growth measurement techniques are widely applied by microbiologists: 1. Petroff-Hauser Counting Chamber - microscopic determination method of bacterial numbers through direct counting - easy, inexpensive and quick; requires a compound microscope! - a special glass chamber (covered by a cover glass) and having an etched counting grid (see Image below) holds a small volume of bacterial suspension - the average number of bacteria in the chamber's 25 squares section of the grid are counted and the cell density is calculated by knowing the dimensions of the chamber as well as the dilution factor, applying the following procedure: 25 squares cover an area of: 1 mm2 the chamber's depth is: 0.02 mm counting chamber volume: 0.02 mm3 = 0.02 l = 2 x 10-5 ml Example: - you put a 1:100 diluted bacterial cell suspension into the chamber - you count 69 bacteria (= nc) within the 25 square section of the chamber What is your cell count given in number nB of bacteria per ml?? General Formula: nB = nc x 100 x 2 x 105 = ____ cells / ml For our example that calculates into: nB = 68 x 100 x 2 x 105 = 1.36 x 109 cells / ml - Disadvantage: difficult to discriminate between live and dead

bacteria; not possible to give a number for viable cells! The Petroff-Hauser Counting Chamber

2. Coulter Counter - sensitive electronic method suitable for measurement of cell count of larger microorganisms, e.g. algae, protozoa, yeast - requires rather expensive electronic equipment - based on the measurement of the electrical resistance of the microbial sample flowing through a narrow electrode-equipped orifice - extensively used in hospital environments and labs for measurement of red and white blood cell counts of patient blood samples Disadvantage: method cannot discriminate between live and dead cells

3. Plating Techniques - rather slow method in which a small sample of a microbial suspension is streaked or plated onto the solid surface of a suitable agar growth plate - the original number of viable microbes in the sample is determined by counting the numbers of developing microbial colonies on the agar surface after a defined incubation period (usually between 24 - 48 hours) - this method allows the determination of viable cell count, usually expressed as colony forming units (CFUs) --> only live bacteria/microbes in the sample will grow and form colonies! Disadvantage: rather slow method requires expensive materials and equipment, e.g. autoclave, media, plates only cultivable microorganisms can be counted with this method! 4. Membrane Filtration Procedure - microbial number determination method based on the use of special vacuum filtration equipment (see Image below), fine pore filtration membranes and subsequent counting of membrane filter-trapped microbes - the pore size of the filters commonly in use is usually 0.22 or 0.45 m - the material of choice for the filter membranes us usually modified cellulose, e.g. nitrocellulose, nylon or PVDF - the filter (after vacuum filtration of the sample) is placed on the solid surface of a suitable agar medium plate and incubated for a defined period of time in an incubator - a count of the visible colonies on the membrane surface gives the number of viable microorganisms in the sample at the time point of sample filtration The Membrane Filtration Procedure

- membrane filters can also be used for direct cell count by using colored or fluorescent cell-specific dyes (see Image below) - e.g. the black background of special polycarbonate membranes allows the use of fluorescent staining dyes to label the trapped bacteria on the membrane with the help of a epifluorescence microscope - commonly used fluorescent dyes are: Acridine orange --> orange or green glowing DNA stain DAPI --> DNA stain Disadvantage: method requires rather expensive equipment and dyes, such as the epifluorescence microscope and fluorescent dyes method cannot discriminate between viable and dead cells Microbial colonies on membrane filters (Membrane Filtration Method)

a = filter & growth on standard nutrient medium b = filter and growth on fecal coliform medium c = filter & enterobacter growth on m-Endo agar d = filter & yeast/molds growth on Wort agar 4. Optical Density (OD) or Turbidity/Microbial Mass Measurement Method - method which is based on the measurement of the increase in biological cell mass over time due to growth of a microbial population in a growth medium-filled vessel - rapid, sensitive technique which is based on the fact that microorganisms scatter light striking them; requires a spectrophotometer (see Figure below) - the degree of scattering and optical density (OD) is proportional to the cell biomass in the vessel - at a cell density of 107 bacteria/ml the medium appears turbid and cloudy - scattering is usually measured in form of light absorption of the cell broth at a

wavelength of 600-660nm - plotting of the measured OD at a constant wavelength (usually 600-660nm) over time allows the establishment of a typical growth curve Measurement of optical density of a bacterial culture --> allows growth curve establishment

5. Bacterial Cultivation Bacteria can be grown in their respective growth media in different ways; the most commonly applied bacterial culture techniques are the batch culture and the continuous flow (chemostat or turbidostat) culture I. Batch culture - closed system growth of bacteria; the nutrients of the medium is not renewed nor metabolic wastes removed - exponential growth last only for a few generations

- bacteria can only be maintained for a short period of time (usually less than 2-3 days) II. Continuous flow culture (CFC) - two different types of CFC are commonly applied: a. Chemostat cultures (see Figure below) - fresh sterile medium is continuously fed into the culture vessel at the same rate as old medium (plus cells) are removed from the reaction vessel - culture medium contains an essential element in limiting quantities - the rate of nutrient exchange is is expressed as dilution factor D D=f/V
D = dilution factor; f = flow rate (ml/h); V = vessel volume (in liter)

- the dilution rate D determines the microbial population density (OD) and the generation time - growth rate is determined by the rate by which the new medium is fed into the vessel - final cell density is determined by the amount of the limiting factor fed into the vessel over time A chemostat continuous flow culture system

b. Turbidostat cultures - culture vessel is equipped with a photocell which allows continuous measurement of the optical density or turbidity in the vessel - flow rate is adjusted in such as way that the OD within the vessel remains unchanged at a predetermined level - dilution rate rather varies - culture medium lacks a limiting nutrient - operates best at high dilution rates Advantage: - provides a constant supply of cells in exponential phase and growing at a constant rate - allows study of microbial growth at very low ("natural") nutrient levels - commonly used in food and industrial microbiology 6. Influence of environmental factors on bacterial growth The growth of bacteria is greatly affected and controlled by the physical and chemical nature of

their surrounding environment; bacteria show remarkable adaptive responses to even the most extreme and inhospitable environments Bacterial growth is strongly influenced by following factors: 1. Solutes and water chemistry & availability - including osmolarity, salinity - includes the osmotolerant and halophiles Effects of NaCl concentration on different bacteria

2. pH - includes the acidophiles, neutrophiles and alkalophiles The pH scale & pH-adapted microbial species

3. Temperature - includes the psychrophiles, psychrotrophs, mesophiles, thermophiles and hyperthermophiles Temperature ranges for different microbial life forms

4. Oxygen availability & concentration 5. Pressure 6. Radiation