Cultivation of Microorganisms Part One PDF

General Microbiology 5) Cultivation of Microorganisms Part One Dr. Lutfi M. Bakar Spring 2024  Nutritional and Physical Requirements, and Enumeration of Microbial Populations Introduction : Bacteria are grown for many pur...

General Microbiology 5) Cultivation of Microorganisms Part One Dr. Lutfi M. Bakar Spring 2024  Nutritional and Physical Requirements, and Enumeration of Microbial Populations Introduction : Bacteria are grown for many purposes, including those of medicine, agriculture, industry and basic research. A wide variety of procedures and nutrient preparations are used to induce microorganisms to grow and reproduce. Different microbes require different environments and nutrients, called culture media. The selection of culture medium, atmospheric conditions and other features essential for isolation are determined by the pathogenic bacterium suspected. Microorganisms are cultivated in containers ranging from test tubes, flasks, and Petri-dishes to huge steel tanks. Appropriate conditions of moisture, pH, temperature, osmotic pressure, atmosphere and nutrients are required for bacterial growth. Nutritional requirements : As do all other living organisms, microorganisms require certain basic nutrients and physical factors for the sustenance of life. However, their particular requirement vary greatly. The main elements required for growth are carbon, hydrogen, oxygen, and nitrogen, with sulphur and phosphorus required in somewhat smaller amounts, and other elements such as sodium, potassium, magnesium, iron, zinc and manganese in considerably smaller amounts necessary for continued efficient performance of varied cellular activities.. The following list illustrates the nutritional diversity that exists among microbes: a- Carbon (energy) source : Carbon is the structural backbone of the organic compounds that make up a living cell. Based on their source of carbon bacteria can be classified as autotrophs or heterotrophs Autotrophs: require only carbon dioxide as a carbon source. An autotroph can synthesize organic molecules from inorganic nutrients. The autotrophic (lithotrophic) bacteria , on the other hand can synthesize all or nearly all of its essential organic materials from inorganic compounds such as carbon dioxide as the sole source of carbon together with ammonium and sulphate ions(i.e., these organisms can be cultivated in a medium consisting solely of inorganic compounds; specifically, they use inorganic carbon in the form of CO2). b-Nitrogen source: Nitrogen is needed for the synthesis of such molecules as amino acids, DNA, RNA and ATP. Depending on the organism, nitrogen, nitrates, ammonia, or organic nitrogen compounds may be used as a nitrogen source. c-Minerals: Are also essential for microbial nutrition. These include phosphorus (are essential for the formation of nucleic acids DNA and RNA and also for synthesis ATP, Phosphate ions are the primary source of phosphorus.), sulfur (Sulfur is needed to synthesize sulfur-containing amino acids and certain vitamins. d-Trace elements: Are elements required in very minute amounts, and like potassium, magnesium, calcium, and iron, they usually function as cofactors in enzyme reactions. They include sodium, zinc, copper,molybdenum, manganese, and cobalt ions. e-Growth factors: Such as amino acids, nucleotides(purines and pyrimidines) monosaccharides, lipids, vitamins and co-enzymes. Certain microorganisms are capable of synthesizing their own required vitamins; others must obtain them from their nutrient medium. The B-complex vitamins play a catalytic role within the cell either as components of coenzymes or as prosthetic groups. Consider the amino acids as an example, 20 amino acids must be provided for protein synthesis and several more for peptidoglycan synthesis in procaryotes. f- Water: All cells require water in the medium so that the low-molecular- weight nutrients can cross the cell membrane. Physical and chemical factors which influence growth: In addition to nutritional factors, growth of bacteria is influenced by genetic factors and by chemical, physical and other environmental factors. Three of the most important physical factors that influence the growth and survival of cells are temperature, hydrogen ion (pH) concentration, and the gaseous environment and also availability of moisture, and osmotic pressure. 1-Gaseous requirements(influence of oxygen and carbon dioxide): a) Obligate aerobes: are organisms that grow only in the presence of oxygen. b) Obligate anaerobes: are organisms that grow only in the absence of oxygen and, in fact, are often inhibited or killed by its presence. c) Facultative anaerobes: are organisms that grow with or without oxygen, but generally better with oxygen. d) Microaerophilic bacteria: are organisms that require a low concentration of oxygen (2% to 10%) for growth 2-Thermal Conditions (influence of temperature): Temperature influence the rate of chemical reactions through its action on cellular enzymes. In general, microorganisms have been shown to grow between the temperatures of about freezing and above 90cْ. The optimum growth temperature for enzymatic activities in all cell types is 20c to 40c. 3-Influence of Hydrogen Ion Concentration: The pH of the extracellular environment greatly affects cell's enzymatic activities. Microorganisms also have certain pH needs as reflected by their growth responses in various media. The scale of values representing this property of solutions commonly extends from 0 to 14. 4- Effect of Osmotic Pressure on Growth : Growth rate vs osmolarity for different classes of procaryotes. Osmolarity is determined by solute concentration in the environment. Osmosis is the diffusion of water across a membrane from an area of higher water concentration (lower solute concentration) to lower water concentration (higher solute concentration). 5-Influence of Moisture and of Desiccation: Four-fifths by weight of the bacterial cell consists of water and as in the case of other organisms, moisture is absolutely necessary for growth. Microbes vary widely in their ability to survive when dried under natural conditions. Thus, the gonococcus , Treponema pallidum and the common cold virus appear to die quickly, whereas the tubercle bacillus , Staph. aureus and the smallpox virus may survive for weeks or months. 6-Influence of Light and other Radiations: Darkness provides a favorable condition for growth and viability. Ultraviolet rays are rapidly bactericidal, which has a lower energy content than ionizing radiations. 7-Influence of Mechanical and Sonic stresses: A bacterial suspension may be largely disintegrated by subjection to very vigorous shaking with fine glass beads or to supersonic or ultrasonic vibration. These measures are used in separating the large molecular components of the cell. Bacterial Growth: Bacterial growth refers to an increase in bacterial numbers not an increase in the size of the individual cells. Bacteria replicate by binary fission, a process by which one bacterium splits into two. Therefore, bacteria increase their numbers by geometric progression whereby their population doubles every generation time.. Geometric progression refers to the population of bacteria doubling every generation time as a result of dividing by binary fission. The first step in division is cell elongation and duplication of the chromosomal DNA. The cell wall and cell membrane than begin to grow inward from all sides at a point between the two regions of the chromosomal DNA. Eventually the ingrown cell wall meet, and two individual cells are formed, each of which is essentially identical to the parent cell. Phases of Growth: Following inoculation of bacterial cells into fresh broth medium, and small samples are taken at regular intervals, a plotting of the data will yield a characteristic growth curve(exhibits lag, exponential and stationary phases, and a final decline phase) as shown on : - Lag Phase. Immediately after inoculation of the cells into fresh medium, the population remains temporarily unchanged. Although there is no apparent cell division occurring, the cells may be growing in volume or mass, synthesizing enzymes, proteins, RNA, etc., and increasing in metabolic activity. - Logarithmic ( exponential) phase: The exponential phase of growth is a pattern of balanced growth wherein all the cells are dividing regularly by binary fission, and are growing by geometric progression. The cells divide at a constant rate depending upon the composition of the growth medium and the conditions of incubation. The rate of exponential growth of a bacterial culture is expressed as generation time, also the doubling time of the bacterial population. - Stationary phase: During this period, the availability of essential nutrients becomes a limiting factor, and there is a balance between cell growth and division and cell death. Population growth is limited by one of three factors: 1. exhaustion of available nutrients; 2. accumulation of inhibitory metabolites or end products(accumulation of toxic waste products(organic acids) or harmful changes in pH and temperature may also play a role). 3. exhaustion of space, in this case called a lack of "biological space". The stationary phase, like the lag phase, is not necessarily a period of quiescence. Bacteria that produce secondary metabolites, such as antibiotics, do so during the stationary phase of the growth cycle (Secondary metabolites are defined as metabolites produced after the active stage of growth). Death phase or Logarithmic decline phase: If incubation continues after the population reaches stationary phase, a death phase follows, in which the viable cell population declines(during this phase, bacterial cells undergo lyses from naturally occurring autolytic enzymes, which reduces the number of viable cells in a geometrical progression that is the reverse of the one in the log growth phase). Bacterial Cultivation: Microscopic examination alone may not be sufficient to differentiate closely related species of bacteria such as Salmonella and E. coli. It is therefore important to grow bacteria and differentiate closely related species by their cultural characteristics, if possible. The cultivation of microorganisms may be necessary for any of the following reasons: - The isolation in pure culture and identification of organisms. - The determination of antibiotic sensitivities of pathogens isolated. - Sterility testing of products destined for human use ( food, water analyses, environmental control, antibiotic and vitamin assays). - Industrial testing and the preparation of biological products such as vaccines. - To preserve bacteria for long periods. - It should be cheap and easy to produce. Basically, all culture media are liquid, semisolid or solid. The culture media can be classified on the basis of physical state into:- 1- liquid media A liquid medium lacks a solidifying agent and is called a broth medium (such as nutrient broth and glucose broth) and are preferred for industrial applications and for automated diagnostic techniques. In this media the bacteria are free to move. Left to Right: - Pellicle on surface(thick, padlike growth on surface) - Sediment (concentration of growth at the bottom of broth) - Uniform Turbidity(finely dispersed growth throughout) - Control (uninoculated nutrient broth tube without growth). 2- Solid media : A solidifying substance or gelling agent is therefore added to the liquid medium without altering its nutritional content A completely solid medium requires an agar concentration of about 1.5% to 2%. When the bacteria grown on solid media (such as nutrient agar and blood agar) they multiply at the site of inoculation and from colonies. The appearance of these colonies is often typical of the species and assist in having bacteria in pure culture. 3- Semisolid media: Which contain a small amount of agar or gelatin in smaller proportions (0.5% or less) than found in solid media and are used for mainly the observation of motility and fermentation tests. Enriched media: Such as blood agar, serum agar, chocolate agar and glucose agar or broth are used for the growth of delicate bacteria that would not grow on the basal medium. - Blood Agar Techniques : Purpose: Blood agar is used both as an enriched medium for growing fastidious bacteria and as a differential medium. Exotoxins called hemolysins cause lysis of the red blood cells. The degree of the hemolysis is an especially useful tool for identification of many of the Gram positive cocci. Differential media: A differential medium is one that supports the growth of various species while providing an environment that makes it easier to distinguish among different organisms. - MacConkey Agar Techniques: MacConkey agar is a widely-used culture medium which is both selective AND differential. Selective media: Containing substances which will inhibit the growth of most organisms other than those for which the media was devised. - Methylene Blue Agar (EMB): This agar is a selective and differential medium used for isolation and differentiation among members of the Enterobacteriaceae. - Mannitol Salt Agar : Mannitol salt agar is both a selective and differential growth medium. It is used to differentiate pathogenic Staphylococcus species from nonpathogenic members of the genus Micrococcus. - Phenylethyl Alcohol Agar (PEA) : This selective medium is used with mixed bacterial cultures to isolate Gram positive bacteria from any Gram negative organisms that may be present. - Hektoen Enteric Agar : This medium is selective, with bile salts added to inhibit Gram positive oganisms. It also differentiates between Salmonella, Shigella, and other Gram negative enteric (gut) bacteria. Margins (smooth, wavy, lobate, Elevations : ( flat, raised, convex, drop- branching, ciliate, wooly). like, hilly, crateriform). Blood Agar Techniques: Purpose: Blood agar is used both as an enriched medium for growing fastidious bacteria and as a differential medium Principle: Hemolysins are grouped in three categories: 1. Beta hemolysins completely lyse the red blood cells and hemoglobin; this results in complete clearing around colonies. 2. Alpha hemolysis refers to the partial lysis of RBC's and hemoglobin and produces a greenish discoloration of the blood agar around the colonies. 3. No hemolysis, sometimes called gamma hemolysis results in no change of the medium. Beta-hemolysis Alpha-hemolysis Gamma-hemolysis Blood agar an enriched medium can be used to differentiate among many bacterial species by their ability to produce different types of haemolysis. (a) Used particularly to distinguish between species of Streptococcus (b) Distinguishes between types of hemolysins – (c) Streptococcus pyogenes produces beta hemolysins which cause clear halos around the colonies (d) Streptococcus pneumoniae produces alpha hemolysins which cause greenish halo around the colonies (e) Other species of Streptococcus produce no hemolysins which results in the absence of a halo MacConkey Agar Techniques: Purpose: MacConkey agar is a widely-used culture medium which is both selective AND differential. The medium is primarily used to differentiate between Gram negative bacteria while inhibiting the growth of most Gram positive bacteria. The medium also differentiates between lactose-fermenting coliforms and lactose nonfermenters, which include potential pathogens Principle: Addition to the nutrient agar base of bile salts and crystal violet will inhibit the growth of most Gram positive bacteria, making MacConkey agar selective. Lactose, a fermentable carbohydrate, and neutral red, a pH indicator, are added to differentiate the lactose positive coliforms from the potentially pathogenic lactose nonfermenters.  Salmonella typhimurium (Gram Negative) on MacConkey Agar: growth, Lactose Fermentation: negative (colorless colonies ) Above : E. coli and a non- Staphylococcus aureus lactose fermentation - (Gram Positive) on MacConkey agar inoculated with MacConkey Agar: no lactose-fermenting Escherichia growth.Fermentation coli (at left) and non-lactose- for S. aureus or other fermenting Proteus (at right). gram positive Lactose-fermenting bacteria organisms cannot be appear bright pink, while non- determined from this lactose-fermenting bacteria test since they do not appear colorless. This grow on MAC agar demonstrates the differential nature of MAC agar.  Enterobacter cloacae (Gram Negative) on Escherichia coli (Gram Negative) on MacConkey Agar: growth with pink colonies MacConkey Agar: growth, with pink colonies ; Lactose Fermenter (pink colonies) (Lactose Fermentation) Salmonella typhimurium (Gram Staphylococcus aureus (Gram Positive) on MacConkey Negative) on MacConkey Agar: growth, Agar: no growth.Fermentation for S. aureus or other Lactose Fermentation: negative gram positive organisms cannot be determined from this (colorless colonies ) test since they do not grow on MAC agar Esculin Techinques: Purpose :This test differentiates between Group D streps and other streps. Group D streps are positive for bile esculin hydrolysis Principle: In the presence of bile or bile salts, Group D streps will hydrolyze the esculin. The by-products of this hydrolysis reacts with the iron salts in the medium, causing the medium to blacken. Bile Esculin Agar - Enterococcus faecalis grows on bile agar and hydrolyzes esculin. Methylene Blue Agar (EMB) Purpose :This agar is a selective and differential medium used for isolation and differentiation among members of the Enterobacteriaceae. Eosin methylene blue agar (EMB) selects for Gram negative bacteria, and differentiates those which ferment lactose (the coliforms ) from the coliforms which do not ferment lactose. Principle: EMB agar contains methylene blue and eosin dyes to inhibit the growth of Gram positive bacteria. It also contains lactose. Small amounts of acid production result in a pink colored growth, while large amounts of acid cause the acid to precipitate on the colony, resulting in a characteristic greenish, metallic sheen. Organisms which do not ferment lactose will be colorless, taking on the color the medium. This medium has been widely used in the past to screen for coliforms in the water.  Thank you For Attention And Good bye