MCB 306 Notes PDF

MCB 306 BY DR. N.U NWOGWUGWU E- NOTES  TABLE OF CONTENTS 1. Cellular composition, macronutrients, micronutrients. 2. Nutritional types, growth factor/accessory nutrients prototrophs and auxotrophs. 3. Cultivation of microorganisms 4. Culture media; types and forms;...

MCB 306 BY DR. N.U NWOGWUGWU E- NOTES  TABLE OF CONTENTS 1. Cellular composition, macronutrients, micronutrients. 2. Nutritional types, growth factor/accessory nutrients prototrophs and auxotrophs. 3. Cultivation of microorganisms 4. Culture media; types and forms; culture vessel; liquid culture, culture on solid media ,tissue cultures. 5. Isolation of pure culture, pure culture techniques- spread, streak and pour plate methods. 6. Colony morphology description.  CELLULAR COMPONENTS All cells have a plasma membrane, ribosomes, cytoplasm and DNA, known as cellular components Plasma membrane surrounds and protects the cells Ribosomes consist of RNA and proteins Cytoplasm is a jelly-like substance that fills a cell DNA is the genetic material of the cell These cell structures are formed from the assemblage of the macromolecules ( e.g nucleic acids, proteins, lipids and polysaccharides)  CELLULAR COMPONENTS CONT’D The cellular components have functions such as: Provide structure for the cell Take in nutrients from the environment Convert nutrients into energy Contain genetic material to make copies of themselves The three main parts of the cell are: the cell membrane the nucleus(nucleolus, DNA and RNA) the cytoplasm  CELLULAR COMPOSITION Cell consists of macromolecules and water 95% macromolecules, proteins most abundant( 55% by dry weight) Four classes of macromolecules: Polysaccharides, Lipids, Nucleic acids and Proteins Macromolecules are polymers formed from repeating units of monomers. Chemical elements of life bond to form these monomers. E.g Glucose C6H12O6 is a monomer, consisting of three elements ; carbon, hydrogen and oxygen bonded together.  CELLULAR COMPOSITION CONT’D Composition of the major chemical elements of life are: Carbon 50% Nitrogen 14% Phosphorus 3% Hydrogen 8% Oxygen 20% Sulfur 1% Macromolecules ( e.g nucleic acids and proteins) formed from monomers( e.g nucleotides and amino acids) are assembled to give cell structures of microorganisms. These monomers or nutrients are required in certain amounts by cells for growth Macronutrients/ macroelements are required in large amounts Micronutrients/ microelements are required in lesser or trace amounts  Macronutrients-C,H,O,N,S,K,P,Na,Ca, Mg, Fe Micronutrients- Mn, Mo, Zn, Cu, Co, Ni, Vanadium,Bo, Cl, Se, Si, Tungsten Carbon –source: Organic compounds serve as Carbon sources CO2 serves as carbon source for autotrophs. Carbon is a major element in all classes of macromolecules. Nitrogen- source:- ( For proteins) In form of ammonia or nitrate Phosphorus- source: ( For nucleic acids and phospholipids) In form of phosphates Sulfur- source: ( In amino acids, vitamins, and coenzyme A) In form sulfates/ sulfides Potassium- Involved in enzyme activity especially in protein synthesis  Magnessium- Required for activities of many enzymes Iron- Plays a role in cellular respiration( a key component of iron- sulfur proteins involved in electron transport. Oxygen- source: In the form of water, CO2 and organic substances In relation to oxygen, three groups of microorganisms existing are: obligate aerobes, obligate anaerobes and facultative anaerobes. Hydrogen –source: Found in organic compounds, as C and O2.  Accessory nutrients/ Growth Factors: Essential cell components or precursors of such components required in small amounts but cannot be synthesized by the microorganism in question. E.g Vitamins, amino acids, purines and pyrimidines. Vitamins- Components of coenzymes or prosthetic groups, function catalytically and needed in small amounts to sustain growth. Vitamins mostly needed are Vit B1, B6 andB12 Lactic acid bacteria(LAB) require vitamins: Streptococcus, Leuconostoc and Lactobacillus species. Amino acids- Needed for protein synthesis Purines and pyrimidines- Needed for nucleic acid synthesis.  Prototrophs-Microorganisms that have the enzymes and biochemical pathways needed to synthesize all cell components using nutrients available. They are independent of accessory nutrients. Auxotrophs- Microorganisms that lack one or more of the enzymes needed to manufacture indispensable constituents. They must obtain these constituents or their precursors from the environment. They have accessory requirements.  Nutritional types of microrganisms are defined based on their energy sources,carbon sources and hydrogen donors or electron sources. Two metabolic types of microorganisms each are identified according to the mechanism of energy conversion; electron source and carbon source. Energy Chemotrophs(Organic/inorganic ) source Phototrophs(light) Electron source Organotrophs (Reduced organic compounds) Lithotrophs (Reduced inorganic molecules)  Carbon source Autotrophs( CO2) Heterotrophs(Organic compounds)  There are five nutritional types of microorganisms based on their primary sources of carbon, energy and electrons. 1) Photolithoautotrophy Carbon CO2 Energy Light Electron Inorganic e- donor Rep. Mco. Purple and green sulfur bacteria, cyanobacteria  2) Photoorganoheterotrophy Carbon Organic carbon/ CO2 Energy Light Electron Organic e- donor Rep Mco. Purple nonsulfur bacteria, green nonsulfur bacteria. 3) Chemolithoautotrophy Carbon CO2 Energy Inorganic chemicals Electron Inorganic e- donor Rep Mco. Sulfur-oxidizing bacteria, hydrogen –oxidizing bacteria, methanogens, nitrifying bacteria, iron-oxidizing bacteria.  4) Chemolithoheterotrophy Carbon Organic carbon/CO2 Energy Inorganic chemicals Electron Inorganic e- donor Rep Mco some sulfur oxidizing bacteria 5) Chemoorganoheterotrophy Carbon Organic carbon Energy Organic chemicals, same as C- source Electron Organic e- donor, same as C- source Rep Mco. Most nonphotosynthetic microbes; pathogens, fungi, protists and many archaea.  Culture vessels: Include plastic/ glass petri dishes, flasks, test tubes, slides etc. Culture media- Nutrient solutions used to grow microorganisms in the laboratory. They can be classified based on the following:  Chemical constituents  Physical nature  Function  Chemical constituents: Defined( synthetic) Complex  Physical nature: Solid, semisolid, liquid  Solid media has 1.5 -2.0% agar as the solidifying agent. Used for the enumeration of microorganisms from samples. For the culture of aerobic microorganisms. Egs Nutrient agar, Potato Dextrose agar etc. Semi solid media has 0.2-0.5% agar, soft, jelly-like. For motility studies of microorganisms. To cultivate microaerophilic bacteria, appear as a thick line. Egs Hugh and Leifson’s oxidation fermantation medium, Stuart’s and Amies media, Mannitol motility media. Liquid media/Broth has no solidifying agent. Grown in flasks on a shaker incubator( for uniform growth) or in a static incubator( to provide the organism with an oxygen gradient). Allow uniform growth of microorganisms and turbid growth at 37°C for 24 hours. Used for profuse growth of microorganisms and fermentation studies. Egs nutrient broth, tryptuc soy broth, phenol red carbohydrate broth, MR-VP broth.  Function: Supportive/ General purpose e.g Nutrient agar, tryptic soy broth and agar. Sustain growth of many microoganisms. Enriched e.g Blood agar for fastidious mcos as S.pyogens and N. gonorrhoeae. Special nutrients are added to encourage their growth. Selective e.g Endo agar, EMB, MacConkey agar. Contain compounds that selectively inhibit growth of some mcos., not others.  Differential e.g MacConkey( Selective and differential), Blood agar( Enriched and differential). Contains indicator ( typically a dye) that allows for differentiation of particular chemical reactions occuring during growth. For distinguishing between species of bacteria; some may carry out this reaction, while others do not, based on biological characteristics of the organisms. a) MacConkey has lactose and neutral red dye, so lactose fermenting mcos appear pink to red distinguishing nonlactose fermenters. b)Blood agar distinguishes between hemolytic and non hemolytic bacteria producing clear zones because of red blood cell destruction. E.g hemolytic Staphylococcus and Streptococcus isolated from throats.  Pure culture: A progeny ( clone) of a single cell. Microorganisms are studied adequately when separated from a mixed population or culture. Techniques:  Spread plate  Streak plate  Pour plate  Spread plate: A small volume of dilute microbial mixture containing around 30-300 colonies is transferred to the center of an agar plate It is spread evenly over the surface with a sterile bent glass rod. The dispersed cells develop into isolated colonies after incubation The number of colonies should equal the number of viable organisms in the sample. This spread plate technique is used to count microbial population.  Streak plate: The microbial mixture is transferred to the edge of an agar plate with a sterile inoculating loop or swab It is streaked out over the surface in one of several patterns. After the first sector is streaked, the inoculating loop is sterilized The inoculum for the second sector is obtained from the first sector, and so on. The cells will develop into separate colonies after incubation.  Pour plate: The original sample is diluted several times to reduce the microbial population sufficiently to obtain separate colonies. Small volume( 0.1 ml) of several diluted samples is mixed with liquid agar that has been cooled to about 45 degrees centigrade It is poured immediately into sterile petri dishes After the agar has hardened, each cell is fixed in place and forms individual colony. The total number of colonies equals number of viable microorganisms in the sample. Used to determine the number of cells in a population , like the spread plate technique.  Colony Morphology Description Colony morphology is a method that scientists use to describe the characteristics of an individual colony of bacteria growing on agar in a petri dish. Morphology of microorganisms is used in their identification. Types of morphology are: Gross and microscopic morphology Gross include: colony shape, size, and surface features Microscopic morphology assigns groups to bacteria such as cocci( round), bacilli(rods) or spirochetes( corkscrew).  Staining helps differentiate bacterial isolates during microscopic examinations e.g Gram staining. Detailed description of colony morphology is as follows:  a) Colony morphology:  i) Colony shape: circular, filamentous(fibrous), rhizoid( thick fibers), irregular.  ii) Colony height/ elevation: raised, flat, convex( rounded),umbonate(peaked) ,craterif orm(indented)  iii) Colony margin(Edge): entire(smooth), undulating( wavy), lobular( finger-like projections),filiform( fibrous projections), curled(swirled) iv) Surface refraction: smooth, dull, glistening(mucoid), rough( ground glass), rugose( wrinkled) V) Opacity and colour: transparent, translucent, opaque, iridescent, buff, red, blue or other color pigmented( dependent on culture media), hemolytic( clear regions surrounding colony on plates containing blood cells)  b) Cellular morphology i) shape: bacilli, cocci, spiral  iii) Inclusions and location: endospores( central, lateral and terminal) iv) Surface features: capsules, flagella.  TISSUE CULTURE Tissue culture is the technique of growing cells and tissues in an artificial medium separate from the organism. It is a method of biological research in which fragments of tissue from plants are transferred to an artificial environment in which they continue to survive and function. The cultures tissue may consist of a single cell , population of cells, or a whole or part of an organ. In tissue culture, fragments of plants are cultured in a laboratory. Growth can be in broth or agar. It is also known as micropropagation. Example of plants grown in tissue culture are oil palm,plantain,banana, date, eggplant, yam, cassava, sweet potato and tomatoes. It is mostly applied as a form of biotechnology in Africa.  Types of tissue culture include: seed culture, embryo culture, callus culture, organ culture, protoplast culture, pollen culture, anther culture, single cell culture, suspension culture, somatic embryogenesis. Applications are in plant science, forestry, biotechnology, horticulture. Hormones used are auxins and cytokinins Duration is 10-14 weeks The bacterium Agrobacterium tumefaciens can infect plant tissues and incorporate part of the DNA into the DNA of the host plant, hence DNA of interest in plants is transferred to a new plant tissue using the microorganism. Stages of micropropagation are initiation stage, multiplication stage, rooting or preplant and acclimatization.  Aseptic condition is essential in micro propagation to achieve success. The four stages: i) Initiation Stage: Piece of plant tissue (an explant) is cut from the plant; disinfested (removal of surface contaminants;) and placed on a medium. The medium contains mineral salts, sucrose, and agar. The objective of this stage is to achieve an aseptic culture, i.e without contaminaing bacteria or fungi.  ii) Multiplication Stage: A growing explant is induced to produce vegetative shoots by including a cytokinin in the medium. Cytokinin is a plant growth regulator that promotes shoot formation from growing plant cells. iii) Rooting or Preplant Stage: Growing shoots induced to produce adventitious roots by including an auxin in the medium. Auxins are plant growth regulators that promote root formation. iv) Acclimatization: A growing rooted shoot can be removed from tissue culture and placed in the soil.  Advantages  Plant cells obtained in a short time with small plant tissue  Disease-free new plants produced  Growth of plants throughout the year, irrespective of the season  Large space not required to grow plants  Speed in the development of new varieties in the market place  Used to produce ornamental plants  Produce plants with desired characteristics through genetic modification  Helps in the conservation of plant biodiversity by the production of endangered plants  The technique can quickly produce plants without tubers, seeds or bulbs  Plant tissues can rejuvenate rapidly, hence produce exact copies of itself known as clones.  Disadvantages:  Requires a standard laboratory set up , with tools and equipment  Greenhouse space is also needed for tissue culture of plants  Strict aseptic conditions are necessary to avoid contamination.  It can be laborious  A high level of expertise is required, as small errors may lead to collapse of the product.  It is costly  There is a chance that the propagated plants will be less resilient to diseases due to the environment they are grown in Recommended Texts Fig1. Description of colony THANK YOU