Microbial Growth Conditions1. Macronutrients
2. Micronutrients
3. Growth factors
4. Environmental factors: temperature; pH; Oxygen etc.
Nutrients: Substances in the environment used by organisms for catabolism and anabolism.
1. Macronutrients: required in large amounts, including: carbon, oxygen, hydrogen, nitrogen, sulfur, phosphorus (Components of carbonhydrates, lipids, proteins, and mucleic acids ); potassium, calcium, magnesium and iron (cations and part of enzymes and cofactors).
2. Micronutrients: Microbes require very small amounts of other mineral elements, such as iron, copper, molybdenum, and zinc; these are referred to as trace elements. Most are essential for activity of certain enzymes, usually as cofactors.
Microorganisms require about ten elements in large quantities, because they are used to construct carbohydrates, lipids, proteins, and nucleic acids. Several other elements are needed in very small amounts and are parts of enzymes and cofactors.
Nutrient requirementsNutrient requirements
Amino acids are needed for protein synthesis,
purines and pyrimidines for nucleic acid synthesis.
Vitamins are small organic molecules that usually make up all or part enzyme cofactors, and only very small amounts are required for growth.
The Common Nutrient Requirements
Macroelements (macronutrients)• C, O, H, N, S, P, K, Ca, Mg, and Fe• required in relatively large amounts
Micronutrients (trace elements)• Mn, Zn, Co, Mo, Ni, and Cu• required in trace amounts• often supplied in water or in media components
Microbial NutritionMicrobial Nutrition
A.A. Nutrient RequirementsNutrient Requirements
B.B. Nutritional types of microorganisms Nutritional types of microorganisms (Nutritional categories)(Nutritional categories)
C.C. Nutrient Transport Processes Nutrient Transport Processes ((How do How do nutrient get into the microbial cell?)nutrient get into the microbial cell?)
D.D. Culture Media Culture Media (How to cultivate (How to cultivate microorganisms?)microorganisms?)
E.E. Isolation of Pure CulturesIsolation of Pure Cultures
Autotroph and Heterotroph
All organisms require Carbon, Hydrogen, and Oxygen. Carbon is needed for the backbone of all organic molecules.
In addition all organisms require a source of electrons. Electrons are involved in oxidation-reduction reactions in the cell, electron transport chains, and pumps that drive molecules against a concentration gradient on cell membranes.
Organic molecules that supply, carbon, hydrogen, and oxygen are reduced and donate electrons for biosynthesis.
Autotrophs
CO2 is used by many microorganisms as the source of Carbon.
Autotrophs have the capacity to reduce it , to form organic molecules.
Photosynthetic bacteria and microalgae are Photoautotrophs that are able to fix CO2 and use light as their energy source.
Heterotrophs
Organisms that use organic molecules as their source of carbon are Heterotrophs. The most common heterotrophs use organic compounds for both energy and their source of carbon.
Microorganisms are versatile in their ability to use diverse sources of carbon. Burkholderia cepacia can use over 100 different carbon compounds.
Methylotrophic bacteria utilize methanol, methane, and formic acid.
Requirements for Nitrogen, Phosphorus, and Sulfur
Needed for synthesis of important molecules (e.g., amino acids, nucleic acids)
Nitrogen supplied in numerous ways Phosphorus usually supplied as inorganic phosphate Sulfur usually supplied as sulfate via assimilatory
sulfate reduction
Requirements for Nitrogen
Nitrogen is required for the synthesis of amino acids that compose the structure of proteins, purines and pyrimidines the bases of both DNA and RNA, and for other derivative molecules such as glucosamine.
Many microorganisms can use the nitrogen directly from amino acids. The amino group ( NH2) is derived from ammonia through the action of enzymes such as glutamate dehydrogenase.
Most photoautotrophs and many nonphotosynthetic microorganisms reduce nitrate to ammonia and assimilate nitrogen through nitrate reduction. A variety of bacteria are involved in the nitrogen cycle such as Rhizobium which is able to use atmospheric nitrogen and convert it to ammonia. ( Found on the roots of legumes like soy beans and clover) These compounds are vital for the Nitrogen cycle and the incorporation of nitrogen into plants to make nitrogen comounds.
Sources of nitrogen
Organic nitrogenOrganic nitrogen• Primarily from the catabolism of amino acids
OxidizedOxidized forms of inorganicinorganic nitrogen• Nitrate (NO
32-) and nitrite (NO
2-)
Reduced inorganic Reduced inorganic nitrogen• Ammonium (NH
4+)
Dissolved nitrogen gas Dissolved nitrogen gas (N2) (Nitrogen
fixation)
Requirements for Phosphorous
Phosphorous is present in phospholipids( membranes), Nucleic acids( DNA and RNA), coenzymes, ATP, some proteins, and other key cellular components.
Inorganic phosphorous is derived from the environment in the form of phosphates. Some microbes such as E. coli can use organophosphates such as hexose – 6-phosphates .
Phosphate sourcePhosphate source• Organic phosphate
• Inorganic phosphate (H2PO4- and HPO4
2-)
Sulfur sourceSulfur source
• Organic sulfur
• Oxidized inorganic sulfur
• Sulfate (SO42-)
• Reduced inorganic sulfur
• Sulfide (S2- or H2S)
• Elemental sulfur (So)
Requirements for Sulfur
Synthesis of proteinSynthesis of protein Synthesis of vitaminsSynthesis of vitamins Involved in cellular respiration and phtosynthesisInvolved in cellular respiration and phtosynthesis
Growth Factors (Growth Factors (Special requirements)Special requirements)
organic compounds essential cell components (or their
precursors) that the cell cannot synthesize must be supplied by environment if cell is
to survive and reproduce
e.ge.g..•Amino acids•Nucleotide bases•Enzymatic cofactors or “vitamins”
Classes of growth factors amino acids
• needed for protein synthesis purines and pyrimidines
• needed for nucleic acid synthesis vitamins
• function as enzyme cofactors
Practical importance of growth factors Development of quantitative growth-response assays for
measuring concentrations of growth factors in a preparation.
Industrial production of growth factors by microorganisms.
Nutritional types of microorganisms Nutritional types of microorganisms (Nutritional categories)(Nutritional categories)
Energy SourceEnergy Source• PhototrophPhototroph (Uses light as an energy source)• ChemotrophChemotroph (Uses energy from the oxidation of
reduced chemical compounds)
Electron (Reduction potential) SourceElectron (Reduction potential) Source• OrganotrophOrganotroph (Uses reduced organic
compounds as a source for reduction potential)• LithotrophLithotroph (Uses reduced inorganic
compounds as a source for reduction potential)
• PrototrophPrototrophA species or genetic strain of microbe capable of growing on a minimal medium consisting a simple carbohydrate or CO2 carbon source, with inorganic sources of all other nutrient requirements
• AuxotrophAuxotrophA species or genetic strain requiring one or more complex organic nutrients (such as amino acids, nucleotide bases, or enzymatic cofactors) for growth
Chemical energy – source organic, Inorganic H/e- donor, Organic carbon source
• MixotrophyMixotrophy
Carbon sourceCarbon source• AutotrophAutotroph (Can use CO
2 as a sole carbon source)
• HeterotrophHeterotroph (Requires an organic carbon source; cannot use CO
2 as a carbon source)
Photolithotrophic autotrophs
Use light energy and have CO2 as their carbon source.
Cyanobacteria uses water as the electron donor and release oxygen
Purple and green sulfur bacteria use inorganic donors like hydrogen and hydrogen sulfide for electrons
Chemoorganotrophic heterotrophs
Use organic compounds as sources of energy,hydrogen, electrons and carbon
Pathogenic organisms fall under this category of nutrition
Photoorganoheterotrophs Common inhabitants of polluted streams. These bacteria
use organic matter as their electron donor and carbon source.
They use light as their source of energy Important ecological organisms
Chemolithotrophic autotrophs Autotrophs Oxidize reduce inorganic compounds such as iron,
nitrogen, or sulfur molecules Derive energy and electrons for biosynthesis Carbon dioxide is the carbon source
Uptake of Nutrients by the Cell
Some nutrients enter by passive(simple) diffusion Most nutrients enter by:
• facilitated diffusion• active transport• group translocation
1. Phagocytosis – Protozoa
2. Permeability absorption – Most microorganisms
C. Nutrient Transport Processes C. Nutrient Transport Processes How do nutrient get into the microbial cell?How do nutrient get into the microbial cell?
D. Culture MediaD. Culture MediaHow to cultivate microorganisms?How to cultivate microorganisms?
preparations devised to support the growth (reproduction) of microorganisms
can be liquid or solid
• Liquid medium• Components are dissolved in water and sterilized
• Semisolid medium• A medium to which has been added a gelling agent• Agar (most commonly used)• Gelatin• Silica gel (used when a non-organic gelling agent is
required)
• solid media are usually solidified with agar
important to study of microorganisms
Microbiological MediaMicrobiological Media Chemically defined vs. complex mediaChemically defined vs. complex media
• Chemically defined media• The exact chemical composition is known
• e.g. minimal media used in bacterial genetics experiments
• Complex media• Exact chemical composition is not known
• Often consist of plant or animal extracts, such as soybean meal, milk protein, etc.
• Include most routine laboratory media, e.g., tryptic soy broth
Synthetic or Defined Media
all components and their concentrations are known
Complex Media
contain some ingredients of unknown composition and/or concentration
Some media components
peptones• protein hydrolysates prepared by partial digestion of
various protein sources extracts
• aqueous extracts, usually of beef or yeast agar
• sulfated polysaccharide used to solidify liquid media
Types of Media
General purpose media• support the growth of many microorganisms
• e.g., tryptic soy agar
Enriched media• general purpose media supplemented by blood or other
special nutrients
• e.g., blood agar
Types of media…Types of media…
Selective media• Favor the growth of some microorganisms and
inhibit growth of others• MacConkey agar
• selects for gram-negative bacteria
• Inhibits the growth of gram-positive bacteria
Types of media…
Differential media• Distinguish between different groups of
microorganisms based on their biological characteristics
• Blood agar• hemolytic versus nonhemolytic bacteria
• MacConkey agar• lactose fermenters versus nonfermenters
Streak plate method• Developed in the 1870s by Koch and his co-workers
• The objective: to obtain isolated colonies – spots of microbial growth that come from a single parent cell
• The method: streak the sample on semisolid medium, containing a gelling agent
• Agar: the most commonly used gelling agent
E. Isolation of Pure CulturesE. Isolation of Pure Cultures
Spread plating & pour plating Limiting dilution
Pure Culture TechniquePure Culture Technique
The Spread Plate and Streak Plate
Involve spreading a mixture of cells on an agar surface so that individual cells are well separated from each other
Each cell can reproduce to form a separate colony (visible growth or cluster of microorganisms)
1. dispense cells ontomedium in petri dish
2. - 3. sterilize spreader
4. spread cellsacross surface
Spread-plate technique
inoculatingloop
Streak plate technique
Isolation of Pure Cultures
Pure culture• population of cells arising from a single cell
Spread plate, streak plate, and pour plate are techniques used to isolate pure cultures
The Pour Plate
Sample is diluted several times
Diluted samples are mixed with liquid agar
Mixture of cells and agar are poured into sterile culture dishes
Colony growth
Most rapid at edge of colony• oxygen and nutrients are more available at edge
Slowest at center of colony In nature, many microorganisms form
biofilms on surfaces
Terms
1. Colony shape and size: round, irregular, punctiform (tiny)2. Margin (edge): entire (smooth), undulate (wavy), lobate (lobed)3. Elevation: convex, umbonate, flat, raised4. Color: color or pigment, plus opaque, translucent, shiny or dull5. Texture: moist, mucoid, dry (or rough).
Preserving Bacterial Cultures
Deep-freezing: –50° to –95°C Lyophilization (freeze-drying): Frozen (–
54° to –72°C) and dehydrated in a vacuum
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