Heading: Topic Summary

Name: Amina I. Solaiman

Subject: Bio-225

Course: B.S. Biology-III Date Submitted: October 18, 2013 ______________________________________________________________________ Nutrition and Culture of Microorganisms Nutrition is a process by which chemical substances called nutrients are acquired from the environment and used in cellular activities such as metabolism and growth (Talaro & Chess, 2012). Metabolism is the general term for all the physical and chemical activities that a cell undergoes. Growth, in the context of microbiology, refers to an increase in cell number. To be able to study microorganisms in the laboratory one must prepare a culture medium and be able to know the growth or nutritional requirements of the microbe in study. Microbial Nutrition All living organisms, which include microorganisms, require nutrients for growth and normal functioning. The amount of which and its required form varies among the different groups of microorganisms. These nutrients that which are taken in by microorganisms may be classified according to the amount required by the certain microorganism (macronutrient or micronutrient), their chemical structure (inorganic or organic) and their importance to the microorganism (essential or nonessential) (Talaro & Chess, 2012) Those that are required to be taken in large amounts and are responsible for cell structure and metabolism are termed as macronutrients. Such are hydrogen, oxygen, carbon, nitrogen, phosphorus, sulfur, and selenium. While those that are required in small amounts and are involved in enzyme function and maintenance of protein structure are termed as micronutrients or trace elements. Such are manganese, zinc, nickel, etc. The micronutrient content of a microbial cell varies among microorganisms and oftentimes can be determined in the laboratory. If one wants to know, it may be feasible, provided that the substance in question must be removed from the growth medium of the microorganism, so as to know if the microorganism can grow even in its absence. Growth factors are like trace elements which are required in small amounts, but is needed to be assimilated from the environment and must be provided when the microorganism is to be cultured in the laboratory. To further know what makes up the nutritional needs of the microorganism, one must know its chemical composition (Talaro & Chess, 2012). Microorganisms may be classified as to their nutritional needs, such as energy source, electron source and carbon assimilation (Anthoniraj, Kannan, & Muthukaruppan, 2004). Microorganisms that oxidize chemical compounds as an energy

source are called chemotrophs while those that utilize radiant energy such as light are called phototrophs. Those that use reduced inorganic compounds as electron donors are called as litotrophs while those that use organic compounds are called organotrophs. Chemotrophs that use reduced inorganic compounds as electron donors are called as chemolitotrophs if they are phototrophs, photolitotrophs. Autotrophs are organisms that use inorganic CO2 as its carbon source while those that utilize preformed organic compounds are heterotrophs. Most heterotrophs are chemoheterotrophs, meaning, they obtain their carbon and energy source from organic compounds. Chemoheterotrophs may be classified into two categories: saprobic (which are free-living organisms that feed on organic detritus from dead organisms) and parasitic organisms (get their nutrition from cells or tissues of a host). Phototrophs that uses inorganic CO2 as its carbon source are called photoautotrophs while chemotrophs that do so are chemoautotrophs (Anthoniraj, Kannan, & Muthukaruppan, 2004). Methanogens are a group of chemoautotrophs that are commonly archaea (that live in an extreme environment with temperatures that reach up to 125C) which produce methane (CH4) from hydrogen gas and carbon dioxide. They play an important role in the recycling of organic nutrients. Culture Media A medium is an environment which supplies the ingredients necessary for the growth of the organism (Anthoniraj, Kannan, & Muthukaruppan, 2004). It is the variation of the needs of the microorganism that different kinds of media are prepared in the laboratory to study and isolate certain microorganisms. A culture media is also helpful to identify the treatment of a certain microorganism that is in study. Culture media can be classified by their physical state, chemical composition and functional types. Physical states of media include liquid, semisolid and solid media (Talaro & Chess, 2012). Liquid media are water-based solutions that do not solidify at temperatures above freezing and that tend to flow freely when the container is tilted. It caters the growth of microorganism that occurs throughout the container and presents a dispersed, cloudy, or flaky appearance. They are prepared by dissolving a certain amount of various solutes in distilled water. An example is a nutrient broth. Semisolid media contain a certain amount of solidifying agent or agar that attributes to its clothlike texture at room temperature and of being thick but does not really produce a firm substrate. Such media are used to determine the motility of a microorganism and localize a reaction at a specific site. Solid media caters microorganisms such as bacteria and fungi as they can form their colonies on the firm substrate that a solid media provides. It can be liquefiable (also known as reversible solid media, which is solid at room temperature and melts at boiling temperature of water. It has a lot of advantages than a nonliquefiable media which would include its flexibility that is attributed to it containing agar and its being not readily digested) and nonliquefiable ( a solid media that which do not melt). By their chemical composition, culture media can be synthetic or nonsynthetic. A synthetic media is that which its chemical composition is defined, that is they can only

be used when the exact nutritional needs of the microorganism to be cultured is known. A nonsynthetic media on the other hand, is that which its chemical composition is not define and presents a rich mixture of nutrients for microbes with complex nutritional needs (Talaro & Chess, 2012). As microorganisms are diverse, microbiologists finds a way to suit their needs, for them to be able to be isolated, studied and cultured in the laboratory. Thus culture media can be classified according to their function. They can be of general purpose, enriched, selective, differential, anaerobic growth, specimen transport and enumeration. (Talaro & Chess, 2012). A general purpose media caters the growth of a wide variety of microorganisms that do not require special growth requirements. An enriched media caters microorganisms that require certain organic compounds and growth factors for them to grow. A selective media contains components that inhibit the growth of microbes but not of a certain microbe. A differential media caters a wide group of microorganisms that bring out visible differences among those microorganisms. These differences include variations in colony size or color, in media color changes, or in the formation of gas bubbles and precipitates. Both the selective and differential media caters special microbial groups and have extensive applications in isolation and identification of microorganisms (Talaro & Chess, 2012). Laboratory Culture of Microorganisms In making laboratory culture of microorganisms, it is important that techniques such as sterile, aseptic and pure culture are to be learned by the one studying the microorganism. Since one cannot pin down wherever microorganisms can be, the best way so as to produce a pure culture (that will only cater the microorganism that the observer want to study), one must sterilize the equipments or tools that is to be used in the laboratory culture of that microorganism. Commonly all glasswares and other tools are being sterilize in the autoclave. Another concern is the possible release of infectious agents from cultures, which is prevented by aseptic techniques. Aseptic technique is a series of steps to prevent contamination during manipulations of cultures and sterile culture media (Clark, Madigan, Martinko, & Stahl, 2012). The aseptic transfer of a culture from one tube of medium to another is typically accomplished with an inoculating loop or needle that has previously been sterilized in a flame. Contamination is a constant problem, so sterile techniques (media, transfer equipment) help ensure that only microbes that came from the sample are present. Many of these techniques revolve around keeping species in the pure culture form for further study, identification, or biotechnology applications (Talaro & Chess, 2012). Microorganisms, as their name suggests are but very small organisms that they cannot be seen with the unaided eye. To study them in the laboratory, various tools are needed. The microscope which is deemed to be one of the most important tools for studying microorganisms is that which enable the observer to have a clearer and closer look of the microorganism; that its invention is a big milestone in studying microorganisms. The two key characteristics of a reliable microscope are magnification which is the ability to make objects appear enlarged, and resolving power which is the

ability to show detail. To observe microorganisms under the microscope, one must prepare a slide specimen and the manner in which it is prepared depends upon (1) the condition of the specimen, either in a living or preserved state; (2) the aims of the examiner, whether to observe overall structure, identify the microorganisms, or see movement; and (3) the type of microscopy available, whether it is bright-field, dark-field, phase-contrast, or fluorescence (Talaro & Chess, 2012). When the specimen is to be viewed in its living state, one is to prepare a wet mount, wherein one can observe natural state and motility of the specimen. When in its dead state, a fixed, stain smear is prepared and can be used for long term study. Smear technique which is developed by Robert Koch is done by spreading a thin film made from a liquid suspension of cells on a slide and air-drying it. Next, the air-dried smear is usually heated gently by a process called heat fixation that simultaneously kills the specimen and secures it to the slide (Talaro & Chess, 2012). The step that follows is to stain the slide, as unstained cells will not produce a distinct image under the microscope. As chemical composition of different microorganisms differ, so is the stain needed for them to produce a distinct image under microscope enough that the overall morphology is seen under the microscope. Two basic types of staining technique are used, depending upon how a dye reacts with the specimen. Most procedures involve a positive stain, in which the dye actually sticks to cells and gives them color. A negative stain, on the other hand, is just the reverse (Talaro & Chess, 2012). Positive stain can be simple, differential or structural stain. Simple stain uses only one dye and is not complicated when done. On the other hand, differential stain uses two dyes, the primary dye and the counter stain. Its procedure is complex and often requires other chemicals to provide a better visual of the microbial cell. Types of differential stain include gram stain, acid fast stain and endospore stain. Gram stain is an important diagnostic staining for bacteria, wherein it distinguishes between differences in the structure of a gram negative or positive cell wall. An acid fast stain is an important diagnostic stain that differentiates acid-fast bacteria (pink) from nonacid-fast bacteria (blue). An endospore stain is similar to the acid-fast method in that a dye is forced by heat into resistant survival cells called spores or endospores. These are formed in response to adverse conditions and are not reproductive. This stain is used to distinguish vegetative cells and spores. Structural stain is used when special cell parts such as capsules, spores and flagella are to be emphasized as these parts does not reveal when conventional staining methods are used. (Talaro & Chess, 2012) Furthermore, to study microorganisms, microbiologists have developed several types of procedures for investigating and characterizing microorganisms. These techniques can be summed up succinctly as the six Is: inoculation, incubation, isolation, inspection, information gathering, and identification, so-called because they all begin with the letter I (Talaro & Chess, 2012). In studying microorganisms in the laboratory, one can use these techniques, and the order in which these will depend on the purpose of the one studying the microorganism. Inoculation, which is usually the first step of culturing, is the process wherein a sample is introduced into a culture media so that they can grow and multiply. This process will also increase visibility. Incubation involves the process whereby the microorganism is exposed to optimal growth conditions for a few hours or days. In the isolation process, individual microbes are separated to isolate individual colonies, so as to have pure cultures that which will be

used for further study. Inspection is the process whereby growth and the cells (to be seen under the microscope) of the isolated microbe is observed. In this step, initial characteristics of microbes are known. Observing the microbes under the microscope, and staining them will reveal their cell type and morphology. Information gathering, as what the name suggests, is the process where testing of cultures are made with procedures that analyze biochemical and enzyme characteristics, immunologic reactions, drug sensitivity, and genetic makeup. With this, one will be able to generalize the profile of the microbe and the results obtained from this step will help in the identification step. In the last process, which is identification, the collected data will be used as a framework in determining the type of microbe present in the sample. In addition, this will help in the further study of the nature of the microbe and will in somehow contribute in various fields of sciences that will help the welfare of society. References: Anthoniraj, S.,Kannan, N. & Muthukaruppan, S. (2004). Microbiology. College road, Chennai: Government of Tamil Nadu. Talaro, K., & Chess, B. (2012). Foundations in Microbiology. New York: McGraw-Hill.

Bacterial Cell Division A dividing bacterial cell must, on average, have precisely twice as much of everything found in a newborn cell (Cooper, 2003). By studying the biosynthesis of cell components during the division cycle, one would know how a bacterial cell ensure that all cell components are duplicated between divisions, and how a cell ensure that it does not divide prior to duplicating all of its components. There is great practical interest in understanding bacterial cell division in great detail because such knowledge could lead to the development of new drugs that target specific steps in the growth of pathogenic bacteria (Clark, Madigan, Martinko, & Stahl, 2012). In this topic summary, cell growth, cell division and the synthesis of peptidoglycan will be discussed. Cell Growth Provided with the nutrients and adequate environmental conditions, microbes become metabolically active and grow (Talaro & Talaro, 2002). Growth, in microbiology means an increase in cell number. Cell growth occurs on two levels: 1) the cell increases in size and synthesizes new cell components and 2) cell number in a population increases. Bacterial cell growth depends upon a large number of cellular reactions of a wide variety of types (Clark, Madigan, Martinko, & Stahl, 2012). Yet, it is the polymerizations that make macromolecules from monomers are the key reactions for the synthesis of cells. Cell wall, cytoplasmic membrane, flagella, ribosomes, enzyme complexes and the like are the new structures from which these macromolecules which are accumulated in the cytoplasm are assembled into which would then eventually lead to cell division. Binary Fission and Cell Division Bacterial cell division occurs mainly through binary, or transverse, fission; binary means that one cell becomes two, and transverse refers to the division plane forming across the width of the cell. During binary fission, the parent cell enlarges, duplicates its chromosome, and forms a central transverse septum that divides the cell into two daughter cells (Talaro & Chess, 2012). Bacterial population increases, as this process is repeated at intervals by each new daughter cell. In a growing culture of a rod-shaped bacterium such as Escherichia coli, cells elongate to approximately twice their original length and then form a partition that constricts the cell into two daughter cells (Clark, Madigan, Martinko, & Stahl, 2012). Septum is the termed used for this kind of partition that result from the inward growth of the cytoplasmic membrane and cell wall from opposing directions. The formation of the septum continues until separation of the two daughter cells happens. As when a mother cell produces two daughter cells, a generation is said to have occurred and the time for one generation to be produced is termed as generation time. Generation time for the different microorganisms varies. In the process, all cellular constituents increase proportionally and are said to be in balanced growth. Each daughter cell receives a chromosome and sufficient copies of ribosomes and all other macromolecular

complexes, monomers, and inorganic ions to exist as an independent cell (Clark, Madigan, Martinko, & Stahl, 2012). The DNA remaining attached to the cytoplasmic membrane during division will dictate the partitioning of the DNA between the two daughter cells with constriction leading to separation of the chromosomes, one to each daughter cell. Fts (filamentous temperature sensitive) proteins, is a series of proteins that is present in all bacteria and is an essential component for cell division to occur. It is at least a key and universal cell division protein which has been revealed in recent genomic studies. Filamentous temperature sensitive describes the properties of cells that have mutations in the genes that encode Fts proteins; these cells form long filamentous cell that do not divide. A divisome is a cell division apparatus that is formed from the interaction fts proteins (Clark, Madigan, Martinko, & Stahl, 2012). The attachment of molecules of FtsZ in a ring around the center of the cell starts the formation of a divisome in rod-shaped cells. This ring prescribes what will eventually become the cell-division plane (Clark, Madigan, Martinko, & Stahl, 2012). Ftsl is an fts protein that which is also present in a divisome and is essential in the synthesis of the peptidoglycan. The new cytoplasmic material and the cell wall which is called the division septum are synthesized by the divisome at the center of the rod-shaped cell until it becomes twice its original length. After which, the two daughter cells is generated from the division of the elongated cell. Before the FtsZ ring forms, DNA already replicates. In the process of duplicating the nucleoids, they effectively block the formation of the FtsZ ring and so the ring forms in the space between the duplicated nucleoids. Min proteins are a series of proteins that facilitate FtsZ to locate the cells actual midpoint, which includes MinC, MinD, MinE. Wherein MinD oscillates moving back and forth from pole to pole and that which forms a spiral structure on the inner surface of the cytoplasmic membrane; it is also required to localize MinC to the cytoplasmic membrane. Ftsz ring is prevented from forming and thus cell division is inhibited when MinC and MinD work together. While MinE sweeps away MinC and MinD as it also oscillates from pole to pole. Consequently, Min proteins ensure that the divisome is not produced elsewhere in the cell but the in the center of the cell (as MinC and MinD is concentrated on the poles and there are no Min proteins that are concentrated on the center of the cell thus allowing for the ring to form). And so, two copies of chromosome are pulled apart, each to a daughter cell as elongation and septum formation begins. Ftsk protein which is an Fts protein and other proteins, facilitate this process. The sealing off of one of the daughter cell from the other and the inward growth of the wall materials to form the septum is triggered by the FtsZ ring that begins to depolymerize as the cell constricts. Guanosine triphosphate (GTP) which is an energy-rich compound is hydrolyze by the enzymatic activity of the FtsZ to yield the energy necessary to fuel the polymerization and depolymerization of the FtsZ ring. In the cell division cycle, there are three categories of molecules (which is synthesized with a unique pattern) that which is needed to be considered to be able to describe the biosynthesis of a cell. The growth pattern of the cell is the sum of these three biosynthetic patterns: 1) cytoplasm is the material enclosed within the cell surface

that is not the genome (which comprises proteins, RNA molecules, ribosomes, small molecules, water, and ions that make up the bulk of the bacterial cell) 2) the genome, which is a one-dimensional linear DNA structure and 3) cell surface which encloses the cytoplasm and the genome and that which is composed of peptidoglycan, membranes, and membrane-associated proteins and polysaccharides (Cooper, 2003). The growth pattern of the cytoplasm is based on the synthesis of its individual components. All parts of the cytoplasm accumulate exponentially during the division cycle, and this pattern is unchanged even when the cell is dividing. The combined rate of the cytoplasm synthesis of the two new daughter cells is equal to that of the mother cell at the instant of division. The cytoplasm increases uneventfully and exponentially during the division cycle. The DNA synthesis pattern during the bacterial division cycle (specifically for E. coli and related bacteria) is composed of one or more periods of constant rates of DNA accumulation (Cooper, 2003). There is a constant rate of DNA synthesis for the first 40 min, with one bidirectional pair of replication forks, followed by a zero rate of synthesis during the last 20 min for cells with a 60 min. interdivision time. For cells, with a 30 min. interdivision time, there is a constant rate of DNA synthesis for the first 10 min, which falls to a constant rate that is two-thirds of the initial rate for the last 20 min. While for cells, with a 20 min. interdivision time, there is a constant rate of DNA synthesis throughout the division cycle. The rate of DNA synthesis during the division cycle is proportional to the extant number of replication points and so the rate of DNA synthesis during any period in the division cycle is constant. As for the cell surface, it grows in response to an increase in the peptidoglycan. The bacterial peptidoglycan grows in the same way as the area of the membrane grows. As the cytoplasm grows exponentially during the division cycle, it is not the case for the synthesis of the cell surface. Cell surface does not grow exponentially because if so, cell volume (cytoplasm and its components) cannot increase exponentially. There are only four discrete events that occur during the division cycle of a growing rod-shaped bacterial cell. They are (1) the initiation of DNA replication, (2) the termination of DNA replication, (3) the initiation of pole formation, and (4) the completion of pole formation (Cooper, 2003)

Peptidoglycan Synthesis Peptidoglycan is a network of polysaccharide chains cross-linked by short peptides that forms the rigid part of bacterial cell walls (Talaro & Talaro, 2002). Beginning at the FtsZ ring small gaps in the wall are made by enzymes called autolysins, enzymes that function like lysozyme to hydrolyze the -1,4 glycosidic bonds that connect N-acetylglucosamine and N-acetylmuramic acid in the peptidoglycan backbone (Clark, Madigan, Martinko, & Stahl, 2012). Across the gaps, the new cell wall material is then added. A wall band is the junction between new and old peptidoglycan forms a ridge on the cell surface of gram-positive bacteria which is analogous to a scar. This new cell wall precursor (N-acetylmuramic acid/N-acetylglucosamine/tetrapeptide units,) that is to be spliced into existing peptidoglycan in a coordinated and consistent manner so as to prevent a breach in peptidoglycan integrity at the splice point is essential in the synthesis of peptidoglycan. Autolysis, a sponataneous cell lysis could result from a breach in the peptidoglycan integrity at the splice point. The controlled

cutting of preexisting peptidoglycan by autolysins together with the simultaneous insertion of peptidoglycan precursors is required in the synthesis of peptidoglycan during growth. Bactoprenol (hydrophobic C55 alcohol) which is a lipid carrier molecule plays an important part in this process, wherein it binds to the N-acetylmuramic acid/Nacetylglucosamine/pentapeptide peptidoglycan precursor. The transport of peptidoglycan precursors across the cytoplasmic membrane is facilitated by bactoprenol by means of making them hydrophobic, enough that will suffice to let them pass through the membrane interior. Bactoprenol will then interact with the enzymes transglycosylases in the periplasm so as that the insertion of the new cell wall precursor in the growing point of the cell wall and the catalysis of the glycosidic bonds formation will proceed. Transpeptidation which is the last step in the synthesis of the cell wall, involves the formation of the peptide cross-links between muramic acid residues in adjacent glycan chains. During the transpeptidation reaction (which is an exergonic reaction , supplies the energy necessary to drive the reaction forward as transpeptidation occurs outside the cytoplasmic membrane where no ATP is available), one of the D-alanine molecule which are the residues of the peptidoglycan precursor is removed and the other remains in the final molecule. In E. coli, the protein FtsI a separate transpeptidase enzyme cross-links peptidoglycan elsewhere in the growing cell. In gram-positive bacteria, where a glycine interbridge is common, cross-links occur across the interbridge, typically from an L-lysine of one peptide to a D-alanine on the other (Clark, Madigan, Martinko, & Stahl, 2012). References: Clark, D. P., Madigan, M. T., Martinko, J. M., & Stahl, D. A. (2012). Biology of Microorganisms (13th ed.). San Francisco: Pearson Education, Inc. Cooper, S. (2003). Bacterial Growth and Division. 28. Talaro, & Talaro. (2002). Foundations in Microbiology. New York: McGraw-Hill. Talaro, K., & Chess, B. (2012). Foundations in Microbiology. New York: McGraw-Hill.

Growth of Bacterial Populations The basis of the growth of bacterial populations is binary fission, a process by which bacterial cell divides. In this process, the parent cell enlarges, duplicates its chromosome, and forms a central transverse septum that divides the cell into two daughter cells (Talaro & Chess, 2012). At intervals, this will be repeated by each new daughter cell and with each successive round of division; the population increases. Because of the numerous factors present in the habitat of microorganisms that prevent microbial cells to divide at their maximum rate, bacterial populations do not increase endlessly. Quantitative laboratory (which employs the use of batch culture method, where it is a closed system with finite nutrients and space (Talaro & Talaro, 2002), studies indicate that a population typically displays a predictable pattern, or growth curve, over time (Talaro & Talaro, 2002). The study of the growth curve of bacterial populations is an essential part of microbiology, and it also contributes to a lot of other fields of microbiology such as microbial control, infection, food microbiology, and culture technology. (Talaro & Chess, 2012). This growth curve represents the entire growth cycle of the bacterial population. Growth Cycle Growth cycle of bacterial populations include the lag phase, exponential (logarithim) phase, stationary phase and death phase. All these phases are only applicable to populations of cells and are not suitable for individual cells. Lag phase is the period when the bacterial population appears not to be growing or is growing less than its exponential rate. The following are the primary reasons why growth lags: 1)the newly inoculated cells require a period of adjustment, enlargement, and synthesis of DNA, enzymes, and ribosomes; 2) the cells are not yet multiplying at their maximum rate; and 3) The population of cells is so sparse or dilute that the sampling misses them (Talaro & Chess, 2012). The duration of this phase differs from one population to another, and will also depend on the condition of the microbes and the medium. If the microbial culture is already exponentially growing, and is transferred into the same medium, given the same conditions of growth (temperature, aeration, etc.), exponential phase begins automatically and there would be no more lag phase. In the event, that the inoculum is taken from a culture that is already old (as in its stationary phase), into the same medium, lag phase will occur. It is because the essential constituents are already used up and time is required for microbial cells to biosynthesize. When the inoculum has cells that have been damage by significant temperature shifts, radiation, or toxic chemicals lag phase occurs because of the time required for the cells to repair the damage (Clark, Madigan, Martinko, & Stahl, 2012). Exponential (logarithim) phase is the period when the rate of cell division is at its maximum, wherein each cell divides to form two cells, each of which also divides to form two more cells, and so on, for a brief or extended period, depending on the availability of nutrients and a favorable environment (Clark, Madigan, Martinko, & Stahl, 2012). Microbial cells in this phase are the ones suitable for studies in the laboratory (of their enzymes and cell components) as they are in their healthiest state. In this phase,

the population fulfills its potential generation time, and growth is balanced and genetically coordinated (Talaro & Chess, 2012). As the length of lag phase or the first stage of growth of microbes differ from one another, so as the rate of their exponential growth. It is influenced by a number of factors including environmental conditions (temperature, composition of the culture medium), as well as by genetic characteristics of the organism itself. Prokaryotes grow faster than eukaryotes. The concept of surfaceto-volume ratio applies here as to the rate of how microbes perform in the exponential phase of their growth. Like for example, smaller eukaryotes grow faster than larger ones. It is due to the increased capacity in nutrient and waste exchange of smaller cells as compared to larger ones, that they grow at a faster rate. Stationary phase is the period when there is no net increase or decrease in microbial cell number. This is the time when exponential growth stops. It is because of several factors which includes an essential nutrient of the culture medium being used up, dropping of oxygen levels, accumulation of a waste product of the organism in the medium and an increased in cell density causes an accumulation of organic acids and other toxic biochemical that will eventually inhibit cell growth (Clark, Madigan, Martinko, & Stahl, 2012). Though the growth rate of the population in this phase is zero, many cell functions (e.g. energy metabolism and biosynthetic process) may still continue. Microbial cells may even divide, but because other cells die out, there is no net increase in cell number. This event where cell growth balances cell death is termed as cryptic growth (Clark, Madigan, Martinko, & Stahl, 2012) As the limiting factors for growth (such as depletion of essential nutrients and oxygen, toxic wastes which populates the environment) intensifies, cells will begin to die. Commonly, the rate of cell death is slower as compared to cell growth in the exponential phase. As lag phase, and exponential phase differs among various groups of microorganism, death rate also varies among bacterial populations. This would depend on the relative resistance of the microbial cell and so are the conditions of the environment that they are in. Death phase may be accelerated by antimicrobial agents, though it is in the exponential phase where these microbial cell are most vulnerable compared to those already in the stationary phase. Though cells are incubated after they have reached the stationary phase, microbial cells could still remain alive, but will eventually die. Most of the time, death of microbial cells is accomplished by actual cell lysis. Growth patterns in microorganisms can account for the stages of microbial infection (Talaro & Talaro, 2002). In the early and middle stages of infection, is the infection more likely to be spread to others or contaminate others than if the infection is at its later stages. The rate of multiplication is a cause of the course of infection a person is handling, if the rate is too fast, it may overwhelm the slower growth rate of the hosts own cellular defenses and thus will be a serious case for the patient (Talaro & Chess, 2012). Being able to comprehend the growth curve of bacterial populations is a handy and a useful tool when handling laboratory cultures. In early stages of growth, it is recommended that an observer must do stains, (with the exception of a spore stain)

and motility tests, as the cells will be able to show their natural size and correct reaction, and motile cells will have functioning flagella (Talaro & Chess, 2012). References: Clark, D. P., Madigan, M. T., Martinko, J. M., & Stahl, D. A. (2012). Biology of Microorganisms (13th ed.). San Francisco: Pearson Education, Inc. Talaro, & Talaro. (2002). Foundations in Microbiology. New York: McGraw-Hill. Talaro, K., & Chess, B. (2012). Foundations in Microbiology. New York: McGraw-Hill.

Measuring Microbial Growth To measure microbial growth, one must track the changes in the number of cells or changes in the level of cellular components (such as proteins, nucleic acids and the dry weight of the cell itself) (Clark, Madigan, Martinko, & Stahl, 2012). The two most common measures of microbial growth are total and viable count and turbidity which will be discussed in this topic summary. Total and Viable Count Total count is the process wherein one counts the number of cells present in a culture or natural sample under the microscope (Clark, Madigan, Martinko, & Stahl, 2012). This method, though simple, is unreliable. The following are several limitations of microscopic cell counting: (1) without special staining techniques dead cells cannot be distinguished from live cells. (2) Small cells are difficult to see under the microscope, and some cells are inevitably missed. (3) Precision is difficult to achieve. (4) A phasecontrast microscope is required if the sample is not stained. (5) Cell suspensions of low density (less than about 106 cells/milliliter) have few if any bacteria in the microscope field unless a sample is first concentrated and resuspended in a small volume. (6) Motile cells must be immobilized before counting and (7) Debris in the sample may be mistaken for microbial cells (Clark, Madigan, Martinko, & Stahl, 2012). Microbial cells can be microscopically counted either by drying the sample or placing samples in liquid. In dried samples, microbial cells are fixed on the glass slide and certain stains are added so as to increase contrast and one can properly distinguish which are the microbial cells that will be counted. As for samples in liquid, there are two ways where one can count microbial cells that are present. One is by using specially designed counting chambers, where there is a grid with squares of known area that is marked on the surface of a glass slide (Clark, Madigan, Martinko, & Stahl, 2012). When the cover slip is placed on the chamber, each square on the grid has a precisely measured volume. The number of cells per unit area of grid can be counted under the microscope, giving a measure of the number of cells per small chamber volume. The number of cells per milliliter of suspension is calculated by employing a conversion factor based on the volume of the chamber sample (Clark, Madigan, Martinko, & Stahl, 2012). Another method to count microbial cells in liquid samples is by using a flow cytometer. Flow cytometer is a machine that employs a laser beam and complex electronics to count individual cells (Clark, Madigan, Martinko, & Stahl, 2012). This method is rarely used in counting microbial cells but has a lot of applications in other fields of study, such as in medical field for counting and differentiating blood cells and other cell types from clinical samples and in microbial ecology where different types of cells are separated for isolation purposes. Viable count is also called plate count because microbial cells are counted in a sample capable of forming colonies on a suitable agar medium. Viable cells are those that are able to divide and produce offspring, and are the very cells that one would like to count when doing studies on microbial growth. And so, these colonies reflect cell

numbers. There are two ways of performing viable or plate count method of counting cells, they are the spread plate and pour plate method. A volume (usually 0.1 ml or less) of an appropriately diluted culture is spread over the surface of an agar plate using a sterile glass spreader in the spread plate method after which, the plate is incubated for the colonies to appear and are counted. The surface of the plate must not be too moist because the added liquid must soak in so the cells remain stationary. Volumes greater than about 0.1 ml are avoided in this method because the excess liquid does not soak in and may cause the colonies to coalesce as they form, making them difficult to count (Clark, Madigan, Martinko, & Stahl, 2012). As for the pour plate method, in a sterile Petri plate a known volume (usually0.11.0 ml) of culture is pipette. Then, a melted agar medium, tempered to just about gelling temperature, is added and mixed by swirling the plate gently. Because the agar medium to be poured is hot (at about 40-55C), the microbial cells to be counted in this method must be able to withstand the high temperature. In this method, colonies will form not only in the surface of the agar medium as with the spread plate method, but all over the media. And so, the counting of colonies must be meticulously done so as not to miss any. Though there are a lot of hard work to be mustered when doing viable counts, it roughly gives the best estimate of the number of viable cells in a sample and so is widely used in many areas of microbiology (Clark, Madigan, Martinko, & Stahl, 2012). One is with the colony number that can change with the length of incubation, culture medium and the incubation conditions. Viable counts will also differ based on the inconsistencies in preparing either the spread plate or the pour plate. Total/microscopic counts and viable counts yield different estimates of the cell numbers, as in total /microscopic counts, dead cells are the ones being counted, while in viable counts, it is viable cells that are counted. And so, plate counts yield lesser numbers than total counts and are unreliable when assessing total numbers of cells of natural samples (Clark, Madigan, Martinko, & Stahl, 2012) Turbidity As microbes grow and divide in a tube of clear nutrient solution, it loses its clarity and becomes cloudy or turbid (Talaro & Talaro, 2002). The turbidity of the solution is due to the microbial cells that scatter light when in a suspension (Clark, Madigan, Martinko, & Stahl, 2012). So, when microbes increase in number, the more the solution appears turbid. This property of being turbid can be used to measure microbial growth. Turbidimetric methods of measuring microbial growth assess measurements of total cell mass which is proportional to cell numbers, though it can only be used for measuring relative amounts of growth, unlike in a total or viable cell count. An instrument that which measures turbidity is a spectrophotometer, by which it passes light through a cell suspension and measures the unscattered light that emerges. It employs a prism or diffraction grating to generate incident light of a specific wavelength. Commonly used wavelengths for bacterial turbidity measurements include 480 nm (blue), 540 nm (green), 600 nm (orange), and 660 nm (red) (Clark, Madigan, Martinko, & Stahl, 2012). Optical density (OD) is the unit of turbidity and is proportional within certain limits to cell

number for unicellular organisms. At high cell concentrations, light scattered away from the spectrophotometers photocell by one cell can be scattered back by another and so, in such situations, OD measurements loses its accuracy due to the loss of one-to-one correspondence of cell number and turbidity. Since cell shape and size differs from one microbe to another, and so as their OD measurements, even though they have the same cell numbers, their OD measurements could vary. So, there is a need to make certain relationships for OD and the actual cell number for each and every organism that is to be studied in the laboratory. Just by observing the turbidity of a solution containing microbes one can already perform a study that involves measuring microbial growth. In using this kind of method, one has not to destroy the sample in order to observe microbial growth. Furthermore, it employs the usage of the same sample and is excellent for monitoring microbial growth. Though this method has a lot of good points, there is also an equal number of disadvantages. As we know it, different groups of microorganisms vary in their nutritional requirements, and are the very reason why some of which are not able to grow in even suspensions in liquid medium. What is more is that OD measurements may become inaccurate for some bacteria that grow from small to large clumps. Continuous Culture A continuous culture caters microorganisms that are always in their exponential phase, wherein microbes grow and multiply at their maximum rate. It is because a continuous culture is an open system unlike a batch culture which is a closed one as it is use to know the growth curve of a certain microorganism. The continuous culture vessel maintains a constant volume to which fresh medium is added at a constant rate while an equal volume of spent culture medium (containing cells) is removed at the same rate (Clark, Madigan, Martinko, & Stahl, 2012). It has the advantage of maintaining the culture in a biochemically active state and preventing it from entering the death phase (Talaro & Chess, 2012). In this kind of culture, the system is in equilibrium, and is said to be in its steady state wherein the chemostat volume, cell number, and nutrient status remain constant. It is done so for the purposes of studying the physiological processes that microorganisms undergo, such as the synthesis of a particular enzyme (Clark, Madigan, Martinko, & Stahl, 2012). A chemostat is a growth chamber with an outflow that is equal to the continuous inflow of nutrient media and is a continuous culture device. This steady-state growth device is used to study such events as cell division, mutation rates, and enzyme regulation (Talaro & Talaro, 2002). In the chemostat, both growth rate and cell density of the culture can be controlled independently and simultaneously (Clark, Madigan, Martinko, & Stahl, 2012). Dilution rate (which is rate at which fresh medium is pumped in and spent medium is removed) and the concentration of a limiting nutrient are two factors that govern growth rate and cell density. Because of this, growth rate (controlled by the dilution rate) and cell number (controlled by the limiting nutrient such as carbon or nitrogen) are controlled independently.

References: Clark, D. P., Madigan, M. T., Martinko, J. M., & Stahl, D. A. (2012). Biology of Microorganisms (13th ed.). San Francisco: Pearson Education, Inc. Talaro, & Talaro. (2002). Foundations in Microbiology. New York: McGraw-Hill. Talaro, K., & Chess, B. (2012). Foundations in Microbiology. New York: McGraw-Hill.

Environmental Effects on Microbial Growth Microorganisms vary a lot amongst each other, their nutritional needs, chemical composition and so as the environment that they live in. As for some microorganisms, they live in an environment that they themselves choose as they have specialized structures that allow motility. But most microorganisms do not have this kind of specialization and so they are left to adapt to their surroundings. This adaptation involves a complex adjustment in biochemistry or genetics that enables long-term survival and growth. The term biologists use to describe the totality of adaptations organisms make to their habitats is niche (Talaro & Chess, 2012). It is the kind of environment that these microorganisms live in, that which fundamentally affects the functions of their metabolic enzymes. Thus, it is a great deal between these enzyme systems of microorganisms and their environment that which will determine their survival. This topic summary will discuss some of the environmental factors which include temperature, pH, osmolarity and oxygen that affect microbial growth. Temperature As microorganisms cannot control their temperature, they should adapt to the temperature of their environment. And so to survive must be able to withstand the temperature changes in their habitat. The ranges of temperature for microbial growth can be expressed as three cardinal temperatures which are minimum, maximum and optimal temperature (Talaro & Chess, 2012). Minimum temperature is the lowest temperature that will allow continued growth and metabolism of the microbe, at temperatures lower than this, activities of the microbe is inhibited. Maximum temperature is the highest temperature for microbial growth and metabolism to proceed, and a slight rise of temperature from this will stop growth. If temperature will continue to rise, the enzymes and nucleic acids of the microbial cell will be permanently deactivated and so the cell will die, a process called denaturation. Optimum temperature on the other hand, is the temperature at which rate of microbial growth and metabolism is the fastest. It is the temperature intermediate between the maximum and the minimum temperature and rarely a single point. Another way to express temperature adaptation is to describe whether an organism grows optimally in a cold (psychrophile), moderate (mesophile), or hot (thermophile) temperature range (Talaro & Chess, 2012). The optimum temperature for psychrophiles is below 15C and is able to grow even at 0C. They cannot grow above 20C and thus laboratory culture is difficult, they must be inoculated in a cold room as room temperature can be lethal to this type of microorganisms. Usually, storage in the refrigerator of laboratory cultures inhibits growth, but it is not the case for psychrophilic laboratory cultures. Natural habitats of psychrophilic bacteria, fungi, algae are lakes, snowfields, polar ice and the deep ocean. It is also very rare that these microorganisms are pathogenic. As for mesophiles, from which most of the medically significant microbes come from, their optimum temperature is at 20C to 40C. They naturally inhabit animals and plants as well as soil and water in temperate, subtropical, and tropical regions. And for thermophiles, their optimum temperature ranges from 45C and

above. Thus they live in soil and water that is associated with very high temperatures, such as volcanoes, in compost piles and in other habitats in which are directly exposed to sun. Generally, they normally grow in temperatures from 45C to 80C. Eukaryotic forms cannot survive temperatures above 60C, though a separate group called hyperthermophiles can grow in temperatures from 80C to 250C (growth in this temperature range is thought to be the limit endured by enzymes and cell structures currently) (Talaro & Chess, 2012). Biotechnology is one of the few areas where these thermophiles are being sought for. Thermus aquaticus is a thermophile species that is, one of the most profitable one, as it can produce enzymes that can make copies of DNA even at high temperatures. Taq polymerase is this enzyme, an essential component of the polymerase chain reaction (PCR), a process that which is used in many areas of medicine, forensics and biotechnology. pH pH is a measure of acidity and alkalinity of a solution that is a number on a scale on which a value of 7 represents neutrality and lower numbers indicate increasing acidity and higher numbers increasing alkalinity and on which each unit of change represents a tenfold change in acidity or alkalinity and that is the negative logarithm of the effective hydrogen-ion concentration in grams equivalent per liter of the solution (Merriam-Webster, 2012). Most organisms live in habitats that have a pH between 6 and 8. It is because a very high (strong base) or low pH (strong acid), would be detrimental to the cells enzymes and other cellular substances. Nearly all microbes live in environments with a neutral pH (7), and are called neutrophiles. Still there are some microbes that live in an environment with an extreme pH, but this kind of environment is not common. Acidic bogs, lakes and alkaline soils are examples of these environments. When an environment that has a very low pH (strongly acidic) favors the microbe to flourish, the microorganism is said to be an acidophile. Example is a Euglena mutabilis which grows at 0 to 1 pH in acid pools. Some acidophiles do not only require low pH for growth, but there are certain bacteria that maintain a low pH internally by releasing strong acid. When it has a very high pH, the microbe is an alkalinophile. Alkalinophiles live in hot pools and soils where levels of basic minerals are very high (pH of 10). Osmolarity Osmosis is the process of diffusion of water across a selectively permeable membrane in the direction of a lower water concentration (Talaro & Chess, 2012); while osmolarity refers to the concentration of an osmotic solution (Merriam-Webster, 2012). Osmotic pressure, in addition, is the pressure associated with osmosis. When a condition is hypotonic, it refers to having lower osmotic pressure than a surrounding medium or the surrounding medium have a lower solute concentration. When the condition is isotonic, it refers to having equal osmotic pressure as the surrounding environment or having equal solute concentrations as the environment. And if the condition is hypotonic, it refers to having a higher osmotic pressure than the environment or has high solute concentration. Though majority of the microorganisms live in hypotonic and isotonic conditions, few live in habitats that are hypertonic and are called osmophiles. A common type of osmophile is halophile. Examples of halophiles

are Halobacterium and Halococcus (some groups of Archaeons) which live in salt lakes, ponds, and other hypersaline habitats. A 25% NaCl solution is the solution in which they can grow optimally, but they only need at least 9% solution along with other salts for growth. These microorganisms have modifications in their cell walls and membranes that allow them to thrive in hypersaline habitats and when exposed to hypotonic habitats, will lyse. On the other hand, some microbes tolerate a wide range of solute concentrations and are called osmotolerant. Oxygen Oxygen gas is one of the atmospheric gases that most influence microbial growth aside from carbon dioxide. Oxygen comprises about 20% of the atmosphere and has a great impact on microbial adaptation. It is an important respiratory and it is also a powerful oxidizing agent that exists in many toxic forms. The ecological need for free oxygen (O2) is based on whether a cell can handle toxic by-products such as superoxide and peroxide. (Talaro & Chess, 2012). Microbes can be classified in three categories regarding the use of oxygen: 1) use oxygen and detoxify it (aerobe) 2) do not use oxygen nor detoxify it and (anaerobe) 3) do not use oxygen but can detoxify it (aerotolerant anaerobe). An aerobe is an organism that uses gaseous oxygen in its metabolism and possesses the enzymes needed to process toxic oxygen products (Talaro & Chess, 2012). An obligate aerobe cannot live without oxygen. Some bacteria, protozoa and algae have strict requirements for oxygen in their metabolism. A facultative anaerobe is an aerobe that does not require oxygen for its metabolism and is capable of growth in the absence of it (Talaro & Chess, 2012). This kind of aerobe metabolizes by aerobic respiration when oxygen is present, but when not present, it is able to survive by adopting an anaerobic mode of metabolism (e.g. fermentation). Gram-negative intestinal bacteria and staphylococci, which are both pathogens, fall into this group of aerobes. Organisms that live in soil, water or the human body, requires a small amount of oxygen (1-15%) are called microaerophile. Organisms that lack the metabolic enzyme systems for using oxygen gas in respiration are called anaerobe. Obligate anaerobes cannot tolerate oxygen because they also lack the enzymes that process toxic oxygen to the extent that they will die if exposed to it. They inhabit strictly reduce habitats, such as deep mud, lakes, ocean and soil. Those organisms that do not utililize oxygen but can grow in its presence are called aerotolerant anaerobes. They possess such mechanisms that allow the breakdown of peroxide and superoxide. An example is a Lactobacili which reside commonly in the intestine inactivate peroxide and superoxide with manganese ions. References: Merriam-Webster Dictionary. (2012). Merriam-Webster Inc. Talaro, K., & Chess, B. (2012). Foundations in Microbiology. New York: McGraw-Hill.