G – N -acetylglucosamine M – N -acetylmuramic acid
Classification – the arrangement of organisms into taxonomic groups (taxa) on the basis of similarities or relationships. - knowledge obtained by experimental as well as observational technique - biochemical, physiologic, genetic, and morphologic properties are often necessary for an adequate description of a taxon. Nomenclature – naming an organism by international rules according to its characteristics. Identification – refers to the practical use of a classification scheme: (1) to isolate and distinguish desirable organisms from undesirable ones; (2) To verify the authenticity or special properties of a culture; or in a clinical setting (3) To isolate and identify the causative agent of a disease (may permit the selection of pharmacologic treatment specifically directed toward their eradiation
Traditional methods of bacterial identification rely on phenotypic identification of the causative organism using gram staining, culture and biochemical methods. However, these methods of bacterial identification suffer from two major drawbacks. First, they can be used only for organisms that can be cultivated in vitro. Second, some strains exhibit unique biochemical characteristics that do not fit into patterns that have been used as a characteristic of any known genus and species. Phenetic - of or relating to taxonomic analysis that emphasizes the overall similarities of characteristics among biological taxa without regard to phylogenetic relationships
The American Type Culture Collection (ATCC) is a private, not-for-profit biological resource center whose mission focuses on the acquisition, authentication, production, preservation, development and distribution of standard reference microorganisms , cell lines and other materials for research in the life sciences . Established in 1914 and originally incorporated by scientists in 1925 to serve as a worldwide repository and distribution center for cultures of microorganisms, the ATCC has developed into the global leader in research and development expertise for identifying, characterizing, preserving and distributing a wide range of cell lines and microbes . Aside from maintaining the biorepository, an R&D program and a product development team, the ATCC also competes for federal grants and contracts and engages in partnerships and collaborations with academic institutions and private companies.
Heterotroph - An organism that cannot manufacture its own food and instead obtains its food and energy by taking in organic substances, usually plant or animal matter. All animals, protozoans, fungi, and most bacteria are heterotrophs. A heterotroph ( Greek ἕτερος heteros = "another", "different" and τροφή trophe = "nutrition", "growth") is an organism that uses organic carbon for growth by consuming other organisms.  This contrasts with autotrophs , such as plants , which can directly use sources of energy such as light to produce organic substrates from carbon dioxide .
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Phototroph – Phototrophy (-process) •Derive energy by photophosphorylation . •Phototrophs may be oxygenic (oxygen-evolving) or anoxygenic (not oxygen-evolving). Chemotroph- process is by respiration •Derive energy by oxidative phosphorylation . •Generally, respirers use molecular oxygen as the external, terminal electron acceptor; this is aerobic respiration . Respirers may also use nitrate or some other "oxygen substitute" in the process of anaerobic respiration . •Certain organisms can only perform anaerobic respiration – for example, the methane producers and many sulfate reducers. Fermentation •Derive energy by substrate-level phosphorylation . Associated with energy production is "reducing power." For biosynthesis, all organisms need reducing power in the form of electrons. Organisms that obtain their electrons from organic compounds are called organotrophs , and those that obtain their reducing power from inorganic compounds are called lithotrophs .
This simplified scheme for use of carbon, either organic carbon or CO2, ignores the possibility that an organism, whether it is an autotroph or a heterotroph, may require small amounts of certain organic compounds for growth because they are essential substances that the organism is unable to synthesize from available nutrients. Such compounds are called growth factors .
Physical and Environmental Requirements for Microbial Growth The procaryotes exist in nature under an enormous range of physical conditions such as O2 concentration, Hydrogen ion concentration (pH) and temperature. The exclusion limits of life on the planet, with regard to environmental parameters, are always set by some microorganism, most often a procaryote, and frequently an Archaeon. Applied to all microorganisms is a vocabulary of terms used to describe their growth (ability to grow) within a range of physical conditions. A thermophile grows at high temperatures, an acidophile grows at low pH, an osmophile grows at high solute concentration, and so on. This nomenclature will be employed in this section to describe the response of the procaryotes to a variety of physical conditions. The Effect of Oxygen Oxygen is a universal component of cells and is always provided in large amounts by H2O. However, procaryotes display a wide range of responses to molecular oxygen O2 (Table 6). Obligate aerobes require O2 for growth; they use O2 as a final electron acceptor in aerobic respiration. Obligate anaerobe s (occasionally called aerophobes ) do not need or use O2 as a nutrient. In fact, O2 is a toxic substance, which either kills or inhibits their growth. Obligate anaerobic procaryotes may live by fermentation, anaerobic respiration, bacterial photosynthesis, or the novel process of methanogenesis. Facultative anaerobes (or facultative aerobes ) are organisms that can switch between aerobic and anaerobic types of metabolism. Under anaerobic conditions (no O2) they grow by fermentation or anaerobic respiration, but in the presence of O2 they switch to aerobic respiration. Aerotolerant anaerobes are bacteria with an exclusively anaerobic (fermentative) type of metabolism but they are insensitive to the presence of O2. They live by fermentation alone whether or not O2 is present in their environment. Microaerophilic organisms are a specific type of microorganism (especially bacteria ) that requires oxygen to survive, but requires environments containing lower levels of oxygen than are present in the atmosphere (~20% concentration). Many microphiles are also capnophiles , as they require an elevated concentration of carbon dioxide. In the laboratory they can be easily cultivated in a candle jar, a container into which a lit candle is introduced before sealing the airtight lid. The flame burns until extinguished by oxygen deprivation, creating a carbon dioxide-rich, oxygen-poor atmosphere.
The Effect of Temperature on Growth Microorganisms have been found growing in virtually all environments where there is liquid water, regardless of its temperature. In 1966, Professor Thomas D. Brock, then at Indiana University, made the amazing discovery in boiling hot springs of Yellowstone National Park that bacteria were not just surviving there, they were growing and flourishing. Brock's discovery of thermophilic bacteria, archaea and other "extremophiles" in Yellowstone is summarized for the general public in an article at this web site Life at High Temperatures . Subsequently, procaryotes have been detected growing around black smokers and hydrothermal vents in the deep sea at temperatures at least as high as 120 degrees. Microorganisms have been found growing at very low temperatures as well. In supercooled solutions of H2O as low as -20 degrees, certain organisms can extract water for growth, and many forms of life flourish in the icy waters of the Antarctic, as well as household refrigerators, near 0 degrees. A particular microorganism will exhibit a range of temperature over which it can grow, defined by three cardinal points in the same manner as pH (Figure 6, cf. Figure 4). Considering the total span of temperature where liquid water exists, the procaryotes may be subdivided into several subclasses on the basis of one or another of their cardinal points for growth. For example, organisms with an optimum temperature near 37 degrees (the body temperature of warm-blooded animals) are called mesophiles . Organisms with an optimum T between about 45 degrees and 70 degrees are thermophiles . Some Archaea with an optimum T of 80 degrees or higher and a maximum T as high as 115 degrees, are now referred to as extreme thermophiles or hyperthermophiles . The cold-loving organisms are psychrophiles defined by their ability to grow at 0 degrees. A variant of a psychrophile (which usually has an optimum T of 10-15 degrees) is a psychrotroph , which grows at 0 degrees but displays an optimum T in the mesophile range, nearer room temperature. Psychrotrophs are the scourge of food storage in refrigerators since they are invariably brought in from their mesophilic habitats and continue to grow in the refrigerated environment where they spoil the food. Of course, they grow slower at 2 degrees than at 25 degrees. Think how fast milk spoils on the counter top versus in the refrigerator. Psychrophilic bacteria are adapted to their cool environment by having largely unsaturated fatty acids in their plasma membranes. Some psychrophiles, particularly those from the Antarctic have been found to contain polyunsaturated fatty acids, which generally do not occur in procaryotes. The degree of unsaturation of a fatty acid correlates with its solidification T or thermal transition stage (i.e., the temperature at which the lipid melts or solidifies); unsaturated fatty acids remain liquid at low T but are also denatured at moderate T; saturated fatty acids, as in the membranes of thermophilic bacteria, are stable at high temperatures, but they also solidify at relatively high T. Thus, saturated fatty acids (like butter) are solid at room temperature while unsaturated fatty acids (like safflower oil) remain liquid in the refrigerator. Whether fatty acids in a membrane are in a liquid or a solid phase affects the fluidity of the membrane, which directly affects its ability to function. Psychrophiles also have enzymes that continue to function, albeit at a reduced rate, at temperatures at or near 0 degrees. Usually, psychrophile proteins and/or membranes, which adapt them to low temperatures, do not function at the body temperatures of warm-blooded animals (37 degrees) so that they are unable to grow at even moderate temperatures. Thermophiles are adapted to temperatures above 60 degrees in a variety of ways. Often thermophiles have a high G + C content in their DNA such that the melting point of the DNA (the temperature at which the strands of the double helix separate) is at least as high as the organism's maximum T for growth. But this is not always the case, and the correlation is far from perfect, so thermophile DNA must be stabilized in these cells by other means. The membrane fatty acids of thermophilic bacteria are highly saturated allowing their membranes to remain stable and functional at high temperatures. The membranes of hyperthermophiles, virtually all of which are Archaea, are not composed of fatty acids but of repeating subunits of the C5 compound, phytane, a branched, saturated, "isoprenoid" substance, which contributes heavily to the ability of these bacteria to live in superheated environments. The structural proteins (e.g. ribosomal proteins, transport proteins (permeases) and enzymes of thermophiles and hyperthermophiles are very heat stable compared with their mesophilic counterparts. The proteins are modified in a number of ways including dehydration and through slight changes in their primary structure, which accounts for their thermal stability. Psychroduric organisms prefer warm temperatures, but can endure very cold or even freezing temperatures. A hyperthermophile is an organism that thrives in extremely hot environments— from 60 degrees C (140 degrees F) upwards. An optimal temperature for the existence of hyperthermophiles is above 80°C (176°F). Hyperthermophiles are a subset of extremophiles , micro-organisms within the domain Archaea , although some bacteria are able to tolerate temperatures of around 100°C (212°F), too. Many hyperthermophiles are also able to withstand other environmental extremes such as high acidity or radiation levels.
Water is the solvent in which the molecules of life are dissolved, and the availability of water is therefore a critical factor that affects the growth of all cells. The availability of water for a cell depends upon its presence in the atmosphere (relative humidity) or its presence in solution or a substance ( water activity ). The water activity (Aw) of pure H2O is 1.0 (100% water). Water activity is affected by the presence of solutes such as salts or sugars, that are dissolved in the water. The higher the solute concentration of a substance, the lower is the water activity and vice-versa. Microorganisms live over a range of Aw from 1.0 to 0.7. The Aw of human blood is 0.99; seawater = 0.98; maple syrup = 0.90; Great Salt Lake = 0.75. Water activities in agricultural soils range between 0.9 and 1.0. The only common solute in nature that occurs over a wide concentration range is salt [NaCl], and some microorganisms are named based on their growth response to salt. Microorganisms that require some NaCl for growth are halophiles . Mild halophiles require 1-6% salt, moderate halophiles require 6-15% salt; extreme halophiles that require 15-30% NaCl for growth are found among the archaea. Bacteria that are able to grow at moderate salt concentrations, even though they grow best in the absence of NaCl, are called halotolerant . Although halophiles are "osmophiles" (and halotolerant organisms are "osmotolerant") the term osmophiles is usually reserved for organisms that are able to live in environments high in sugar. Organisms which live in dry environments (made dry by lack of water) are called xerophiles . The concept of lowering water activity in order to prevent bacterial growth is the basis for preservation of foods by drying (in sunlight or by evaporation) or by addition of high concentrations of salt or sugar.
The Effect of pH on Growth The pH, or hydrogen ion concentration, [H+], of natural environments varies from about 0.5 in the most acidic soils to about 10.5 in the most alkaline lakes. Appreciating that pH is measured on a logarithmic scale, the [H+] of natural environments varies over a billion-fold and some microorganisms are living at the extremes, as well as every point between the extremes! Most free-living procaryotes can grow over a range of 3 pH units, about a thousand fold change in [H+]. The range of pH over which an organism grows is defined by three cardinal points : the minimum pH , below which the organism cannot grow, the maximum pH , above which the organism cannot grow, and the optimum pH , at which the organism grows best. For most bacteria there is an orderly increase in growth rate between the minimum and the optimum and a corresponding orderly decrease in growth rate between the optimum and the maximum pH, reflecting the general effect of changing [H+] on the rates of enzymatic reaction (Figure 4). Microorganisms which grow at an optimum pH well below neutrality (7.0) are called acidophiles . Those which grow best at neutral pH are called neutrophiles and those that grow best under alkaline conditions are called alkaliphiles . Obligate acidophiles, such as some Thiobacillus species, actually require a low pH for growth since their membranes dissolve and the cells lyse at neutrality. Several genera of Archaea, including Sulfolobus and Thermoplasma , are obligate acidophiles. Among eukaryotes, many fungi are acidophiles, but the champion of growth at low pH is the eukaryotic alga Cyanidium which can grow at a pH of 0. In the construction and use of culture media, one must always consider the optimum pH for growth of a desired organism and incorporate buffers in order to maintain the pH of the medium in the changing milieu of bacterial waste products that accumulate during growth. Many pathogenic bacteria exhibit a relatively narrow range of pH over which they will grow. Most diagnostic media for the growth and identification of human pathogens have a pH near 7.
Figure 1. Bacterial growth by binary fission. Most bacteria reproduce by a relatively simple asexual process called binary fission: each cell increases in size and divides into two cells. During this process there is an orderly increase in cellular structures and components, replication and segregation of the bacterial DNA, and formation of a septum or cross wall which divides the cell into two progeny cells The process is coordinated by the bacterial membrane perhaps by means of mesosomes. The DNA molecule is believed to be attached to a point on the membrane where it is replicated. The two DNA molecules remain attached at points side-by-side on the membrane while new membrane material is synthesized between the two points. This draws the DNA molecules in opposite directions while new cell wall and membrane are laid down as a septum between the two chromosomal compartments. When septum formation is complete the cell splits into two progeny cells. The time interval required for a bacterial cell to divide or for a population of bacterial cells to double is called the generation time. Generation times for bacterial species growing in nature may be as short as 15 minutes or as long as several days. Electron micrograph of Streptococcus pyogenes by Maria Fazio and Vincent A. Fischetti, Ph.D. with permission. The Laboratory of Bacterial Pathogenesis and Immunology , Rockefeller University.
The Bacterial Growth Curve In the laboratory, under favorable conditions, a growing bacterial population doubles at regular intervals. Growth is by geometric progression: 1, 2, 4, 8, etc. or 20, 21, 22, 23.........2n (where n = the number of generations). This is called exponential growth . In reality, exponential growth is only part of the bacterial life cycle, and not representative of the normal pattern of growth of bacteria in Nature. When a fresh medium is inoculated with a given number of cells, and the population growth is monitored over a period of time, plotting the data will yield a typical bacterial growth curve (Figure 3 below). Figure 3. The typical bacterial growth curve. When bacteria are grown in a closed system (also called a batch culture), like a test tube, the population of cells almost always exhibits these growth dynamics: cells initially adjust to the new medium (lag phase) until they can start dividing regularly by the process of binary fission (exponential phase). When their growth becomes limited, the cells stop dividing (stationary phase), until eventually they show loss of viability (death phase). Note the parameters of the x and y axes. Growth is expressed as change in the number viable cells vs time. Generation times are calculated during the exponential phase of growth. Time measurements are in hours for bacteria with short generation times. Four characteristic phases of the growth cycle are recognized. 1. 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. The length of the lag phase is apparently dependent on a wide variety of factors including the size of the inoculum; time necessary to recover from physical damage or shock in the transfer; time required for synthesis of essential coenzymes or division factors; and time required for synthesis of new (inducible) enzymes that are necessary to metabolize the substrates present in the medium. 2. Exponential (log) 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. Generation time (G) is defined as the time (t) per generation (n = number of generations). Hence, G=t/n is the equation from which calculations of generation time (below) derive. 3. Stationary Phase . Exponential growth cannot be continued forever in a batch culture (e.g. a closed system such as a test tube or flask). Population growth is limited by one of three factors: 1. exhaustion of available nutrients; 2. accumulation of inhibitory metabolites or end products; 3. exhaustion of space, in this case called a lack of "biological space". During the stationary phase, if viable cells are being counted, it cannot be determined whether some cells are dying and an equal number of cells are dividing, or the population of cells has simply stopped growing and dividing. 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). It is during the stationary phase that spore-forming bacteria have to induce or unmask the activity of dozens of genes that may be involved in sporulation process. 4. Death Phase . If incubation continues after the population reaches stationary phase, a death phase follows, in which the viable cell population declines. (Note, if counting by turbidimetric measurements or microscopic counts, the death phase cannot be observed.). During the death phase, the number of viable cells decreases geometrically (exponentially), essentially the reverse of growth during the log phase.
Culture Media for the Growth of Bacteria For any bacterium to be propagated for any purpose it is necessary to provide the appropriate biochemical and biophysical environment. The biochemical (nutritional) environment is made available as a culture medium , and depending upon the special needs of particular bacteria (as well as particular investigators) a large variety and types of culture media have been developed with different purposes and uses. Culture media are employed in the isolation and maintenance of pure cultures of bacteria and are also used for identification of bacteria according to their biochemical and physiological properties. The manner in which bacteria are cultivated, and the purpose of culture media, varies widely. Liquid media are used for growth of pure batch cultures, while solidified media are used widely for the isolation of pure cultures, for estimating viable bacterial populations, and a variety of other purposes. The usual gelling agent for solid or semisolid medium is agar , a hydrocolloid derived from red algae. Agar is used because of its unique physical properties (it melts at 100 degrees and remains liquid until cooled to 40 degrees, the temperature at which it gels) and because it cannot be metabolized by most bacteria. Hence as a medium component it is relatively inert; it simply holds (gels) nutrients that are in aqueous solution.
Liquid media = used for growth of pure batch cultures Semi-solid = used for biochemical/motility testing Solid media = used widely for the isolation of pure cultures, for estimating viable bacterial populations, and a variety of other purposes. Agar = cannot be metabolized by most bacteria, a medium component relatively inert; simply holds (gels) nutrients that are in aqueous solution.
Types of Culture Media Culture media may be classified into several categories depending on their composition or use. A chemically-defined (synthetic) medium (Table 4a and 4b) is one in which the exact chemical composition is known. A complex (undefined) medium (Table 5a and 5b) is one in which the exact chemical constitution of the medium is not known. Defined media are usually composed of pure biochemicals off the shelf; complex media usually contain complex materials of biological origin such as blood or milk or yeast extract or beef extract, the exact chemical composition of which is obviously undetermined. A defined medium is a minimal medium (Table 4a) if it provides only the exact nutrients (including any growth factors) needed by the organism for growth. The use of defined minimal media requires the investigator to know the exact nutritional requirements of the organisms in question. Chemically-defined media are of value in studying the minimal nutritional requirements of microorganisms, for enrichment cultures, and for a wide variety of physiological studies. Complex media usually provide the full range of growth factors that may be required by an organism so they may be more handily used to cultivate unknown bacteria or bacteria whose nutritional requirement are complex (i.e., organisms that require a lot of growth factors, known or unknown). Complex media are usually used for cultivation of bacterial pathogens and other fastidious bacteria. Table 4a. Minimal medium for the growth of Bacillus megaterium. An example of a chemically-defined medium for growth of a heterotrophic bacterium. ComponentAmountFunction of component sucrose10.0 gC and energy sourceK2HPO42.5 gpH buffer; P and K sourceKH2PO42.5 gpH buffer; P and K source(NH4)2HPO41.0 gpH buffer; N and P sourceMgSO4 7H2O0.20 gS and Mg++ sourceFeSO4 7H2O0.01 gFe++ sourceMnSO4 7H2O0.007 gMn++ Sourcewater985 mlpH 7.0
Methods for Measurement of Cell Numbers Measuring techniques involve direct counts, visually or instrumentally, and indirect viable cell counts. 1. Direct microscopic counts are possible using special slides known as counting chambers. Dead cells cannot be distinguished from living ones. Only dense suspensions can be counted (>107 cells per ml), but samples can be concentrated by centrifugation or filtration to increase sensitivity. A variation of the direct microscopic count has been used to observe and measure growth of bacteria in natural environments. In order to detect and prove that thermophilic bacteria were growing in boiling hot springs, T.D. Brock immersed microscope slides in the springs and withdrew them periodically for microscopic observation. The bacteria in the boiling water attached to the glass slides naturally and grew as microcolonies on the surface. 2. Electronic counting chambers count numbers and measure size distribution of cells. For cells the size of bacteria the suspending medium must be very clean. Such electronic devices are more often used to count eucaryotic cells such as blood cells. 3. Indirect viable cell counts , also called plate counts , involve plating out (spreading) a sample of a culture on a nutrient agar surface. The sample or cell suspension can be diluted in a nontoxic diluent (e.g. water or saline) before plating. If plated on a suitable medium, each viable unit grows and forms a colony. Each colony that can be counted is called a colony forming unit (cfu) and the number of cfu's is related to the viable number of bacteria in the sample. Advantages of the technique are its sensitivity (theoretically, a single cell can be detected), and it allows for inspection and positive identification of the organism counted. Disadvantages are (1) only living cells develop colonies that are counted; (2) clumps or chains of cells develop into a single colony; (3) colonies develop only from those organisms for which the cultural conditions are suitable for growth. The latter makes the technique virtually useless to characterize or count the total number of bacteria in complex microbial ecosystems such as soil or the animal rumen or gastrointestinal tract. Genetic probes can be used to demonstrate the diversity and relative abundance of procaryotes in such an environment, but many species identified by genetic techniques have so far proven unculturable. Advantages of the technique are its sensitivity (theoretically, a single cell can be detected), and it allows for inspection and positive identification of the organism counted. Disadvantages are (1) only living cells develop colonies that are counted; (2) clumps or chains of cells develop into a single colony; (3) colonies develop only from those organisms for which the cultural conditions are suitable for growth. The latter makes the technique virtually useless to characterize or count the total number of bacteria in complex microbial ecosystems such as soil or the animal rumen or gastrointestinal tract. Genetic probes can be used to demonstrate the diversity and relative abundance of procaryotes in such an environment, but many species identified by genetic techniques have so far proven unculturable.
In 1951, Freeman made the remarkable discovery that pathogenic (toxigenic) strains of C. diphtheriae are lysogenic, (i.e., are infected by a temperate Beta phage), while non lysogenized strains are avirulent. Subsequently, it was shown that the gene for toxin production is located on the DNA of the Beta phage. In the early 1960s, Pappenheimer and his group at Harvard conducted experiments on the mechanism of a action of the diphtheria toxin. They studied the effects of the toxin in HeLa cell cultures and in cell-free systems, and concluded that the toxin inhibited protein synthesis by blocking the transfer of amino acids from tRNA to the growing polypeptide chain on the ribosome. They found that this action of the toxin could be neutralized by prior treatment with diphtheria antitoxin. Subsequently, the exact mechanism of action of the toxin was shown, and the toxin has become a classic model of an ADP-ribosylating bacterial exotoxin.
All cocci are gram (+) except Neisseria, Branhamella, Moraxella and Veilonella. All bacilli are gram (-) except Mycobacterium, Corynebacterium, Lactobacillus, Listeria, Clostridium, Bacillus, Erysipelothrix, and Nocardia. All spirals are gram (-)
3 TYPES OF COLONY (consistency) Mucoid - slimy/watery/glistening Smooth – uniform texture easily emulsified with NSS Rough – granulated & rough not easily emulsified
Mannitol Salt Agar 7.5% NaCl ( Staphylococcus spp .) Hektoen enteric Agar Bile salts (Gram (-) rods)
ANAEROBIC GROWTH Thioglycollate broth absorbs O2 and slows down its penetration demonstrates O2 requirement Mannitol Salt Agar contains mannitol fermented only by S. aureus Lactose broth shows fermentation of lactose indicated by gas trapped in Durham tubes
ASSAY test the effectiveness of antimicrobial drugs asses the effects of disinfectant, antiseptic, cosmetic, preservative on the growth of microbes ENUMERATION used to count the number of microbes in milk, water, food, soil
Introduction to Microbiology Describe the historical development of microbiology Enumerate the development of science with emphasis on person/scientists and their contributions Explain the divisions of microbiology Define terms
Historical Development: The beginnings…Robert Hooke “Micrographia” Compound microscope and its uses History of cell biologyAnton van Leeuwenhoek First to observe live microorganisms (animalcules) Single-lens microscope
HISTORICAL DEVELOPMENT:The Transition PeriodCarolus Linnaeus Binomial nomenclature : genus & specific epithet. Are italicized or underlined. Are “Latinized” and used worldwide. May be descriptive or honor a scientist. Staphylococcus aureus Escherichia coli
Abiogenesis versus BiogenesisSpontaneous Generation / Abiogenesis: that living organisms arise from nonliving matter a “vital force” forms life.Biogenesis: that the living organisms arise from pre- existing life.
Abiogenesis / Spontaneous Generation Aristotle’s hypothesis:. Decaying living material animals Jan Baptista van Helmont’s _______________: dirty a few grains shirt or + of wheat / = rags wheat bran
Evidence Pro and ConFrancesco Redi 1st real experiment to dispute abiogenesis CONDITIONS RESULTS 1st : 3 sealed jars : 3 open jars 2nd: 3 jars covered w/ fine net
Evidence Pro and ConJohn Needham put boiled nutrient broth into covered flasks. CONDITIONS RESULTS Nutrient broth heated, cooled then placed in sealed flask
Evidence Pro and ConLazzaro Spallanzani Showed that fluids heated in sealed flasks did not contain microbes
Rudolf Virchow / Theodor Schwann / Matthias Schleiden Cell theoryLouis Pasteur Is there a “life force” present in air that can cause microbes to develop by spontaneous generation? Is there a means of allowing air to enter a container but not the bacteria that are present in it?
CONCLUSION: Conditions ResultsNutrient broth placed in longnecked-flasks, heated, then sealed
Historical Development : THE GOLDEN AGE OF MICROBIOLOGYPasteur’s WorkMicrobes are responsible for fermentation andspoilage of food.Vinegar is produced when bacteria fermentsethanol in wine.Spoilage bacteria could be killed heat that wasnot hot enough to evaporate alcohol in wine.
The Germ Theory of Disease 1835: Agostino Bassi discovered a silkworm disease caused by a ________. 1865: ________ discovered another silkworm disease caused by a protozoan. 1840s: Ignaz Semmelweis advocated _____________ to prevent spread of puerperal fever. 1860s: ____________ used phenol to prevent surgical wound infections.
1876: Robert Koch provided experimental steps (__________) used to prove that a specific microbe causes a specific disease. disease Microbial Etiology of Important diseases established Koch : •Vibrio cholerae •Mycobacterium tuberculosis •Bacillus anthracis
Exceptions to Koch’s Postulates1. Many healthy people carry pathogens but do not exhibit symptoms of the disease. These “carriers” may transmit the pathogens to others who then may become diseased (hospital-acquired infections, typhoid fever, diphtheria) .2. Some microbes are very difficult or impossible to grow in vitro in artificial media (viruses, rickettsias, chlamydias, M. leprae, T. pallidum) .
3. To induce a disease from a pure culture, the experimental animal must be susceptible to that pathogen. Many animals are very resistant to microbial infections. Many pathogens are species-specific. Use of human volunteers are difficult to find and ethical considerations limit their use.4. Certain diseases develop only when an opportunistic pathogen invades a weakened host. These opportunists cause disease in a person who is ill or recovering from another disease. (pneumonia, ear infections following influenza)
Vaccination 1796: Edward Jenner inoculated a person with cowpox virus resulting to protection from smallpox.
The Bir th of ModernChemotherapy agents used to treat Chemotherapeutic infectious disease can be synthetic drugs or antibiotics. _________ are chemicals produced by bacteria and fungi that inhibit or kill other microbes. 1910: Paul Ehrlich developed a synthetic arsenic drug, __________, to treat syphilis. 1930s: Sulfonamides were synthesized. 1928: Alexander Fleming discovered the 1st antibiotic, ___________.
HISTORICAL DEVELOPMENT:Modern Developments in Microbiology Bacteriology Mycology Parasitology Virology Genomics, the study of an organism’s genes, have provided new tools for classifying microorganisms.
___________ is the study of immunity. Who proposed the use of immunology to identify some bacteria according to serotypes?__________________ Vaccines and IFs are being investigated to prevent and cure viral diseases.
Paul Berg (1960s) introduced ____________ by inserting animal DNA into bacterial DNA resulting in the production of an animal protein. Recombinant DNA technology or genetic engineering involves microbial genetics and molecular biology.
Using microbes George Beadle and Edward Tatum (1942) showed that genes encode a cell’s enzymes Oswald Avery, Colin MacLeod, and Maclyn McCarty (1944) showed that DNA was the hereditary material. Francois Jacob and Jacques Monod (1961) discovered the role of mRNA in protein synthesis
HISTORICAL DEVELOPMENT:Microbes and HumanWelfareBioremediation Bacteria degrade organic matter in sewage & detoxify pollutants (eg. oil, mercury)Biological Insecticides ___________________ infections are fatal in many insects but harmless to other animals, humans & plants.
HISTORICAL DEVELOPMENT:Microbes and HumanWelfareModern Biotechnology Genetic engineering - a biotechnologic technique. Bacteria and fungi can produce a variety of proteins (vaccines, enzymes). ____________ : replacement of missing/defective genes. Genetically modified bacteria - used to protect crops from insects and freezing.
HISTORICAL DEVELOPMENT:Microbes and HumanDis eas e Flora, microflora = microbes were once classified as plants ______________ = new term for microbes Normal Microbiota They prevent growth of pathogens. They produce growth factors. Resistance
Emerging Infectious Diseases :BACTERIALInvasive group A Streptococcus Rapidly growing bacteria cause extensive tissue damage.Anthrax Bacillus anthracis Veterinarians and agricultural workers (at risk).Escherichia coli O157:H7 Enterotoxigenic E. coli Leading cause of diarrhea worldwide.
Superbugs:CRKP pneumonia, meningitis, UTI, wound infections and blood infectionsMRSA boils and abscesses resembling infected bug bites, but can also present as pneumonia or flu-like symptoms.
VIRAL West Nile encephalitis West Nile Virus Bovine Spongiform Encephalopathy Prions Also causes Creutzfeldt-Jakob disease Ebola hemorrhagic fever Ebola virus fever, hemorrhaging, and blood clotting
Hantavirus pulmonary syndrome Hantavirus cause hemorrhagic fever U.S.,1995: A disease with respiratory symptoms was seen Hantavirus Sin Nombre virus (U.S., carried by rats) Acquired immunodeficiency syndrome (AIDS) Human immunodeficiency virus (HIV) STD; pandemic identified in 1981. infecting 40 million; 14,000 new infections daily.
Bacterial Structure,Classification and Growth Requirements
Part I.Bacterial Structures LEARNING OBJECTIVES• Classify microorganisms according to their morphology and distinct characteristics• Differentiate between prokaryote and eukaryote• Identify the different structures and functions of a bacterial cell• Describe bacterial morphology• Point out ways of classifying bacteria
DIVISIONS OF MICROBIOLOGY1. VIROLOGY : Viruses smallest intact infectious agents intracellular reproduction only consist of: RNA or DNA core Protein coat glycoprotein envelope
3. MYCOLOGY : Fungi• 2 Forms: _______, _______• thick cell wall• Develop from spores or fragments of hyphae4. PHYCOLOGY : ________• Mainly aquatic• contain chlorophyll• Some produce neurotoxins which can concentrate in fish / shellfish and cause poisoning in humans
5. BACTERIOLOGY : Bacteria• Unicellular, Prokaryotic• Free living• Contain both RNA and DNA• Multiply by _____________• Eubacteria ; Archaebacteria
BACTERIA ARCHAEA• Prokaryotic • Prokaryotic• Peptidoglycan cell • Lack peptidoglycan walls • Live in extreme• characterized by environments shape, motility & metabolism Figure 1.1a
Comparison Between Prokaryotic and Eukaryotic Cells Characteristic Prokaryotes EukaryotesSize of cell Typically 0.2-2.0 µ m in diameter Typically 10-100 µ m in diameterNucleus No nuclear membrane or nucleoli True nucleus, consisting of (nucleoid) nuclear membrane & nucleoliMembrane-enclosed organelles Absent Present; examples include lysosomes, Golgi complex, endoplasmic reticulum, mitochondria & chloroplastsFlagella Consist of two protein building Complex; consist of multiple blocks microtubulesGlycocalyx /Capsule Present as a capsule or slime Present in some cells that lack a layer cell wallCell wall Usually present; chemically When present, chemically complex (typical bacterial cell simple wall includes peptidoglycan)Plasma membrane No carbohydrates and generally Sterols and carbohydrates that lacks sterols serve as receptors presentCytoplasm No cytosketeton or cytoplasmic Cytoskeleton; cytoplasmic streaming streamingRibosomes Smaller size (70S) Larger size (80S); smaller size (70S) in organellesChromosome (DNA) arrangement Single circular chromosome; Multiple linear chromosomes lacks histones with histonesSexual reproduction No meiosis; transfer of DNA Involves meiosis fragments only (conjugation)
A. Flagella PARTS filament Hook basal body Flagella rotates to move Flagellin (H Ags) Purpose: ___________________
Flagellar Arrangement1. Monotrichous : _______ flagellum at one end2. ____________ : small bunches arising from one end of cell3. Amphitrichous: ____________________4. ____________ : flagella dispersed over surface of cell Figure 4.7
Internal Flagella• Also known as: ___________________• Periplasmic filaments• enclosed between cell wall & cell membrane of spirochetes• motility
B. Appendages for Attachment FIMBRIAE • fine hairlike bristles from the cell surface • function in adhesion to other cells and surfaces
Pili• Appendages for Mating rigid tubular structure• Made up of: __________• in ____________ cells onlyFunctions: joins bacterial cells for DNA transfer adhesion
C. Bacterial Surface CoatingGlycocalyx• external to the cell wall• Made of sugars and/or proteinsFUNCTIONS ________________ ________________ ________________
Gram Negative Cell Wall Consists of: outer membrane with lipopolysaccharide thin shell of peptidoglycan periplasmic space inner membrane LPS endotoxin Lose crystal violet and may function as stain _______ from receptors and blocking _________ . immune response Protective structure while contains ________ providing some flexibility proteins in upper layer and sensitivity to lysis
The Gram Stain• Differential stain Gram-negative Gram-positive• Important basis of bacterial classification and identification• Practical aid in diagnosing infection and guiding drug treatment
Atypical Cell Walls• Some bacteria lack typical cell wall structure Mycobacterium and Nocardia Gram-positive cell wall structure with lipid ____________. • basis for ____________________• Some have no cell wall Mycoplasma cell wall is stabilized by ___________
Cytoplasm• dense gelatinous solution of sugars, amino acids, & salts• 70-80% water• serves as solvent for materials used in all cell functions
Chromosome Plasmids• single, circular, • double-stranded DNA molecule• contains all the genetic information required by a cell• DNA is tightly coiled around a protein
Inclusions & Ribosomes Granules • intracellular storage• prokaryotic differ from bodies eukaryotic ribosomes in size & number of proteins Examples: Glycogen• site of ________ synthesis gas vesicles carboxysomes• all cells have ribosomes Polyphosphate granules
Endospores• Resting cells• Resistant to heat, radiation & chemicals• Examples: _________, ____________________: Endospore formation___________: Return to vegetative state
Endospores• resistance linked to high levels of calcium & some acids• longevity verges on immortality• Control: pressurized steam at 120oC for 20-30 minutes
Part II. Classification of BacteriaLearning outcomesStudents should be able to:• Understand the basic principles of microbial classification systems.• Be familiar with structural and biological characteristics to classify bacteria
Interrelated areas of taxonomy:• Classification – the arrangement of organisms into taxonomic groups on the basis of similarities or relationships.• ________________ - naming an organism by international rules according to its characteristics.• Identification (1) to isolate and distinguish desirable organisms from undesirable ones; (2) To verify the authenticity or special properties of a culture, or in a clinical setting; (3) To isolate and identify the causative agent of a disease
Classification SystemsNumerical Taxonomy• the computer clusters different strains based on the frequency with which they share traits.Phylogenetic Classification System • Groups reflect genetic similarity and evolutionary relatednessPhenetic/Phenotypic Classification System • Groups are based on convenient, observable characteristics.
Levels of Classification• Kingdom (not used by most bacteriologists)• Phylum/Division• Class• Order• Family• Genus (plural: Genera)• Species (both singular & plural)
Species: Classic definition: A collection of microbial strains that share many properties and differ significantly from other groups of strains. Species are identified by comparison with known “type strains” -- well-characterized pure cultures - references for the identification of unknowns.
Strain: • A population of microbes descended from a single individual or pure culture. • Different strains represent genetic variability within a species. _________: Strains that differ in biochemical or physiological differences. _________: Strains that vary in morphology. _________: Strains that vary in their antigenic properties
NomenclatureScientific name (Systematic Name) • Species name is never abbreviated. • A genus name may be used alone to indicate a genus group. • A species name is never used alone. • Common or descriptive names (trivial names)
Bergey’s Manual of Systematic Bacteriology• main resource for determining the identity of bacteria species, utilizing every characterizing aspect.• Use successive "key" features to narrow down identification• Primary emphasis is phylogenetic, not phenetic
Part III. Physical and Nutritional Growth Requirements of Bacteria• Identify the growth requirements of bacteria• Appreciate the importance of the physiological and nutritional requirements for bacterial growth
PHYSIOLOGY study of vital life processes of organisms & how these processes normally function in living organismsNUTRITIONAL REQUIREMENTSsubstances required for energy generation and cellular biosynthesis.chemicals and elements of the environment that are utilized for bacterial growth
Table 1. Major elements, their sources and functions in bacterial cells. % ofElement dry Source Function weight organic compounds orCarbon 50 CO2 Main constituent of cellular material H2O, organic compounds, Constituent of cell material and cell water; O2 is electron acceptor inOxygen 20 CO2, and O2 aerobic respiration NH3, NO3, organicNitrogen 14 Constituent of amino acids, nucleic acids nucleotides, and coenzymes compounds, N2 H2O, organic compounds,Hydrogen 8 Main constituent of organic compounds and cell water H2 inorganic phosphates Constituent of nucleic acids, nucleotides, phospholipids, LPS, teichoicPhosphorus 3 (PO4) acids SO4, H2S, So, organicSulfur 1 Constituent of cysteine, methionine, glutathione, several coenzymes sulfur compoundsPotassium 1 Potassium salts Main cellular inorganic cation and cofactor for certain enzymesMagnesium 0.5 Magnesium salts Inorganic cellular cation, cofactor for certain enzymatic reactions Inorganic cellular cation, cofactor for certain enzymes and a component ofCalcium 0.5 Calcium salts endospores Component of cytochromes and certain nonheme iron-proteins and aIron 0.2 Iron salts cofactor for some enzymatic reactions
TYPES OF ORGANISMS BASED ON PHYSIOLOGIC REQUIREMENTS: Nutritional type Energy sourcePhototrophC Inorganic chemicals Organic chemicalsAutotrophHeterotropha
Metabolic Diversity Among Organisms Nutritional Energy Carbon Example type source sourcePhotoauto- Light CO2 Oxygenic:troph ______________ Anoxygenic: ______________Photohetero- Light Green, purpletroph nonsulfur bacteria.Chemoauto- Chemical CO2 Iron-oxidizing, sulfur,troph hydrogen, nitrifying bacteria.Chemohetero- Chemical Most bacteria,troph fermentative bacteria, animals, protozoa, fungi.
Growth Factors• are essential substances that the organism is unable to synthesize• required in small amounts for biosynthesis CATEGORIES: Purines and pyrimidines Amino acids Vitamins
Water• the solvent in which the molecules of life are dissolved• Supply depends on: relative humidity and water activity (Aw).• Aw = affected by the presence of solutes that are dissolved in the water.• The higher the solute concentration of a substance, the lower is the Aw and vice-versa.
Water and Salinity• SALT: only common solute in nature that occurs over a wide concentration range• ____________: microorganisms that require some NaCl for growth. • Mild halophiles • Moderate halophiles • Extreme halophiles• _____________ = grows at moderate salt concentrations, even though they grow best in the absence of NaCl.• Xerophiles = organisms which live in ___________ .
Movement across membranes:_________diffusion :• Movement of a solute from an area of high concentration to an area of low concentration_________ diffusion :• Solute combines with transporter protein in membrane
Movement Across MembranesOsmosis – Mov’t of H2O across a selectively permeable membrane from an area of ↑ H2O conc’n to an area of ↓ H2O.Osmotic pressure – pressure req’d. to stop H2O mov’t across the membrane. Figure 4.18a
pH REQUIREMENT the acidity or alkalinity of a solution.• ACIDOPHILE• NEUTROPHILE• ALKALIPHILE
Nutritional and PhysicalRequirements for Bacteria Growth.• Major and trace elements• Carbon and energy sources• Growth factors• Oxygen, Carbon dioxide• Temperature• Water / Moisture• pH requirement
Bacterial Growth, Genetics andControl of Bacterial Growth
Part I: Bacterial Growth• Describe the different phases of the bacterial growth curve• Choose the appropriate culture media for bacterial growth• Synthesize the importance of the processes involved in culturing bacteria
Bacterial GrowthGrowth = an orderly increase in the quantity of cellular constituents.____________ = asexual reproduction. increase in cell mass & number of ribosomes duplication of bacterial chromosome cell wall & plasma membrane synthesis chromosome partitioning & septum formation cell division.
Generation Time the time it takes for an organism todouble its number time required for a cell to divideIf a single bacterium reproduce every 20minutes, how many would there be in 2 hrs?If S. aureus has a generation time of 60minutes, how many cells would be in 7 hrs?
A. Lag phase Organism absorbs nutrients, synthesizes enzymes & prepares for reproductionA. __________ cellular reproductive stage; number doubles with each generation timeB. Plateau phase organisms maintain their greatest population densityC. ___________ rapid decline in cells until there is complete cessation of reproduction.
Culture Media• Nutrients for microbial growth• Inoculum: Microbes introduced into medium• Culture: Microbes growing in/on a medium • ___________: Contains only one species or strain • _________: A population of cells arising from a single cell or spore.
Types of Culture Media According to Consistency: Liquid media Semi-solid Solid mediaAccording to Composition: Synthetic medium Non-synthetic medium
According to Function/Purpose : General purpose Enriched Selective Differential Anaerobic growth Specimen transport Assay Enumeration
Types of Colonies• Mucoid colony• Smooth colony• Rough colony
Measurement of Cell Numbers1. Direct microscopic counts Dead cells cannot be distinguished from living ones. Samples must be concentrated by centrifugation or filtration to increase sensitivity.2. Electronic counting chambers Count numbers and measure size distribution of cells.3. Indirect viable cell counts spreading a sample of culture on a nutrient agar surface. On a suitable medium, each viable unit grows and forms a colony.
Part II: BACTERIAL GENETICSEach student will be able to:• Describe the characteristics of a bacterial genome;• Identify mechanisms of gene transfer;• Recognize the great clinical importance of the ability of bacteria to transfer genes, especially genes for antibiotic resistance, to other bacteria both within and between species.
The Bacterial GenomeThe Chromosome• single, long piece of circular, double- stranded DNA• Carry all of the essential genes and many nonessential genes of the bacterium• Contain 2000 to 4000 genes
The Bacterial GenomePlasmids• Small DNA circles• Replicate independently (1 or more)• Genes for toxins, proteins that promote transfer
GENETIC TRANSFER•____________ Gene Transfer – When organisms replicate their genomes and provide copies to descendants•____________ Gene Transfer – Acquisition of genes from other microbes of the same generation, – can be a different species, or even a different genus than the donor.
HORIZONTAL GENE TRANSFER 1) Conjugation 1) Transformation 1) Transduction
BACTERIOPHAGE (phage)• a virus that replicates inside a bacterial cell• Consists of a nucleic acid encapsulated in a protective protein coat• DNA or RNA, single/double stranded, 3000-200,000 bases
LYSOGENIC CONVERSION LYSOGENY Cells are immune to reinfxn by same phage. Cells may exhibit new properties. Allows phage to take a bit of the adjacent bacterial DNA
GENETIC VARIATIONA. _____________ any change in the DNA base sequenceB. MOBILE GENETIC ELEMENTS ______________ = DNA segments that have the ability to move from place to place on the chromosome and into and out of plasmids Probably responsible for most of the genetic variability & the spread of antibiotic resistance genes
Part III Control of Microbial Growth• Discuss concepts, principles & significance of infection control & laboratory safety• Discuss various of sterilization & disinfection methods with emphasis on temperature, time, principles/ mechanism involved, advantages & disadvantages
TERMINOLOGIES: Sterilization: Killing or removing ________________ in a material or an object. Commercial Sterilization: Heat treatment that kills endospores of _______________, the causative agent of botulism. _____________: Reducing the number of pathogenic microbes to the point where they no longer cause diseases.
Disinfectant: Applied to ______________Antiseptic: Applied to ______________Degerming: Mechanical removal of most microbes in a limited area._________: Use of chemical agent on food- handling equipment to meet public health standards and minimize chances of diseasetransmission.
Sepsis: Indicates bacterial contamination.Sepsis________: Absence of significant contamination.Aseptic techniques: prevent contamination of techniques surgical instruments, medical personnel, and the patient during surgery.Bacteriostatic Agent: An agent that ________ Agent the growth of bacteria, but does not necessarily kill them.Germicide: An agent that kills certain microbes.Germicide
Factors influence the effectiveness ofantimicrobial treatment:1. Number of Microbes2. Type of Microbes3. Environmental influences4. Time of Exposure
Physical Methods of Microbial Control:A. HEAT: Kills microorganisms by _____________ ________________________________. Thermal Death Point: Lowest T° at which all of the microbes in a liquid suspension will be killed in 10 minutes. Thermal Death Time: Minimal length of time in which all bacteria will be killed at a given T° . Decimal Reduction Time: Time (mins.) at which 90% of bacteria at a given T° will be killed.
A.1. Moist Heat: BOILING: Heat to 100oC or more. Kills vegetative forms of bacteria, most viruses, fungi (and spores) within 10 mins or less. Endospores & some viruses are not easily destroyed.
AUTOCLAVE: Chamber which is filled with hot steam under pressure. steam T° reaches ___oC at ___ psi for ___ minutes.PASTEURIZATION: reduces microbes responsible for spoilage of beverages. Classic Method of Pasteurization High T° Short Time Pasteurization Ultra High T° Pasteurization
A.2. Dry Heat: Kills by ____________ effects. Direct Flaming Incineration Hot Air SterilizationB. Filtration: Removal of microbes by passage of a liquid or gas through a screen-like material with small pores. High Efficiency Particulate Air Filters (HEPA) Membrane Filters
C. Low Temperature REFRIGERATION: 0 to 7oC. Reduces metabolic rate of most microbes so they cannot reproduce or produce toxins. FREEZING: below 0oC. Flash Freezing Slow Freezing
D. ___________: without water, microbes cannot grow or reproduce, but some may remain viable for years.E. Osmotic Pressure: increased concentrations Pressure of salt & sugar in substances can increase osmotic pressure & create a ____________ environment. ________________: As water leaves the cell, plasma membrane shrinks away from cell wall. Cell may not die, but usually stops growing.
F. Radiation: ________________ Radiation:• Dislodge electrons from atoms and form ions.• Cause mutations in DNA and produce peroxides.• Sterilize pharmaceuticals & disposable medical supplies. ________________ Radiation:• Damages DNA by producing thymine dimers, which cause mutations.• Used to disinfect ORs, nurseries, cafeterias.
Microwave Radiation: Heat is absorbed by water molecules. May kill vegetative cells in moist foods. Bacterial endospores are not damaged by microwave radiation. Solid foods are unevenly penetrated by microwaves. Trichinosis outbreaks have been associated with pork cooked in microwaves.
Chemical Methods of Microbial ControlA. PHENOLS & PHENOLICS: Phenol = Rarely used today because it is a skin irritant and has strong odor. Phenolics = chemical derivatives of phenol Cresols Biphenols Triclosan.
B. HALOGENS: Effective alone or in compounds. Iodine: Tincture of iodine Iodophors Chlorine: When mixed in water forms ________________: Cl2 + H2O ------> H+ + Cl- + HOCl to disinfect drinking water, pools, and sewage. Chlorine is easily inactivated by organic materials.
C. ALCOHOLS: Kill bacteria, fungi, but not endospores or naked viruses. Used to mechanically wipe microbes off skin before injections or blood drawing. Not good for open wounds, because cause proteins to coagulate.
D. HEAVY METALS: Oligodynamic actionSilver: 1% AgNO3Mercury: merthiolate , mercurochromeCopper Copper sulfateSeleniumZinc Zinc chloride, Zinc oxide
E. QUATERNARY NH4 COMPOUNDS (Quats) : surface active agents; cationic detergents. Effective against GPB, less effective against GNB bacteria. Also destroy fungi, amoebas, and enveloped viruses.Advantages: Strong antimicrobial action, colorless, odorless, stable, and nontoxic.Diasadvantages: Organic matter interferes with effectiveness. Neutralized by soaps & anionic detergents.
F. ALDEHYDES:Formaldehyde gas Excellent disinfectant. ____________ (37% aqueous solution) Irritates mucous membranes, strong odor.Glutaraldehyde Less irritating, more effective than formaldehyde. sterilizing agent. Commonly used to disinfect hospital instruments.
G. Gaseous Sterilizers: Denature proteins, by replacing functional groups with alkyl groups.Ethylene Oxide: Kills all microbes & endospores Exposure Time : ____________. Toxic and explosive in pure form.
H. Peroxygens (Oxidizing Agents): Oxidize cellular components of treated microbes. Disrupt membranes and proteins. Ozone:• Highly reactive form of ________• Used along with chlorine to disinfect water.• More effective killing agent than chlorine, but less stable and more expensive.• Made by exposing oxygen to __________ or ____________.
Hydrogen Peroxide: Used as an antiseptic. Effective in disinfection of inanimate objects. Sporicidal at higher temperatures. Benzoyl Peroxide: Used in acne medications. Peracetic Acid: effective liquid sporicide Sterilant does not leave toxic residues.
LEARNING OBJECTIVES:Each student is able to:• Describe the general principles for specimen handling;• Select the appropriate specimen collection, transport and processing method;• Relate the significance of specimen collection and handling in the pre-analytical steps in the accurate diagnosis of infectious diseases.
Specimen Collection: General GuidelinesObtain specimen before treatmentCollect material from the appropriate siteCollect specimen during acute stage of illnessCollect specimen properly & asepticallyCollect sufficient quantityLabel all specimenTransport the specimen immediatelySpecimen should be accompanied with arequest form
MAJOR APPROACHES TO AVOID CONTAMINATION• Disinfectant the area• Bypass area with normal flora• Culture for specific pathogen only• Quantitate by colony counting• Sufficient quantity of specimen• Prompt delivery to the laboratory
Successful Recovery ofEtiologic Agents Depends on:Advanced planningCollection of appropriate andadequate specimenCorrect packaging and rapid transportto the laboratoryAbility of the laboratory to accuratelyperform the diagnostic tests
BLOODGENERAL CONSIDERATIONS: Collect blood during febrile episode During a chill A (+) Blood culture depends on the pathogenic process of the organism. Obtain 2-3 simultaneous blood cultures from different sites per 24 hours. A single (-) blood culture does not rule out bacteremia. Collect blood aseptically.
VOLUME :Young Children: ______in ____ml brothAdults: __________in 50ml brothIf Px is on penicillin, administerpenicillinase Not effective against methicillin, cloxacillin, nafcillinHold for 2 weeks before reporting as (-)
BLOOD CULTURES CAN BE OBTAINED IN THE FOLLOWING CONDITIONS : In acute illnesses Fever of unknown origin In cases of patients with acute infective endocarditis In cases of suspected bacterial endocarditis
Indications of a (+) Blood Culture:TurbidityHemolysisColony or pellicle formationPresence of gas or bubble
MEDIA USED:Trypticase Soy Broth Columbia Broth Brain–Heart Infusion Brucella Broth Castaneda medium Thioglycollate BrothANTICOAGULANT:Sodium Polyanethol Sulfonate Neutralizes bactericidal effect of serum Prevents phagocytosis Inactivates some antimicrobial agents
COMPONENT OF BLOOD CULTURE BROTH1 % Gelatin enhance growth of Neisseria meningitidis0.1 % Bacto-agar enhance growth of anaerobic organism anticoagulant0.025 % Sodium Polyanetholsulfonate inhibit activity of complement & lysosyme prevents phagocytosis inactivates therapeutic concentration of aminoglycosides
Blood Culture Technique Find the puncture site, if arm veinis selected, apply a tourniquet Clean the skin with iodine, followedby 70% alcohol Clean the rubber stopper withalcohol swab Collect the required amount ofblood
PROCESSING OF BLOOD CULTURES Inoculate blood in broth in a ratio of 1:10 Incubation Temperature: 350C - 370C Incubation Time: 7 days Subculture on BAP & CAP: after 14-17 hrs, 3 days, 5 days, 7 days
BLOOD COMPONENTS• Organisms found in fatal transfusion reactions are psychrophilic.• Gram (-) bacilli• PLATELET
RESPIRATORY TRACT SPECIMENSA. Upper Respirator yTract Nasopharyngeal swab Throat swab POSSIBLE PATHOGENS OF THE URT : S. pneumoniae, S. pyogenes S.aureus C. diphtheriae Haemophilus influenzae Klebsiella spp. Other Enterobacteriacae
Throat Swabbing MEDIA for URT specimen: H. influenzae: CAP or BAP w/ Staph N. meningitidis: CAP/Thayer Martin B. pertussis: Charcoal CephalexinCulture must include anaerobic conditions for β streptococcus.
Collection & Transport of SputumUse a dry, wide-mouthed bottleCollect sample early morningTransport ASAPmay be refrigerated but examinedw/in 2–3 hrs
PROCEDURE: Make a direct smear Do concentration technique Culture
Suitability of Sputum for CultureSputum Classification Based on WBCs & Squamous Epithelial Cell DensitiesCell numbers per x 100 (low power) fieldGroup WBCs Epithelial Cells 6 <25 <5 5 >25 <10 4 >25 10-25 3 >25 >25 2 10-25 >25 1 <10 >25
URINEContainer: Sterile wide mouth glass or plastic jar with tight-fitting lids.Method: Suprapubic aspiration Cystoscopy or catherization Clean - catch midstreamCollection: Early morningProcessing: Within 2 hrs after collection
GS of uncentrifuged urinePROCEDURE: Inoculate spx in BAP, EMB, Mac using a calibrated loop Incubate overnight Observe for 48 hrs before reporting negative
Actual # of Calibrationcolonies x of loop = POUR PLATE METHOD: • 1:100 dilution of urine w/ sterile H2O • incubate for 18–24 hrs • colony count x 100 = bacteria / mL
POSSIBLE PATHOGENS Enterobacteriacae S. aureus S. saprophyticus Enterococcus spp. Yeast
CEREBROSPINAL FLUIDThe specimen should be taken beforeantibiotics are administered.Antigen tests may give a specificdiagnosis even after antibiotic treatment.CSF should always be taken frompatients with suspected purulentmeningitis, regardless of antibiotictreatment.
PROCEDURE: Centrifuge CSF; Make a smear for GS and india ink Culture CSF on recommended mediaMEDIA: Trypticase Soy Broth / thioglycollate BAP for Gram (+) cocci CAP for Gram (-) cocci EMB/Mac for Gram (-) bacilli
• CSF for bacterial culture: Incubate for not > 12 hrs OR; Stand at room T° not > 1 hour DO NOT REFRIGERATE!• CSF for viral culture: Refrigerate immediately If held for more than 24 hrs, freeze specimen at –700C
Order of Tests for Scanty Sample: 1) Culture 2) Bacterial Antigen Detection 3) Gram Stain 4) Cell Count 5) Other clinical pathology tests
MAJOR PATHOGENSStreptococcus pneumoniaeHaemophilus influenzae type bNeisseria meningitidis
STOOLIdeal specimen: Freshly collected stool on the early stage of a disease Rectal swab may be usedAmount: _______________Container: Clean, wide mouth with lid .Transport time : 2 hours after collectionTransport medium : _____________________
PROCEDURE: Put rectal swab in enrichment broth or transport medium GS is NOT usually done but helps in identifying etiologic agents • Gram + cocci in clusters • Gram – comma-shaped bacilli • Gram + bacilli in large numbers • Gram – bacilli
Stool Culture : 1st day Inoculate the spx and incubate it overnight MEDIA: • Differential • Selective • Enrichment
Stool Culture : 2nd day Check diff’l. media for LFs & NLFs With growth: Subculture and do biochemical tests No growth: inoculate culture from enrichment media into EMB or Mac
Stool Culture : 3rd day Note patterns of biochemical reactions If suggestive of Salmonella, Shigella, Vibrio, DO serological typing If growth occurs after doing step 3 (2 nd day), DO biochemical test Incubate overnight and perform step 1 of day 3
COLLECTION1. Discharge / Fluids : best to aspirate a. Dry wound – moisten swab w/ NSS before collecting b. Skin lesion – remove crust of pustule/ vesicle cap then gently swab lesion Punch biopsy Tzanck smear
c. Endocervical : use swabd. Urethra : use swab or scrape mucosa of anterior urethrae. Anorectal: insert swab about 4-5 cm. into the anal canal
Possible Pathogens InAnogenital Specimen: T. pallidum N. gonorrheae C. trachomatis C. albicans G. vaginalis
C. Differential Stains: GRAM STAINING Basis: thickness & chemical composition of the cell wall Chemical Gram + Gram – Crystal violet Purple Purple Gram’s Iodine Purple Purple Acetone alcohol Purple Colorless Safranin
GENERAL RULES:• All cocci are gram (+) EXCEPT Neisseria, Branhamella/Moraxella, & Veilonella.• All bacilli are gram (-) EXCEPT Mycobacterium, Corynebacterium, Lactobacillus, Listeria, Clostridium, Bacillus, Erysipelothrix, and Nocardia.• All spiral bacteria are gram (-) when stained.
THEORIES OF GRAM STAINING GRAM (+) GRAM (-)Mg-RNA MgRNA + CV - I2 MgRNA isTheory complex = insoluble absent compd.Benian Less permeable cell Permeable cellTheory walls wallsStearn & isoelectric pt isoelectric pt Stearn making them acidic; making them Theory bind well w/ basic basic; bind well dye w/ acidic dye Lipid lipid content but lipid content;content teichoic acid no teichoic acid
ERRORS IN GRAM STAINING Gram (+) becomes Gram (-) becomes Gram (-) Gram (+)Using acidic Gram’s Inadequateiodine decolorizationAging, dying, autolysis Thick smearsRemoval of MgRNA w/precipitation from bilesalts in mediaoverdecolorization
Non–Stain System to Determine True Gram Stain Reaction• L – alanine, 4 – nitroanilite (LANA) • Turns yellow_________ when touched to a colony of gram negative bacteria• 3% KOH • Formation of string–like material indicates _____________organisms
ACID FAST STAINING• MYCOLIC ACID – responsible for the acid fastness of mycobacteria GENERAL RULE: All organisms are non-acid fast EXCEPT: • Mycobacterium • Nocardia spp. • Corynebacterium spp.
Chemicals AFB NON- AFBCarbolfuchsin Red RedHeat Red RedAcid-alcohol Red ColorlessMethyleneBlue
Ways To Facilitate AFB staining: Using steam Increase concentration of phenol & basic fuchsin Prolonged contact time Adding wetting agent
TYPES OF ACID FAST STAINING1. ZIEHL – NEELSEN METHOD uses Heat (hot stain)2. KINYOUN METHOD Uses wetting agents3. PAPPENHEIM’S Differentiates M. tb from M. lacticala and M. smegmatis4. BAUMGARTEN Differentiates blue M. tb & red M. leprae
Physical Form:SOLID MEDIA LIQUEFIABLE NON – LIQUEFIABLEDISTRIBUTION•TUBED•PLATED – sterile petri dish
Functional Type:GENERAL PURPOSE MEDIA• Designed to grow a broad spectrum of microbes• Examples: – Nutrient agar – Nutrient broth – Trypticase Soy Agar
Functional Type: Functional Type:ENRICHED ENRICHMENT• with substances that • Similar w/ enriched allow growth of except that the fastidious organisms media is liquid• blood, serum, Hgb, vitamins, AA
Functional Type:SELECTIVE• contains agents that inhibit growth of some microbes• permits preliminary identification of genus or spp.Examples: Mannitol Salt Agar Hektoen Enteric Agar
SELECTIVE MEDIUM USES AGENT Mueller Potassium Isolation of Tellurite tellurite C.diphtheriaeEnterococcus Na azide Isolation of fecalfaecalis broth Tetrazolium EnterococciPhenylethanol Phenylethanol Isolation of agar chloride Staph & StrepTomato juice Tomato juice, Isolation of agar acid Lactobacilli Isolation of MacConkey Bile, crystal violet Gram (-) enteric
SELECTIVE MEDIUM USES AGENT Isolation of Bile, citrate, SSA Salmonella & brilliant green dye Shigella Isolation &Lowenstein – Malachite green maintenance ofJensen agar dye MycobacteriaSabouraud’s Isolation of pH 5.6 agar fungi
Functional Type:DIFFERENTIAL• displays visible differences among microbesExamples: MacConkey agar Neutral red Spirit blue agar Spirit blue dye
Substance for DifferentiatesMEDIUM differentiation between Types of BAP Intact RBCs hemolysis Mannitol, phenol red, Species of MSA 7.5% NaCl Staphylococcus Bromthymol blue, acid Salmonella, fuchsin, salicin, citrate, HEA sucrose, thiosulfate, Shigella, other LFs & NLFs ferric ammonium, bile Fat-utilizing SBA Spirit blue dye,oil bacteria from those that do not
Substance for DifferentiatesMEDIUM differentiation betweenUrea Urea-hydrolyzing Urea, phenol redbroth bacteria SIM Thiosulfate, Fe H2S gas production Triple sugars, Fe, Sugar fermentation TSI & H2S production phenol red dye Enterobacter, Xylose, lysine, Fe, Escherichia, XLD thiosulfate, phenol Proteus, agar red Providencia, Salmonella, Shigella
Functional Type:MISCELLANEOUS1. ANAEROBIC GROWTH Thioglycollate broth Mannitol Salt Agar Lactose broth2. SPECIMEN TRANSPORT Stuart’s medium Amie’s medium
Functional Type:MISCELLANEOUSC. ASSAY • Mueller – Hinton agar • BAPD. ENUMERATION • Nutrient Agar • Chromocult Coliform Agar • Differential Coliform Agar
INOCULATION OF CULTURE MEDIAA. TUBED MEDIA LIQUID SEMI – SOLID SOLID
A. CARBOHYDRATE FERMENTATION TEST CULTURE MEDIA COMPOSITION Triple Sugar Iron Kligler’s Iron Agar Russel’s Double AgarTSI: Determines if a GNB utilizes glucose, lactose or sucrose fermentatively & forms H2S.
Triple Sugar Iron Agar• INDICATORS : A. Phenol Red • phenolsulfonphthalein (PSP) B. Ferrous Ammonium Sulfate • H2S indicator • (+) blackening of the medium
TSI REACTIONS: (Slant over Butt)K = alkaline; NC = No change; H2S = blackeningA = acidic; Gas = bubbles, in agar cracks in agar SLANT/BUTT COLOR SUGAR REACTION FERMENTEDK/K; K/NC RED/REDK/A RED/YELLOWA/A YELLOW/ YELLOWA/K YELLOW/RED
TSI : Carbohydrate Fermentation Test• Quality Control A/A Gas+: Escherichia coli K/A H2S+: Salmonella typhi K/NC or K/K: Pseudomonas aeruginosa
B. IMViC TESTTESTS: INDOLE METHYL RED VOGUES – PROSKAUER TEST CITRATEMEDIA USED: • Tryptophan Broth or SIM • MRVP or Clark & Lubs Dextrose Broth • Simmon’s Citrate Slant
INDOLE TESTQUALITY CONTROL:A.KOVAC’S method (+): E. coli (-): K. pneumoniaeB. EHRLICH’S method (+): Elizabethkingia meningoseptica (-): Paracoccus yeeii sp.nov (CDC group EO-2)
2. METHYL RED / VOGES-PROSKAUERDetermines an organism’s ability to… produce & maintain stable acid end products produce neutral end productsQUALITY CONTROL:METHYL RED VOGES-PROSKAUER (+): E. coli (+): E. cloacae (-): Enterobacter (-): E. coli cloacae
METHYL RED TEST FermentationGLUCOSE large amount of acidMETHODS:•MRVP medium•Buffered Peptone Glucose Broth
VOGES – PROSKAUER TESTGLUCOSE Acids & addfermentation acetoin α-naphthol and KOH Barritt’s method for GNB: •Solution A (α -naphthol) •Solution B (KOH)
3. CITRATE TEST utilize Sodium produce citrate & inorganic NH4 saltsMEDIUM: Simmon’s Citrate slantpH INDICATOR: Bromthymol Blue (+) prussian blue (-) yellow green
C. CATALASE• Differentiates Staphylococcus species from Streptococcus species catalase 30% H202 O2 + H2OMETHOD: • Place a drop of 30% H2O2 onto the slide • Mix in the inoculum.
D. OXIDASE TEST• To determine the presence of ____________________by oxidation of oxidase reagent to __________, a ____________-colored end product.• Detects the ability of organisms to oxidize aromatic amines in the presence of air.