<h5 id="61-historical-perspective">6.1 Historical Perspective</h5> <ul> <li>The development of microbiology began with the invention of the microscope in the mid-1600s. Anton van Leeuwenhoek, a Dutch merchant, made significant contributions to the field by observing microscopic organisms.</li> <li>In the late 1800s, Louis Pasteur and Robert Koch made groundbreaking discoveries in microbiology. Pasteur disproved the theory of spontaneous generation and postulated the germ theory of disease, while Koch proved the germ theory by isolating pure cultures of Bacilli and identifying their role in causing diseases.</li> <li>The period from the late 1800s to the early 1900s is referred to as the Golden Age of Microbiology, during which various causative agents responsible for microbial diseases were discovered.</li> <li>The use of solid media instead of liquid medium proved to be an important milestone in the development of bacteriology. Agar replaced gelatin as it provided solid support for microbiological media.</li> <li>Microorganisms can be categorized based on the means of obtaining their carbon and energy. Heterotrophs obtain their carbon from organic molecules, while autotrophs obtain their carbon from carbon dioxide. Autotrophs can be further divided into chemoautotrophs and photoautotrophs.</li> </ul> <p>======</p> <h5 id="62-nutritional-requirements-and-culture-media">6.2 Nutritional Requirements and Culture MEDIA</h5> <ul> <li>Microorganisms require various nutrients for energy production, growth, and multiplication, including macronutrients (C, O, H, N, S, P, K, Ca, Mg, Fe) and micronutrients (Mn, Zn, Co, Mo, Ni, Cu).</li> <li>Major components required for nutrient media are: a) Carbon source (glucose, lactose, sucrose, starch, glycogen, cellulose, etc.), b) Nitrogen, phosphorous, and sulfur sources (ammonium salts, urea, animal tissue extracts, amino acid mixtures, etc.), c) Growth factors (certain amino acids, purines, pyrimidines, vitamins), d) Anti-foams (olive oil, sunflower oil, silicones), and e) Water.</li> <li>Carbon source provides carbon, oxygen, and hydrogen atoms for the skeleton or backbone of all organic molecules.</li> <li>Nutrient media are essential as most micronutrients are already present with various regular media components and not supplied separately.</li> <li>The specific composition of a suitable medium depends on the particular microorganism species, requiring prior knowledge of its habitat and nutrient requirements.</li> </ul> <p>======</p> <h5 id="types-of-media">Types of media</h5> <ul> <li>Media can be classified into two types based on chemical composition:</li> </ul> <ol> <li> <p>Synthetic or chemically defined media: Contains known chemical components, used in research, prepared by mixing various components according to the requirements for experimental microorganisms. Examples include M9 media for E. coli and BG11 media for blue green algae.</p> </li> <li> <p>Complex media: Contains some constituents with unknown chemical composition, useful for meeting the nutritional requirements of various microorganisms. Undefined components include peptones, beef extract, and yeast extract. Examples include nutrient broth, tryptic soy broth, and Luria-Bertani (LB) medium.</p> </li> </ol> <ul> <li>Based on consistency, media can be of three types:</li> </ul> <ol> <li> <p>Liquid media or broth: Used for the growth of microorganisms without agar, after inoculation and incubation, the growth of cells becomes visible in the broth.</p> </li> <li> <p>Solid media: Required for surface cultivation of microorganisms, prepared by solidifying liquid media with the addition of 1.0-2.0% agar, used for making agar slants, slopes, and agar stabs.</p> </li> <li> <p>Semi-solid agar media: Prepared by adding 0.5% of agar in the media, used for promoting the growth of one organism and retarding the growth of others.</p> </li> </ol> <ul> <li>Based on application and functions, media can be classified into:</li> </ul> <ol> <li> <p>Selective media: Favors the growth of a particular type of microorganism, provides nutrients that increase the growth of a particular type of microorganism and suppresses all other microorganisms that are present in the culture.</p> </li> <li> <p>Differential media: Used to distinguish different groups of microorganisms on the basis of appearance on the medium as well as their biological characteristics.</p> </li> <li> <p>Enrichment media: Adjusts the nutritional environment to selectively enhance the growth of certain microorganisms within the mixed inoculum.</p> </li> </ol> <p>======</p> <h5 id="63-sterilisation-methods">6.3 Sterilisation Methods</h5> <ul> <li>Sterilisation is the process of killing/removing all microorganisms, including bacterial spores, while disinfection eliminates most pathogenic microorganisms excluding bacterial spores.</li> <li>Decontamination eliminates contaminating pathogenic microorganisms from surfaces and equipment using sterilisation or disinfection.</li> <li>Sanitisation reduces microbial population to safe levels applicable in public health systems.</li> <li>Sterilisation by heat is effective and rapid for heat-resistant articles, achieved through boiling, autoclaving, pasteurisation, and heating/flaming.</li> <li>Sterilisation by radiation uses ionising and non-ionising radiations, with UV rays useful for surface disinfection and electromagnetic rays for sterilising materials with high penetrative power.</li> <li>Sterilisation by filtration separates microbes based on size, used for heat-labile substances, and HEPA filters are 99.97% efficient for removing particles >0.3μm in diameter.</li> <li>Sterilisation/disinfection can be achieved using chemicals like alcohols, aldehydes, heavy metals, and hydrogen peroxide solution.</li> </ul> <p>======</p> <h5 id="64-pure-culture-techniques">6.4 Pure Culture Techniques</h5> <ul> <li>Bacteria can be cultured using liquid or solid media. Solid media are used for the isolation and long-term storage of bacteria, while liquid media are used for large-scale production.</li> <li>A pure culture is a culture consisting of a single strain of organism, obtained through methods like streak plating or pour plating.</li> <li>In streak plating, an inoculating loop is used to streak out a bacterial suspension or colony on a solid agar plate, creating progressively more dilute streaks. After incubation, visible bacterial colonies are formed from a single cell.</li> <li>The pour-plate method involves diluting the original microbial sample and pouring small volumes of each dilution onto a Petri plate. Melted medium with agar is then poured and mixed gently. After incubation, isolated cells grow into colonies.</li> <li>The spread-plate technique involves transferring a small volume of diluted bacterial mixture to the center of a solidified agar plate and spreading evenly with a sterile L-shaped glass spreader. After incubation, dispersed cells develop into isolated colonies.</li> </ul> <p>======</p> <h5 id="65-factors-affecting-microbial-growth">6.5 Factors Affecting Microbial Growth</h5> <ul> <li>Temperature: Microbial growth occurs in a range of 25-45°C, with optimal growth at 20-4</li> </ul> <p>======</p> <h5 id="66-the-growth-curve">6.6 The Growth Curve</h5> <ul> <li>The growth of cells in nutrient media depends on cell concentration and results in cellular reproduction. Unicellular organisms divide by binary fission, leading to an exponential increase in the number of cells.</li> <li>The growth of cultured microorganisms can be analyzed by plotting the logarithm of the number of viable cells versus the incubation time, resulting in a curve with four distinct phases: lag phase, exponential phase, stationary phase, and decline phase.</li> <li>During the exponential phase, microorganisms grow and divide at the maximal rate, doubling in number at regular intervals. The doubling time can be expressed as $t_d=0.693/μ$, where $μ$ is the specific growth rate.</li> <li>During the stationary phase, the total number of viable microorganisms remains constant due to a balance between cell division and cell death. Nutrient limitation can cause the population to enter the stationary phase.</li> <li>During the death phase, unfavorable environmental conditions, such as nutrient deprivation and accumulation of toxic wastes, lead to a decline in the number of viable cells. The death phase is often logarithmic, similar to the exponential phase.</li> </ul> <p>======</p>