BTE 102 Lecture 4 (Summer 2024).pptx.pdf

Lecture 4 BTE 102 Microbial World Course Instructor Nabiha Tasneem Khan Microbial Growth Microbial Growth Microbial growth is defined as the process of an increase in the number of microorganisms through cell division or replica...

Lecture 4 BTE 102 Microbial World Course Instructor Nabiha Tasneem Khan Microbial Growth Microbial Growth Microbial growth is defined as the process of an increase in the number of microorganisms through cell division or replication, resulting in an exponential increase in the population size under favorable conditions. Microbial growth is increase in number of cells, not cell size  The Requirements for Growth: Physical Requirements Temperature Most microorganisms grow well at the temperatures that human favors. Psychrophiles – optimum growth temperature of 15 °C Mesophiles – optimum growth temperature of 30– 40°C Thermophiles – optimum growth temperature of 50–60°C Temperature Psychrophiles Vs Psychrotrophs Psychrophiles are extremophilic bacteria or archaea which are cold-loving, having an optimal temperature for growth at about 15°C or lower, a maximal temperature for growth at about 20°C and a minimal temperature for growth at 0°C or lower. Psychrotrophs are cold-tolerant bacteria or archaea that have the ability to grow at low temperatures, but have optimal and maximal growth temperatures above 15 and 20°C, respectively.  The Requirements for Growth: Physical Requirements pH pH refers to the acidity or alkalinity of a solution Most bacteria grow best in a narrow pH range, between pH 6.5 and 7.5 ❑ Acidophiles – tolerant of acidity ❑ Alkaliphiles – tolerant of alkanity  The Requirements for Growth: Physical Requirements Osmotic Pressure Hypertonic environments, increase salt or sugar, cause plasmolysis Extreme or obligate halophiles require high osmotic pressure Facultative halophiles tolerate high osmotic pressure Most microorganisms, however, must be grown in a medium that is nearly isotonic  The Requirements for Growth: Chemical Requirements Carbon Structural organic molecules, energy source Half the dry weight of a typical bacterial cell is carbon Chemoheterotrophs use organic carbon sources Autotrophs use CO2  The Requirements for Growth: Chemical Requirements Nitrogen Nitrogen makes up about 14% of the dry weight of a bacterial cell. DNA, RNA and protein synthesis require nitrogen Most bacteria decompose proteins to get nitrogen Some bacteria use NH4+ or NO3- A few bacteria use N2 (nitrogen fixation) Nitrogen fixation is the process by which atmospheric nitrogen (N2) is converted into biologically usable forms, such as nitrates (NO3-) and ammonium (NH4+), that can be taken up by plants and used for growth and reproduction.  The Requirements for Growth: Chemical Requirements Sulfur In amino acids, thiamine, biotin Most bacteria decompose sulfur-containing amino acids Some bacteria use SO42- or H2S Phosphorus In DNA, RNA, ATP, and membranes PO43- is a source of phosphorus  The Requirements for Growth: Chemical Requirements Trace Elements (iron, copper, molybdenum, and zinc etc.) Inorganic elements required in small amounts Usually as enzyme cofactors  The Requirements for Growth: Chemical Requirements Organic Growth Factors Organic compounds obtained from the environment Vitamins, amino acids, purines, pyrimidines Vitamins and other organic growth factors are provided by meat extracts or yeast extracts. The soluble vitamins and minerals from the meats or yeasts are dissolved in the extracting water, which is then evaporated so that these factors are concentrated. (These extracts also supplement the organic nitrogen and carbon compounds.) Culture Media Culture Medium: Nutrients prepared for microbial growth Inoculum: Introduction of microbes into medium Culture: Microbes growing in/on culture medium What criteria must the culture medium meet? It must contain the right nutrients It should also contain sufficient moisture Properly adjusted pH Suitable level of oxygen (?!) The medium must be sterile Incubation at the proper temperature. Agar Complex polysaccharide Used as solidifying agent for culture media in Petri plates, slants, and deeps Generally not metabolized by microbes Liquefies at 100°C Solidifies ~40°C Culture Media Chemically defined media: autotrophic bacteria Complex Media: for heterotrophic bacteria and fungi Extracts and digests of yeasts, meat, or plants The exact chemical composition varies from batch to batch In complex media, the energy, carbon, nitrogen, and sulfur requirements of the growing microorganisms are provided primarily by protein. Partial digestion by acids or enzymes reduces proteins to shorter chains of amino acids called peptones. If a complex medium is in liquid form, it is called nutrient broth. When agar is added, it is called nutrient agar. Anaerobic Culture Methods Media for growth of anaerobic bacteria: Anaerobic bacteria require absence of oxygen to grow, hence they must be placed in a special medium called a reducing medium. These media contain ingredients, such as sodium thioglycolate, that chemically combine with dissolved oxygen and deplete the oxygen in the culture medium.  Many clinical laboratories have special carbon dioxide incubators Desired CO2 levels are maintained by electronic controls. High CO2 levels are also obtained with simple candle jars Microbes that grow better at high CO2 concentrations are called capnophiles. The low-oxygen, high- CO2 conditions resemble those found in the intestinal tract, respiratory tract, and other body tissues where pathogenic bacteria grow. Anaerobic Culture Anaerobic chamber Selective Media Designed to suppress the growth of unwanted bacteria and encourage the growth of the desired microbes Bismuth sulfite agar is one medium used to isolate the typhoid bacterium, the gram-negative Salmonella typhi from feces. Bismuth sulfite inhibits gram positive bacteria and most gram-negative intestinal bacteria (other than S. typhi), as well. Eosin-methylene blue (EMB) agar is selective for gram-negative bacteria against gram-positive bacteria. In addition, EMB agar is useful in isolation and differentiation of the various gram-negative bacilli and enteric bacilli, generally known as coliforms and fecal coliforms respectively. Enrichment Media Encourages growth of desired microbe Enrichment media are typically liquid in their consistency. Selective media is a term used to describe agar-based media that fulfil the same function. Assume a soil sample contains a few phenol-degrading bacteria and thousands of other bacteria - Inoculate phenol-containing culture medium with the soil and incubate Only phenol-metabolizing bacteria will be growing Differential Media Differential media, also known as indicator media, is a type of media (usually of a solid or semi-solid consistency) used to distinguish between bacterial cultures based on their biochemical properties. Isolation of Pure Culture A pure culture contains only one species or strain A colony is a cluster of bacteria growing together. A colony is a population of cells arising from a single cell or spore or from a group of attached cells. The colony forming unit (CFU) is a measure of viable colonogenic cell numbers in CFU/mL. CFU/ml is equal to the total number of colonies multiplied by the dilution factor and this is divided by the volume of the culture plate. CFU/ml = (Number of colonies*dilution factor) / volume of culture plate. https://youtu.be/EjnQ3peWRek?t=368 Streak Plate Streak Plate Method Procedure Sterilize all the instruments, flasks and media that are required for the streaking procedure. Clean your work area using a disinfectant to minimize any contamination. Set up the bunsen burner in your work area carefully. Wash your hands with an antiseptic solution before handling any microbial solution. Label the petri dish with all important information, such as your name, date, media used and the culture being inoculated. To pick up the sample, you can use either a metal loop or disposable plastic loops. A loopful of sample is streaked on the first quadrant in a back-and-forth motion on the agar plate. Sterilize the loop by heating it in the bunsen burner if using a metal loop. Streak the other three quadrants by a similar method. Close the lid of the plate after streaking, and store the dish upside down in an incubator with optimal temperature. Spread plate technique Spread Plate Method It is used for evenly spreading cells to ensure growth of the isolated separate colonies. Further, it can be used for serial dilutions. The spread plate method is used for enrichment, screening and selection of microorganisms. Onto the agar media, with the help of a sterile spreader, inoculate the clinical specimen where we spread the bacteria gently on the whole culture media surface. This is done by rotating the plate while spreading it backwards and forward. Refrain from allowing the spreader to touch the edges of the plate Substitute the lid and ensure the plate is standing in an upright position for drying (10-12 minutes) Now incubate the spread agar plate at the optimum temperature with the lid at the base (inverted) The biggest advantage of a spread plate method is that the morphology of the isolated bacteria can be seen vividly. The only disadvantage is that sometimes fungal colonies may grow. Pour Plate method The pour plate method is a plating technique that is commonly used for obligate anaerobic bacteria. In this method, the liquid sample is poured into the petri dish before the solidification of the agar medium. After solidification, colonies grow both inside and on the surface of the medium. However, the colonies growing inside the medium are confluent; those on the surface are used for viable counting. Pour Plate Method Sterilize all the instruments, flasks, and media that are required for the streaking procedure. Clean your work area using a disinfectant to minimize any contamination. Set up the bunsen burner in your work area carefully. Wash your hands with an antiseptic solution before handling any microbial solution. Label the petri dish with all important information, such as your name, date, media used, and the culture being inoculated. Sample Preparation: If the sample is in semisolid or solid form, suspend it in sterile water or broth to prepare a liquid solution. If the sample is already in liquid form, prepare serial dilutions of the sample to lessen the load of microbial colonies in the range of 20-300 CFU/ml. For inoculation, open the lids of the Petri dishes and pour 1 ml of the diluted sample. Take the molten agar, heat it a little and pour around 15-18 ml of it onto the sample. Keep in mind that the agar should not be either too hot or too cold. Close the lid of the dish and swirl it slowly. Another method for inoculation is to mix the diluted sample in the agar medium, mix it gently, and then pour it into the petri dish. However, this method is less commonly used. Let the plate solidify. Invert the plate and incubate it at an optimal temperature (usually 37℃) for 24-48 hours. Preserving Bacteria Culture Cryopreservation and lyophilization (Freeze-drying) both are well-known methods for long-term preservation of microbial cultures. Use of ultra-low temperature with different cryoprotectants (Ethylene glycol, dimethyl sulfoxide (DMSO), glycerol) for preservation of microorganism is a traditional practice in every microbiological laboratory. Bacteria can be stored for months and years if they are stored at -80 °C and in a high percentage of glycerol. Bacterial Growth Bacteria are unicellular organisms that tend to reproduce asexually by the means of binary fission. Bacterial growth is the increase in the number of bacterial cells rather than the increase in their cell size. The growth of these bacterial cells takes place in an exponential manner, i.e., one cell divides into 2, then 4, then 8, 16, 32 and so on. Binary fission is a form of asexual reproduction in single-celled organisms by which one cell divides into two cells of the same size. Bacterial Growth The time taken for a bacterial cell to double is called generation time. The generation time varies among different species of bacteria based on the environmental conditions they grow in. Clostridium perfringens is the fastest growing bacteria that has a generation time of 10 minutes while Escherichia coli has a doubling time of 20 minutes. Mycobacterium tuberculosis is one of the slowest growing bacteria, taking about 12 to 16 hours to double. Binary Fission Growth Curve In a closed system with enough nutrients, a bacteria shows a predictable growth pattern that is the bacterial growth curve. Phases of the Bacterial Growth Curve Upon inoculation into a new nutrient medium, there are four basic phases of growth: ❖ Lag phase ❖ Log phase ❖ Stationary phase ❖ Death phase Bacterial Growth Curve Lag Phase The bacteria upon introduction into the nutrient medium take some time to adapt to the new environment. In this phase, the bacteria does not reproduce but prepares itself for reproduction. The cells are active metabolically and keep increasing in size. The cells synthesize RNA, growth factors and other molecules required for cell division. Log Phase Soon after the lag phase, i.e., the preparation phase, the bacterial cells enter the log phase. The log phase is also known as the exponential phase. This phase is marked by the doubling of the bacterial cells. The cell number increases in a logarithmic fashion such that the cell constituent is maintained. The log phase continues until there is depletion of nutrients in the setup. The stage also comes to a stop if toxic substances start to accumulate, resulting in a slower growth rate. The cells are the healthiest at this stage and researchers prefer to use bacteria from this stage for their experimental processes. Stationary Phase In the stationary phase, the rate of growth of the cells becomes equal to its rate of death. The rate of growth of the bacterial cells is limited by the accumulation of toxic compounds and also depletion of nutrients in the media. The cell population remains constant at this stage. Plotting this phase on the graph gives a smooth horizontal linear line. Death Phase This is the last phase of the bacterial growth. At this stage, the rate of death is greater than the rate of formation of new cells. Lack of nutrients, physical conditions or other injuries to the cell leads to death of the cells. Quorum Sensing Quorum sensing or quorum signaling is the process of cell-to-cell communication which allows bacteria the ability to detect and respond to cell population density by gene regulation. Quorum sensing signaling molecules are usually secreted at a low level by individual bacteria. At low cell density, the molecules may just diffuse away. At high cell density, the local concentration of signaling molecules may exceed its threshold level, and trigger changes in gene expression. Quorum sensing is involved in several biological activities, such as biofilm formation, bioluminescence, sporulation, motility, genetic exchange, etc. Biofilm Bacterial biofilms are clusters of bacteria that are attached to a surface and/or to each other and embedded in a self-produced matrix. A biofilm forms when certain microorganisms (for example, some types of bacteria) adhere to the surface of some object in a moist environment and begin to reproduce. The microorganisms form an attachment to the surface of the object by secreting a slimy, glue-like substance. Example of Quorum Sensing: Bioluminescence Bioluminescence is the production and emission of light by a living organism. Aliivibrio fischeri / Vibrio fischeri The bioluminescent bacterium A. fischeri is the first organism in which Quorum sensing was observed. It lives as a mutualistic symbiont in the photophore (or light-producing organ) of the Hawaiian bobtail squid. When A. fischeri cells are free-living, the autoinducer is at low concentration, and, thus, cells do not show luminescence. 11 However, when the population reaches the threshold in the photophore (about 10 cells/ml), transcription of luciferase is induced, leading to bioluminescence. In A. fischeri bioluminescence is regulated by AHLs (N-acyl-homoserine lactones) which is a product of the LuxI gene. LuxR transcriptional regulator is a key player in Quorum Sensing (QS), coordinates the expression of a variety of genes including luciferase. Bioluminescence occurs when an enzyme, known as a luciferase, oxidizes a small-molecule substrate, known as a luciferin. LuxR works only when AHLs binds to the LuxR. The squid has evolved to live in a symbiotic relationship with the bioluminescent bacteria, which serves to protect the squid from its predators and prevent it from being seen by its prey. https://youtu.be/mQ43fuJJW7M?t=178 Biosafety Level Basic microbiology teaching laboratory might be BSL-1. BSL-2 could be used for organisms that present a moderate risk of infection. BSL-3 labs are intended for highly infectious airborne pathogens such as the tuberculosis agent. BSL-4 labs are popularly known as “the hot zone.” Most dangerous microbes are handled.