CSI161 NOTES_240709_115937.PDF

LECTURE 1 SAFETY IN CLINICAL MICROBIOLOGY LABORATORY SBGA_260324 Mar-Aug24  General laboratory and personal safety, possible working hazard and the use of laminar flow and biological safety cabinet when culturing organisms from human (or animal) sources. Implem...

LECTURE 1 SAFETY IN CLINICAL MICROBIOLOGY LABORATORY SBGA_260324 Mar-Aug24  General laboratory and personal safety, possible working hazard and the use of laminar flow and biological safety cabinet when culturing organisms from human (or animal) sources. Implement proper aseptic techniques.  What is laboratory (microbiology) safety? important in the prevention of infection that might be caused by the microorganisms being studied. in addition, many of the reagents, equipment, and procedures used are potentially hazardous. Attention to proper procedures and prudent laboratory practices are required for your safety and protection.  Biosafety (microbes, cells, toxins etc.) Chemical Safety (acid, corrosive etc.) Radiation Safety (x-rays, gamma-rays) Fire Safety (prevent fires, minimize the risk of fire-related accidents) Hazardous Waste Removal (treated by chemical, thermal, biological, physical methods) Injury Reporting and Occupational Health  2 1 1 3 8 9 4 5 10 7 https://www.thoughtco.com/important-lab-safety-rules-608156 Keep long or fluffy hair tied up and out of the way. Hair can contaminate and be contaminated by microbial cultures. Wear a lab coat in lab. We will be working with a variety of materials that can cause permanent stains on some fabrics. Also, a lab coat can help protect from accidental contamination by microorganisms. No eating or drinking during lab. Many pathogens spread by ingested food and drink. In addition, food can carry microorganisms that might contaminate laboratory cultures. Always wear shoes in lab. Thoroughly wash your hands with soap and water before and after lab. Thorough and frequent hand washing easily and effectively controls the spread of many pathogens. Clean the lab bench with disinfectant before and after lab. This helps to prevent contamination of cultures, books, clothing, etc. Keep the lab bench free of unnecessary materials. Don't use the lab bench as a storage area for coats, books, etc. Do not take cultures from the lab area. Dispose all contaminated materials in autoclave bags. When in doubt, ask the instructor. Immediately report all accidents and spills to the instructor. Cover spills with disinfectant-soaked paper towels for at least 15 minutes before disposing them. Read all assigned materials before the lab session. Experiments will go smoother and have greater chances of success when you know what you will be doing ahead of time.  Laminar flow vs. Biosafety cabinet Which is which?  Exercise: List the additional parts for BSC compared to laminar flow. Biosafety cabinet A Laminar Flow Hood (LFH) is not a biological safety cabinet. These devices do not provide any protection to the worker. They are designed to provide a sterile environment to protect the product. Air potentially contaminated with infectious agents may be blown towards the worker. A biological safety cabinet (BSC) is a primary engineering control used to protect personnel against biohazardous or infectious agents and to help maintain quality control of the material being worked with as it filters both the inflow and exhaust air.  What is a biosafety cabinet used for? A BSC is a primary containment device used with biological material. While handling biological agents, it is the biological equivalent of using hazardous chemicals inside a fume hood. Like a chemical fume hood, a biosafety cabinet protects the user from hazardous material using directional air flow. Fume Hood vs. Biosafety Cabinet https://www.phe.gov/s3/BioriskManagement/biocontainment/Pages/BSC-vs-Fume-Hoods.aspx  Workspace in the Biosafety Cabinet  Items in your biosafety cabinet should be properly set up with the necessary items required for your experiment:  Biosafety Cabinet (classes) vs. Biosafety Levels There are 3 classes There are 4 levels of of BSC BSL Type A1 Type A2 Type B1 Type B2 4 Biosafety Lab Levels_cdc.govorrinfographicsbiosafety  Aseptic technique aseptic. / (əˈsɛptɪk, eɪ-) / adjective. free from living pathogenic organisms; sterile. aiming to achieve a germ-free condition. Method that involves target-specific practices and procedures under suitably controlled conditions to reduce the contamination from microbes. It is a compulsory laboratory skill to conduct research related in the field of microbiology. Routine practices used in microbiology laboratories to prevent contamination of samples and cultures throughout the analysis.  Principle of aseptic technique 1. Wearing appropriate personal protective equipment (PPE). 2. Using a biological safety cabinet. 3. Cleaning and sterilizing surfaces and equipment. 4. Handling samples and cultures carefully.  Inoculating loop petri dish It can be used to perform aseptic manipulations, such as transferring cultures from the bottle to the petri dish, without the risk of contamination. Spray using 70% alcohol Samples and cultures should be handled using sterile pipettes and other equipment to minimize the risk of contamination. It is important to avoid touching samples and cultures with bare hands, as the skin is a natural reservoir for microorganisms. Examples of commonly used PPE for radiation protection from X-rays and gamma rays include: Lead aprons or vests. Wearing lead aprons can reduce a worker's radiation dose. https://safetyculture.com/topics/radiation-safety/ Fire safety is a set of procedures which aim to reduce the amount of damage and injuries caused by fires. These include risk assessments to help identify and reduce areas of fire risk and formulate an emergency and evacuation plan in the event that a fire does break out. Hazardous waste can be treated by chemical, thermal, biological, and physical methods. Chemical methods include ion exchange, precipitation, oxidation and reduction, and neutralization. Among thermal methods is high-temperature incineration, which not only can detoxify certain organic wastes but also can destroy them.  Microbiology: Unseen World Shape/ morphology Exercise: Look for bacterial species names according to their shape. Example: Staphylococcus aureus, Gram positive, cocci.  Microbiology: Unseen World Growth media Solid growth media is used in the following forms: agar plates, agar slants (commonly used to generate stocks of bacteria), and agar deeps. To make agar deeps or agar slants, melted agar https://www.youtube.com/watch?v=9cy9VqVUODo is poured into a test tube and then allowed to solidify vertically (agar deep), or at a slant (agar slant). Agar plates are made by pouring melted agar into a petri dish. End of Lecture 1  LECTURE 2 NORMAL FLORA & HOST-PARASITE RELATIONSHIP SBGA_020424 Mar-Aug24  Host/microbe interaction Mechanism pathogenicity, virulence and toxigenicity. Normal flora and opportunistic microorganisms of human body. Routes of transmission.  What is microbe? What counts as a microbe? Microscopic organism (microorganism) – too small to be seen with the naked eye, especially one that causes disease. *Bacteria *Fungi Archaea Protist *Viruses *Parasites (Toxoplasma, Plasmodium, Babesia etc.) Microbes and diseases (*pathogen).  e.g. Bacteria – TB, cholera, typhoid Fungi – Ringworm, histoplasmosis, candidiasis Viruses – cold, dengue, Covid-19 Parasites – malaria, Chagas disease, toxoplasmosis, amebiasis Exercise: List example of various types of disease-causing pathogens https://www.acko.com/health-insurance/diseases/diseases-caused-by- microorganisms/  Definition Pathogen - an organism causing disease to its host, with the severity of the disease symptoms referred to as *virulence. Pathogenicity - the ability of an organism to cause disease (i.e., harm the host). This ability represents a genetic component of the pathogen and the overt damage done to the host is a property of the host-pathogen interactions. Commensals and opportunistic pathogens lack this inherent ability to cause disease. Pathogenesis - the mechanisms by which it develops, progresses, and either persists or is resolved. Pathology – study and diagnosis of disease. Clinical pathology involves the examination of surgically removed organs, tissues (biopsy samples), bodily fluids, and in some cases the whole body (autopsy). Chemical pathology (also known as clinical biochemistry) involves the biochemical investigation of bodily fluids such as blood, urine and cerebrospinal fluid. Chemical pathology I (CSI212) Chemical pathology II (CSI262)  Definition (short words 😁) Patho - Relating to disease Pathogen - agents (microorganisms) able to cause disease Pathogenesis - development of a disease Pathology - study of disease Pathogenicity - ability to infect a host and cause disease and depends on a number of factors: ability of pathogen to invade a host ability of pathogen to multiply in the host ability of pathogen to avoid host defences (evasion of host immune response (IR)) the number of infectious organisms that enter the body Degree of pathogenicity is called VIRULENCE, with highly virulent pathogens being more likely to cause disease in a host (pathogens varying in their ability) https://microbenotes.com/factors-affecting-bacterial-pathogenicity/  invade D39 evasion of host IR D39 Pathogenicity factors D39 CAMφ SBGA© Activated state multiply bacteria MΦ  Definition *Virulence - a pathogen's or microorganism's ability to cause damage to a host. In most, especially in animal systems, virulence refers to the degree of damage caused by a microbe to its host. The pathogenicity of an organism - its ability to cause disease which determined by its virulence factors. Virulence factor (VF) - the molecules that assist the bacterium colonize the host at the cellular level. Virulence factor (VF)  Virulence Factor (VF) characteristics that contribute to virulence are called virulence factors (VF): physical structures that the bacterium has (pili, flagellum etc.). chemical substances that the bacterium can produce (toxins). the genes that code for virulence factors are commonly found clustered on the pathogen’s chromosome or plasmid DNA, called pathogenicity islands (PI). PI facilitate the sharing of virulence factors between bacteria due to horizontal gene transfer (HGT), leading to the development of new pathogens over time. Often the genes for virulence factors are controlled by quorum sensing (QS), to ensure gene activation when the pathogen population is at an optimal density. Triggering the genes too soon could alert the host’s immune system to the invader, cutting short the bacterial infection.  Transformation is the uptake of 'free' DNA from the environment Transduction is the transfer of DNA by bacteria-specific viruses called bacteriophage Conjugation is the transfer of circular DNA called plasmids through cell to cell contact Horizontal DNA (gene) transfer is the exchange of genes between two cells of the same generation, as opposed to from parent to progeny  Virulence Factor (Mechanism of QS) QS – where bacteria “talk” to one another Autoinducers (signalling molecules – to measure population) bind to signalling receptors (on the surface or cytoplasm) becoming autoinducer/ receptor complex (ARC) Activate QS sensing genes ARC Multicellular population ARC activates the transcription of QS-controlled genes multicellular population When these autoinducers reach a critical, threshold level, they activate bacterial quorum sensing genes that enable the bacteria to behave as a multicellular population rather than as individual single-celled organism. The autoinducer/receptor complex is able to bind to DNA promoters and activate the transcription of quorum sensing-controlled genes in the bacterium. In this way, individual bacteria within a group are able to benefit from the activity of the entire group. https://bio.libretexts.org/Bookshelves/Microbiology/Book%3A_Microbiology_(Kaiser)/Unit_2%3A_Bacterial_Genetics_and_the_Chemical_C ontrol_of_Bacteria/3%3A_Bacterial_Genetics/3.2%3A_Bacterial_Quorum_Sensing%2C_Pathogenicity_Islands%2C_and_Secretion_Systems_(I njectosomes) Some pathogens are much more virulent than others Less cell numbers (102)of S. pneumoniae (Gram POS) killed 100% mice than high cell numbers S.p S.t (106)of S. typhimurium (Gram NEG) - S. pneumoniae more virulence than S. typhimurium Low dose of Strain A increases mortality than higher dose of Strain B - Strain A more virulence than Strain B https://slideplayer.com/slide/6281198/  In summary, pathogenicity vs. virulence Pathogenicity is the ability of an organism to infect a host and cause disease Aggressiveness is the ability of the pathogen to invade and establish within the host Virulence is the severity of the disease in infected hosts (or degree of pathogenicity) Ecological relationship https://www.thoughtco.com/commensalism-definition-and-examples-4114713  Normal flora vs. opportunistic mirobes Normal flora - microbes that colonize the body and usually do not cause disease. Opportunistic pathogens - microbes that normally do not cause disease, but may under certain circumstances.  Normal Flora microorganisms that live on another living organism (human or animal) or inanimate object without causing disease. *red – more resistant bacteria http://www.microbiologynutsandbolts.co.uk/normal-flora.html Opportunistic pathogen - harmless microbe that typically infects a host that is compromised in some way, either by a weakened immune system or breach to the body’s natural defences (skin, mucous membranes, tears, earwax, mucus, and stomach acid), such as a wound https://yakult.com.sg Quick think 1: Is human body sterile? No. We become colonised by bacteria from the moment we are born Quick think 2: What is the normally sites in the human body? Brain; Central nervous system Blood; Tissues; Organ systems Sinuses; Inner and Middle Ear Lower Respiratory Tract: Larynx; Trachea; Bronchioles (bronchi); Lungs; Alveoli Kidneys; Ureters; Urinary Bladder; Posterior Urethra Uterus; Endometrium (Inner mucous membrane of uterus ); Fallopian Tubes; Cervix and Endocervix Occurs when defect or breach in the natural defenses that creates a portal of entry Knowledge of the normal flora of the human body allows: Prediction of the pathogens causing infection as bacteria tend to grow in specific body sites e.g. Streptococcus pneumoniae from the upper respiratory tract causing pneumonia or Staphylococcus aureus from the skin causing intravenous cannula infections. Investigation for underlying abnormalities in specific areas of the body when bacteria are isolated from normally sterile sites e.g. Escherichia coli isolation from blood cultures indicates probable intra-abdominal pathology because Escherichia coli is part of the normal gastrointestinal flora, or isolation of a Viridans Streptococcus in blood cultures may indicate infective endocarditis as a result of poor dentition as Viridans Streptococcus are part of the normal mouth flora.  Brushing teeth linked to the lower risks of atrial fibrillation and HF. Malas gosok gigi selepas makan dan sebelum tidur menyebabkan pelbagai penyakit seperti penyakit jantung…  What is host-pathogen (microbe) interaction? Defined as how microbes sustain themselves within host organisms on a molecular, cellular, organismal or population level. Routes of transmission = https://www.sciencedirect.com/topics/immunology-and-microbiology/host-pathogen-interaction  Causes damage/ disease/ to the hosts: Host- pathogens pathogen interactions Hosts Hosts  Mechanisms of (bacterial) pathogenicity, virulence & toxigenicity Bacterial pathogens express a wide range of molecules that bind host cell targets to facilitate a variety of different host responses. The molecular strategies used by bacteria to interact with the host can be unique to specific pathogens or conserved across several different species. 1 2 3  Host Microbes (Pathogens) Interactions Direct: Direct contact Droplet Indirect: virulence vs. Host Resistant Airborne number of microbes able to fight off the Feco-oral infection before Vector borne Mechanical causes disease disease Biological signs/symptoms develop eyes, mouth, nose, or The expelling urogenital openings, of bacteria from the or through wounds or body. Important routes bites that breach the include the respiratory skin barrier tract, genital tract, and intestinal tract. A primary host that harbors the pathogen but shows no ill effects and serves as a source of infection  Adherence, Colonisation & Invasion Bacterial pathogens must be able to grab onto host cells or tissue, and resist removal by physical means (such as sneezing) or mechanical means (such as movement of the ciliated cells that line our airway). Adherence can involve polysaccharide layers made by the bacteria, such as a capsule or slime layer, which provide adhesion to host cells as well as resistance from phagocytosis. Adherence can also be accomplished by physical structures such as a pilus or flagellum. Once cells are successfully adhering to a surface, they increase in number, utilizing resources available at the site. This colonization is important for pathogen survival and invasion to other sites, which will yield increased nutrients and space for the growing population. Adhesion  Invasion Invasion refers to the ability of the pathogen to spread to other locations in the host, by invading host cells or tissue. It is typically at this point when disease or obvious signs/symptoms of illness will occur. While physical structures can still play a role in invasion, most bacterial pathogens produce a wide array of chemicals, specifically enzymes that effect the host’s cells and tissue. Enzymes such as collagenase, which allows the pathogen to spread by breaking down the collagen found in connective tissue. Or leukocidins, which destroy the host’s white blood cells, decreasing resistance. Hemolysins lyse the host’s red blood cells, releasing iron, a growth-limiting factor for bacteria. Bacteria in the bloodstream, a condition known as bacteraemia, can quickly spread to locations throughout the host. This can result in a massive, systemic infection known as septicaemia, which can result in septic shock and death, as the host becomes overwhelmed by the bacterial pathogen and its products.  bacteraemia septicaemia septic shock Septic shock is a life-threatening condition that happens when your blood pressure drops to a dangerously low level after an infection.  Invasion – cont. Once bacteria have successfully colonised their host, they need a way to invade these tissues in order to proliferate and cause disease. Invasion can be mediated by: Toxins which penetrate and damage cells, usually to aid the producing bacterium.  adhesion Successful establishment of infection by bacterial pathogens requires adhesion to host cells, colonization of tissues, and in certain cases, cellular invasion—followed by intracellular multiplication, dissemination to other tissues, or persistence. dissemination Toxins (VF)  Toxins (VF) Toxins are a very specific virulence factor produced by some bacterial pathogens, in the form of substances that are poisonous to the host. Toxigenicity refers to an organism’s ability to make toxins. For bacteria, there are two categories of toxins, the exotoxins and the endotoxins. 1. Exotoxins are heat-sensitive soluble proteins that are released into the surrounding environment by a living organism. There are many different bacteria that produce exotoxins, causing diseases such as botulism, tetanus, and diphtheria.  Toxins There are three categories of exotoxins: 1.Type I: cell surface-active – these toxins bind to cell receptors and stimulate cell responses. One example is superantigen, that stimulates the host’s T cells, an important component of the immune system. The stimulated T cells produce an excessive amount of the signalling molecule cytokine, causing massive inflammation and tissue damage. 2.Type II: membrane-damaging – these toxins exert their effect on the host cell membrane, often by forming pores in the membrane of the target cell. This can lead to cell lysis as cytoplasmic contents rush out and water rushes in, disrupting the osmotic balance of the cell (example pneumolysin (Ply) produced by S. pneumoniae). 3.Type III: intracellular – these toxins gain access to a particular host cell and stimulate a reaction within the target cell. One example is the AB-toxin – these toxins are composed of two subunits, an A portion and a B portion. The B subunit is the binding portion of the toxin, responsible for recognizing and binding to the correct cell type. The A subunit is the portion with enzymatic activity. Once delivered into the correct cell by the B subunit, the A subunit enacts some mechanism on the cell, leading to decreased cell function and/or cell death. An example is the tetanus toxin produced by the bacterium Clostridrium tetani. Once delivered to a neuron, the A subunit will cleave the cellular synaptobrevin, resulting in a decrease in neurotransmitter release. This results in spastic paralysis of the host. Each AB-toxin is associated with a different disease. Others include V. cholera toxins  Toxins – cont. 2. Endotoxins are made by Gram negative bacteria, as a component of the outer membrane of their cell wall. The outer membrane contains lipopolysaccharide or LPS. A massive release of endotoxin in a host can cause endotoxin shock, which can be deadly. Routes of transmission  Host Microbes (Pathogens) Interactions The transmission of microorganisms can be divided into: Exercise: Example of some microorganisms that can be transmitted by more than one route. Transmission Regardless of the reservoir, transmission must occur for an infection to spread. First, transmission from the reservoir to the individual must occur. Then, the individual must transmit the infectious agent to other susceptible individuals, either directly or indirectly. Pathogenic microorganisms employ diverse transmission mechanisms. Contact transmission includes: direct contact (person-to-person). e.g. touching, kissing, sexual intercourse, or droplet sprays. indirect contact - involves inanimate objects called *fomites that become contaminated by pathogens from an infected individual or reservoir. *fomites - objects or materials which are likely to carry infection, such as clothes, utensils, and furniture. Contaminated doorknobs, towels, and syringes are all common examples of fomites https://courses.lumenlearning.com/suny-microbiology/chapter/modes-of-disease-transmission/ Vehicle transmission: refers to transmission of pathogens through vehicles such as water, food, and air. e.g. water contamination through poor sanitation methods leads to waterborne transmission of disease. dust and fine particles known as aerosols, which can float in the air, can carry pathogens and facilitate the airborne transmission of disease. https://link.springer.com/article/10.1007/s10867-020-09562-5 Transmission over distances greater than one meter is called airborne transmission. Tuberculosis is often transmitted via airborne transmission when the causative agent, Mycobacterium tuberculosis, is released in small particles with coughs. Tuberkulosis (TB) atau batuk kering In summary https://link.springer.com/article/10.1007/s10311-023-01579-1?fromPaywallRec=false Vector transmission: transmitted by a mechanical or biological vector, an animal (typically an arthropod) that carries the disease from one host to another. Mechanical transmission is facilitated by Biological transmission occurs when the a mechanical vector, an animal that carries a pathogen reproduces within a biological pathogen from one host to another without being vector that transmits the pathogen from one infected itself (food poisoning - diarrhoea, dysentery) host to another (malaria, dengue etc.) https://courses.lumenlearning.com/suny-microbiology/chapter/modes-of-disease-transmission/  You will learn some of these vectors in CSI153 https://courses.lumenlearning.com/suny-microbiology/chapter/modes-of-disease-transmission/  You will learn some of these vectors in CSI153 https://courses.lumenlearning.com/suny-microbiology/chapter/modes-of-disease-transmission/  You will learn some of these vectors in CSI153 https://courses.lumenlearning.com/suny-microbiology/chapter/modes-of-disease-transmission/ Exercise: Self-learning): 1. Barrier defences (skin, hair, mucus) 2. Internal defences (immune cells (cellular) defences) End of Lecture 2  LECTURE 3 BACTERIAL ANATOMY & PHYSIOLOGY SBGA_160424 Mar-Aug24  Functions of the capsule, cell wall, cytoplasmic membrane, cytoplasm, nuclear body, flagella, pili, inclusion granules, endospore and plasmids. Cellular morphology and arrangement of bacteria. Bacterial growth. Bacterial genetic. Prokaryotes vs. Eukaryotes  Prokaryotes vs. Eukaryotes Characteristics Prokaryotes Eukaryotes Composition Unicellular Multi-celled (more than one cell; collection of cells) Size Smaller Larger Nucleus & Membrane- Lack/ absence Possess/ presence encased (bound) genetic material DNA in organised into chromosomes organelles prokaryotes is not bound More/ highly structured within a nucleus Less structured Examples Bacteria, yeast Animals (human), plants, fungi, protist (unicellular, eukaryotic) – algae, protozoa Recap: 1. What is organelles? a subcellular structure that has one or more specific jobs to perform in the cell, much like an organ does in the body. 2. What is chromosomes? thread-like structure made up of DNA. Chromosomes are found in the nucleus of each cell. What is the basic hierarchy within a cell DNA chromosomes nucleus https://www.civilsdaily.com/biotechnology-basics-of-cell-nucleus- chromosomes-dna-genes-etc/ In the nucleus of each cell, the DNA molecule is packaged into thread-like structures called chromosomes. Each chromosome is made up of DNA tightly coiled many times around proteins called histones that support its structure.  Prokaryotes vs. Eukaryotes *Plasmid - a small molecule of DNA that can reproduce independently Containing DNA Is cytoplasm and plasma membrane the same? Both prokaryotic and eukaryotic cells have a plasma membrane, a double layer of lipids that separates the cell interior from the outside environment. In eukaryotic cells, which have a nucleus, the cytoplasm is everything between the plasma membrane and the nuclear envelope. In prokaryotes, which lack a nucleus, cytoplasm simply means everything found inside the plasma membrane. Read more at: https://www.khanacademy.org/science/biology/structure-of-a-cell/prokaryotic-and-eukaryotic- cells/a/plasma-membrane-and-cytoplasm Bacterial Anatomy  Bacterial Anatomy Capsule - many prokaryotes secrete on their surface a substance called glycocalyx (meaning sugar coat; substances that surround cells). The bacterial glycocalyx is a viscous (sticky), gelatinous polymer that is external to the cell wall and composed of polysaccharide, polypeptide, or both. It is made inside the cell and secreted to the cell surface. If the substance is organized and is firmly attached to the cell wall, the glycocalyx is described as a capsule. The presence of a capsule can be determined by using negative staining. If the substance is unorganized and only loosely attached to the cell wall, the glycocalyx is described as a slime layer. Capsules confer resistance to phagocytosis. The capsule is found most commonly among Gram- Some Gram-positive bacteria may also have a capsule: negative bacteria: Bacillus megaterium Escherichia coli (in some strains) Bacillus anthracis Neisseria meningitidis Streptococcus pyogenes Klebsiella pneumoniae Streptococcus pneumoniae has at least 91 different capsular Haemophilus influenzae serotypes Pseudomonas aeruginosa Streptococcus agalactiae Salmonella Staphylococcus epidermidis Acinetobacter baumannii Staphylococcus aureus Lactococcus garvieae The yeast Cryptococcus neoformans, though not a bacterium, has a similar capsule. The background is formed with india ink or nigrosin or congo red. India ink is difficult to obtain nowadays; however, nigrosin is easily acquired. A positive capsule stain requires a mordant that precipitates the capsule. By counterstaining with dyes like crystal violet or methylene blue, bacterial cell wall takes up the dye. Capsules appear colorless with stained cells against dark background. https://microbeonline.com/bacterial-capsule-structure-and-importance-and-examples-of-capsulated-bacteria/ A bacterial capsule has a semi-rigid border that follows the contour of the cell. The capsule excludes India Ink when dyed. A slime layer is a non-rigid matrix that is easily deformed and is not able to exclude India Ink. Biofilms are composed of many cells and their outer barriers. The primary functions of both capsules and slime layers are for protection and adhesion.  Bacterial capsule prevents the direct access of lysosome contents with the bacterial cell, preventing their killing. Capsules may protect bacteria from complement activation. As a result, encapsulated bacteria are not immediately recognized as invaders by the phagocytes. Capsulated Strep. pneumoniae resist engulfment by macrophages and PMNs and are virulent; however, non-capsulated strains are easily phagocytosed and are avirulent. Capsulated, encapsulated, non-capsulated https://microbeonline.com/bacterial-capsule-structure-and-importance-and-examples-of-capsulated-bacteria/  Bacterial Anatomy Cell wall - Both Gram-positive and Gram-negative bacteria possess cell wall peptidoglycans, which confer the characteristic cell shape and provide the cell with mechanical protection. There are two main types of bacterial cell walls, those of gram-positive bacteria and those of gram-negative bacteria, which are differentiated by their Gram staining characteristics.  The major difference between Gram-positive and Gram-negative peptidoglycan involves the thickness of the layers surrounding the plasma membrane. https://byjus.com/biology/composition-of-bacterial-cell-wall/ https://microbenotes.com/differences-between-gram-positive-and-gram-negative-bacteria/ https://emedicodiary.com/que/505/gram-staining-procedure Chemicals that damage bacterial cell walls, or interfere with their synthesis, often do not harm the cells of an animal host because the bacterial cell wall is made of chemicals unlike those in eukaryotic cells. Thus, cell wall synthesis is the target for some antimicrobial drugs. https://courses.lumenlearning.com/suny-microbiology/chapter/mechanisms-of-antibacterial-drugs/  Bacterial Anatomy Plasma (cytoplasmic) membrane (PM) (or inner membrane). thin structure lying inside the cell wall and enclosing the cytoplasm of the cell. PM of prokaryotes consists primarily of phospholipids. prokaryotic PMs are less rigid than eukaryotic membranes. PM serve as a selective barrier through which materials enter and exit the cell, breakdown of nutrients and production of energy. Because the plasma membrane is vital to the bacterial cell, it is not surprising that several antimicrobial agents exert their effects at this site. In addition to the chemicals that damage the cell wall and thereby indirectly expose the membrane to injury, many compounds specifically damage plasma membranes. https://courses.lumenlearning.com/suny-microbiology/chapter/mechanisms-of-antibacterial-drugs/  Bacterial Anatomy The nucleoid contains a single long, continuous, and frequently circularly arranged thread of double-stranded DNA called the bacterial chromosome. This is the cell’s genetic information, which carries all the information required for the cell’s structures and functions. Unlike the chromosomes of eukaryotic cells, bacterial chromosomes are not surrounded by a nuclear envelope (membrane) and do not include histones. The nucleoid can be spherical, elongated, or dumbbell-shaped. Plasmid - a small molecule of DNA In addition to the bacterial chromosome, bacteria often contain that can reproduce independently small usually circular, double-stranded DNA molecules called plasmids. These molecules are extrachromosomal genetic elements; that is, they are not connected to the main bacterial chromosome, and they replicate independently of chromosomal DNA.  Bacterial Anatomy Flagella (singular: flagellum) are long filamentous appendages that propel bacteria. Not all bacteria have flagella. Locomotor organelle/ drive cell locomotion. They thrust cells in liquids (swimming) or on surfaces (swarming) so that cells can move toward favorable environments. Types: Atrichous (without projection; lack flagella) Peritrichous (distributed over the entire cell) Plasmid - a small molecule of DNA Polar (at one or both poles or ends of the cell): that can reproduce independently Monotrichous (a single flagellum at one point) Lophotrichous (a tuft of flagella coming from one pole) Amphitrichous (flagella at both poles of the cell).  Proteus sp. – an opportunistic bacterial pathogen – classification, swarming growth, clinical significance and virulence factors DOI: 10.2478/fobio-2013-0001 https://www.nature.com/articles/nrmicro2405  E.coli, B. subtilis Exercise/ Self-learning: Look for the species examples.  Bacterial Anatomy Pili (also known as fimbriae) are proteinaceous, filamentous polymeric organelles expressed on the surface of bacteria. Their functions include mediation of cell-to-cell interactions, motility, and DNA uptake. Plasmid - a small molecule of DNA that can reproduce independently https://bio.libretexts.org/Bookshelves/Microbiology/Microbiology_(Kaiser)/Unit_1%3A_Introduction_to_Microbiology_and_Prokaryotic_ Cell_Anatomy/2%3A_The_Prokaryotic_Cell_Bacteria/2.5%3A_Structures_Outside_the_Cell_Wall/2.5C%3A_Fimbriae_and_Pili Long conjugation pili, also called "F" or sex pili. The conjugation pilus enables conjugation (the transfer of DNA from one bacterium to another by cell-to-cell contact). In gram-negative bacteria it is typically the transfer of DNA from a donor or "male bacterium" with a sex pilus to a recipient or "female bacterium" to enable genetic recombination.  Bacterial Anatomy Inclusions – lie within the cytoplasm of prokaryotic cells. are several kinds of reserve deposits: Metachromatic granules Polysaccharide granules Lipid inclusions Sulfur granules Carboxysomes Gas vacuoles Magnetosomes Exercise/ Self-learning: Look for the species examples. Microbiology an Introduction_13th Ed_Tortora_2021_pg. 117-118  Bacterial Anatomy Endospores – when essential nutrients are depleted, certain Gram-positive bacteria, such as those of the genera Clostridium and Bacillus, form specialized “resting” cells called endospores. some members of the genus Clostridium cause diseases such as gangrene, tetanus, botulism, and food poisoning. some members of the genus Bacillus cause anthrax and food poisoning. unique to bacteria, endospores are highly durable dehydrated cells with thick walls and additional layers. they are formed internal to the bacterial cell membrane. when released into the environment, they can survive extreme heat, lack of water, and exposure to many toxic chemicals and radiation.  Endospores are important from a clinical viewpoint and in the food industry because they’re resistant to processes that normally kill vegetative cells. Such processes include heating, desiccation, use of chemicals, and radiation. Whereas most vegetative cells are killed by temperatures above 70°C, endospores can survive in boiling water for several hours or more. Endospores of thermophilic (heat-loving) bacteria can survive in boiling water for 19 hours. Endospore-forming bacteria are a problem in the food industry because they are likely to survive under processing, and, if conditions for growth occur, some species produce toxins and disease.  Bacterial Growth – How microbes grow? the bacterial cell cycle involves the formation of new cells through the replication of DNA and partitioning of cellular components into two daughter cells. In prokaryotes, reproduction is always asexual, although extensive genetic recombination in the form of horizontal gene transfer (HGT) takes place. Most bacteria have a single circular chromosome; however, some exceptions exist. For example, Borrelia burgdorferi, the causative agent of Lyme disease, has a linear chromosome.  Bacterial Growth – Binary fission most common mechanism of cell replication in bacteria. Before dividing, the cell grows and increases its number of cellular components. Next, the replication of DNA starts at a location on the circular chromosome called the origin of replication, where the chromosome is attached to the inner cell membrane. Replication continues in opposite directions along the chromosome until the terminus is reached.  Bacterial Growth – Generation time time it takes for a population of bacteria to double in number. In eukaryotic organisms, the generation time is the time between the same points of the life cycle in two successive generations. For example, the typical generation time for the human population is 25 years. This definition is not practical for bacteria, which may reproduce rapidly or remain dormant for thousands of years. In prokaryotes (Bacteria and Archaea), the generation time is also called the doubling time and is defined as the time it takes for the population to double through one round of binary fission.  Bacterial Growth – Generation time For many common bacteria, the generation time is quite short, 20-60 minutes under optimum conditions. For most common pathogens in the body, the generation time is probably closer to 5-10 hours.  Bacterial Growth – The Growth Curve Microorganisms grown in closed culture (also known as a batch culture), in which no nutrients are added and most waste is not removed, follow a reproducible growth pattern referred to as the growth curve.  Bacterial Growth – The Growth Curve The Lag Phase: the beginning of the growth curve represents a small number of cells, referred to as an inoculum, that are added to a fresh culture medium, a nutritional broth that supports growth. cells are gearing up for the next phase of growth. The number of cells does not change during the lag phase; however, cells grow larger and are metabolically active, synthesizing proteins needed to grow within the medium. If any cells were damaged or shocked during the transfer to the new medium, repair takes place during the lag phase. The duration of the lag phase is determined by many factors, including the species and genetic make-up of the cells, the composition of the medium, and the size of the original inoculum. The Log Phase: in the logarithmic (log) growth phase, sometimes called exponential growth phase, the cells are actively dividing by binary fission and their number increases exponentially. for any given bacterial species, the generation time under specific growth conditions (nutrients, temperature, pH, and so forth) is genetically determined, and this generation time is called the intrinsic growth rate.  Bacterial Growth – The Growth Curve The Stationary Phase: as the number of cells increases through the log phase, several factors contribute to a slowing of the growth rate. waste products accumulate and nutrients are gradually used up. In addition, gradual depletion of oxygen begins to limit aerobic cell growth. This combination of unfavorable conditions slows and finally stalls population growth. The total number of live cells reaches a plateau referred to as the stationary phase. in this phase, the number of new cells created by cell division is now equivalent to the number of cells dying; thus, the total population of living cells is relatively stagnant. during the stationary phase, cells switch to a survival mode of metabolism. As growth slows, so too does the synthesis of peptidoglycans, proteins, and nucleic-acids; thus, stationary cultures are less susceptible to antibiotics that disrupt these processes. in bacteria capable of producing endospores, many cells undergo sporulation during the stationary phase. Secondary metabolites, including antibiotics, are synthesized in the stationary phase. In certain pathogenic bacteria, the stationary phase is also associated with the expression of virulence factors, products that contribute to a microbe’s ability to survive, reproduce, and cause disease in a host organism. For example, quorum sensing in Staphylococcus aureus initiates the production of enzymes that can break down human tissue and cellular debris, clearing the way for bacteria to spread to new tissue where nutrients are more plentiful.  Bacterial Growth – The Growth Curve The Death Phase: as a culture medium accumulates toxic waste and nutrients are exhausted, cells die in greater and greater numbers. Soon, the number of dying cells exceeds the number of dividing cells, leading to an exponential decrease in the number of cells. this is the aptly named death phase, sometimes called the decline phase. Many cells lyse and release nutrients into the medium, allowing surviving cells to maintain viability and form endospores. a few cells, the so-called persisters, are characterized by a slow metabolic rate. Persister cells are medically important because they are associated with certain chronic infections, such as tuberculosis, that do not respond to antibiotic treatment. Bacterial Growth – Sustaining Microbial Growth A chemostat is a culture vessel fitted with an opening to add nutrients (feed) and an outlet to remove contents (effluent), effectively diluting toxic wastes and dead cells. The addition and removal of fluids is adjusted to maintain the culture in the logarithmic phase of growth. If aerobic bacteria are grown, suitable oxygen levels are maintained.  Bacterial Genetic The central dogma states that DNA encodes messenger RNA, which, in turn, encodes protein. Bacterial Genetic – Asexual in Prokaryotes (horizontal gene transfer - HGT) naked DNA is genes are transferred use of a hollow tube called a taken up from the between cells in a conjugation pilus to transfer environment virus (bacteriophage) genes between cells https://microbenotes.com/bacteria/ End of Lecture 3  LECTURE 4 BACTERIAL TAXONOMY & SCIENTIFIC NOMENCLATURE SBGA_160424 Mar-Aug24  Definitions: family, genus, species, strain, biotype (biovar), serotype (serovar). Structural and physiological differences among bacteria, fungi, mycoplasmas, rickettsiae, chlamydiae, viruses Taxonomy – naming, describing and classifying organisms Taxonomy (which literally means “arrangement law”) is the science of classifying organisms to construct internationally shared classification systems with each organism placed into more and more inclusive groupings. The taxonomic classification system (also called the Linnaean system after its inventor, Carl Linnaeus, a Swedish botanist, zoologist, and physician) uses a hierarchical model. Pongo pygmaeus Biovar or Biotype: a group of strains that have a characteristic biochemical pattern. The organisms sharing a specified genotype. Serovar or Serotype: a distinct variation within a species of bacteria or virus or among immune cells of different individuals. Serotype is a serologically distinguishable strain of a microorganism. It is a distinct variation within a species of bacteria or virus or among immune cells of different individuals. Biotype is a group of organisms having same genotype. Taxonomy of Clinically Relevant Microorganisms https://courses.lumenlearning.com/suny-microbiology/chapter/taxonomy-of-clinically-relevant-microorganisms/ Taxonomy of Clinically Relevant Microorganisms https://courses.lumenlearning.com/suny-microbiology/chapter/taxonomy-of-clinically-relevant-microorganisms/ Taxonomy of Clinically Relevant Microorganisms https://courses.lumenlearning.com/suny-microbiology/chapter/taxonomy-of-clinically-relevant-microorganisms/ Taxonomy of Clinically Relevant Microorganisms https://courses.lumenlearning.com/suny-microbiology/chapter/taxonomy-of-clinically-relevant-microorganisms/ End of Lecture 4  LECTURE 5 BACTERIAL METABOLISM & PHYSIOLOGY SBGA_230424 Mar-Aug24  Growth requirement of bacteria. Nutritional categories of bacteria. Metabolic pathways used by bacteria.  Definitions Metabolism - from the Greek term metaballein, meaning change, pertains to all chemical workings of cells. Physiology - the branch of biology that deals with the normal functions and activities of life or of living matter. Enzymes - the proteins that help speed up metabolism, or the chemical reactions in our bodies.  Microbial Metabolism – The Chemical Crossroads of Life a shorthand term for encapsulating almost any activity or behaviour of an organism, from the general to the specific, including growth, synthesis, transport, digestion, energy release and consumption and movement. Most of the chemical reactions can be placed into: Catabolism - larger molecules are degraded or broken down into smaller molecules, usually with the release of energy (e.g. breakdown of glucose). Anabolism (biosynthesis) - larger molecules are built from smaller ones, which results in the formation of cell structures (e.g. protein synthesis), require energy. Exercise: read more at: https://byjus.com/biology/differences-between-catabolism-and-anabolism/ Simplified model of metabolism. Chemical reactions that occur in cells fall into two major categories. Catabolism involves the breakdown of complex organic molecules to extract energy and form simpler end products. Anabolism uses the energy to synthesize necessary macromolecules and cell structures from simple precursors, increasing chemical complexity. The two systems interact in a continuous cycle that maintains the metabolic balance inside cells. Talaro’s_Foundation of Microbiology_page 230  Microbial Metabolism – Bacteria rely on enzymes Endoenzymes Exoenzymes produced work within the inside the cell and then cells transported to the outside (intracellular) (work outside the cell; https://charatoon.com/cat/? extracellular) id=133#google_vignette For bacteria, enzymes needed for metabolic reactions are either endoenzymes, which work within the cell, or exoenzymes, which are produced inside the cell and then transported to the outside where they facilitate the preliminary digestion of high molecular weight substrates that do not pass readily through the cell membrane. Most exoenzymes have digestive function, breakdown the molecule. Talaro’s_Foundation of Microbiology_page 235  All of this chemistry results in the production of biomolecules and waste, much of which is excreted by the cells into the surrounding environment. Detecting and identifying the biochemical products of metabolism provides us with a way to learn more about the physiologic and growth capabilities of bacteria, and also give us a way to differentiate among and/or identify species. Biochemical tests are used to identify bacterial species by differentiating them on the basis of biochemical activities. The difference in protein and fat metabolism, carbohydrate metabolism, enzyme production, compound utilization ability, etc. are some factors that aid in bacterial identification. 1 https://www.labtestsguide.com/biochemical-tests 2 Overview of Biochemical tests for differentiating Gram positive cocci https://microbeonline.com/overview-of-biochemical-tests-used-to-identify-bacteria-in-microbiology-laboratory/ 3 https://microbenotes.com/biochemical-test-of-bacteria/  The definitive reference book on bacteria, determinations of identity or taxonomic group are based on many criteria. Gram stain – 1st criteria Biochemical characteristics as the 2nd criteria: Aerobic/ anaerobic respiration Sugar fermentation Amino acids and protein degradation Other cellular events Intermediates or end products of these varied metabolic activities can be detected by performing biochemical assays on a bacterial culture. The results of these tests provide a biochemical profile, or “fingerprint,” that can be used to classify or even identify the bacterial species. The outcomes of laboratory tests can also provide insight into physiology and what is needed to encourage and support bacterial growth.  Microbial Physiology & Growth Characteristics Growing bacteria in culture requires consideration of their nutritional and physical needs. Food, provided in the media, is broken down by cells and used for energy and building biomass. Unlike eukaryotic cells, bacteria have options when it comes to making energy, which depend not only on the type of organic molecules in the food but also on the availability of oxygen as a final electron acceptor for respiration. Respiration is the pathway in which organic molecules are sequentially oxidized to strip off electrons, which are then deposited with a final electron acceptor. Along the way, ATP is made. For many types of bacteria, oxygen serves as the final electron acceptor in respiration. Remarkably, oxygen is not always a requirement for respiration. For bacteria that live in environments with no air, alternative electron acceptors may take the place of oxygen. Microbial Physiology & Growth Characteristics - RESPIRATION Fermentation and anaerobic respiration are anaerobic processes - meaning that no oxygen is required for ATP production. Based on whether oxygen is required for growth, bacteria can be considered to be either aerobes or anaerobes. However, because some bacteria may use more than one pathway, there are ‘additional categories’ that describe a culture’s requirement for oxygen in the atmosphere. The three major categories are: Obligate  Microbial Physiology & Growth Characteristics Overlapping categories include:  AEROBES VERSUS ANAEROBES Bog - an area of wet muddy ground that is too soft to support a heavy body that devoid of oxygen The increased aeration allows for increased degradation and increased pathogen removal https://sswm.info/factsheet/aerated-pond The rumen (the first compartment of a cow’s stomach), which provides an oxygen-free incubator for methanogens and other obligate anaerobic bacteria.  AEROBES vs. ANAEROBES microaerophiles (aerobes) CHARACTERISTICS OF COCCI Microbial Physiology & Growth Characteristics – DETECTING END-PRODUCTS CATALASE TEST - detects the ability of bacteria to produce an enzyme called catalase which is found in cells that live where there is air. All Staph. are Use of hydrogen CATALASE positive peroxide (H2O2), the – breakdown to substrate of the hydrogen peroxide catalase enzyme into water that which converts produces bubbles hydrogen peroxide to water and oxygen Microbial Physiology & Growth Characteristics – DETECTING END-PRODUCTS OXIDASE TEST - identifies bacteria that produce cytochrome oxidase or indophenol oxidases, which are redox enzymes in the electron transport system that shuttle electrons to oxygen. If blue/ purple colour appears in 5 to 10 seconds, then the microorganisms are oxidase positive Microbial Physiology & Growth Characteristics – DETECTING END-PRODUCTS NITRATE REDUCTION TEST - detects reduced forms of nitrate, which occurs when bacteria use nitrate (NO3) as a substitute for oxygen (O2) during respiration. Nitrate reduction is demonstrated by adding chemicals that react with nitrite and noting development of a red colour, which will occur if the bacteria reduced nitrate to nitrite Microbial Physiology & Growth Characteristics – DETECTING END-PRODUCTS TRIPLE SUGAR IRON (TSI) - a slant medium with two growth environments: aerobic (on the slant) and anaerobic (in the “butt”). The results of this test are reported as appearance of the slant/appearance of the butt, using A to indicate acid reaction (yellow colour), K to indicate an alkaline reaction, and NC to indicate no change in the medium. H2S (detected as a blackening in the media) and the production of gas (CO2) as a by-product of fermentation are also reported if observed Microbial Physiology & Growth Characteristics – DETECTING END-PRODUCTS TRIPLE SUGAR IRON (TSI) - a slant medium with two growth environments: aerobic (on the slant) and anaerobic (in the “butt”). Differentiating Among Bacterial Species Based on Phenotypic Characteristics  “Staph” is Latin for grape-like Strepto - "twisted" in Latin, and clusters, and “coccus” is Latin cocco-, "seed." Definitions of for round, meaning that this is strep. spherical Gram-positive a group of bacteria that appear bacteria occurring in pairs or under the microscope as chains; cause e.g. scarlet fever round, grape-like clusters. and tonsillitis.  Differentiating Among Staphylococcus spp. – COAGULASE TEST For differentiating S. aureus from other staphylococci (S. epidermidis, S. saprophyticus). ability to produce the enzyme coagulase, which induces blood clot formation, along with other cell surface antigens such as Protein A. Staphylococcal food poisoning may result from ingesting food contaminated with either the bacteria or a heat-stable enterotoxin produced by the bacteria. Add drop of human or rabbit plasma  Serum vs. Plasma? Quick think: What is antisera? a serum containing antibodies Differentiating Among Staphylococcus spp. – MANNITOL SALT AGAR (MSA) a selective medium for staphylococci and other halotolerant bacteria because the high concentration of salt (7.5%) inhibits the growth of bacteria susceptible to the effects of osmotic stress. In addition, the medium contains mannitol, which is a fermentable substrate for some bacteria, and phenol red as an indicator for acid. The test is performed by streaking the bacteria over the surface of an MSA plate and incubating. Positive (left side) and negative (right side) results for mannitol fermentation are shown on the MSA plate.  Differentiating Among Staphylococcus spp. – HAEMOLYSIS Some bacteria are known to produce enzymes that break down phospholipids and cause the cell membranes of red blood cells to rupture. Haemolytic bacteria then scavenge the haemoglobin released from the cell, typically to utilize the iron or other “growth factors” from inside the cell. Haemolysis can be observed by streaking bacteria across the surface of a Blood Agar Plate (BAP), which contains intact red blood cells.  S. aureus S. epidermidis Beta-haemolytic on blood agar Gamma (non) haemolytic on blood agar coagulase-positive coagulase-negative Mannitol positive (yellow colonies) Mannitol negative Golden (pigment White staphyloxanthin) colonies on Tryptic colonies on *Tryptic Soy Agar (TSA) Soy Agar (TSA) *TSA - general growth medium for the isolation and cultivation of microorganisms http://www.bacteriainphotos.com/ https://www.microbiologyinpictures.com/  Differentiating Among streptococcal species – BILE ESCULIN AGAR medium used to isolate and identify enterococci. many species inhabit the oral cavity and upper respiratory tract, while others are found in the GI tract. Philosophically, these two diverse habitats prompted taxonomists to split the GI dwelling streptococci into a separate genus, Enterococcus. Those species that inhabit the mouth remain in the genus Streptococcus https://www.coursehero.com/tutors-problems/Microbiology/27103960-View-the-video-how-to-develop-and-use-a-dichotomous-key-httpsbitl/ CHARACTERISTICS OF BACILLI  Some bacilli are short and plump little rods, while others are extremely slender and long. Curved and spiral shapes are common. A few species of bacilli produce endospores or other types of cellular inclusion bodies, such as metachromatic granules and parasporal crystals. Gram positive bacilli are further subdivided according to whether they form endospores, have filamentous growth or hyphae, and if they are acid-fast. Gram-negative bacilli are typically distinguished by size, shape, motility, and oxygen growth categories.  Many of the Gram negative bacteria residing in the GI tract are members of the Family Enterobacteriaceae. Bacteria in this family are facultative anaerobes that are usually oxidase-negative and ferment glucose, which distinguishes them from aerobic species of Gram negative rods. To differentiate among them, a set of tests with the acronym IMViC, which stands for: Indole, Methyl red, Voges-Proskauer, and Citrate. The TSI (triple sugar iron agar) and urease tests and others may also be performed at the same time to improve the identification process.  Differentiating Among Bacilli – IMViC test * * *Remel 5% Alpha Naphthol (VP A) *40% potassium hydroxide (KOH) (VP B)  Differentiating Among Bacilli – IMViC test - Indole Sulfide–Indole–Motility (SIM): This is another medium in which a number of different reactions may occur. In fact, this medium is used to determine if:  the bacterium reduces sulfate and produces H2S, which is evidence of anaerobic respiration  the culture oxidizes the amino acid tryptophan and produces indole  the bacterium is motile. Bacteria are inoculated in this medium with an inoculating needle (as opposed to a loop) and stabbing the bacteria deep into the soft agar. Motility test: This is a differential medium used to determine whether an organism is equipped with flagella and thus capable of ‘swimming away’ from a stab mark. The results of motility agar are often difficult to interpret.  Differentiating Among Bacilli – IMViC test – MR-VP Methyl Red (MR) Bacteria utilizing a mixed acid fermentation produce stable organic acid end products. After the bacteria grow, the pH indicator methyl red (which is red below pH 4) is added to the culture. If the broth is red after methyl red is added, the result is considered a positive test. For bacteria that produce alcohols and other less acidic metabolites, the indicator (and therefore the medium) turns orange to yellow, which is a negative result. Voges-Proskauer (VP) Some bacteria use a butanediol fermentation pathway when glucose is the fermented substrate. The end products of this type of fermentation include a variety of both acidic and non-acidic end products, including ethanol, butanediol, and acetoin. If acetoin is present, chemical reagents added to the broth will react with it and form a reddish-brown coloured compound, which is considered a positive test result.  Differentiating Among Bacilli – IMViC test – Citrate Citrate Some bacteria have an enzyme (citrate permease) that facilitates the transport of citric acid into the cell, and another enzyme, citrase, for citrate catabolism. These bacteria have the ability to grow on a medium (Simmons’ citrate agar) that contains nothing more than citric acid, the first intermediate in the Krebs cycle of aerobic respiration, as a food source. Metabolism of citric acid releases carbon dioxide, which reacts with sodium and water in the medium to form a compound with a basic (alkaline) pH. In the presence of a base, the pH indicator in the medium (bromothymol blue) changes colour from green to blue, which is a positive result for this test It’s time to say hello to your brain End of Lecture 5  LECTURE 6 PRINCIPLES & FUNCTIONS OF CULTURE MEDIA SBGA_300424 Mar-Aug24  Importance of media for culturing microbe. Methods of obtaining pure cultures. Liquid, solid and semisolid media. Types of media.  Media Used for Bacterial Growth The primary objective of cultivating microorganisms, specifically bacteria, is to maintain viable populations of these organisms under controlled laboratory environments. Challenging process due to highly specific nutritional and environmental requirements and the diversity of these requirements among different species.  Nutritional Requirements All-purpose medium - tryptic soy broth (TSB). Specialised media - are used in the identification of bacteria and are supplemented with dyes, pH indicators, or antibiotics. e.g. enriched media - contains growth factors, vitamins, and other essential nutrients to promote the growth of fastidious (fussy) organisms, organisms that cannot make certain nutrients and require them to be added to the medium. Chemically defined medium - complete chemical composition of a medium is known. e.g. EZ medium Complex media - contain extracts and digests of yeasts, meat, or plants, the precise chemical composition of the medium is not known. e.g. nutrient broth, TSB, brain heart infusion (BHI). BHI is made by combining an infusion from boiled bovine or porcine heart and brain with a variety of other nutrients. BHI broth is often used in food safety, water safety, and antibiotic sensitivity tests.  Nutritional Requirements Selective medium - inhibit the growth of unwanted microorganisms and support the growth of the organism of interest by supplying nutrients and reducing competition. e.g. MacConkey agar that contains bile salts and crystal violet, which interfere with the growth of many gram-positive bacteria and favour the growth of gram-negative bacteria, particularly the Enterobacteriaceae. Entero – the intestine; Enterobacteriaceae species (enterics) reside in the intestine that adapted to bile salt. Quick think 1: Bile salt are stored in the _______. What is lobe? part of an organ that appears to be separate in some way from the rest.  Lactose-fermenting organisms, such as E. coli and Klebsiella spp, grow as pink to red colonies with or without a zone of precipitated bile. Do E. coli & Klebsiella ferment lactose? Lactose-non-fermenting organisms, such as Salmonella, Shigella and Proteus spp., form colourless or clear colonies. MUCOID is resembling mucus. microbenotes.com/macconkey-agar/ https://microbiologyinfo.com/macconkey-agar-composition- principle-uses-preparation-and-colony-morphology/  Nutritional Requirements Enrichment medium - general purpose enrichment agar which can nourish and support the growth of gram-positive as well as gram-negative bacteria. e.g. blood agar (5% defibrinated mammalian blood – horse, sheep, human) is added to the autoclaved basal media (TSA or Columbia Agar). Why enriched? Supports the growth of fastidious bacteria and inhibits the growth of some bacteria like Neisseria and Haemophilus. e.g. chocolate agar - nonselective, enriched growth medium used for isolation of pathogenic bacteria. It is a variant of the blood agar plate, containing red blood cells that have been lysed by slowly heating to 80°C. as a result, the cell lysis releases intracellular nutrients such as hemoglobin, hemin (“X” factor), and the coenzyme nicotinamide adenine dinucleotide (NAD or “V” factor) into the agar which is utilized by fastidious bacteria. Red blood cell lysis gives the medium a chocolate-brown coloration when prepared from which the agar gets its name. The most common bacterial pathogens that require this enriched medium for growth include Neisseria gonorrhoeae and Haemophilus species. https://microbenotes.com/chocolate-agar/ https://microbeonline.com/x-v-factor-test-haemophilus-principle-procedure-results/  Nutritional Requirements Differential medium e.g. Eosin Methylene Blue (EMB) - slightly inhibits the growth of Gram-positive bacteria and provides a color indicator distinguishing between organisms that ferment lactose (e.g., E. coli) and those that do not (e.g., Salmonella, Shigella). Certain lactose-fermenting bacteria produce flat, dark colonies with a green metallic sheen (most E. coli strains). Other lactose fermenters produce larger, mucoid colonies, often purple only in their centre. Non-lactose fermenter demonstrated colourless colonies. https://microbenotes.com/eosin-methylene-blue-emb-agar/ https://www.labtestsguide.com/eosin-methylene-blue-agar-emb-agar  Mueller Hinton Agar (MHA) commonly used for the routine susceptibility testing of non-fastidious microorganism by the Kirby-Bauer disk diffusion technique. “ZOI” What is your comment? Smaller zone vs. larger zone? Which concentrations indicate more sensitive/ resistant results? How about if no ZOI observed? Fungal Culture Media https://microbeonline.com/common-fungal-culture-media-uses/ Exercise https://microbiologyinfo.com/list-of-culture-media-used-in-microbiology-with-their-uses/ Autoclave  Obtaining Pure Culture is a laboratory culture containing a single species of organism. a pure culture is usually derived from a mixed culture (one containing many species) by transferring a small sample into new, sterile growth medium in such a manner as to disperse the individual cells across the medium surface or by thinning the sample many fold before inoculating the new medium. obtaining a pure culture of bacteria is usually accomplished by spreading bacteria on the surface of a solid medium so that a single cell occupies an isolated portion of the agar surface. This single cell will go through repeated multiplication to produce a visible colony of similar cells, or clones.  Solid vs. semi solid vs. liquid media other than nutrients, a solidification agent (agar, an inert substance extracted from sea algae. It does not show any nutritional value) is used during the preparation of solid and semi solid media. semi solid media (deep culture) are microbial culture media that are prepared to add less amount of agar (solidifying agent at 0.2 to 0.5 %) to observe motility of bacteria. Are the results macroscopic? Exercise: Compare between solid and semi solid media.  Solid vs. semi solid vs. liquid media broth culture is any type of liquid used to grow bacteria. Nutrient agar (NA) vs. nutrient broth (NB) culture: NA - contains solidifying agent NB – remains in liquid form  Spread Plate Technique is the method of isolation and enumeration of microorganisms in a mixed culture and distributing it evenly. The technique makes it easier to quantify bacteria in a solution. https://microbeonline.com/spread-plate-technique/ Spread Plate Technique  Pour Plate Technique The pour plate method is a microbiological laboratory technique for isolating and counting the viable microorganisms present in a liquid sample, which is added along with or before molten agar medium prior to its solidification. A spread-plate assay produces a plate with colonies distributed across the agar surface, while a pour plate assay produces a mixture of colonies embedded within the agar layer and colonies presenting at the agar surface End of Lecture 6 The main distinction between enriched (diperkayakan) and enrichment (pengayaan) media is that enriched media supports the growth of nutritionally demanding bacteria, whereas enrichment media prevents the growth of undesired or contaminated microorganisms. Foundation for Medical Microbiology: Koch Postulates The father of bacteriology Antonie van Leeuwenhoek - the father of microbiology Joseph Lister - the father of modern surgery/ antiseptic surgery Louis Pasteur - the father of modern microbiology Watch - Father of Microbiology, Modern Micro, Bacteriological Tech., Antiseptic Surgery, Immuno,Soil Micro (https://www.youtube.com/watch?app=desktop&v=a02KpOfPWmw) 1. Recognizing the presence of an agent in all cases of disease 2. Isolating that organism 3. Re-introducing the organism causes similar symptoms 4. The organism can be isolated again. Koch's postulates were limited: viral pathogens were not culturable in early microbiology labs and symptomatic carriers break this protocol. some pathogens do not exhibit the same presentation in mice (no animal model of infection with that particular bacteria). Mycobacterium leprae cannot be “grown in pure culture” in the lab. How about harmless bacteria? Can they cause a disease? Y/ N?  It has acquired extra virulence factors making it pathogenic.  It gains access to deep tissues via trauma, surgery, an IV line, etc.  It infects an immunocompromised patient.  LECTURE 7 SELECTIVE CULTIVATION OF MICROORGANISMS SBGA_140524 Mar-Aug24  Use of the following in the selective cultivation of microorganisms Chemicals Dyes Antimicrobial agents pH Temperature of incubation Atmospheric conditions  Selective Cultivation (medium) A selective culture medium is used to isolate a particular bacterial species or genus. After the addition of a number of inhibitors to the culture medium, the objective of this type of medium is to eliminate unwanted microbial flora. The selective medium is composed of a basic medium to which antibiotics, antiseptics, chemicals, dyes, sodium salts or phages can be added. What is phage? a virus that infects and replicates within bacteria and archaea. Bacteriophage (phage) https://antibioticguardian.com/antimicrobials-amr-ams/ https://byjus.com/chemistry/antimicrobial-agents/  1. Antibiotics more commonly used selective agents in culture media. Molecules that targets Gram-positive bacteria - penicillin G, bacitracin, vancomycin. Molecules that targets Gram-negative bacteria – colistin, polymixin B. Molecules that targets yeast and fungi – Amphotericin B, cycloheximide, nystatin. 1. Antibiotics https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6961714/  2. Antiseptics more rarely used in culture media. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6961714/  Disinfectants are used to kill germs on non-living surfaces. disinfection – the act of disinfecting something by using disinfectant to destroy, inactivate or reduce the concentration of pathogenic agents. Antiseptics kill microorganisms on your skin: washes (hand/ body washes/ soaps). rubs (leave-on products, or hand “sanitisers”, as well as antiseptic wipes).  3. Chemical substances used to inhibit certain bacteria. Potassium tellurite and bile salt inhibit Gram-positive bacteria. Lithium chloride inhibits Gram-negative bacteria. 3. Chemical substances https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6961714/  4. Dyes used as a colour indicator in a culture medium or as a selective agent against certain bacteria. e.g.: Crystal violet (CV) inhibits the growth of Gram-positive bacteria by inducing DNA lesions. Malachite green inhibits Gram-positive and –negative bacteria. Methylene blue inhibits Gram-positive bacteria and some yeast by making use of toxic dyes. 4. Dyes https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6961714/  5. Sodium salts known for their inhibitory properties. sodium chloride is used to select halophilic (salt-loving) bacteria that resist very high amounts of salts. 5. Sodium salts https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6961714/  6. Phage Bacteriophages are specific viruses of bacteria that can infect and even destroy bacteria, in the case of lytic phages. In order to isolate Mycobacterium tuberculosis, the use of phage lysin decontaminates the sputum of other bacteria present in the pulmonary microbiota. T1, T4 and φX174-like phages reduced E. coli population. Overview of studies using phage-antibiotic combinations against pathogenic bacteria, separated by type of study and experimental design. https://www.frontiersin.org/articles/10.3389/fcimb.2019.00022/full End of Lecture 7  LECTURE 8 PREPARATION AND STORAGE CULTURE MEDIA SBGA_140524 Mar-Aug24  Methods for storage and quality control of media. Methods for storing both dehydrated and prepared culture media. Maintaining a collection of bacterial and fungal stock cultures Microscopic examination. Inoculation and incubation of media Examination of specimens Methods for establishing and maintaining a culture collection. Components of Microbial Culture Media Components of Microbial Culture Media Preparation of Culture Media https://www.clinicalmicrobiologyandinfection.com/article/S1198-743X(21)00239-1/fulltext  Preparation of Culture Media Calculation & Weighing 16 grams of BAB medium (Sigma-Aldrich, UK) was added to 400ml of distilled water and autoclaved at 15psi (103kPa) for 30 minutesAfter autoclaving, the media were cooled to ~56oC, 20ml of Sterile Defibrinated Horse Blood (Sigma-Aldrich, UK) (5% v/v) was added and gently mixed. 14.8 grams of BHI medium (Sigma-Aldrich, UK) was added to 400ml of distilled water and autoclaved. Following autoclaving, the ‘straw’ coloured medium was stored at room temperature for up to 4 weeks. https://microbeonline.com/preparation-of-culture-media/  Preparation of Culture Media Mixing with water & boiling Why we have to use distilled water not the tap water? Tap water will affect selectivity and pH.  Preparation of Culture Media Sterilisation (autoclaving) Most media require sterilisation, so only bacteria from patient specimens will grow and not contaminants from water or powdered media.  Some media cannot be autoclaved (e.g., SS agar, Cary Blair agar). Liquid media are distributed to individual tubes or bottles before sterilization. Autoclave a medium only when the ingredients are completely dissolved. Do not tighten the lids or caps completely. Agar media are sterilised in large flasks or bottles capped with either plastic screw caps or plugs before being placed in an autoclave. Items are positioned with enough space for the steam to pass through  Nev's Ink autoclave tape will change from white to black to indicate that the autoclave's interior temperature is high enough for sterilisation. https://www.researchgate.net/figure/An-autoclave-tape-Brand-Surgical-System-A-bright-zebra-stripe-indicates-a-complete_fig4_349482812 How do we sterilise components that cannot withstand steam sterilisation (e.g. serum, certain carbohydrates and antibiotics, other heat-labile substances)? Membrane filtration with pores ranging in size from 0.2 to 0.45µm in diameter. However, virus will not be removed but does effectively remove most bacterial and fungal contaminants.  Preparation of Culture Media Fine-tuning (supplements and pH) If other ingredients are to be added (e.g., supplements such as sheep blood or specific vitamins, nutrients, growth promoters, or antibiotics), they should be incorporated when the molten agar has cooled, just before distribution to plates.  Preparation of Culture Media Fine-tuning (supplements and pH)  Preparation of Culture Media Dispensing Cool down the culture media in a water bath (45-50°C) or hot plate stirrer before dispensing to minimize condensation. Dispensing at too high temperature leads to excessive evaporation. If media stay too long in a water bath, it may cause precipitation (reheat, but do not overheat). Do not use cold water to cool down agar media, as it may lead to flakes or cloud formation.  Preparation of Culture Media Dispensing Mueller-Hinton Agar (MHA)  Preparation of Culture Media Dispensing  Culture Media Storage Guideline Must be kept in an optimum environment.  Light - should be stored away from hot lights. In addition, exposure to direct sunlight should be avoided if possible.  Heat - should be stored away from heat sources such as drying ovens, autoclaves and incubators.  Temperature - sealed, unopened containers should be stored at a temperature of 10 - 30°C. Certain sensitive media should be stored at 2-8 °C. Culture media should not be stored at or below 0°C as freezing could damage the media performance.  Humidity - culture media are hygroscopic in nature where they tend to absorb moisture from the surrounding. Sealed plastic containers are unaffected by normal laboratory humidity. However, frequently opened and closed containers of media will deteriorate in storage. Avoid storing media at media preparation rooms where the humidity is exceedingly high. https://www.fcbios.com.my/blogs/news-insight/culture-media-storage-guidelines  Culture Media Storage Guideline Wrap the plates in sealed, labelled plastic bags, a maximum of ten plates per bag, to avoid moisture. Store them upside down, at 2-8°C, in the dark following manufacturer’s instructions. At 2-8°C, in fridge (refrigerator) or freezer? A refrigerator popularly referred to as a fridge is a cooling appliance that is typically used for the storage and preservation of food. In most cases, it usually consists of two sections: a refrigerator section and a freezer compartment. The freezer compartment is designed to store frozen food while the refrigerator part is used to keep food and beverage from spoiling. Shelf life  Using Culture Media Bring the culture media to room temperature before use. Ensure there are no visible drops of water on the agar surface or inside the lid. If seen, do not shake off condensation water from the lid. Instead, dry plates for 20-30 minutes at 35-37°C with agar plates upside down and agar base resting at an angle on the lid (if necessary, dry plates at 20-25°C overnight). Do not over-dry plates (cracks in surface, surface wrinkled). Visual sterility check before use is a mandatory step. Check the plates for contamination or growth of colonies. Most common errors & possible causes Most common errors & possible causes Most common errors & possible causes Most common errors & possible causes Exercise: Please watch at https://www.youtube.com/watch?v=oe7AY0QTcD4&t=377s End of Lecture 8  LECTURE 9 MAINTAINING BACTERIAL & FUNGAL STOCK SBGA_040624 Mar-Aug24  Purpose of maintaining the microorganims After a microorganism has been isolated and cultivated in pure culture, it becomes essential to preserve its purity and viability by preventing contamination of the pure culture. Microbiology laboratory has to maintain quality control (QC) stocks obtained from the ATCC or commercial vendors. ATCC - The American Type Culture Collection. Collection of bacterial strains, animal and plant viruses, protozoans, yeasts and fungi. 2024  2 In laboratories, the pure cultures are transferred 1 periodically onto or into a fresh medium (subculturing) to allow continuous growth and viability of microorganisms. The transfer is always subject to aseptic conditions to avoid contamination. Since repeated subculturing is time-consuming, it becomes difficult to maintain a large number of pure cultures successfully for a long time. In addition, there is a risk of genetic changes as well as contamination. Therefore, it is now being replaced by some modern methods that do not need frequent subculturing. These methods include refrigeration, paraffin method, cryopreservation, and lyophilization (freeze-drying).  Short term Long term storage storage  Periodic Transfer to Fresh Media  Freezing at -70°C  Refrigeration  Cryopreservation  Preservation in Glycerol at -20°C  Lyophilization (Freeze-Drying)  Stab Cultures Recovery of Bacteria from  Cooked-meat medium lyophilized storage condition (anaerobes)  Paraffin Method  preparing a fresh culture from the previous stock. the temperature and the medium chosen should support a slow rather than a rapid rate of growth so that the time interval between transfers can be as long as possible. common heterotrophs remain viable for several weeks or months on a medium like nutrient agar. disadvantage of failing to prevent changes in the characteristics of a strain due to the development of variants and mutants. Troph refers to nourishment, food. An autotroph is an Heterotroph is an organism that can organism that does not produce its own food have the ability to using light, water, chemically produce (i.e. carbon dioxide (as sole synthesise) its own food source of carbon), or from inorganic molecules. other chemicals.  Autotrophs are producers Heterotrophs are consumers who who prepare their own depend on other sources for their food. food. Photoautotrophs - utilise solar energy and carbon dioxide to prepare their food by the process of photosynthesis. Chemoautotrophs - utilise energy obtained from a chemical reaction involving oxidation in order to prepare their food.  Pure cultures can be successfully stored at 0-4°C in refrigerators or cold rooms. This method is applied for a short duration (2-3 weeks for bacteria and 3-4 months for fungi) because the metabolic activities of the microorganisms are significantly slowed down but not stopped. Thus their growth continues slowly, nutrients are utilised, and waste products are released into the medium. This finally results in the microbes’ death after some time.  Grow a pure culture on an appropriate solid medium. Glycerol is used as a cryoprotectant When the culture is fully developed, scrape it off with a as it disrupts the hydrogen bonding loop. between water molecules, hence preventing the formation of ice crystals Suspend small clumps of the culture in sterile neutral during freezing. This protects yeast glycerol. and bacterial cells from ice crystal damage, and maintains their viability for future re-culture. Distribute in quantities of 1–2 ml in screw-capped tubes or vials. 10-15% glycerol - MINIMISES the Store in a freezer at -20°C (for several months) and -80°C damage during freezing. (for many years). Most labs store bacteria in 15- Avoid repeated freezing and thawing. Transfer after 12–18 25% glycerol. months. Exercise: Read more at https://www.wikihow.com/Prepare-Glycerol-Stock  Stab cultures are similar to agar plates, but are formed by solid agar in a test tube. Used for non-fastidious organisms only (Staphylococci & Enterobacteriaceae). Bacteria is introduced via