PST NTA Level 5

NTA Level 5 • Semester 1 • PST05103 Antiseptics and Disinfectants Pharmaceutical Microbiology • Source Session/Topic 43 Full source-text version: all educational wording from the extracted learning source is retained; only presenter/tutor metadata and web-layout noise are removed, while formatting is improved for readability. Session 43: Antiseptics and Disinfectants Total Session Time: 120 minutes Pre-requisites • Human anatomy and physiology Students Learning Tasks By the end of this session students are expected to be able to: • List criteria for selection of antiseptics • Explaincriteria for selection of disinfectants • Identify factors affecting action of antiseptics and disinfectants • Describe chemical groups of antiseptics and disinfectants Resources Needed: • Flip charts, marker pens, and masking tape • Black/white board and chalk/whiteboard markers SESSION OVERVIEW |Step |Time |Activity/ |Content | | | |Method | | |1 |5 minutes |Presentation |Introduction, Learning Tasks | |2 | |Presentation |General Properties of Antiseptics | | |15 minutes | |and Disinfectants | |3 |50 minutes |Presentation |Chemical Agents Used as | | | | |Antiseptics and Disinfectants | |4 |20 minutes |Presentation |Factors affecting action of | | | | |antiseptics and disinfectants | |5 |20 minutes | |Criteria for selection of | | | |Presentation |antiseptics and disinfectants | |6 |5 minutes |Presentation |Key Points | | 7|5 minutes |Presentation |Evaluation | SESSION CONTENTS STEP 1: Presentation of Session Title and Learning Tasks (5 minutes) READ or ASK students to read the learning tasks and clarify ASK students if they have any questions before continuing STEP 2: General Properties of Antiseptics and Disinfectants (15 minutes) Antiseptics • Antiseptics are chemicals that are applied to animate surfaces (skin, mucous membranes and wounds) to kill or inhibit the growth of microorganisms • The ideal antiseptic has to have similar properties as an ideal disinfectant • But the primary importance for antiseptics is the selective toxicity o Toxicity to microorganisms but not to human cells • The degree of selectivity of the antiseptic agents can change depending on the tissues they contact • Chemical used as antiseptics have sufficiently low toxicity for host cells that they can be used directly on skin, mucous membranes or wounds • Ideal Characteristics of Antiseptics o They must have adequate antimicrobial activity o The must not be toxic or irritating for skin o Antiseptics are mostly used to reduce the microbial population on the skin before surgery or on the hands to help prevent spread of infection by this route. Antiseptics are often lower concentrations of the agents used for disinfection • Uses of antiseptics o The treatment of skin infections o Prevention of infections in cuts and wounds o Cleaning the skin area of surgery from microorganisms o Prophylaxis and treatment of infections in mucosal areas such as mouth, nose and vagina that are open to environment o As a scrub for surgeons and the medical personnel Disinfectants • Disinfectants are used on inanimate surfaces • They may be the same agents used for antisepsis but at higher concentrations • Ideal characteristics • Should be soluble in various solvent especially water. • Should have high potency. • Should be compatible with organic matters. • Should be stable on storage. o Effective at room temperature o Non-corrosive and nontoxic o Inexpensive STEP 2: Chemical Agents Used as Antiseptics and Disinfectants (50 minutes) • Classes of chemicals used as antiseptics and disinfectants o Alcohols o Acids and Esters o Aldehydes o Biguanides o Halogens o Hydrogen peroxide and peroxygen compounds o Phenols • Alcohols o The aliphatic alcohols (ethanol and isopropanol) are used for disinfection and antisepsis o They are bactericidal against vegetative forms, including Mycobacterium species, but are not sporicidal o Cidal activity drops sharply below 50% concentration o Alcohols have poor penetration of organic matter and their use is therefore restricted to clean conditions o They possess properties such as a cleansing action and volatility, are able to achieve a rapid and large reduction in skin flora and are widely used for skin preparation before injection or other surgical procedures o Ethanol (CH3CH2OH) ▪ Ethanol is widely used as a disinfectant and antiseptic ▪ The presence of water is essential for activity; hence 100% ethanol is ineffective ▪ Concentrations between70% and 90% are best bactericidal and a 70% solution is usually employed for the disinfection of skin, clean instruments or surfaces ▪ At higher concentrations, e.g. 90%, ethanol is also active against most viruses, including HIV ▪ Ethanol is also a used in pharmaceutical preparations and cosmetic products as a solvent and preservative o Isopropyl alcohol (isopropanol, CH3. CHOH.CH3) ▪ Has slightly greater bactericidal activity than ethanol but is also about twice as toxic ▪ It is less active against viruses, particularly nonenveloped viruses, and should be considered a limited-spectrum virucide ▪ Used at concentrations of 60–70%, it is an acceptable alternative to ethanol for preoperative skin treatment and is also employed as a preservative for cosmetics • Aldehydes o Many aldehydes have antimicrobial properties, including sporicidal activity, o Glutaraldehyde (CHO(CH2)3CHO) ▪ It is the most widely used for disinfection ▪ It is a highly effective biocide and it is also used as ‘chemosterilant’ ▪ It has a broad spectrum of antimicrobial activity and rapid rate of kill, most vegetative bacteria being killed within a minute of exposure, although bacterial spores may require 3 hours or more. ▪ It is not affected significantly by organic matter ▪ The glutaraldehyde molecule possesses two aldehyde groupings which are highly reactive and their presence is an important component of biocidal activity ▪ At a pH of 8, biocidal activity is greatest but stability is poor due to polymerization ▪ In contrast, acid solutions are stable but considerably less active ▪ In practice, glutaraldehyde is generally supplied as an acidic 2% or greater aqueous solution, which is stable on prolonged storage ▪ This is then ‘activated’ before use by addition of a suitable alkalizing agent to bring the pH of the solution to its optimum for activity ▪ The activated solution will have

NTA Level 5 • Semester 1 • PST05103 Sterilization Methods and Criteria for Selection Pharmaceutical Microbiology • Source Session/Topic 44 Full source-text version: all educational wording from the extracted learning source is retained; only presenter/tutor metadata and web-layout noise are removed, while formatting is improved for readability. Session 44: Sterilization Methods and Criteria for Selection Total Session Time: 120 minutes Pre-requisites • Human anatomy and physiology Students Learning Tasks By the end of this session students are expected to be able to: • List methods used for sterilization • Explain (dry and moist) heat sterilization • Explain gaseous sterilization • Explain radiation sterilization • Explain sterilization by filtration • List the criteria for selecting sterilization method Resources Needed: • Flip charts, marker pens, and masking tape • Black/white board and chalk/whiteboard markers • Handout 48.1: Sterilization Methods SESSION OVERVIEW |Step |Time |Activity/ |Content | | | |Method | | |1 |5 minutes |Presentation |Introduction, Learning Tasks | |2 |15 minutes |Presentation |Introduction to Sterilization | |3 |30 minutes |Presentation |Heat sterilization | |4 |20 minutes |Presentation |Gas sterilization | |5 |15 minutes |Presentation |Radiation sterilization | |6 |15 minutes |Presentation |Sterilization by filtration | |7 |10 minutes |Presentation |Criteria for selecting | | | | |sterilization method | |8 |5 minutes |Presentation |Key Points | | 9|5 minutes |Presentation |Evaluation | SESSION CONTENTS STEP 1: Presentation of Session Title and Learning Tasks (5 minutes) READ or ASK students to read the learning tasks and clarify ASK students if they have any questions before continuing STEP 2: Introduction to Sterilization (15 minutes) • Microorganisms cause contamination, infection, and decay • In order to prevent contamination of pharmaceuticals and infection, it is necessary to remove microorganisms • Sterilization is the process of killing or removing all viable organisms by by physical, chemical and mechanical means. • Physical sterilization employs the use of heat and radiation to kill the microorganisms • Chemical sterilization uses chemical agents in form of liquid or gases to kill the microorganisms • Mechanical methods involve removal of microorganisms by filtration • In sterilization all micro-organisms both pathogenic and non- pathogenic including spores are killed or removed. • All sterilization methods must be validated and monitored e.g. bacillus stearothermophilus is used to monitor moist heat sterilization process STEP 3: Heat Sterilization (30 minutes) Heat Sterilization • Heat sterilization is the killing of all organisms from an item by the use of heat. • Heat sterilization is the safest and most common method of sterilization • Types of heat sterilization o Dry heat sterilization o Moist heat sterilization Dry Heat Sterilization • Dry heat sterilization involves use of dry heat to kill microorganisms. Organisms are killed by oxidation effects • Dry heat sterilization is divided into flaming, incineration and hot air sterilization o Flaming ▪ It is the oldest and the simplest method of sterilization ▪ The item to be sterilized is held in an open flame. ▪ Example of sterilization by flaming is the heating of a loop wire to a red glow on a Bunsen flame before using it to transfer microorganisms [pic] ▪ Advantages of Flaming Method • Flaming is a convenient way of killing many, but not necessarily all, microorganisms • It can be used to kill all microorganisms in meat ▪ Disadvantages of Flaming • Does not necessarily kill all microorganisms o Incineration ▪ This is an excellent method of destroying or disposing of materials such as heavily contaminated cloth, animal carcasses and pathological materials [pic] ▪ Advantages • Incineration destroys 100% of microorganisms and reduces the volume of waste significantly. • It's also the ideal way to prevent contaminated items from being reuses ▪ Disadvantages • Incineration completely the object/item being incinerated • It is expensive method (repair, maintenance and operation) o Hot air sterilization (Hot air oven) ▪ Is a sterilization method in which organisms are killed by oxidation effects ▪ In this process, the various items need to be sterilized are duly kept in an electric oven, preferably with a stainless- steel chamber inside, and duly maintained at 170°C for a duration of approximately 2 hours (to ensure complete sterilization) ▪ Dry heat sterilization requires raising the temperature of an object to a controlled 170° C for approximately 2 hours to kill all microorganisms ▪ The items to be sterilized is exposed at higher temperature for prolonged duration of time ▪ Dry heat is the ideal way to kill microorganisms attached to fats, oils and powders, which can't be sterilized in an autoclave because they either resist water or are destroyed by it. ▪ The Hot Air Oven is the equipment used for this type of sterilization. This oven has; • A thermostat controlling the temperature • Double walled insulation keeps the heat in and conserves energy ▪ Hot air oven is used for sterilization of; • Medical equipment resistant to heat e.g. forceps, scissors, scalpels, swabs • Thermal stable and moisture-labile pharmaceuticals products o Nonaqueous Liquids (Liquid paraffin, fats and grease o Solids (powders) Moist Heat Sterilization • This is a sterilization method that uses heat in moisture to kill microorganisms • In this method microorganisms are killed by coagulating or denaturation of their proteins • Moist heat sterilization methods include boiling, autoclaving and pasteurization • Boiling o Boiling at 100°C at 760 mm atmospheric pressure kills particularly several varieties of vegetative states of microbial strains, many viruses and fungi usually within a period of 10 minutes o This method cannot destroy endospores and certain viruses o This method is mainly used for making food products safer for human consumption • Autoclaving o This is a sterilization method that is operated in a special equipment called Autoclave o The process of moist heat sterilization is also known as Autoclaving o Items to be sterilized are heated and maintained at temperatures of 120 ± 2°C and high positive pressure in an autoclave o Autoclaving is used for items/products that are not sensitive to moisture and thermostable An autoclave [pic] Schematic diagram

NTA Level 5 • Semester 1 • PST05103 Introduction to Immunology Pharmaceutical Microbiology • Source Session/Topic 45 Full source-text version: all educational wording from the extracted learning source is retained; only presenter/tutor metadata and web-layout noise are removed, while formatting is improved for readability. Session 45: Introduction to Immunology Total Session Time: 60 minutes Pre-requisites • Human anatomy and physiology Students Learning Tasks By the end of this session students are expected to be able to: • Explain e terms used in immunology and immunization • Describe types of immunity Resources Needed: • Flip charts, marker pens, and masking tape • Black/white board and chalk/whiteboard markers SESSION OVERVIEW |Step |Time |Activity/ |Content | | | |Method | | |1 |5 minutes |Presentation |Introduction, Learning Tasks | |2 |10 minutes |Presentation |Common Terminologies used in | | | | |Immunity | |3 |10 minutes |Presentation |The Immune System | |4 |25 minutes |Presentation |Types of Immunity | |5 |5 minutes |Presentation |Key Points | | 6|5 minutes |Presentation |Evaluation | SESSION CONTENTS STEP 1: Presentation of Session Title and Learning Tasks (5 minutes) READ or ASK students to read the learning tasks and clarify ASK students if they have any questions before continuing STEP 2: Common Terminologies Used in Immunology (10 minutes) • Immunity o Immunity is the ability of the body to defend itself against diseases • Immunology o Immunology is the study of immunity • Immune response o A response generated against a potential pathogen • Granulocytes o Are leukocytes that contain densely staining granules • Monocytes o Are small leukocytes that circulate in the blood • Phagocytosis o Is a process whereby cells of the immune system known as phagocytic cell e.g. a neutrophil, recognize, ingests and destroys a pathogen • Antigens o Are substances that are capable of stimulating the immune system to produce specific immune response against them • Antibodies o Are specific substances produced by the immune system in response to exposure to an antigen STEP 3: The Immune System (10 minutes) • The immune system is the system of specialized cells and organs that protect the body from diseases • The immune system functions to protect the body against bacterial and viral infections and destroy cancer cells and foreign substances • When the immune system is weakened, its ability to defend the body also weakens. This allows pathogens to grow and flourish in the body, and cause diseases • The immune system also performs surveillance of tumour cells, and immune suppression has been reported to increase the risk of certain types of cancer • The immune system protects the body from infection through various lines of defence. • The immune system is a complex system of structures and processes that work together to protect the body • The immune system is made up of two components derived from the central (bone marrow and thymus) and peripheral (spleen, lymph nodes and lymphatic channels, tonsils, adenoids, Peyer's patches, and appendix) lymphoid organs; o The molecular component ▪ This component uses specific and nonspecific molecules to protect the body against foreign substances and pathogens ▪ Molecules of the immune system; • Immunoglobulins • Lymphokines o The cellular components ▪ This component consists of immune cells that protect the body ▪ Cells of the immune system are white blood cells • Granulocytes • Lymphocytes • Monocytes • Macrophages STEP 4: Types of Immunity (25 minutes) • There are basically two types of immunity, innate immunity and acquired immunity • Innate immunity o It is an immunity that an individual is born with, it is pre- existing in the body o It is the first line of defence o It is non-specific and non-adaptive ▪ It produces the same responses for all potential pathogens o Innate immunity includes; ▪ Physicochemical barriers (e.g. skin, saliva, lysozymes etc.) • These physical barrier kills or prevent entry of pathogens ▪ Cells (e.g. macrophages, neutrophils, basophils, mast cells etc.) • These cells attack and kills foreign organisms and toxins ▪ Processes • Processes such as inflammatory reactions and phagocytosis limit spread of infection o These components protect the body for the first few days of an infection o Sometimes innate immunity is enough to clear the pathogen • Acquired immunity o This is the type of immunity that is not present at birth o It is the second line of defence which involves building up memory of encountered infections so can mount an enhanced response specific to the pathogen or foreign substance o Acquired immunity is adaptive o It involves ▪ Antibodies • which generally target foreign pathogens roaming free in the bloodstream ▪ T cells • which are directed especially towards pathogens that have colonised cells and can directly kill infected cells or help control the antibody response o Acquired immunity has two components; humoral immunity and cell- mediated immunity o Humoral immunity is mediated by antibodies while cell-mediated immunity is mediated by immune cells o Acquired immunity is either active or passive o Active acquired immunity; ▪ It is induced after contact with a foreign substance or pathogen (antigen) ▪ After exposure the body actively produces antibodies and immune cells that will fight against the pathogens ▪ Active acquired immunity is acquired either artificially e.g. by injection of antigen (e.g. vaccines) or naturally e.g. after a clinical or subclinical infection ▪ Advantage: • Produces long term immunity ▪ Disadvantages; • Slow onset of immunity • Requires prolonged contact with antigen o Passive acquired immunity ▪ Passive acquired immunity is naturally acquired from the mother or artificially produced e.g. by injection of an antibody containing preparation ▪ Advantage: • Immediate action ▪ Disadvantages • Short lived • Hypersensitivity reaction STEP 5: Key Points (5 minutes) • Immunity is the ability of the body to defend itself against invading pathogens or toxin • The immune system consists of innate and acquired components of the immunity • The innate component is pre-existing, comprised of physical barriers such as skin and mucous membrane together

NTA Level 5 • Semester 1 • PST05103 Antigens and Antibodies Pharmaceutical Microbiology • Source Session/Topic 46 Full source-text version: all educational wording from the extracted learning source is retained; only presenter/tutor metadata and web-layout noise are removed, while formatting is improved for readability. Session 46: Antigens and Antibodies Total Session Time: 120 minutes Pre-requisites • Human anatomy and physiology Students Learning Tasks By the end of this session students are expected to be able to: • Differentiate between antigens and antibodies • Explain sources of antibodies • Classify antibodies Resources Needed: • Flip charts, marker pens, and masking tape • Black/white board and chalk/whiteboard markers SESSION OVERVIEW |Step |Time |Activity/ |Content | | | |Method | | |1 |5 minutes |Presentation |Introduction, Learning Tasks | |2 | 35 minutes|Presentation |Antigens | |3 |35 minutes |Presentation |Antibodies | |4 |25 minutes |Presentation |Classification of antibodies | |5 |10 minutes |Presentation |Differences between antigens and | | | | |antibodies | |6 |5 minutes |Presentation |Key Points | | 7|5 minutes |Presentation |Evaluation | SESSION CONTENTS STEP 1: Presentation of Session Title and Learning Tasks (5 minutes) READ or ASK students to read the learning tasks and clarify ASK students if they have any questions before continuing STEP 2: Antigens (35 minutes) Definition of Antigens • Antigens are substances that induce a specific immune response and subsequently react with the products of a specific immune response. • An antigen is a molecule that stimulates an immune response. • The definition encompasses all substances that can be recognized by the adaptive immune system. • Antigens differ from allergens • An allergen is a substance that causes the allergic reaction. The (detrimental) reaction may result after exposure via ingestion, inhalation, injection or contact with skin • The name “antigen” is derived from the term “ANTI’body GEN’erator” • Superantigens are class of molecules that can interact with antigen presenting cells and T-cells in nonspecific way e.g. staphylococcal exotoxins, Toxic shock syndrome toxins and some viral proteins Classification of Antigens • Antigens can be classified in order of their origins into exogenous antigens, endogenous antigens and autoantigens. o Exoantigens ▪ Exogenous antigens are antigens that have entered the body from the outside, for example by inhalation, ingestion, or injection. ▪ These antigens enter the body by endocytosis or phagocytosis • The antigens are taken into the antigen-presenting cells (APCs) and processed into fragments. o Antigen-presenting cells (APCs) are a heterogeneous group of immune cells that mediate the cellular immune response by processing and presenting antigens for recognition by certain lymphocytes such as T cells. Classical APCs include dendritic cells, macrophages, Langerhans cells and B cells. o Endogenous antigens ▪ Endogenous antigens are antigens that have been generated within the cell, as a result of normal cell metabolism, or because of viral or intracellular bacterial infection. o Autoantigens ▪ An autoantigen is usually a normal protein or complex of proteins (and sometimes DNA or RNA) that is recognized by the immune system of patients suffering from a specific autoimmune disease. ▪ These antigens should under normal conditions not be the target of the immune system, but due to mainly genetic and environmental factors the normal immunological tolerance for such an antigen has been lost in these patients. o There are antigens that are known as tumor antigens ▪ Tumor antigens are those antigens that are presented by the major histocompatibility complex class I (MHC I) molecules on the surface of tumor cells. ▪ These antigens can sometimes be presented only by tumor cells and never by the normal ones. In this case, they are called tumor-specific antigens (TSAs) and typically result from a tumor specific mutation. • Antigens can also be classified as Thymus-dependent antigens (TD-Ag) and Thymus independent antigens (TI-Ag) • Characteristics of Antigens o Immunogenicity ▪ The capacity to stimulate the production of antibodies or cell- mediated immune responses ▪ It is the ability of the antigen to stimulate an immune response from the immune system o Antigenicity ▪ The ability to bind antibody ▪ An incomplete antigen that cannot elicit an immune response is known as a hapten o Foreignness ▪ this an antigen being recognized as non-self. It is important feature because self-responsive cells are eliminated during lymphocyte activation leaving only cells that respond to non- self o Specificity ▪ This is the uniqueness of an antigen. ▪ Antigen specificity depends on epitopes • An epitope is the region or part of the antigen that is active immunogically i.e. it binds to antibodies and products of immune response o Chemical and structural complex ▪ Proteins are the most potent immunogens ▪ Polysaccharides are less immunogenic ▪ Nucleic acids and lipids do not elicit good immune response ▪ Molecules with simple chemical structures e.g. a simple sugar are less immunogenic than complex molecules o Molecular size ▪ Large molecular weight proteins are highly antigenic ▪ Smaller molecular weight molecules are less antigenic o Stability ▪ Highly stable and non-degradable substance are not immunogenic o Biological systems ▪ Some substances are immunogenic in one individual but not immunogenic in others o Dose of antigen ▪ Very low dose does not stimulate immune response ▪ Repeated accumulation may be needed to stimulate immune response in some cases e.g. booster doses in immunization such as in diphtheria o Route of entry ▪ Antigens entering through parenteral route produce good levels of antibodies o Adjuvants ▪ A substance that increases immunogenicity of an antigen when mixed with that antigen and then injected ▪ Adjuvants increase the strength and duration of immune response • Aluminium potassium sulfate STEP 3: Antibodies (35 minutes) • An antibody (or an Immunoglobulin) is a specialized immune protein produced because of the introduction of an antigen into the body, and which possesses the ability to combine with the very antigen that triggered its production. • The production of antibodies is a major function of the immune system and is carried out by B-lymphocytes • Antibodies can be triggered by and directed at

NTA Level 5 • Semester 1 • PST05103 Activation of Lymphocytes Pharmaceutical Microbiology • Source Session/Topic 47 Full source-text version: all educational wording from the extracted learning source is retained; only presenter/tutor metadata and web-layout noise are removed, while formatting is improved for readability. Session 47Activation of Lymphocytes Total Session Time: 120 minutes Pre-requisites • Human anatomy and physiology Students Learning Tasks By the end of this session students are expected to be able to: • Explain types and functions of lymphocytes Resources Needed: • Flip charts, marker pens, and masking tape • Black/white board and chalk/whiteboard markers SESSION OVERVIEW |Step |Time |Activity/ |Content | | | |Method | | |1 |5 minutes |Presentation |Introduction, Learning Tasks | |2 |15 minutes |Presentation |Lymphocytes | |3 |20 minutes |Presentation |Major Histocompatibility Complex | |4 |40 minutes |Presentation |Activation of Lymphocytes | |6 |5 minutes |Presentation |Key Points | | 7|5 minutes |Presentation |Evaluation | SESSION CONTENTS STEP 1: Presentation of Session Title and Learning Tasks (5 minutes) READ or ASK students to read the learning tasks and clarify ASK students if they have any questions before continuing STEP 2: Lymphocytes (15 minutes) • Lymphocytes are a type of white blood cell (leukocyte) that are of fundamental importance in the immune system • Lymphocytes determine the specificity of the immune response to infectious microorganisms and other foreign substances • In adult humans lymphocytes make up roughly 20 to 40 percent of the total number of white blood cells • Lymphocytes are found in the circulation and in central lymphoid organs and tissues where initial immune response occurs • There are two primary types of lymphocytes; o B lymphocytes and T lymphocytes, or B cells and T cells • Both types of lymphocytes originate from stem cells in the bone marrow and are initially similar in appearance • The B cells produce antibodies that are used to attack invading bacteria, viruses, and toxins • The T cells destroy the body's own cells that have themselves been taken over by viruses or become cancerous STEP 3: Major Histocompatibility Complex (20 minutes) • Major Histocompatibility Complex (MHC) is a group of genes that code for proteins found on the surfaces of cells that help the immune system recognize foreign substances. The genes are located on chromosome 6 in human beings • Each gene of MHC has an unusually large number of alleles therefore it is very rare for two persons to have the same set of MHC molecules (tissue types) • MHC proteins are found in all higher vertebrates. In human beings the complex is also called the human leukocyte antigen (HLA) system • There two classes of MHC, class I and class II • MHC Class I o Molecules of MH Class I are found on membranes of almost all cells o These help the immune system to recognize heathy cells from infected cells and pathogens • MHC Class II o In MHC class II molecules are restricted to macrophages and lymphocytes o This class help cells of the immune system to interact and communicate with one another • The MHC also contains genes that code for other proteins e.g. the complement proteins, cytokines and enzymes. These proteins are called Major Histocompatibility Class III molecules • MHC molecules are important components of the immune system because they allow T lymphocytes to detect cells, such as macrophages, that have ingested infectious microorganisms • When a macrophage engulfs a microorganism, it partially digests it and displays peptide fragments of the microbe on its surface, bound to MHC molecules • The T lymphocyte recognizes the foreign fragment attached to the MHC molecule and binds to it, stimulating an immune response • In uninfected healthy cells, the MHC molecule presents peptides from its own cell (self-peptides), to which T cells do not normally react STEP 4: Activation of Lymphocytes (40 minutes) • Activation of lymphocytes occurs o in the thymus to form T-cells or T-lymphocytes o in bone marrow to form B-cells or B-lymphocytes • Most lymphocytes are short-lived, with an average life span of a week to a few months • A few lymphocytes live for years, providing a pool of long-lived T and B cells o These cells account for immunologic “memory,” a more rapid, vigorous response when the same antigen enters the body • Activation process involves macrophages engulfing and digesting pathogens o Through receptor molecules on their surfaces, lymphocytes are able to bind antigens and help remove them from the body o Each lymphocyte bears receptors that bind to a specific antigen o The ability to respond to virtually any antigen comes from the enormous variety of lymphocyte populations that the body contains, each of them with a receptor capable of recognizing a unique antigen o When macrophages engulf and digest pathogens, they take the antigenic components of that pathogen and display them on their membrane as antigens by attaching them onto major histocompatibility complex class II (MHC class II) ▪ This is antigen presentation and the macrophages are antigen presenting cells (APCs) o T-lymphocytes such as inactivated helper T cells can now bind onto these antigen complexes by using special receptors of their own (T cell receptors that contain the glycoprotein CD4) o Once bound, they begin releasing various chemicals such as interleukin-1, which activates that helper T cell o The helper T cell can then detach and bind to B-lymphocytes that contain that same antigenic piece, which causes the two cells to begin releasing cytokines and lymphokines o This causes the cloning process in which the cells divide mitotically to form many identical clones o Some of these B-cell clones differentiate into plasma cells and memory B cells while other T-cell clones differentiate into cytotoxic T cells. o The plasma cells produce antibody molecules which are released into the blood and lymph o Antibodies then bind to the target antigen and initiate its neutralization or destruction o Antibody production continues for several days or months, until the antigen has been overcome

NTA Level 5 • Semester 1 • PST05103 Complement System Pharmaceutical Microbiology • Source Session/Topic 48 Full source-text version: all educational wording from the extracted learning source is retained; only presenter/tutor metadata and web-layout noise are removed, while formatting is improved for readability. Session 48 Complement System Total Session Time: 120 minutes Pre-requisites • Human anatomy and physiology Students Learning Tasks By the end of this session students are expected to be able to: • Describe complement system Resources Needed: • Flip charts, marker pens, and masking tape • Black/white board and chalk/whiteboard markers SESSION OVERVIEW |Step |Time |Activity/ |Content | | | |Method | | |1 |5 minutes |Presentation |Introduction, Learning Tasks | |2 |20 minutes |Presentation |Characteristics of complement | | | | |system | |3 |25 minutes |Presentation |Biological effect of complement | | | | |system | |4 |30 minutes |Presentation |Regulation of compliment system | |5 |5 minutes |Presentation |Key Points | |6 |5 minutes |Presentation |Evaluation | SESSION CONTENTS STEP 1: Presentation of Session Title and Learning Tasks (5 minutes) READ or ASK students to read the learning tasks and clarify ASK students if they have any questions before continuing STEP 2: Characteristics of complement system(20minutes) • The complement system is a well regulated system that enhances (complements) the ability of antibodies and phagocytic cells to clear microbes and damaged cells from an organism, promotes inflammation, and attacks the pathogen's cell membrane • The complement system includes serum and membrane-bound proteins that participate in both innate and adaptive immunity • These proteins are highly regulated and interact through a series of proteolytic cascades • Complements are soluble proteins and glycoproteins mostly produced by hepatocytes. • More than 20 types of complements are present in serum, found circulating normally in human body in inactive forms (called as zymogens or proenzymes) • Complement activation is triggered by an antibody when it is bound to the antigen • It can also be triggered by some components of innate immunity • Thus the complement system works in both innate and acquired immunity • Complements are activated only during inflammatory reactions • During the inflammation, more amount of complements reaches to the interstitial area of the infected tissue through dilated blood vessels, which are then activated by proteolytic cleavage; this exposes the active site of the complements. • Complements are mainly denoted by the capital letter C with numbers; like, C1, C2, C3, and so on. Some have only alphabet, like, B, D. Some are simply represented by names, like, homologous restriction factor. • C1 has three sub-units; C1q, C1r and C1s • C2-C5 have two components, a and b • Larger subunits are denoted by b and the smaller are denoted by a (except C2a, which is larger than C2b) • • STEP 3:Biological effect (Functions) of the Complement system • (40 minutes) • Opsonization and phagocytosis o C3b, bound to immune complex or coated on the surface of pathogen, activate phagocytic cells o These proteins bind to specific receptors on the phagocytic cells to get engulfed. • Cell lysis o Membrane attack complex formed by C5b6789 components ruptures the microbial cell surface which kills the cell. • Chemotaxis o Complement fragments attract neutrophils and macrophages to the area where the antigen is present o These cell surfaces have receptors for complements, like C5a, C3a, thus, run towards the site of inflammation, i.e. chemotaxis. • Activation of mast cells and basophils and enhancement of inflammation (Anaphylatoxins) o The proteolytic complement fragments, C5a, C4a, and C3a induce acute inflammation by activating mast cells and neutrophils. All three peptides bind to mast cells and induce degranulation, with the release of vasoactive mediators such as histamine. These peptides are also called anaphylatoxins because the mast cell reactions they trigger are characteristic of anaphylaxis. Binding to specific complement receptors on cells of the immune system, they trigger specific cell functions, inflammation, and secretion of immunoregulatory molecules. • Production of antibodies o B cells have receptor for C3b. When C3b binds to B-cell, it secretes more antibodies o Thus C3b is also an antibody producing amplifiers which converts it into an effective defence mechanism to destroy invading microorganism. • Immune clearance o The complement system removes immune complexes from the circulation and deposits them in the spleen and liver o Thus it acts as anti-inflammatory function. Complement proteins promote the solubilization of these complexes and their clearance by phagocytes. • The complement activation occurs via three pathways; which are: o Classical pathway ▪ activated by antigen-antibody reaction o Alternative pathway ▪ activated on microbial cell surfaces o Mannose binding Lectin (MBL) pathway ▪ activated by a plasma lectin that binds to mannose residues on microbes. • The Classic Pathway o The classical pathway begins with the formation of antigen- antibody complex (immune complex) o When an antigen enters the body, the antibody (IgM/IgG) binds to it o This induces conformational changes in the Fc portion of the antibody which exposes a binding site for C1 protein o Hence, the antibody activates the complement system only when bound to an antigen. o C1 is a large, multimeric, protein complex composed of one molecule of C1q and two molecules each of C1r and C1s subunits o C1q binds to the antigen bound antibody (Fc portion). C1r and C1s are proteases which help to cleave C4 and C2 o The immune complex bound to C1 calls another protein C4 which is cleaved into C4a and C4b o C4a goes away whereas activated C4b attaches to the target surface near C1q o Now, C4b attracts C2 which is also cleaved into C2a and C2b. C2a binds C4b forming the C4b2a complex whereas C2b goes away o The active C4bC2a activates C3. The C4b2a complex is also known as C3 convertase as this converts C3 into an active form by separating C3a and C3b o One molecule of C4b2a can cleave a large number of C3 molecules. C3b binds to the microbial surface or to the convertase

NTA Level 5 • Semester 1 • PST05103 Immunological Preparations Pharmaceutical Microbiology • Source Session/Topic 49 Full source-text version: all educational wording from the extracted learning source is retained; only presenter/tutor metadata and web-layout noise are removed, while formatting is improved for readability. Session 49: Immunological Preparations Total Session Time: 120 minutes Pre-requisites • Human anatomy and physiology Students Learning Tasks By the end of this session students are expected to be able to: • List characteristics of immunological preparations • List immunological preparations • Define vaccines and sera • Differentiate between vaccine and sera • List components of vaccines and sera • Classify vaccines and list their characteristics Resources Needed: • Flip charts, marker pens, and masking tape • Black/white board and chalk/whiteboard markers SESSION OVERVIEW |Step |Time |Activity/ |Content | | | |Method | | |1 |5 minutes |Presentation |Introduction, Learning Tasks | |2 |20 minutes |Presentation |Immunological Preparations | |3 |15 minutes |Presentation |Vaccines and Sera | |4 |40 minutes |Presentation |Classification of Vaccines and | | | | |Sera | |5 |30 minutes |Presentation |Composition of Vaccines and Sera | |6 |5 minutes |Presentation |Key Points | | 7|5 minutes |Presentation |Evaluation | SESSION CONTENTS STEP 1: Presentation of Session Title and Learning Tasks (5 minutes) READ or ASK students to read the learning tasks and clarify ASK students if they have any questions before continuing STEP 2: Immunological Preparations (20 minutes) • Immunological preparations o Immunological preparations are a group of pharmaceutical preparations with diverse origins but with a common pharmacological purpose of modifying the immune status of a recipient, either to provide immunity to infectious disease or to help in detection (diagnosis) of a disease o Types of immunological preparations ▪ Vaccines ▪ Immune sera ▪ In-vivo diagnostics ▪ Immunoglobulins • Characteristics of Immunological Preparations o They modify the immune system o They contain antigens or antibodies/immunoglobulins o They produce specific responses STEP 3: Vaccines and Immune sera (15 minutes) • Vaccine o Vaccine is a biological preparation that consists of either a whole organism (killed or live attenuated) or part of an organism introduced into an individual to induce adequate antibody production against the organism so that the individual is protected against infection caused by that particular organism o Vaccines contain antigens that stimulate the immune system of the recipient to produce T-cells or antibodies that attack and destroy the infectious agent • Immune Sera o Immune sera (singular serum) also known as antisera are amber- coloured, protein-rich liquid that separates out when blood coagulates o Blood serum of an animal is used to provide immunity to a pathogen or toxin by inoculation or used as a diagnostic agent o The sera contain antibodies against specific disease and used to provide passive immunity to that diseases. Sera do not stimulate production of antibodies o Immune sera are prepared by injecting repeated doses of an antigen into horse or other suitable animal o The animal is injected until high titre of antibodies are produced by the body of the animal o The animal must be in good health and free from infections o Blood is obtained from the animal and processed to produce serum • Human Immunoglobulins o Are preparations of immunoglobulins principally IgG subclasses that are present in human blood o They are derived from plasma of donated blood and from plasma obtained from plasmapheresis o Specific immunoglobulins are prepared from smaller pools of plasma obtained from individuals who have suffered recent infections or who have undergone recent immunization and who have high titre of a particular antibody ▪ Plasma containing antibodies against hepatitis B and HIV or any plasma capable of transmitting infections to recipient are excluded o The immunoglobulins are presented as freeze dried or liquid preparation at suitable concentration (10-20 times higher than that in the plasma) o Glycine may be added as a stabilizer and thiomersal as a preservative STEP 4: Classes of vaccines and Sera (40 minutes) • Vaccines can be classified into two broad groups; o Vaccines containing killed or inactivated pathogens o Vaccines containing live or attenuated pathogens • Vaccines containing live or attenuated pathogens o Live vaccines ▪ Live vaccines are made up of live bacteria, viruses, or other agents which when administered by appropriate route cause subclinical or mild infections ▪ In the course of such infection the antigenic components in the vaccine evoke an immune response which provide protection against the more serious natural disease o Attenuated vaccines ▪ Attenuated vaccines are vaccines containing pathogens manipulated in the laboratory to produce strain of the pathogen with reduced virulence • Attenuation can be attained by mutant strain polio virus, consecutive passage through an anima which is not a host of the pathogen e.g. small pox vaccine passage in calf and passage through a series of cultures e.g. rabies virus in human cell culture ▪ The attenuated organisms multiply in the host and provoke and immune response o Examples of live vaccines include Bacille Calmette-Guerin (BCG), oral polio vaccine, yellow fever vaccine, measles vaccine etc. o Advantages of live vaccines include; ▪ Single dose is often enough ▪ Produce strong immunity o Disadvantage of live vaccines ▪ The organism may cause full fledge infection • Vaccines containing killed or inactivated pathogens (Inactivated vaccines) o These vaccines do not replicate as the pathogens are killed or only their components are present in the vaccines o They require adjuvants to enhance immunogenicity o They are toxic and thus require multiple doses o Most viral vaccines are killed or inactivated o Killed vaccines ▪ Killed vaccines are suspensions of whole bacteria, viruses or other pathogenic agents that have been killed by heat or by disinfectants such as phenol or formaldehyde ▪ The killed organisms cannot replicate to cause infection ▪ Because all the components of the microorganisms are present in the vaccine, the vaccine may be toxic to the body ▪ Therefore, killed vaccines are usually divided into initial and booster doses give at regular intervals of

NTA Level 5 • Semester 1 • PST05103 Storage and Delivery of Vaccine Total Session Time: 60 minutes Pharmaceutical Microbiology • Source Session/Topic 50 Full source-text version: all educational wording from the extracted learning source is retained; only presenter/tutor metadata and web-layout noise are removed, while formatting is improved for readability. Session 50: Storage and Delivery of Vaccine Total Session Time: 60 minutes Pre-requisites • Human anatomy and physiology Students Learning Tasks By the end of this session students are expected to be able to: • Define cold chain • List components of cold chain • List equipment for cold chain • Explain the procedure for arrangement of vaccines in the refrigerator • List tools for monitoring cold chain • Explain strategies for vaccine delivery • List factors affecting quality of vaccines Resources Needed: • Flip charts, marker pens, and masking tape • Black/white board and chalk/whiteboard markers SESSION OVERVIEW |Step |Time |Activity/ |Content | | | |Method | | |1 |5 minutes |Presentation |Introduction, Learning Tasks | |2 | 20 minutes|Presentation |The Pharmaceutical cold chain | |3 |10 minutes |Presentation |Components of a pharmaceutical | | | | |cold chain | |4 |50 minutes |Presentation |Cold Chain Equipment | |5 |25 minutes |Presentation |Quality and vaccine delivery | |6 |5 minutes |Presentation |Key Points | | 7|5 minutes |Presentation |Evaluation | SESSION CONTENTS STEP 1: Presentation of Session Title and Learning Tasks (5 minutes) READ or ASK students to read the learning tasks and clarify ASK students if they have any questions before continuing STEP 2: Pharmaceutical Cold Chain (20 minutes) • Cold chain is the system of transporting and storing vaccines (or other temperature sensitive items) at recommended temperature from the point of manufacture to the point of use • Common temperature range for a cold chain in pharmaceutical industries is 2 to 8 °C • Cold chains are important because; o Vaccines are biological product which lose potency with time o They ensure that maximum benefits are obtained from immunization i.e. reduce wastage of vaccines o They ensure quality of vaccines is maintained in order to gain confidence of the public on immunization programmes o They ensure compliance with manufacturers’ recommendation STEP 3: Components of Pharmaceutical Cold Chain (10 minutes) • Components of the cold chain are; o Personnel ▪ Trained to run the cold chain ▪ They need to be dedicated people who appreciate the delicate nature and importance of delivering safe vaccines to save lives o Equipment ▪ There two main types of equipment • Equipment for transportation of vaccines • Equipment for storage of vaccines • The equipment must be maintained in good condition to ensure safety and quality of vaccines STEP 4: Cold Chain Equipment (50 minutes) • Different levels of the health care system need different equipment for transporting and storing vaccine and diluent at the correct temperature • Central and regional stores need cold rooms, freezers, refrigerators and cold boxes (for transportation) • District stores need freezers, refrigerators and cold boxes • Primary health facilities need refrigerators, cold boxes and vaccine carriers • Equipment for storage of vaccines o Walk-in cold rooms ▪ These are large specialized rooms (in the warehouse) in which low temperature is maintained for storage of large quantities of vaccines ▪ They are used at the regional level ▪ Maintains products at low temperatures for a duration of up to 3 months o Deep freezers ▪ They maintain temperature from -15°C to -25°C ▪ Used at primary health care for preparation of ice packs o Ice lined refrigerators ▪ These are used at district and primary health care levels ▪ They maintain temperature from +2°C to +8°C ▪ The refrigerators are top opening and they can hold cold air inside better than front opening refrigerators ▪ For front opening refrigerators; • The refrigerators should not be used for storage of anything other than vaccines • Should have uninterrupted power supply e.g. having alternative sources of power • Should not be more than 50% full • Should not be place in direct sunlight • Should not store vaccine for more than 1 month at primary health facility • Should not be used for foods or medical specimens • Nothing should be place in the fridge door • Items should be arranged away from fridge walls and cold air vents • Equipment for Transportation of Vaccines o Refrigerated vehicles ▪ Used for long-term transportation of vaccines o Cold boxes ▪ Cold boxes are insulated containers that can be lined with frozen ice packs to keep vaccines and diluent cold ▪ Used for transporting vaccines ▪ Fully frozen ice packs are placed at the bottom and sides ▪ DPT, TT and DT should not be kept in direct sunlight ▪ Cold boxes are used by health centre staff to collect and transport monthly vaccine supplies from district stores ▪ They are also used to store vaccines when the refrigerator is out of order or being defrosted ▪ The most suitable cold box for a particular health centre is determined by: • the vaccine storage capacity needed • the cold life needed, depending on the longest time that vaccine will be stored in the box • its weight, this depending on how the box will be transported, e.g., by motor vehicle or bicycle Figure56.1: A small vaccine cold box [pic] o Vaccine carriers ▪ Vaccine carriers are insulated containers that can be lined with frozen ice packs to keep vaccines and diluents cold ▪ They are smaller than cold boxes and easier to carry when walking, but they stay cold for as long as 24-72 hours ▪ These are used to carry small quantities of vaccines (16-20 vials) ▪ Four ice packs are used ▪ Vaccine carriers are used to transport vaccine and diluent to outreach sites and for temporary storage during health centre immunization sessions Figure 56.2: A large vaccine carrier [pic] o Ice packs ▪ Ice packs are flat, square plastic bottles that can be filled with water