Clinical Bacteriology 2024-2025 1st Semester PDF

CLINICAL BACTERIOLOGY BS MEDICAL LABORATORY SCIENCE ACADEMIC YEAR 2024-2025 (1st Semester) PROFESSOR: Ethel Marie Mangada INTRODUCTION: BRIEF HISTORY OF MICROBIOLOG...

CLINICAL BACTERIOLOGY BS MEDICAL LABORATORY SCIENCE ACADEMIC YEAR 2024-2025 (1st Semester) PROFESSOR: Ethel Marie Mangada INTRODUCTION: BRIEF HISTORY OF MICROBIOLOGY OUTLINE In one of his poems, he also described syphilis as “french disease,” he coined this term for syphilis. I INTRODUCTION II THE FIRST OBSERVATIONS HIPPOCRATES III SPONTANEOUS GENERATION IV THEORY OF BIOGENESIS No diseases are not from supernatural causes but natural V THE GOLDEN AGE OF MICROBIOLOGY causes. VI GERM THEORY OF DISEASE VII IMMUNOLOGY: ADVENT OF VACCINATION Theory of Humors: VIII THE BIRTH OF MODERN CHEMOTHERAPY: DREAMS OF A Blood “MAGIC BULLET” Phlegm IX THE FIRST SYNTHETIC DRUGS Yellow bile X A FORTUNATE ACCIDENT - ANTIBIOTICS Black bile XI TABLE: NOBEL LAUREATES According to Hippocrates, for an individual to maintain his or health, there should be balance between those four humors. For one to be able to treat their INTRODUCTION sickness, the four humors should be balanced. Microbiology - a branch of science that studies microorganisms. They are a large and diverse group FRANCESCO STELLUTI (1577-1652) of microscopic organisms made of a single cell or cluster of cells. They are very minot in size, so we Made the earliest observations on bees and weevils using need the aid of a microscope to observe them. a microscope supplied by Galileo. Examples of microorganisms made of a single cell: bacteria, parasitology (protozoans). ROBERT HOOKE Examples of microorganisms made of clusters of cells: fungi, other parasites like tapeworms. Reported to the world that life’s smallest structural units In microbiology, we also study viruses, though they’re were “little boxes,” or “cells” not made of a cell. During this time, he first observed the blood cell, it is said that it is the same with the animal cell. THE FIRST OBSERVATIONS Marked the beginning of cell theory. LUCRETIUS (98-55 B.C) AND GIROLAMO FRACASTORO CELL THEORY (1478-1553) All living things are composed of cells. o Published in his book, “Micrographia” Suggested that diseases were caused by “invisible living creatures.” o This is the start of spontaneous generation theory, telling that microorganisms came from non-living things. GIROLAMO FRACASTORO Contagion theory Wrote a book about contagion and contagious disease, the disease experienced by humans are caused by contagion, it started contagion theory. It gave scientists the idea that diseases were caused by something that they called that time “contagion” Made statement about disease, contagion, and Figure 1. Anton van Leewenhoek was inspired by this infection publication by Robert Hooke entitled, “Micrographia: or He had an idea about the mode of transmission. Some Physiological Descriptions of Minute Bodies Made According to his book, diseases can be transferred by Magnifying Glasses. With Observations and Inquiries from one thing to another, they called that the Thereupon” “fomites.” Contagion disease can be transferred through direct contact or from an inanimate object to the person or through other things, from one thing to another. He studied the start of epidemiology, it was during the time that he studied about syphilis, epidemiology is the study and analysis of the determinants and patterns of health and disease. CANCERAN, LACAMBRA | 3A - MLS 1  TRANS: INTRODUCTION: BRIEF HISTORY OF MICROBIOLOGY Figure 3. Robert Hooke’s Microscope (1665) Figure 2. Robert Hooke’s Microscope (1665) Bacteria I ig Protozoa ANTON VAN LEEUWENHOEK (1632-1723) Sperm cells Blood cells Microscopic worms Microscopicworms Considered as the “first true microbiologist” He observed live microorganisms through his magnifying glass that’s about 500x LEEUWENHOEK’S MICROSCOPE magnification. First actually to observe live microorganisms through the magnifying lenses of more than 400 microscopes he constructed. First type of cells he observed were the red blood cells, protozoa, and sperm cell Wrote a series of letters to the Royal Society of London describing the “animalcules” he saw through his simple, single-lens microscope (50x to 300x) some would tell the magnification were up to 500x Made detailed drawings of “animalcules” in rainwater, in his own feces, and in material scraped from his teeth. Giardialamblia ANIMALCULES Tiny living and moving cells. Figure 4. Also called “wee beasties” 3” to 4” microscope Observed in droplets of water, which may also refer to Required good lighting and patience body fluids. SPONTANEOUS GENERATION “Diba very cutesy, very demure” - mam ethel 2k24 The first to propose this was actually Aristotle, but many scientists tried to support this theory. Believed that some life forms could arise spontaneously from non-living matter. How did this theory emerge? During that time, they observed that when cheese with bread wrapped in a rag and was placed in a dark place, they said that it produced mice. So they believed that the bread and cheese suddenly produced the mice. Toads, snakes, and mice could be born of moist soil. Flies could emerge from manure Maggots, the larvae of flies, could arise from decaying corpses. ARISTOTLE (384-322 B.C) Mentioned that simple invertebrates could arise from spontaneous generation. This belief was proven wrong until the time of Louis Pasteur. FRANCESCO REDI Demonstrate that maggots did not arise spontaneously from decaying meat (1668) Results of his investigation invalidated the long-held belief that life forms could arise from non-living things. Father of experimental biology. CANCERAN, LACAMBRA | 3A - MLS 2  TRANS: INTRODUCTION: BRIEF HISTORY OF MICROBIOLOGY 2nd experiment (bottom): The sealed flask did not manifest any bacterial growth, but then when he opened the flask, he observed the growth of bacteria. ANTON LAURENT LAVOISIER Showed the importance of oxygen to life. THEORY OF BIOGENESIS RUDOLF VIRCHOW (1821-1902) Challenged the case for spontaneous generation with the concept of biogenesis. He started the study of Histology, he observed that living Figure 6. Redi’s Experiment cells are from pre-existing living cells, they are not from non-living matter. JOHN NEEDHAM (1731-1781) BIOGENESIS Observed that a boiled mutton broth eventually became Living cells can arise only from pre-existing living cells. cloudy after pouring it into a flask that was then sealed tightly. THEODOR SCHWANN (1810-1882) Found that even after he heated nutrient fluids (chicken broth and corn broth) before pouring them into covered flasks, the cooled solutions were soon teeming with Observed that no growth occurred in a flask that contained microorganisms. The reason for the growth of a nutrient solution after allowing the air to pass through a microorganisms in the sealed, boiled broth, is that the heated tube. boiling time was less than the required time to kill the Why? The heat may have killed the microorganisms that microorganisms or the bacterial spores that are already may have possibly entered the flask. The idea of Aseptic present in the broth. technique is slowly emerging. Claimed that microbes developed spontaneously from the fluids. HEINRICH SCHRODER (1810-1885) AND THEODORE VON DUSCH Asserted that organic matter possessed a “vital force” that (1824-1890) could give rise to life. LAZZARO SPALLANZANI Noticed that no growth occurred after allowing the air to pass through a sterile cotton wool placed on a flask of heat-sterilized medium. Suggested that microorganisms from the air probably had entered Needham's solutions after they were boiled proposed that air carried microorganisms to the culture LOUIS PASTEUR (1822-1895) medium showed that nutrient fluids heated after being sealed in a disproved the doctrine of spontaneous generation flask did not develop microbial growth demonstrated that microorganisms are present in the air and can contaminate sterile solutions, but that air itself does not create microbes. It’s not the air that creates microbes, it’s just the place where they thrive but it is not where they arise from. Figure 6. Spallanzani’s Experiment 1st experiment (top): After some time, the broth became turbid or cloudy owing to the growth of Figure 7. Pasteur’s Experiment bacteria, so the broth is contaminated. The By coiling or curving the long neck of the flask, he microorganisms might be in the air because it was left was able to block the access of the air-borne open. microorganisms, that’s why there’s no growth in the broth. CANCERAN, LACAMBRA | 3A - MLS 3  TRANS: INTRODUCTION: BRIEF HISTORY OF MICROBIOLOGY showed that microorganisms can be present in nonliving GERM THEORY OF DISEASE matter-on solids, in liquids, and in the air. demonstrated conclusively that microbial life can be Microorganisms might have relationships with plants and destroyed by heat and that methods can be devised to animals---specifically, that microorganisms might cause block the access of airborne microorganisms to nutrient disease environments. form the basis of Aseptic Techniques. AGOSTINO BASSI ASEPTIC TECHNIQUES had proved that another silkworm disease was caused by techniques that prevent contamination by unwanted a fungus. microorganisms, which are now the standard practice in laboratory and many medical procedures IGNAZ SEMMELWEIS (1816-1865) JOHN TYNDALL demonstrated that physicians, who at the time did not disinfect their hands, routinely transmitted infections Showed that dust carry germs that could contaminate a (puerperal, or child -birth, fever) from one obstetrical sterile broth patient to another demonstrated that routine hand washing can prevent the TYNDALLIZATION spread of disease is a form of sterilization in the 19th century that uses moist heat for 3 consecutive days to eradicate vegetative cells JOSEPH LISTER (1821-1912) and endospores For the first day, heat at 100C or less than that then introduced the system of antiseptic surgery in Britain cooled, on the second day, heated and cooled again, 3rd applied the germ theory to medical procedures day, same process. Then on the next consecutive days, began treating surgical wounds with a phenol solution there will be no growth seen on vegetative cells and pioneered in promoting among surgeons handwashing endospores. before and after an operation, the wearing of gloves, Tyndallization is not often used now, autoclaving na sterilization of surgical instruments. ngaun. FERDINAND COHN (1828-1898) ROBERT KOCH (1843-1910) Discovered that there are bacteria that could withstand a First to show irrefutable proof that bacteria indeed series of heating and boiling because of heat resistant cause disease structures known as endospores. discovered Bacillus anthracis in the blood of cattle That’s why they were able to develop a sterilization that had died of anthrax (1876) technique that would kill the endospores. Discovered Mycobacterium tuberculosis (1882) first to cultivate bacteria on boiled potatoes, gelatin, THE GOLDDEN AGE OF MICROBIOLOGY meat extracts and protein (1857-1914) established a sequence of experimental steps for directly relating a specific microbe to a specific FERMENTATION AND PASTEURIZATION disease: Koch's Postulates THEODOR SCHWANN KOCH’S POSTULATE 1. The microorganism should be found in all cases stated that yeast cells are responsible for the conversion of the disease in question, and its distribution in of sugars to alcohol the body should be in accordance with the lesions observed. If this is disease A, and we have 3 patients with LOUIS PASTEUR disease A, we are sure that tall of these patients have the bacteria A, and that if we are going to isolate Found that microorganisms called yeasts convert the microorganism, for example if that disease produce sugars to alcohol in the absence of air: FERMENTATION. lesions in the patient, we can be sure that that Pasteur's solution to the spoilage problem was to heat the bacteria A is present in the lesions of the patients with beer and wine just enough to kill most of the bacteria that disease A. caused the spoilage: PASTEURIZATION 2. The microorganism should be grown in pure Stated Souring and spoilage are caused by different culture in vitro (or outside the body of the host) microorganisms called bacteria; in the presence of air, for several generations. bacteria change the alcohol in the beverage into vinegar 3. When such a pure culture is inoculated into (acetic acid) susceptible animal species, the typical disease must result. Assignment!! Pure culture - it has no other microorganisms. Look into the different techniques used in the sterilization of If this is the culture media, we will grow bacteria A canned goods, read articles. that we derived from the lesion of disease A, and we grow that in a pure culture, and when we get an inoculum from that culture and inoculate or introduced CANCERAN, LACAMBRA | 3A - MLS 4  TRANS: INTRODUCTION: BRIEF HISTORY OF MICROBIOLOGY that in a susceptible host, the susceptible host will microorganisms other than the microorganism of also have the disease A. interest. 4. The microorganism must again be isolated from the lesions of such experimentally produced IMMUNOLOGY: ADVENT OF VACCINATION disease. From the new host/patient (#3) inoculated with the EDWARD JENNER (1749-1823) disease A, we should also be able to isolate the same bacteria from his/her lesion, still the bacteria A, same bacteria type that we previously isolated from the first embarked on an experiment to find a way to protect patient with disease A. people from smallpox introduced the concept of vaccination Koch’s postulate tells us that specific bacteria cause a specific disease. PHYSICIANS IN CHINA immunized patients by removing scales from drying pustules of a person suffering from a mild case of smallpox, grinding the scales to a fine powder, and inserting the powder into the nose of the person to be protected Variolation - practice done before the birth of the concept of vaccination. LOUIS PASTEUR (1822-1895) AND PIERREPAUL EMILE ROUX (1853-1933) Pasteur used the term vaccine for an attenuated culture both made a series of experiments to produced attenuated strains of bacteria Figure 8. Koch’s Postulate prove that when attenuated strains are introduced into a healthy host, the latter remains protected and healthy COLLABORATORS OF KOCH against the virulent agent. FANNY HESSE (1850-1934) Pasteur discovered that attenuated (weakened) bacterial cultures could produce immunity. suggested the use of agar, a solidifying agent, in the Bacillus anthracis attenuation- heating the bacterial preparation of the culture media suspension at 42 °C the bacteria loses its endospore - nonvirulent form of anthrax JULIUS RICHARD PETRI (1852-1921) Cholera attenuation- serial passages (serial subcultures) developed the Petri Dish, which is a circular glass or plastic plate for holding the culture media. Rabies vaccine- by drying the spinal cords of rabbits Petri dishes (plastic plate) are disposable. using the material to prepare a series of 14 injections In our lab we used circular glass petri dishes so that we of increasing virulence. can autoclave them after we use. MARTINS BEIJERINK (1851-1931) AND SERGEI WINOGRADSKY CHARLES CHAMBERLAND (1851-1908) (1856-1953) created a porcelain bacterial filter and developed the developed the enrichment-culture technique and the use anthrax vaccine together with Pasteur of selective media Sometimes they also enrich it with blood so that is why They developed vaccines not just for bacteria but also we have what we call blood agar plates and chocolate for pathogenic viruses. agar plates.They incorporate nutrients and other additional cultures to encourage the growth of microorganisms that EMIL VON BEHRING (1854-1917) require certain nutrients for them to multiply. Types of culture media: Inhibitory media and Selective media They allow the growth of a certain microorganism to prepared antitoxins for diphtheria and tetanus inhibit other microorganisms from growing. Adding enrichment to the culture media can also add ELIE METCHNIKOFF (1845-1916) inhibitory substances to avoid growth of other CANCERAN, LACAMBRA | 3A - MLS 5  TRANS: INTRODUCTION: BRIEF HISTORY OF MICROBIOLOGY first to described the immune system cells and the process of phagocytosis Mesangial cells - macrophages found in kidney Kupffer cells - macrophages found in liver MHC II - CD4+ cells MHC I - CD8 cytotoxic cells THE BIRTH OF MODERN CHEMOTHERAPY: DREAMS OF A "MAGIC BULLET“ CHEMOTHERAPY treatment of disease by using chemical substances chemical treatment of non-infectious diseases, such as cancer ANTIBIOTICS chemicals produced naturally by bacteria and fungi to act against other microorganisms. Example: penicillin SYNTHETIC DRUGS chemotherapeutic agents prepared from chemicals in the laboratory THE FIRST SYNTHETIC DRUGS PAUL EHRLICH (1854-1915) speculated about a “bullet" that could hunt down and destroy a pathogen without harming the infected host found a chemotherapeutic agent called Salvarsan (Arsphenamine), an arsenic derivative effective against syphilis SELMAN WAKSMAN (1888-1973) HOWARD FLOREY (1898-1968) AND ERNST CHAIN discovered streptomycin and neomycin antibiotics (1906-1979) regarded as “Father of Antibiotics” by some historians. A FORTUNATE ACCIDENT - ANTIBIOTICS Made the purification process for penicillin and clinical trials to humans ALEXANDER FLEMING (1881-1955) EDWARD ABRAHAM (1913-1999) accidentally discovered Penicillin mold was later identified as Penicillium notatum (later First to propose the correct biochemical structure of renamed Penicillium chrysogenum Penicillin. CANCERAN, LACAMBRA | 3A - MLS 6  TRANS: INTRODUCTION: BRIEF HISTORY OF MICROBIOLOGY NOBEL LAUREATES YEAR OF COUNTRY CONTRIBUTION PRESENTATION OF BIRTH Emil A. Von Behring 1901 Germany Developed a diphtheria antitoxin Ronald Ross 1902 England Discovered how malaria is transmitted Robert Koch 1905 Germany Cultured tuberculosis bacteria Paul Erlich 1908 Germany Developed theories on immunity Elie Metchnikoff 1908 Russia Described phagocytosis, the intake of solid materials by cells Alexander Fleming, 1945 Scotland Discovered penicillin Ernst Chain, and England Howard Florey England Selman A. Walksman 1952 Ukraine Discovered streptomycin Hans A. Krebs 1953 Germany Discovered chemical steps of the Krebs Cycle in carbohydrate metabolism John F. Enders, 1954 United States Cultured poliovirus in cell cultures Thomas H. Weller, and Frederick C. Robbins Joshua Lederberg, 1958 United States Described genetic control of biochemical George Beadle, reactions Edward Tatum Frank Macfarlane 1960 Australia Discovered acquired immune tolerance Burnet and Peter Great Britain Brian Medawar James D. 1962 United States Identified the physical structure of DNA Watson.Frances England H.C. Crick, and New Zealand Maurice A.F. Wilkins Francois Jacob, 1965 France Described how protein synthesis is Janques Monod, regulated in bacteria and Andre Lwoff Peyton Rous 1966 United States Discovered cancer causing viruses Max Delbruck, Alfred D. 1969 Germany Described the mechanism of viral infection Hersey, and United States of bacterial cells Salvador E. Luria Italy Gerald M. Edelman and 1972 United States Described the nature and structure of Rodney R. Porter England antibodies Renato Dulbeco, 1975 United States Discovered reverse transcriptase and Howard Temin, and described how RNA could cause David Baltimore cancer Daniel Nathans, 1978 United States Described the action of restriction enzymes Hamilton Smith, United States (now used in recombinant DNA) and Werner Arber Switzerland Peter Mitchell 1978 England Described the chemiosmotic mechanism for ATP synthesis Paul Berg 1980 United States Performed experiments in gene splicing Aaron Klug 1982 South Africa Described the structure of tobacco mosaic virus (TMV) Barbara McClintock 1983 United States Discovered transposons (small segments of DNA that can move from one region of a DNA molecule to another) Cesar Milstein, George 1984 Argentina Developed a technique for producing J.F. Kohler,and Germany monoclonal antibodies (single pure Niels Kai Jerne Denmark antibodies) Susumu Tonegawa 1987 Japan Described the gebetics of antibody production Johan Deisenhofer, 1988 Germany Described the structure of bacterial Rober Huber, and photosynthetic pigments Hartmut Michel CANCERAN, LACAMBRA | 3A - MLS 7  TRANS: INTRODUCTION: BRIEF HISTORY OF MICROBIOLOGY J. michael Bishop and 1989 United States Discovered cancer causing genes called Harold E. Varmus oncogenes Joseph E. Murray and 1990 United States Perform the first successful organ E. Donnall Thomas transplants by using immunosuppressive agents Edmond H. Fisher and 1992 United States Discovered protein kinases, enzymes that Edwin G. Krebs regulae cell growth Richard J. Roberts and 1993 Great Britain Discovered that a gene can be separated Philip A. Sharp United States onto different segments of DNA Kary B.Mullis 1993 United States Discovered the polymerase chain reaction to amplify (make multiple copies) of DNA Peter C. Doherty and 1996 Australia Discovered how cytotoxic T cells Rolf M. Zinkernagel Switzerland recognized virus-infected cells prior to destroying them Stanley B. Prusiner 1997 United States Discovered and named proteinaceous infectious particles (prions) and demonstrated a relationship between prions and deadly neurological diseases in humans and animals Peter Agre and 2003 United States Discovered water and ion channels in Roderick plasma membranes MacKirron Aaron Ciechanover, 2004 Israel Discovered how cells dispose of unwanted Avran Hershko, Israel proteins in proteasomes and Irwin Rose United States Barr Marshall and J. 2005 Australia Discovered that Helicobacter pylori causes Robin Warren peptic ulcers Andrew Fire and Craig 2006 United States Discovered RNA interference (RNAi)or Mello gene silencing by double stranded RNA Harald zur Hausen 2008 Germany Discovered that human papilloma viruses cause cervical cancer Francoise 2008 France Discovered human immunodeficiency virus Barre-Sinoussi and (HIV) Luc Montagnier Table 1. Contributors in the development of microbiology CANCERAN, LACAMBRA | 3A - MLS 8  CLINICAL BACTERIOLOGY BS MEDICAL LABORATORY SCIENCE ACADEMIC YEAR 2024-2025 (1st Semester) PROFESSOR: Ethel Marie M. Mangada [TRANS] UNIT 1.2: MICROBIAL TAXONOMY OUTLINE B. KINGDOM contains similar divisions or phyla I MICROBIAL TAXONOMY A Classification C. PHYLUM i. Domain ii. Kingdom contains similar classes; equivalent to the Division taxa iii. Phylum iv. Class D. CLASS v. Order vi. Family contains similar orders vii. Genus viii. Species E. ORDER ix. Subspecies contains similar families B Nomenclature C Identification D Identification Methods F. FAMILY i Genotypic Characteristics contains similar genera ii Phenotypic Characteristics group of organisms that may contain multiple genera and E Major Characteristics Used in Taxonomy consists of organisms with a common attribute i Classical Characteristics ii Molecular Characteristics Example: Enterobacteriaceae, Streptococcaceae F Classification By Cellular Type: Prokaryotes, Eukaryotes, and Archaeobacteria G. GENUS i Prokaryotes contains similar species ii Eukaryotes based on various genetic and phenotypic characteristics iii Archaea (Archaeobacteria) shared among the species Example: Streptococcus TAXONOMY H. SPECIES area of biologic science comprising three distinct but highly interrelated disciplines: specific epithet o classification most basic of the taxonomic groups and can be defined as a collection of bacterial strains that share common o nomenclature (naming) physiologic and genetic features and differ notably from o identification of organisms other microbial species ▪ it's important to identify organisms for us to Example: Streptococcus pyogenes know where to classify them orderly classification and grouping of organisms into taxa I. SUBSPECIES (categories) taxonomic subgroups within a species based on similarities and differences in genotype and species subdivided based on phenotypic the following phenotype differences: o Genotype: genetic makeup o Biotype o Phenotype: physical characteristics, biochemical ▪ considered the same species with the same reactions, etc. Carl von Linne (Carolus Linnaeus) Linnean characteristic genetic makeup that displays Taxonomy differential physiologic characteristics o Father of Modern Biosystematics/Taxonomy o laid down the basic rules for taxonomic categories ▪ based on structure or biochemical test result differences CLASSIFICATION ▪ Example: Staphylococcus aureus biotype method for organizing microorganisms into groups or taxa may be different: S. aureus that is either based on similar morphologic, physiologic, and genetic resistant or susceptible to penicillin traits hierarchical classification system consists of the following o Serotype taxa designations: ▪ based on serologic differences A. DOMAIN Phage typing (based on susceptibility to specific bacterial Bacteria, Archaebacteria, Eukarya phages) has also been used for this purpose o Bacteria: scope of Bacteriology o Eukarya: organisms that are considered eukaryotes, such as parasites and fungi TUMBALI & VALLES | 3A - MLS 1  TRANS: MICROBIAL TAXONOMY NOTE: o Examples: macroscopic (colony morphology on Diagnostic microbiologists traditionally emphasize media) and microscopic (size, shape, arrangement placement and naming of bacterial species into three into groups or chains of organisms) morphology, (occasionally four or five) categories: staining characteristics (gram-positive or o the family gram-negative), nutritional requirements, o a genus physiologic and biochemical characteristics, and o a species susceptibility or resistance to antibiotics or chemicals Example: Family of Micrococcaceae, Genus Staphylococcus, and Specie Staphylococcus aureus Species definitions are distinguished using DNA profiling, including a nearly complete 16S rRNA sequence in combination with phenotypic traits NOMENCLATURE Naming of microorganisms according to established rules and guidelines set forth in the International Code of Nomenclature of Bacteria (ICNB) or the Bacteriological Code (BC) Genus designation - first letter is always capitalized Species designation - first letter is always lower case o Two components are used simultaneously and are MAJOR CHARACTERISTICS printed in italics or underlined in script USED IN TAXONOMY o Example: Streptococcus pneumoniae, Streptococcus pyogenes, Streptococcus agalactiae, Classical Characteristics and Streptococcus bovis o Useful in routine identification of phylogenetic When bacteria are referred to as a group, their names are information neither capitalized nor underlined (e.g., staphylococci) o Phylogenetic and phyletic classification is based on evolutionary relationships instead of general IDENTIFICATION resemblance o Example: morphology, physiology and metabolism, Process by which a microorganism’s key features are ecology and genetic analysis Molecular Characteristics MPMEGT delineated Process of discovering and recording the traits of o Based on the study of nucleic acid composition and organisms so that they may be placed in an overall proteins taxonomic scheme Organism can then be assigned to the most appropriate CLASSIFICATION BY CELLULAR TYPE: taxa (classification) and can be given appropriate genus PROKARYOTES, EUKARYOTES, AND and species names (nomenclature) ARCHAEOBACTERIA IDENTIFICATION METHODS Organisms fall into three distinct groups based on type of cell organization and function: prokaryotes, eukaryotes, Genotypic Characteristics and archaeobacteria o Relate to an organism’s genetic makeup, including Taxonomists have placed all organisms into three domains the nature of the organism’s genes and constituent that have replaced some kingdoms: Bacteria, Archaea nucleic acids and Eukarya o Examples: base sequencing of DNA or RNA and Each of these domains is divided into kingdoms based on DNA base composition ratio to measure the degree the similarities of RNA, DNA, and protein sequences of relatedness of two organisms PROKARYOTES Archaea and Bacteria (Eubacteria) EUKARYOTES Fungi, algae, protozoa, animals, and plants FAPAP ARCHAEA (ARCHAEOBACTERIA) Closely related to eukaryotic cells than to prokaryotic cells Phenotypic Characteristics Found in microorganisms that grow under extreme o Based on features beyond the genetic level and environmental conditions include both readily observable characteristics and Cell walls lack peptidoglycan but they mostly contain a characteristics that may require extensive analytic protein or glycoprotein wall structure —“S-layer” procedures to be detected Can stain gram-positive and gram-negative TUMBALI & VALLES | 3A - MLS 2  TRANS: MICROBIAL TAXONOMY Cellular structure include the cell wall, plasma membrane, ribosomes and flagella Do not contain a nucleus CPRFand membrane-bound organelles Produce through binary fission, fragmentation or budding Examples: Methanospirillum, Halobacterium, Sulfolobus MAS TUMBALI & VALLES | 3A - MLS 3  CLINICAL BACTERIOLOGY BS MEDICAL LABORATORY SCIENCE ACADEMIC YEAR 2024-2025 (1st Semester) PROFESSOR: Ethel Marie M. Mangada UNIT 3: CONTROL OF MICROORGANISMS OUTLINE CONTROL OF MICROORGANISMS I CONTROL OF MICROORGANISMS IMPORTANCE OF THE PRACTICE OF A Sterilization vs Disinfection STERILIZATION AND DISINFECTION B Terminologies to reduce the chance of transmission of disease C Factors That Influence the Degree of Killing in Bacteriology, it is important for us to make sure that Microorganisms our isolated organism is specific to the disease that D Methods of Disinfection and Sterilization we’re trying to diagnose from our patients i. Physical Methods a. Heat i. Moist Heat STERILIZATION VS DISINFECTION 1. Boiling 2. Autoclaving STERILIZATION 3. Flowing Steam 4. Inspissation complete removal or destruction of all forms of life, 5. Pasteurization / Particle including bacterial spores Sterilization chemical or physical methods ii. Dry Heat 1. Flaming or Direct Heating DISINFECTION 2. Hot Air Oven 3. Incineration process that eliminates a defined scope of 4. Cremation microorganisms b. Low / Cold Temperature process of killing or removing microorganisms in inanimate i. Freezing surfaces through the use of chemical agents (e.g. use of c. Desiccation and Lyophilization alcohol) d. Filtration destroys pathogenic organisms, but not necessary all i. Depth Filters microorganisms or spores ii. Membrane Filter (Circular Filter) 1. Liquid Filtration most are chemical substances 2. Air Filtration iii. Filtration of Bacteria, Yeasts and Molds TERMINOLOGIES iv. Critical Sterilizing e. Radiation i. Ionizing Radiation STERILE ii. Non Ionizing Radiation free of life of every kind iii. Ultrasonic and Sonic Vibrations sometimes used relative to the method used in sterilizing ii. Chemical Methods or disinfecting a. Alcohol b. Aldehydes / Cold / Chemical Sterilants i. Formaldehyde BACTERIOSTATIC ii. Glutaraldehyde having the property of inhibiting bacterial growth or c. Halogens multiplication i. Iodine ii. Chlorine and Chlorine Compounds BACTERICIDAL d. Quaternary Ammonium Compounds (Quats) / Detergents having the property of killing or destroying bacteria e. Phenolics precipitates bacterial protein (H2SO4, HCl) i. Chlorhexidine Gluconate o bactericidal substances cannot be used to human ii. Hexachlorophene tissues iii. Chloroxylenol (Parachlorometaxylenol [PCMX]) iv. Triclosan GERMICIDE OR DISINFECTANT f. Heavy Metals chemical substance used to kill infection producing g. Gas microorganisms on surface but too toxic to be applied h. Hydrogen Peroxide and Peracetic Acid directly on tissues i. Acid and Alkaline Solutions E Disinfectant Screening Test: Phenol Coefficient F Laboratory Safety SEPTIC i Chain of Infection characterized by the presence of pathogenic microbes in ii Engineering Controls living tissue a. Biological Safety Cabinet iii CDC List of Infectious Diseases that may be Acquired ASEPTIC In Healthcare Facilities characterized by the absence of pathogenic microbes iv Classification of Biological Agents based on Hazard ANTISEPTIC ACCAD, HORCAJO | 3A - MLS 1  UNIT 3: CONTROL OF MICROORGANISMS chemical substance which opposes sepsis or putrefaction ○ example: sulfuric acid, hydrochloric acid either by killing microorganism or preventing their growth applied topically to living tissues o example: Phisohex, Hexachlorophene, Tincture of F. Contact Time CT alcohol and iodine preparations (betadine) - at least 1 Iodine / Povidone Alcohol (Iodophor) to 2 minutes to kill microorganisms the longer the contact time, the more bacteria are DECIMAL REDUCTION TIME (DRT/D/D) killed time in minutes to reduce the bacterial population or spores by 90% at a specified temperature G. Temperature T the higher the temperature, the more the disinfectant is effective FACTORS THAT INFLUENCE THE DEGREE OF KILLING MICROORGANISMS H. pH plt not all disinfectants are effective when exposed to pH ○ pH can neutralize or remove the A. Types of Organisms Typeof Bacterial Spores - resistant organin effectiveness of disinfectants ○ presence of spores helps us determine the appropriate method to use I. Biofilms BF groups or population of bacteria in layers ○ if dealing with bacterial spores, avoid using ○ the bacteria has multiplied and became low grade disinfectants, instead, use cecile or permanently attached to the surface sterilization techniques such as moist heat ○ biofilms have slimy surface that protects Acid-Fast Bacilli them ○ example: Mycobacterium tuberculosis is resistant to techniques like desiccation J. Compatibility of Disinfectants and Sterilants because it is lipid-rich; lipid protects CDOS there are disinfectants that when are used together, microorganisms from drying they become ineffective Non-enveloped Viruses do not mix / combine disinfectants or sterilants ○ pore-resistant compared to enveloped together viruses combination of disinfectants might deactivate / ○ very resistant to disinfectants neutralize their effectivity Vegetative Bacteria Enveloped Viruses NOTE: PRIONS HER most resistant to the actions of heat, chemicals, and p radiation o resistant to sterilization techniques and disinfectants kg o highly infectious: more infectious than bacteria, virus, parasites naked pieces of protein without nucleic acid degenerative diseases of the nervous system (transmissible spongiform encephalopathy - mad cow disease, Creutzfeldt-Jakob disease) Figure 1. Different Types of Organisms and their B. Number of Organisms (Microbial Load / Microbial Burden) NO Resistance to Killing Agents higher numbers of organisms require longer exposure times METHODS OF DISINFECTION AND STERILIZATION C. Concentration of Disinfecting Agent COPA the higher concentration of disinfecting agent, the more effective ○ example: alcohol however in betadine or iodine, the lesser the concentration, the more effective D. Presence of Organic Material blood, mucus, and pus poom they act as protection they cover microorganisms and they inactivate the disinfectants; the disinfectants loose its effectivity in doing the disinfection Figure 2. Device Classification and Methods of Effective Disinfection E. Nature of Surface to be Disinfected NSD not all disinfectants are safe when applied to skin ACCAD, HORCAJO | 3A - MLS 2  UNIT 3: CONTROL OF MICROORGANISMS PHYSICAL METHODS I. HEAT most common method used for the elimination of microorganisms most reliable and universally applicable method of sterilization A. Moist Heat kills microorganisms rapidly than dry heat ○ requires shorter time and lower FIE temperature compared to dry heat which requires longer time and higher temperature lethal effect is attributed to denaturation and coagulation of protein and Figure 2. Gravity Displacement Autoclave Illustration degradation of nucleic acid fastest and simplest physical method of 3. Flowing Steam sterilization FS sterilizing heat-sensitive culture media sterilization method of choice for heat-stable objects Carbs containing carbohydrates Fractional/Intermittent Sterilization/Tyndallization 1. Boiling materials are exposed for 30 minutes for 3 100 C for 15-30 minutes (20 minutes successive days minimum) ○ Day 1: Vegetative Cells surgical instruments, needles, hypodermic ○ Day 2: Sporulated Cells Dots syringes, rubber stoppers kills all vegetative organism but not all spores or viruses ○ Day 3: Remaining Cells destroys vegetative cells and spores after ii three consecutive days ○ for disinfection only, not for instrument: Arnold Sterilizer - use flowing sterilization steam 100 C for 30 minutes 2. Autoclaving sterilize biohazardous trash and heat-stable 4. Inspissation objects BT Hso materials used in ○ example: principle: thickening through evaporation sterilize high protein containing media that bacteriology cannot withstand high temperature all microorganisms (except for prions) and their endospores are destroyed within approximately 15 minutes of exposure LTD example: LJ, Leoffler’s and Dorsey egg medium 70 C - 80 C for 2 hours for 3 consecutive Autoclave - chamber which is filled with hot days Steam Under Pressure (principle) two common sterilization temperatures: 5. Pasteurization / Partial Sterilization ○ 121 C (250 F), 15 psi for 15 partially sterilizing organic solutions by heat MY minutes - media, liquids, and instruments without altering their chemical properties used to sterilize milk, dairy products, and ○ 132 C (270 F), 15 psi for 30-60 IMW minutes - infectious medical waste alcoholic beverages eliminates food-borne pathogen and BIOLOGIC INDICATOR: Geobacillus organisms responsible for food spoilage stearothermophilus (B. Gps cannot eliminate bacterial endospores stearothermophilus) incubated at 56 C Three Types of Pasteurization Gravity Displacement Type A. Low Temperature Holding (LTH) / Batch most commonly used steam sterilizer in the microbiology laboratory 43 Method destroys milk-borne pathogens GPT 63 C for 30 minutes B. High Temperature Short-Time (HTST) / HF Flash Pasteurization 72 C for 15 seconds quick heating and immediate cooling UH C. Ultra-high Temperature 140 C for 3 seconds cooled very quickly in a vacuum chamber advantage: milk can be stored at RT for 2 months without affecting its flavor ACCAD, HORCAJO | 3A - MLS 3  UNIT 3: CONTROL OF MICROORGANISMS B. Dry Heat ○ Mycobacterium tuberculosis - viable for requires longer exposure times (1.5 to 3 several months hours) and higher temperatures than moist ○ Bacillus and Clostridium - viable for 10 heat (160 C - 180 C) years does not require water sterilization of glasswares, oil products or IV. FILTRATION PD powders method of choice for antibiotics solution, toxin lethal effects: protein denaturation, chemicals, radioisotopes, vaccines and Be oxidative damage and toxic effects of carbohydrates, serum, plasma, urea (heat-sensitive elevated levels of electrolytes solutions) may be used with both liquid and air substances 1. Flaming or Direct Heating Flaming with a Bunsen Burner - flaming Types of Filters mouth of culture tubes or slides A. Depth Filters Burning with a Bunsen Burner - wire made up of fibrous or granular materials loops, forceps and straight wire GM examples: ○ Berkefield Filter - diatomaceous 2. Hot Air Oven BD earth most widely used type of dry heat ○ Chamberland Filter - unglazed GM I used for glasswares, certain metals and oils porcelain 2 hours for 160 C - 180 C kill organisms ○ Seitz (Compressed Asbestos) - including all spore formers 98% effective QC: Bacillus subtilis var. Niger (Bacillus ○ Membrane Filter (Swinney) atrophaeus) at 35 C - 37 C Millipore 0.22 um - 100% bacterial sterility 3. Incineration principle: burning of materials into ashes B. Membrane Filter (Circular Filter) at 300 C - 400 C porous membranes (almost 0.1 um thick) most common method of treating infectious composed of cellulose acetate or waste and infected laboratory animals destruction of sputum cups, garbage and SUG used dressings E polycarbonate sterilize pharmaceuticals, ophthalmic solutions, culture media, antibiotics and oil 870 C - 980 C - hazardous material POCA products 4. Cremation 1. Liquid Filtration burning dead human bodies control the pulling the solution through cellulose acetate spread of communicable disease or cellulose nitrate membrane with a vacuum II. LOW / COLD TEMPERATURE 2. Air Filtration considered bacteriostatic - reduces the rate of uses high-efficiency particulate air filters metabolism remove microorganisms larger than 0.3 um exposure to 2 C for 72 hours kills the agent of from isolation rooms, operating rooms, and syphilis (Treponema pallidum) biologic safety cabinets (BSCs) 1. Freezing C. Filtration of Bacteria, Yeasts, and Molds not reliable method of sterilization uses 0.45 and 0.80 um pores of membrane repeated freezing and thawing are much filters more destructive than prolonged freezing 0.2 to 0.45 um in diameter - remove most preservation of bacterial culture via bacteria as well as fungi but not viruses Me lyophilisation or freeze-drying ○ maintaining culture using powder Lethal Effects: protein denaturation, toxic 0.01 um are capable of retaining small viruses effects of increased intracellular electrolyte D. Critical Sterilizing concentration 0.22 um critical sterilizing (parental solutions and III. DESICCATION AND LYOPHILISATION alcohol) 1. Desiccation remove vegetative cells but not viruses disruption of metabolism that involves removing of water from microbes V. RADIATION (bacteriostatic) principle: when radiation passes through the cells, free hydrogen and hydroxyl radicals and some 2. Lyophilisation peroxidase are created which in turn cause different changes in proteins and chemical reactions intracellular damage BIOLOGIC INDICATOR: Bacillus pumilus NOTE: bacteria which remain active in dry environment A. Ionizing radiation (Cold Sterilization) ○ Neisseria gonorrhoeae - viable for 1 hour gamma rays (1500 - 2500 radiation), electron beams, x-rays ACCAD, HORCAJO | 3A - MLS 4  UNIT 3: CONTROL OF MICROORGANISMS short wavelength and high energy chloride -SH groups; toxic at high used for plastic syringes, catheters, sutures, (HgCl2) lyses cell concentration gloves, hormone solutions and antibiotics membrane s causes mutation in the DNA and produces peroxidase Detergents Quaternary Disrupt cell Skin destroys vegetative cells and endospores ammonium membranes antiseptics; compounds disinfectants B. Non Ionizing Radiation damage to cellular DNA by producing Phenolics Phenol, Denature Disinfectants Thymine dimers carbolic acid, proteins; at high ultraviolet rays (10 um to 400 um) in which lysol, disrupt cell concentration 260 um is the most lethal hexachloroph membranes s; used in long wavelength (>1 um) and low energy ene soaps at low poor penetrability concentration used to disinfect exposed surfaces, s operating rooms, nursery rooms control of airborne infection Gases Ethylene Alkylating Sterilization oxide agent of C. Ultrasonic and Sonic Vibrations heat-sensitive no practical value in sterilization and objects disinfection since there are many survivors after the treatment I. ALCOHOL excellent in vitro bactericidal activity against most CHEMICAL METHODS gram- positive and gram-negative bacteria Used mainly as disinfectants also kill Mycobacterium tuberculosis, various fungi, and certain enveloped viruses Chemosterilizers not sporicidal and have poor activity against Chemical agents used to sterilize (eg. glutaraldehyde) certain nonenveloped viruses inactivated by the presence of organic material Chemical agents exert their killing effect by the following should be used in concentrations between 60% and mechanisms: 90% (best concentration is 70%) Reaction with components of the cytoplasmic denaturation of proteins and dissolution of lipid membrane membranes Denaturation of cellular proteins used as both antiseptic and disinfectant(bactericidal Reaction with the thiol (-SH) groups of enzymes and fungicidal) Damage of RNA and DNA allowed to evaporate from the surface to achieve complete antisepsis Table 1. Chemical Agents Commonly Used as Disinfectants Isopropanol and Ethanol and Antiseptics APPLICATIONS II. ALDEHYDES/COLD/CHEMICAL STERILANTS ACTION(S) TYPE AGENT(S) memorize AND inactivation of proteins and nucleic acids PRECAUTIONS commonly used in sterilizing medical instruments Alcohols Ethanol, Denature Skin 8% formaldehyde and 2% glutaraldehyde (50%-70%) isopropanol, proteins; make antiseptics benzyl lipids soluble A. Formaldehyde used as formalin, a 37% alcohol BIE aqueous solution or formaldehyde gas Aldehydes Formaldehyd React with Disinfectants; Formaldehyde gas often used to (in e (8%), NH2, -SH, and kill disinfectant biosafety hoods, HEPA filters solution) glutaraldehyd -COOH endospores; Mycobacteria:3%-8% formalin is used e (2%) groups toxic to with a contact time of at least 30 minutes GF humans Irritability factor and its potential carcinogenicity Halogens Tincture of TI iodine (2% in Inactivates proteins Skin disinfectants B. Glutaraldehyde (Pseudomonacidal, 70% alcohol), Tuberculocidal, Fungicidal and Virucidal) saturated five-carbon dialdehyde Chlorine and Reacts with Used to Inactivation of DNA and RNA through chlorine water to form disinfect alkylation of sulfhydryl and amino groups compounds hypochlorous drinking broad-spectrum activity and rapid killing acid (HCIO); water; action and remains active in the presence of oxidizing surface organic matter CC agents disinfectants Extremely susceptible to pH changes because it is active only in an alkaline Heavy Silver bitrate Precipitates Eye drop (1% environment metals (AgNO3) proteins solution) 2% solution germicidal in approximately 10 minutes and sporicidal in 3 to 10 hours Mercuric Reacts with Disinfectant ACCAD, HORCAJO | 3A - MLS 5  UNIT 3: CONTROL OF MICROORGANISMS does not penetrate organic material well when used as a sterilant 1 : 10 dilution of 5.25% sterilizer of choice for medical equipment recommended by CDC for cleaning up blood that is not heat- stable and cannot be spill autoclaved as well as for material that cannot be sterilized with gas NOTE: safe, high-level disinfectant for most plastic Contact time: 3 minutes and longer if organic material is and rubber items present and 10-30 minutes for mycobacteria effective against HIV and HBV with a Disadvantage: ineffective use in the presence of large minimum of 10 minutes’ exposure at a amount of protein temperature between 20°C and 30°C 2% solution at 25°C to 30°C 100% tuberculocidal IV. QUATERNARY AMMONIUM COMPOUNDS Cold Sterilants sporicidal in a minimum of derived by substitution of the four-valence ammonium 10 hours’ exposure at room temperature ion with alkyl halides cationic, surface-active agents, or surfactants, that III. HALOGENS work by reducing the surface tension of molecules in destroys through oxidation process a liquid Chlorine, Iodine, Fluorine, Bromine, Astatine CIFBA tincture of iodine and iodophor---effective antiseptics effectiveness is reduced by hard water and soap, and they are inactivated by excess organic matter 1:10 NaOCl disruption of the cellular membrane, resulting in ○ effective bleach leakage of cell contents ○ freshly prepared every day with 10-30 not sporicidal or tuberculocidal minutes of contact time---effective disinfection of noncritical surfaces such as benchtops tuberculocide and floors Halozone Pseudomonas aeruginosa - resistant to quaternary ○ parasulfone dichloraminobenzoic acid ammonium compounds ○ contains Cl and is used to disinfect drinking Example: Zephiran (Benzalkonium Chloride and water Ceepryn) and cetylpyridinium chloride A. IODINE V. PHENOLICS TINCTURE molecules of phenol (carbolic acid) that have been alcohol and iodine solutions chemically substituted, typically by halogens, alkyl, used mainly as antiseptics phenyl, or benzyl groups 2% iodine in 70% alcohol Ortho-phenylphenol and ortho-benzyl-para-chlorophenol IODOPHOR Not sporicidal combination of iodine and a neutral stable, biodegradable, and relativelyactive polymer(Detergent) carrier that increases the in the presence of organic material solubility of the agent Disruption of cell walls, resulting in precipitation of less irritating, non staining, and more stable proteins used as antiseptics or disinfectants able to disrupt enzyme systems---lower concentration slow and continuous release of free iodine found in germicidal soaps Free iodine degrades microbial cell walls and cytoplasm, denatures enzymes, and A. CHLORHEXIDINE GLUCONATE coagulates chromosomal material disrupts the microbial cell membrane and Bactericidal action is due to the oxidative precipitates the cell contents effects of molecular iodine (I2) and hypoiodic more effective against gram-positive than acid (HOI) Contact time: 30-60 seconds gram-negative bacteria and has less activity onto the skin prior to blood collection against fungi and tubercle bacilli (0.5% to 4%) B. CHLORINE AND CHLORINE COMPOUNDS inactive against bacteria spores except hypochlorite---liquid sodium at elevated temperatures hypochlorite (household bleach) and solid binds to the skin and remains active for at calcium hypochlorite least 6 hours oxidative effects of hypochlorous acid, pH-dependent: 5.5 - 7.0 formed when chloride ions are dissolved in water Lipid enveloped viruses (herpesvirus, HIV, respiratory not used as sterilants because of the long viruses, influenza virus, cytomegalovirus) --> rapidly exposure time required for sporicidal action inactivated inactivated by organic matter Concentrated bleach solutions should not Nonenveloped viruses (rotavirus, adenovirus, be used for disinfection—Corrosive enteroviruses) --> not inactivated influenced by the pH of the surrounding medium B. HEXACHLOROPHENE effective against gram-positive bacteria 0.5% to 1% Sodium Hypochlorite chlorinated bisphenol used for disinfection stable for no longer than 30 days ACCAD, HO