INTRODUCTION OF INTRODUCTION OF MICROBIOLOGYMICROBIOLOGY

Laboratory of MicrobiologyLaboratory of Microbiology

Faculty of MedicineFaculty of Medicine

Brawijaya UniversityBrawijaya University

microbiologymicrobiology

The branch of biology that deals with The branch of biology that deals with microorganisms and their effects on other living microorganisms and their effects on other living organismsorganismsMany branches of Microbiology :Many branches of Microbiology :

- Environmental Microbiology- Environmental Microbiology - Space Microbiology- Space Microbiology - Marine Microbiology- Marine Microbiology - Agricultural Microbiology- Agricultural Microbiology - Food Microbiology ……. etc.- Food Microbiology ……. etc. - - Medical MicrobiologyMedical Microbiology

First SessionFirst Session

A BRIEF HISTORY OFA BRIEF HISTORY OF

MICROBIOLOGYMICROBIOLOGY

MICROSCOPEMICROSCOPE

A BRIEF HISTORY OF A BRIEF HISTORY OF MICROBIOLOGYMICROBIOLOGY

THE FIRST OBSERVATION

THE DEBATE OVER SPONTANEOUS GENERATION

THE GOLDEN AGE OF MICROBIOLOGY

THE BIRTH OF MODERN CHEMOTHERAPY : DREAMS OF A “MAGIC BULLET”

MODERN DEVELOPMENTS IN MICROBIOLOGY

THE FIRST OBSERVATION

1665 – An Englishman, Robert Hooke, reported to the world that life’s smallest structural units were “little boxes” or “cells” Using his improving version of a compound microscope he was able to see individual cells beginning of the cell theory that all living things are composed of cells

1673 – 1723, Antony van Leeuwenhoek The Dutch merchant and amateur scientist was the first to actually observe live microorganism through his simple single lens microscope the “animalcules” in rainwater, in liquid in which peppercorn had soaked, and in material scraped from his teeth He drew the basic form of bacteria as coccus, rods and spiral

coccus

rods

spiral

THE DEBATE OVER SPONTANEOUS GENERATION Until the second half of the 19th century many scientists and philosophers (Aristoteles, Samson, Virgil) believed that some form of life could arise spontaneously from nonliving matter spontaneous generation

People commonly believed that toads, snakes, and mice could be born of moist soil; that flies could emerge from manure; and that maggots could arise from decaying corpses

Evidence Pro and Con 1668 – Fransesco Redi, the Italian physician, a strong opponent of spontaneous generation demonstrate that maggots do not arise spontaneously from decaying meat Redi filled three jars with decaying meat and sealed them tightly. Then he arranged three other jars similarly but left them open. Maggots appeared in the open jars.Sealed containers showed no signs of maggots

Redi’s antagonists were not convinced; they claimed that fresh air was needed for spontaneous generation

THE DEBATE OVER SPONTANEOUS GENERATION Redi set up a second experiment three jars were covered with a fine net instead of being sealed. No larvae appeared in the gauze-covered jars

Many scientists still believed that small organisms (van Leeuwenhoek’s animalcules) were simple enough to be generated from nonliving materials

1745 : spontaneous generation seem to be strengthened, when John Needham, an Englishman, found that even after he heated nutrient fluid before pouring them into covered flasks, the cooled solution were soon teeming with microorganisms. Needham claimed that microbes developed spontaneously from the fluid

Twenty years later : Lazzaro Spallanzani, an Italian scientist, suggested that microorganisms from the air probably had entered Needham’s solutions after they were boiled. Spallanzani showed that nutrient fluids heated after being sealed in a flask did not develop microbial growth

Needham responded by claiming the “vital force” necessary for sponta- neous generation had been destroyed by the heat and was kept out of the flasks by the sealed

THE DEBATE OVER SPONTANEOUS GENERATION

Laurant Lavoisier showed the importance of oxygen to life.

Spallanzani’s observations were criticized on the grounds that there was not enough oxygen in the sealed flasks to support microbial life

The Theory of Biogenesis

Rudolf Virchow challenged spontaneous generation with the concept of biogenesis, he claim that living cells can arise only from preexisting living cells

Arguments about spontaneous generation continued until 1861, when the issue was resolved by the French scientist Louis Pasteur

With a series of ingenious experiments, Pasteur demonstrate that microorganisms are present in the air and can contaminate sterile solutions, but air itself does not create microbes

Pasteur’s experiments disproving the theory of spontaneous generation

(1) Pasteur first poured beef broth into a long-necked flask. (2) Next he heated the neck of the flask and bent it into an S-shaped curve; then he boiled the broth for several minutes. (3) Microorganisms did not appear in the cooled solution, even after long periods

Pasteur showed that microorganisms can be present in nonliving matter on solids, in liquids, and in the air

He demonstrated that microbial life can be destroyed by heat form the basis of aseptic techniques

The debate of Spontaneous Generation disproved

However still have a problem of “spores” resistant to heat John Tyndall (1820-1893) TYNDALLIZATION Spontaneous Generation Theory totally finished

The Golden Age Of MicrobiologyThe Golden Age Of Microbiology1857 - 19141857 - 1914

Pasteur & Robert Koch, led to the establishmentPasteur & Robert Koch, led to the establishment

of microbiology as of microbiology as a sciencea science

Discoveries during these years included both the Discoveries during these years included both the agents of many diseases and the role of agents of many diseases and the role of immunity in the prevention and cure of diseaseimmunity in the prevention and cure of disease

Fermentation and PasteurizationFermentation and Pasteurization

The Germ Theory of DiseaseThe Germ Theory of Disease

VaccinationVaccination

Fermentation & PasteurizationFermentation & Pasteurization

A group of French merchants asked Pasteur to find out why A group of French merchants asked Pasteur to find out why wine and beer soured wine and beer soured a method that would prevent a method that would prevent spoilage ?spoilage ?

Many scientist believed that air converted the sugars in Many scientist believed that air converted the sugars in these fluids into alcoholthese fluids into alcohol

Pasteur found instead that microorganisms called Pasteur found instead that microorganisms called yeastsyeasts convert the sugars to alcohol in the absence of air. This convert the sugars to alcohol in the absence of air. This process, called process, called fermentationfermentation, is used to make wine and , is used to make wine and beerbeer

Souring and spoilage are caused by different micro-Souring and spoilage are caused by different micro-organisms called organisms called bacteriabacteria. In the presence of air, bacteria . In the presence of air, bacteria change the alcohol in the beverage into vinegar (acetic acid)change the alcohol in the beverage into vinegar (acetic acid)

The Germ Theory of DiseaseThe Germ Theory of Disease

Before the time of Pasteur, effective treatments for many Before the time of Pasteur, effective treatments for many diseases were discovered by trial and error, but the causes diseases were discovered by trial and error, but the causes of the disease were unknownof the disease were unknownThe realization that yeasts play a crucial role in The realization that yeasts play a crucial role in fermentation was the first link between the activity of a fermentation was the first link between the activity of a microorganism and physical and chemical changes in microorganism and physical and chemical changes in organic materialsorganic materialsThis discovery alerted scientists to the possibility that This discovery alerted scientists to the possibility that microorganisms might have similar relationships with plants microorganisms might have similar relationships with plants animals - specifically, that microorganisms might cause animals - specifically, that microorganisms might cause disease. This idea was known as disease. This idea was known as the germ theory of the germ theory of diseasedisease

The Germ Theory of DiseaseThe Germ Theory of Disease

1840 – 1840 – Ignaz SemmelweisIgnaz Semmelweis a Hungarian physician, had a Hungarian physician, had demonstrated that physician, who at the time did not disinfect demonstrated that physician, who at the time did not disinfect their hands, routinely transmitted infections (puerperal, or their hands, routinely transmitted infections (puerperal, or childbirth fever) from one obstetrical patient to anotherchildbirth fever) from one obstetrical patient to another1860 – 1860 – Joseph ListerJoseph Lister an English surgeon, applied the germ an English surgeon, applied the germ theory to medical procedure. Disinfectants were not used at theory to medical procedure. Disinfectants were not used at the time, but Lister knew that phenol (carbolic acid) kills the time, but Lister knew that phenol (carbolic acid) kills bacteria, so he began treating surgical wounds with a phenol bacteria, so he began treating surgical wounds with a phenol solutionsolution1876 – 1876 – Robert KochRobert Koch, a German physician, proved that , a German physician, proved that bacteria actually cause disease bacteria actually cause disease discovered rod-shaped discovered rod-shaped bacteria now known as bacteria now known as Bacillus anthracisBacillus anthracis in the blood of in the blood of cattle that had died of anthrax cattle that had died of anthrax

The Germ Theory of DiseaseThe Germ Theory of Disease

Koch’s research provides a framework for the study of the Koch’s research provides a framework for the study of the etiology of any infectious disease etiology of any infectious disease Koch Postulates :Koch Postulates :

1. The same pathogen must be present in every case of 1. The same pathogen must be present in every case of diseasedisease 2. The pathogen must be isolated from the diseased host2. The pathogen must be isolated from the diseased host and grown in pure cultureand grown in pure culture 3. The pathogen from the pure culture must cause the 3. The pathogen from the pure culture must cause the disease when it is inoculated into a healthy, susceptibledisease when it is inoculated into a healthy, susceptible laboratory animallaboratory animal 4. The pathogen must be isolated from the inoculated 4. The pathogen must be isolated from the inoculated animal and must be shown to be the original organismanimal and must be shown to be the original organism

VACCINATIONVACCINATION1796 – 1796 – Edward JennerEdward Jenner, a young British physician, embarked , a young British physician, embarked on an experiment to find a way to protect people from on an experiment to find a way to protect people from smallpox :smallpox :

- A young milkmaid informed Jenner that she couldn’t get- A young milkmaid informed Jenner that she couldn’t get smallpox because she already had been sick from cowpoxsmallpox because she already had been sick from cowpox - First Jenner collected scrapings from cowpox blisters. Then - First Jenner collected scrapings from cowpox blisters. Then he inoculated a healthy 8-year-old volunteer with thehe inoculated a healthy 8-year-old volunteer with the cowpox material by scratching the person’s arm with a cowpox material by scratching the person’s arm with a pox-contaminated needle. In a few days, the volunteer pox-contaminated needle. In a few days, the volunteer became mildly sick but recovered and never againbecame mildly sick but recovered and never again contracted either cowpox or smallpox. The process was contracted either cowpox or smallpox. The process was called called vaccinationvaccination (vacca=cow) (vacca=cow) - The protection from disease provided by vaccination is- The protection from disease provided by vaccination is called called immunityimmunity

VACCINATIONVACCINATION1880 – Pasteur discovered why vaccination work. He found 1880 – Pasteur discovered why vaccination work. He found

that the bacteria that causes that the bacteria that causes fowl cholera fowl cholera lost its ability lost its ability

to cause disease after it was grown in the laboratory forto cause disease after it was grown in the laboratory for

long periods. However it – and other microorganisms withlong periods. However it – and other microorganisms with

decreased virulence – was able to induce immunity againstdecreased virulence – was able to induce immunity against

subsequent infections by its virulent counterpart.subsequent infections by its virulent counterpart.

Some vaccines are still produced from avirulent microbialSome vaccines are still produced from avirulent microbial

strains that stimulate immunity to the related virulent strain. strains that stimulate immunity to the related virulent strain. Other vaccines are made from killed virulence microbes, Other vaccines are made from killed virulence microbes,

from isolated components of virulent micoorganisms, or by from isolated components of virulent micoorganisms, or by genetic engineering techniquesgenetic engineering techniques

The Birth of Modern Chemotherapy :The Birth of Modern Chemotherapy :Dreams of a “ Magic Bullet “Dreams of a “ Magic Bullet “

After the relationship between microorganisms andAfter the relationship between microorganisms and disease was established, medical microbiologists nextdisease was established, medical microbiologists next focused on the search for substances that could destroyfocused on the search for substances that could destroy pathogenic microorganisms without damaging the pathogenic microorganisms without damaging the

infected animal or humaninfected animal or humanTreatment of disease by using chemical substances isTreatment of disease by using chemical substances is

called called chemotherapychemotherapyChemotherapeutic agents prepared from chemicals inChemotherapeutic agents prepared from chemicals in

the laboratory are called the laboratory are called synthetic drugssynthetic drugsChemicals produced naturally by bacteria or fungi to act Chemicals produced naturally by bacteria or fungi to act against other microorganisms are called against other microorganisms are called antibioticsantibiotics

The First Synthetic DrugsThe First Synthetic Drugs

Paul EhrlichPaul Ehrlich, a German physician, was the imaginative , a German physician, was the imaginative thinker who fired the first shot in the chemotherapy thinker who fired the first shot in the chemotherapy revolution. Ehrlich speculated about a “magic bullet” that revolution. Ehrlich speculated about a “magic bullet” that could destroy pathogen without harming the infected hostcould destroy pathogen without harming the infected hostIn 1910, he found a chemotherapeutic agent called In 1910, he found a chemotherapeutic agent called salvarsansalvarsan, an arsenic derivative effective against syphylis, an arsenic derivative effective against syphylisBy the late 1930s, researchers had developed several other By the late 1930s, researchers had developed several other synthetic drugs, mostly were derivatives of dyes that could synthetic drugs, mostly were derivatives of dyes that could destroy microorganisms destroy microorganisms In addition, In addition, Domagk (1935)Domagk (1935) discovered that discovered that prontosilprontosil had had dramatically effect against streptococcal infections dramatically effect against streptococcal infections in the in the body was changed into body was changed into sulfanilamidesulfanilamide that analog with that analog with PABAPABA

A Fortunate Accident - AntibioticsA Fortunate Accident - Antibiotics

The first antibiotic was discovered by accidentThe first antibiotic was discovered by accident

1928 – 1928 – Alexander FlemingAlexander Fleming, a Scottish physician and , a Scottish physician and bacteriologist, looked at the curious pattern of growth on the bacteriologist, looked at the curious pattern of growth on the contaminated plates. There was a clear area around the mold contaminated plates. There was a clear area around the mold where the bacterial culture had stopped growingwhere the bacterial culture had stopped growing

The mold was later identified as The mold was later identified as Penicillium notatumPenicillium notatum, and , and Fleming named the mold’s active inhibitor as Fleming named the mold’s active inhibitor as penicillinpenicillin..

The enormous usefulness of penicillin was not apparent until The enormous usefulness of penicillin was not apparent until the 1940s the 1940s Florey & ChainFlorey & Chain

1939 – 1939 – ReneRene´́ Dubos, Dubos, a French microbiologist, discovered a French microbiologist, discovered two antibiotics called two antibiotics called gramicidin gramicidin and and tyrocidine.tyrocidine. Both were Both were produced by a bacterium, produced by a bacterium, Bacillus brevisBacillus brevis, cultured from soil. , cultured from soil.

Modern Development in MicrobiologyModern Development in Microbiology

BacteriologyBacteriology

MycologyMycology

ParasitologyParasitology

ImmunologyImmunology

VirologyVirology

Recombinant DNA technologyRecombinant DNA technology

VIROLOGYVIROLOGY

The study of virus, actually originated during the Golden Age of The study of virus, actually originated during the Golden Age of MicrobiologyMicrobiology

1892 – 1892 – Dmitri IwanowskiDmitri Iwanowski reported that the organism that cause reported that the organism that cause mosaic disease of tobacco was so small that it passed through filter mosaic disease of tobacco was so small that it passed through filter fine enough to stop all known bacteriafine enough to stop all known bacteria

1935 – 1935 – Wendell Stanley Wendell Stanley demonstrated that the organism, called demonstrated that the organism, called tobacco mosaic virus (TMV)tobacco mosaic virus (TMV), was fundamentally different from other , was fundamentally different from other microbes and so simple and homogeneous that it could be cristallized microbes and so simple and homogeneous that it could be cristallized like chemical compoundlike chemical compound

1940 – Since the development of the electron microscope, 1940 – Since the development of the electron microscope, microbiologists have been able to observe the structure of viruses in microbiologists have been able to observe the structure of viruses in detail.detail.

Recombinant DNA TechnologyRecombinant DNA Technology

Until the 1930s, all genetic research was based on the study of plant and Until the 1930s, all genetic research was based on the study of plant and animal cellsanimal cells

In the 1940s, the scientists turned to unicellular organisms, primarily In the 1940s, the scientists turned to unicellular organisms, primarily bacteriabacteria

In 1941 – In 1941 – George W.Beadle George W.Beadle and and Edward L.Tatum Edward L.Tatum demonstrated the demonstrated the relationship between genes and enzymesrelationship between genes and enzymes

1944 – 1944 – Oswald Avery, Colin McLeodOswald Avery, Colin McLeod, and , and Maclyn McCarty Maclyn McCarty DNA was DNA was established as the hereditary materialestablished as the hereditary material

1946 – 1946 – Joshua Lederberg Joshua Lederberg and and Edward L.TatumEdward L.Tatum discovered that genetic discovered that genetic material could be transferred from one bacterium to another by a process material could be transferred from one bacterium to another by a process called conjugationcalled conjugation

1953 – 1953 – James WatsonJames Watson and and Francis Crick Francis Crick proposed a model for the proposed a model for the structure and replication of DNAstructure and replication of DNA

MICROSCOPEMICROSCOPE

MICROSCOPEMICROSCOPE

Compound Light MicroscopyCompound Light Microscopy

Darkfield MicroscopyDarkfield Microscopy

Phase-contrast MicroscopyPhase-contrast Microscopy

Fluorescence MicroscopyFluorescence Microscopy

Confocal MicroscopyConfocal Microscopy

Electron MicroscopyElectron Microscopy

Compound Light MicroscopeCompound Light Microscope

Has a series of lenses and use visible light as its source Has a series of lenses and use visible light as its source of illuminationof illuminationA series of finely ground lenses forms a clearly focused A series of finely ground lenses forms a clearly focused image that is many time larger than the specimens itselfimage that is many time larger than the specimens itselfThis magnification is achieved when light rays from an This magnification is achieved when light rays from an illuminator illuminator condensor condensor specimen specimen objective lenses objective lenses ocular lens ocular lensTotal magnification = objective lens magnification x Total magnification = objective lens magnification x ocular lens magnificationocular lens magnificationObjective lens : 10 x (low power), 40 x (high power), and Objective lens : 10 x (low power), 40 x (high power), and 100 x (oil immersion)100 x (oil immersion)Ocular lens : 10 xOcular lens : 10 x

Compound Light MicroscopeCompound Light Microscope

The total magnifications would be 100 x for low power, The total magnifications would be 100 x for low power, 400 x for high power , and 1000 x for oil immersion. 400 x for high power , and 1000 x for oil immersion. Some compound light microscopes can achieve a Some compound light microscopes can achieve a magnification of 2000 x with the oil immersion lens.magnification of 2000 x with the oil immersion lens.

The oil immersion has the same refractive index as The oil immersion has the same refractive index as glass, so the oil becomes part of the optics of the glass glass, so the oil becomes part of the optics of the glass of the microscope.of the microscope.

Unless immersion oil is used, light rays are refracted as Unless immersion oil is used, light rays are refracted as they enter the air from the slidethey enter the air from the slide

Compound Light MicroscopeCompound Light Microscope

Darkfield MicroscopyDarkfield Microscopy

Is used for examining live microorganisms that either are Is used for examining live microorganisms that either are invisible in the ordinary light microscope, cannot be invisible in the ordinary light microscope, cannot be stained by standard methods, or are so distorted by stained by standard methods, or are so distorted by staining that their characteristics then cannot be identifiedstaining that their characteristics then cannot be identified

A darkfield microscope uses a darkfield condensor that A darkfield microscope uses a darkfield condensor that contain an opaque disc contain an opaque disc the specimen appears light the specimen appears light against a black backgroundagainst a black background

One use for darkfield microscopy is the examination of One use for darkfield microscopy is the examination of very thin spirochetes, such as very thin spirochetes, such as Treponema pallidumTreponema pallidum, the , the causative agent of syphiliscausative agent of syphilis

Phase-Contrast MicroscopyPhase-Contrast Microscopy

Is especially useful because it permits detailed examination Is especially useful because it permits detailed examination of internal structures in living microorganismsof internal structures in living microorganisms

The principle of phase-contrast microscopy is based on the The principle of phase-contrast microscopy is based on the wave nature of light rays, and the fact that light rays can be wave nature of light rays, and the fact that light rays can be in phase in phase (their peaks and valleys match) or (their peaks and valleys match) or out of phaseout of phase..

In phase-contrast microscopy, one set of light rays comes In phase-contrast microscopy, one set of light rays comes directly from the light source. The other set comes from light directly from the light source. The other set comes from light that is reflected or diffracted from particular structure in the that is reflected or diffracted from particular structure in the specimenspecimen

Phase-contrast microscope is provided with diffraction platePhase-contrast microscope is provided with diffraction plate

Brightfield Darkfield Phase-contrastBrightfield Darkfield Phase-contrast

Fluorescence MicroscopyFluorescence Microscopy

The object is stained with one of a group of fluorescent dyes The object is stained with one of a group of fluorescent dyes called called fluorochromesfluorochromes ( (primolin, acridine orange R,

thiazo yellow-G, auramine O, fluorescein isothiocyanate)

The light source Ultraviolet light

The principal use of fluorescence microscopy is a diagnostic technique called fluorescence-antibody (FA) technique, or immunofluorescence

Confocal MicroscopyConfocal Microscopy

A fairly recent development in light microscopyA fairly recent development in light microscopy

Like fuorescent microscopy, specimens are stained with Like fuorescent microscopy, specimens are stained with fluorochromesfluorochromes

The light source The light source laser laser

Most confocal microscopes are used in conjunction with Most confocal microscopes are used in conjunction with computers to construct three-dimensional imagescomputers to construct three-dimensional images

Can be used to evaluate cellular physiology by Can be used to evaluate cellular physiology by monitoring the distributions and concentrations of monitoring the distributions and concentrations of substances such as ATP and calcium ions.substances such as ATP and calcium ions.

Electron MicroscopyElectron Microscopy

Object smaller than about 0.2 Object smaller than about 0.2 µm, such as virus, or the µm, such as virus, or the internal structures of cels must be examined with an internal structures of cels must be examined with an electron microscopeelectron microscopeA beam of electrons is used instead of lightA beam of electrons is used instead of lightObjects are generally magnified 10,000 – 100,000 xObjects are generally magnified 10,000 – 100,000 xInstead of using glass lenses, an electron microscope Instead of using glass lenses, an electron microscope uses electromagnetic lenses to focus a beam of uses electromagnetic lenses to focus a beam of electrons onto specimenelectrons onto specimen There are two types of electron microscope :There are two types of electron microscope :

- The transmission electron microscope- The transmission electron microscope - The scanning electron microscope- The scanning electron microscope