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Perkembangan Mikrobiologi
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
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1stObservation
Robert hooke
Antony van Leewenhoek
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Spontaneous Generation
Fransesco Redi
Louis Pasteur
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The Golden Age Of Microbiology
1857 - 1914
Fermentation & Pasteurization -- Pasteur The Germ Theory of Disease
Ignaz Semmelweis dokter vs bidan
Joseph Lister phenol untuk luka
Robert Koch postulat koch kec M.lepare
Vaccination
Edward Jenner pox virus
Pasteur
Magic Bullet Paul Erlich aslvarsan
Domagk prontosilsulfa
Alexander Fleming Penicillin
Rene
Dubos gramicidin and tyrocidine
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Koch Postulates
1. Mikroorganisme tertentu selalu ditemukan berasosiasi
dengan penyakit yang ditimbulkan
2. Mikroorganisme dapat diisolasi dan ditumbuhkan
sebagai biakan murni di laboratorium
3. Biakan murni tersebut bila diinjeksikan pada binatang
yang sesuai dapat menimbulkan penyakit4. Mikroorganisme tersebut dapat diisolasi kembali dari
hewan yang telah terinfeksi tersebut.
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The Birth of Modern Chemotherapy :
Dreams of a Magic Bullet
Treatment of disease by using chemical substances is
called chemotherapy
Chemotherapeutic agents prepared from chemicals
in the laboratory are called synthetic drugsChemicals produced naturally by bacteria or fungi toact against other microorganisms are calledantibiotics
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The First Synthetic Drugs
Paul Ehrlichspeculated about a magic bullet
In 1910, he found salvarsan, an arsenic derivative effective against
syphylis
Domagk (1935)discovered that prontosilhad dramatically
effect against streptococcal infectionsin the body waschanged into sulfanilamidethat analog with PABA
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There was a clear area around the mold
Penicillium notatum
The first antibiotic was discovered by accident
(Alexander Fleming)(1928)
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1939ReneDubos, a French microbiologist, discovered two
antibiotics called gramicidin and tyrocidine.Both were produced
by a bacterium, Bacillus brevis, cultured from soil.
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Microscope
Compound Light Microscopy
Darkfield Microscopy examining live microorganisms
Phase-contrast Microscopy permits detailed examination of internal structures
Fluorescence Microscopy stained with fluorescentUV light
Confocal Microscopy stained with fluorochromesLASER3D
Electron Microscopy Object smaller than about 0.2 m (virus), or the internal
structures of cells
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Brightfield Darkfield Phase-contrast
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KINDS OF STAINING TECHNIQUES
Simple Stains :
- methylene blue
- saffranin
Differential Stains :
GramGram (+) / Gram ( - )
Acid Fastacid fast/non acid fast bacilli
Special Stains :
- Metachromatic staining
- Spore staining
- Capsule staining
- Flagella staining
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SIMPLE STAINS
Also said as progressive staining
Use a single basic dye only
Simple stains commonly used :
- methylene blue
- saffranin- crystal violet
Primary purposeto see themorphology of bacteria (cellularshape and basic structures arevisible)
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Gram : KLAS
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BTA
Primary stainfuchsinredCounterstainmethylene blueChemical intensifiercarbolPhysical intensifierheat
Kinds of acid fast staining :- Ziehl Neelsen- Tan Thiam Hok- Kinjoun carbolfuchsinBacteria : Mycobacterium tuberculosis,
Mycobacterium leprae
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Special Staining
Endopsore (spore) stainingSchaeffer Fulton
malachite green -- safranin
Capsule stainingNegative staining
Flagella stainingGray, Leiffson
Metachromatic stainingNeisser
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The Taxonomy Hierarchy
DOMAIN : BACTERIA
Phylum : PROTEOBACTERIA
Class : Gamma-proteobacteria Order : Enterobacteriales
Family : Enterobacteriaceae
Genus : Escherichia Species : Escherichia coli
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(a) Normal DNA molecule (c) Nonsense mutation
(b) Missense mutation (d) Frameshift mutation
Type of Mutation
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GENETIC TRANSFER
INVOLVE DONOR CELLS AND RECIPIENT CELLS
TYPES :
1.TRANSFORMATION
2.TRANSDUCTION
3. CONJUGATION
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GRIFFITHS EXPERIMENT
TRANSFORMATION
Streptococcus pneumoniae
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TRANSDUCTION
Bacteriophage
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TRANSDUCTION
Bacteriophage
sex pilus
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CONJUGATION
plasmid
Hfr cell
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Plasmid
Extrachromosomal genetic elements
Replicate independently of chromosomal
DNA
Usually contain from 5100 genes
May carry genes for such activities as
antibiotic resistance, tolerance to toxicmetals, the production of toxin, and the
synthesis of enzymes
Can be transferred from one bacterium to
another
Is used for gene manipulation in
biotechnology Examples :
- F plasmids
- Penicillinase plasmid
- R factors : RTF + r-determinant
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Transposon / Jumping Genes
The simplest transposon, also called insertionsequences (IS), contain only a gene that codesfor an enzyme (transposase) and recognitionsites
Complex transposonsalso carry other genesnot connected with the transposition process,for examplemay contain genes forenterotoxin or for antibiotic resistance
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GENE CLONING
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1. ISOLATION OF DNA CONTAINING GENE
2. RESTRICTION ENZYMES (ENDONUCLEASES)
- produced by bacteria (> 2000 enzymes)- molecular scalpelcut DNA on the
particular sites
- recognize particular nucleotide base sequences
(4,6,8 bp)
- cutting point :
- blunt-ended fragments
- sticky ends
Hae IIIGG CC EcoR IG AATTC
CC GG CTTAA G
3 GENE IS INSERTED INTO CLONING VECTOR
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3. GENE IS INSERTED INTO CLONING VECTOR
CLONING VECTOR:
- must has origin of replication
- has 1 cloning site- has gene that code for product to differentiate :
transformed cells and untransformed cells
Types :- PLASMID
> circular DNA moleculesextrachromosomal
> small size (5000 bp)
> replicate autonomously
- COSMID
- BACTERIOPHAGE
- PHAGE M 13
- etc
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By using the same restriction enzyme DNA vectorand DNA that contain gene of interest are cut
Gene of interest inserted into pd DNA vector + ligase
Recombinant DNA
Recombinant DNA
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4. TRANSFORMATION
Vector + DNA of interest bacteria competence cells
CaCl2
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5. SELECTING A CLONE
BLUE-WHITE SCREENING
Plasmid contain :
- gen ampR resistant to penicillin- gen lacZ -galactosidase
-galactosidaseis a protein encoded by the lacZgene
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Metode seleksi recombinant bacteria
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X-Gal, a colourless analog of lactose that may be cleaved by
-galactosidase to form 5-bromo-4-chloro-indoxyl, which
then spontaneously dimerizes and oxidizes to form a bright
blue insoluble pigment 5,5'-dibromo-4,4'-dichloro-indigo.
This results in a characteristic blue colour in cells containing
a functional -galactosidase.
Blue colonies therefore show that they may contain a
vector with an uninterrupted lacZ (therefore no insert),
while white colonies, where X-gal is not hydrolyzed,
indicate the presence of an insert in lacZ which disrupts
the formation of an active -galactosidase.
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Physical Requirements
Temperature Psychrophiles (cold loving)
Mesophiles (moderate-temperature loving)
Thermophiles (heat-loving)
pH Most bacteria grow best at pH 6.5 - 7.5
Acidophilestolerant to acidity
Osmotic pressure
They require water for growth and made up of 80-90% water In hypertonic solution,most microbes undergo plasmolysis
Halophiles,can tolerate high salt concentrations
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Chemical Requirements
Carbon
Carbon is the structural back-bone of living matter
Half the dry weight of bacterial cell is carbon
Chemoheterotrophs, use an organic molecule
Autotroph(chemoautotroph or photoautotroph)derive theircarbon from carbon dioxide
Nitrogen
for protein and nucleic acid synthesis
from the decomposition of proteins or from NH4+or NO3-
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Chemoheterotrophs unable to form their own organic compounds
derives energy and carbon from the oxidation ofpreformed organic compounds
Photoautotrophs autotrophs that makes food using the sun light
Chemoautotrophs autortophs that makes food using chemical
compounds such as sulfite
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Chemical Requirements
Oxygen obligate aerobes, facultative anaerobes, aerotolerant
anaerobes, and microaerophilic
Aerobes, facultative anaerobes, and aerotolerant anaerobesmust have the enzymes superoxide dismutase(2O2
-+ 2H+O2+ H2O2)
catalase(2H2O22H2O + O2)
peroxidase(H2O2 + 2H22H2O)
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Phases of Growth
f
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Measurement of
Microbial Growth
Direct
Indirect
Di M f
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Direct Measurement of
Microbial Growth
Colony forming units (CFU) Filtration
Most probable number (MPN)
Petroff-Hausser cell counter
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Colony Forming Units (CFU)
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A standard plate count reflects the number of viablemicrobes and assumes that each bacterium grows into
a single colony. Because it is impossible to say thateach colony actually arose from an indivual cell (cellsclump, fact of life) plate counts are reported as thenumber of colony-forming units (CFU) instead of the
number of cells.
If the concentration of bacteria is too great thecolonies will grow into each other and the plate will be
uncountable.
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Filtration
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Most Probable Number (MPN)
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The most probable number (MPN) method can be
used for microbes that will grow in a liquid medium;
it is a statistical estimation.
A dilution series to no growth is prepared and the
combination of positives is used to look the most
probable number up in a table (see MPN Table). Used for microbes that won't grow on solid media or
are grown in differential liquid media for
identification purposes
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Petroff-Hausser cell counter
Indirect Meas rement of
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Indirect Measurement of
Microbial Growth
Turbidity
Metabolic Activity
Dry Weight
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Turbidity McFarland standard
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Metabolic Activity Acid production or oxygen consumption
Dry Weight
For filamentous organisms such as fungi,measuring dry weight is a convenient methodof growth measurement
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Axial Filament inSpirochaeta
Spirochaeta
Treponema
Leptospira
Borrelia
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Flagella
A-Monotrichous
B-Lophotrichous
C-Amphitrichous
D-Peritrichous
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Detection of virus-infected cells :
Cytopathic effects (morphologic changes in the cells :cell lysis or necrosis, inclusion formation, giant cellformation, cytoplasmic vacuolization)
Appearance of virus-encoded protein(hemagglutinin)
Adsorption of erythrocytes to infected cells(hemadsorbtion)
Interference
Morphologic transformation (loss of contact
inhibition) Viral growth in an embryonated chick egg (as
described previously)
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