Biologi Mikroba

7/22/2019 Biologi Mikroba

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Biologi Mikroba


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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
http://en.wikipedia.org/wiki/Beta-galactosidasehttp://en.wikipedia.org/wiki/Beta-galactosidasehttp://en.wikipedia.org/wiki/Beta-galactosidasehttp://en.wikipedia.org/wiki/Beta-galactosidasehttp://en.wikipedia.org/wiki/Beta-galactosidase


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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
http://id.wikipedia.org/wiki/Spirocheteshttp://id.wikipedia.org/wiki/Spirocheteshttp://id.wikipedia.org/wiki/Spirocheteshttp://id.wikipedia.org/wiki/Spirocheteshttp://id.wikipedia.org/wiki/Spirocheteshttp://id.wikipedia.org/wiki/Spirocheteshttp://id.wikipedia.org/wiki/Spirocheteshttp://id.wikipedia.org/wiki/Spirochetes


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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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