Transcript Metode Mikrobiologis - Selamat Datang di Komunitas e

Metode Mikrobiologis-1
1. Penanganan Mikrobia
2. Metode Kultur murni dan Teknik
Aseptis
3. Isolasi, Purifikasi
4. Penumbuhan mikrobia: Culture
media
5. Sterilisasi dan desinfeksi
6. Mikroskopi
7. Pengecatan mikrobia
8. Enumerasi mikrobia
9. Karakterisasi dan Identifikasi
1. Penanganan mikrobia
Safe handling microbes:
• Sebagian besar mikrobia bermanfaat
• Sebagian kecil mikrobia merupakan agensia penyakit
e.g. HIV, Neisseria meningitis
• Kultur mikrobia tidak terlepas ke lingkungan
• Kultur mikrobia tidak terkontaminasi oleh mikrobia lain
• Teknik saftey harus dipenuhi dalam bekerja dengan
mikrobia
• Bahan dan alat dan tempat bekerja harus steril (teknik
aseptis)
• Kultur mikrobia yang tidak dipakai lagi harus
dimusnahkan
2.Metode kultur murni (Pure culture)
• Mikrobia yang dipelajari harus dalam bentuk
kultur murni
• Kultur murni: kumpulan sel yang merupakan
keturunan sel tunggal
• Strain : kumpulan sel kultur murni yang
berasal dari satu sel tunggal
• Untuk memperoleh kultur murni → Teknik
Aseptis
• Teknik Aseptis: semua bahan, alat, dan
tempat bekerja yang digunakan serta cara
kerja harus bebas dari kontaminasi (steril)
Kultur murni mikrobia
Inokulasi
Koloni
A pure culture is a culture consisting of only
one kind of microorganism
 Aseptic technique is technique which
involves avoiding any contact of the pure
culture, using sterile medium, and using
sterile surfaces of the growth vessel with
contaminating microorganisms
 How to accomplish it?
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1. Sterilization of work area
2. Sterilization of working instruments and medium
3. Work must be done quickly and efficiently
Method used to isolate specific groups of
microorganism
 Culture medium and incubation
conditions are designed to support
target microorganism
 Microorganisms other than the target will
grow poorly or unable to grow in
enrichment medium
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Isolation of pure cultures involves:
› Separating samples of microorganisms into
individual cells
› Allowing the individual cells to reproduce
and form clones of single microorganism
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The success of isolation depends on:
› Ability to do physical separation of the
microorganism
› Ability to maintain viability and growth of
pure culture of microorganism
Samples
Enrichment
Dilution
Platting
Streak Plate
Spread Plate
Maintenance
Pour Plate
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A suitable culture media for
microorganisms growth must:
› Containing substances which support their
needs. Substances (nutrients) divided into
three types: macronutrients (C, N, P, S, K, Mg,
Ca, Na, Fe), micronutrients(Cr, Co, Cu, Mn,
Mo, Ni, Se, W, V, Zn), and growth factors
(vitamins, amino acids, purines, pirimidines)
› Having suitable environment conditions (pH,
temperature, oxygen level)
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Defined media: media in which all
components are known( eg. Azotobacter
medium, BG-11 Medium)
Complex media: media in which the
components are not totally known and may
vary (eg. NA, PDA)
Selective media: media which only favor the
growth of specific microorganisms (eg.
Salmonella-Shigella Agar)
Differential media: media which contain
substances that can detect microorganisms
with specific metabolic activitise (eg,
MacConkey Agar)
Maintenance of microorganism is
important to preserve cultures which
have special features
 The purposes of maintenance are to
maintain viability and genetic stability of
the microorganisms
 Several methods of maintenance are:
subculture, drying, freeze-drying, and
freezing
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5. Sterilisasi dan disinfeksi
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Steril: bebas dari segala bentuk kehidupan
Sterilisasi: upaya untuk mencapai keadaan steril
Bactericidal
Fungicidal
Viricidal (seolah virus jasad hidup !) - inactivator
Germicides
Bacteristatic
Fungistatic
Antibiotics
5. Sterilisasi dan disinfeksi
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Desinfection
Desinfectan
Antisepsis
Antiseptic
Sanitation
Metode Sterilisasi
Metode Fisikawi:
• Mekanis : filtrasi
• Pemanasan:
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Pemijaran: e.g. pemijaran ose
Udara panas kering: e.g. oven
Uap air panas: e.g. Arnold –steam sterilizer
Uap air panas bertekanan: e.g. Autoclave
• Penyinaran :
 Radiasi UV, gamma
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Sterilization is a process to eliminate
contaminant microorganisms
Chemical sterilization (alcohol, xylol, H2O2
etc) --> work surface and working
instrument
Radiation sterilization (UV, gamma, etc) -->
work surface and working instrument
Filtration --> heat-sensitive growth medium
Heating (autoclave, oven) --> working
instrument, heat-resistant growth medium
Metode Sterilisasi
Metode Kimiawi:
• Disinfectant: HgCl2, betadine
• Antiseptics: fenol, cresol
• Antibiotics
• Ozonization (ozon)
6. Mikroskopi
Light microscopy:
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Bright-field microscopy
Dark-ground microscopy
Phase-contrast microscopy
Fluorescence microscopy
Electron microscopy:
 Scanning Electron Microscopy
(SEM)
 Transmission Electron
Microscopy (TEM)
Microscopy is the use of microscope to
view objects too small to be visible to
naked eye
 Type of microscope:
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› Light microscope (brightfield, darkfield,
ultarviolet, fluorescence, phase contrast,
nomarski differential interference, confocal)
› Electron microscope (Transmission Electron
Microscope, Scanning Electron Microscope)
Confocal microscopy
 Confocal scanning laser microscopy (CSLM)
 Memakai sinar LASER
 Bayangan tiga dimensi dan lebih jelas
Scaning Probe Microscopy
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Scanning tunneling microscope (1980)
G. Binnig & H. Rohrer : Nobel Prize (1986)
Perbesaran 100 juta kali
Dapat melihat atom pada permukaan padat
Atomic force microscope (lebih baru)
Spesimen yang non-konduktor: interaksi protein, letak
restriction site pada plasmid
7. Pengecatan mikrobia
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Simple staining
Gram staining
Ziehl-Neelsen staining
Granules staining
Negative staining
Spore staining
Staining is important to discerns object
from its background, hence it is become
viewable under light microscope
 Staining procedure:
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› Simple staining (positive staining, negative
staining)
› Differential staining (Gram staining, acid-fast
staining, endospore staining)
Single stain is used
 All cells and structures generally stain the
same color
 Positive staining: stain attracted to the
cell
 Negative staining: stain repelled by the
cell, background takes on the color
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Multiple staining reactions are employed
Differentiate types of cells or cell’s structures
base on their staining reactions, hence given
different color
The specimen must be “fixed” by heating or
chemical
Examples:
› Gram stain: staining based on composition of lipids
and peptidoglycan in bacterial cell wall
› Acid-fast stain: staining based on composition of
mycolic acid, fatty acids, waxes, and complex lipids
in bacterial cell wall
› Endospore stain: staining to visualize bacterial
endospore
Gram staining
Acid-fast staining
Acid-fast Staining
Gram Staining
Endospore Staining