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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?
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

Isolation of pure cultures involves:


Separating samples of microorganisms into

individual cells
Allowing the individual cells to reproduce
and form clones of single microorganism

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

Sample
s
Enrichme
nt

Diluti
on
Plattin
g

Streak
Plate

Spread
Plate

Maintenanc
e

Pour
Plate

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)

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

5. Sterilisasi dan disinfeksi

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

Desinfection
Desinfectan
Antisepsis
Antiseptic
Sanitation

Metode Sterilisasi
Metode Fisikawi:
Mekanis : filtrasi
Pemanasan:
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

Sterilization is a process to eliminate


contaminant microorganisms
Chemical sterilization (alcohol, xylol, H 2O2
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:

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:
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


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

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:
Simple staining (positive staining, negative

staining)
Differential staining (Gram staining, acidfast 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

Multiple staining reactions are employed


Differentiate types of cells or cells 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

Endospore Staining

Gram
Staining