6 PEWARNAAN BAKTERI

8/16/2019 6 PEWARNAAN BAKTERI

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BACTERIAL STAINING

RENITA YULIANA

STAINING?Kenapa

bakteri harus

diwarnai?

Cara

mewarnainya

?


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PEWARNAAN BAKTERI

KeyMagnification (perbesaran) & Resolution

Contrast

Untuk melihat bentuk sel bakteri (warna)

spesimen harus kontras dengan latar belakang

dari lapang pandang mikroskop

sitoplasma pada dasarnya transparan,

sehingga sulit untuk dilihat dengan mikroskop

tanpa pewarnaan/ pengecatan

1 pengecatan dapat menentukan/ membedakanmorfologi sel, ukuran, dan susunan

PEWARNAAN BAKTERI

Prinsip : zat warna akan bergabung secara

kimiawi dengan protoplasma bakteri

Fungsi : mengamati morfologi, struktur dan sifat

bakteri

Cat pewarna stains) larutan yang berisi

pelarut (akuades atau ethanol) dan molekul

pewarna (biasanya derivat dari benzena) yang

disebut Chromogen

Bagian chromogen yang dapat mewarnai sel di

sebut Chromophore

Auxochrome bagian chromogen bermuatan


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STAIN CATEGORIES

Morphological – size, shape, arrangement

• Simple stain

• Negative stain

Differential – cell wall composition

• Gram stain

• Acid-fast stain

Structural – cell structures

• Endospore stain• Capsule stain

• Flagell stain

• Mycoplasma

SIMPLE STAIN

Tujuan Untuk membedakan bentuk, ukuran, dansusunan sel bakteri

Prinsip:

Surface of mostbacterial cell

Hanya menggunakan 1 macam cat pewarna

Methylene Blue

Safranin

Crystal violet


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NEGATIVE STAIN

Teknik untuk menentukan morfologi dansususan sel bakteri yang tidak tahan panas

(fiksasi), ex : Treponema

Dipakai untuk mengamati bakteri yang sukar

terwarnai secara langsung

Dipakai untuk menentukan ukuran sel teknik

ini dapat meminimalkan penyusutan sel

Menggunakan zat warna nigrosin (tinta cina),congo red

Prinsip : pewarna asam akan mewarnai latar

belakang, sedangkan sel tidak akan terwarnai

NEGATIVE STAIN Prinsip:

Chromogen acidmembawa muatan negatif

Muatan negatif di permukaan sel bakteri menolak

muatan negatif chromogen sel tidak terwarnai,

background yang terwarnai


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GRAM STAIN

Untuk membedakan antara bakteri Gram positifdan Gram negatif

Untuk menentukan morfologi sel, susunan sel,

dan ukuran sel bakteri

Dapat digunakan sebagai dugaan awal dalam

suatu identifikasi

Primary stain crystal violet

Mordant iodine Decolorizer Alkohol/ acetone

Counter stain safranin


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GRAM STAIN

Dasar Pembeda struktur dinding sel

GRAM STAIN Gram negative cell walls have a higher lipid content and

a thinner peptidoglycan layer than Gram positive cell

walls the decolorizer extracts the lipid, making the

Gram negative wall more porous and incapable of

retaining the crystal violet-iodine complex

THE DECOLORIZATION step is the MOST CRUCIAL and

most likely source of Gram stain inconsistency

Age of the culture also affects gram stain consistency Older cultures may lose their ability to resist

decolorization and give artifactual Gram negative result

Cultures 24 hours old or less are the best for this

procedure


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Time Frame

1) 1 minute

2) 1 minute

3) 15 seconds4) 1 minute


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ACID FAST STAIN

Purpose:

To identification of acid-fast bacili Preliminary diagnosis of tbc

Principle

The presence of mycolic acids in the cell walls of acid

fast organisms in the cytological basis for this

differential stain

Mycolic acid is waxy substance that gives acid fast cells

a higher affinity for the primary stain and resistance to

decolorization by an acid alcohol solution

Method:

Ziehl-Neelsen (ZN) method

Kinyoun (K) method

Beda:

ZN menggunakan pemanasan

K cold stain


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ACID FAST STAIN

ZN Method:

Phenolic compound carbolfuchsin (as the primarystain lipid soluble and penetrates the waxy cell wall)

Staining by carbolfuchsin is further enhanched by steam

heating the preparation to melt the wax and allow the

stain to move into the cell Decolorizer acid alcohol (decolorize non acid fast

cells) Counterstain methylene blue

K-Method: Same with ZN methode, but without the use of heat

Less sensitive than ZN method Counterstain brilliant green or methylene blue


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ACID FAST STAIN

Other Staining Fluorescent dyes, such as auramine or rhodamine

Actually preferable to traditional carbolfuchsin stains

for examination of direct smears because of their

higher sensitivity

The fluorochrome combines specifically with mycolic

acid

Acid alcohol is used for decolorization

Potassium permanganate is the counterstain

When observed under the microscope with UV

illuminations, acid-fast cells are yellow against a dark

background and nonacid fast cells are not seen


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ENDOSPORE STAIN

Purpose:

To detect the presence and location of spores in

bacterial cells (ex: Bacillus & Clostridium )

Principle

Spore are resistant to heat and chemicals because of

tough outer covering made of the protein keratin

proses pemanasan akan membuka lapisan spora danmemudahkan masuknya zat warna, impermeabilitas dari

spora akan melindungi zat yang mewarnai spora dari

perlakuan pelunturan atau penambahan zat pewarna sel

vegetatif


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ENDOSPORE STAIN

Schaeffer Fulton method Primary stainmalachite green is forced into the spore

by steaming the bacterial emulsion (alternatif biarkan

sampel terendam MG selama 15 menit atau lebih)

MG is water soluble and has a low affinity for cellular

material, so vegetative cell can be decolorized with

water and counter stain (safranin)


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CAPSULE STAIN

Purpose:

To detect cells capable of producing an extracelluar

capsule

Capsule production increase virulence in some

bacteria, by making them less vulnerable to

phagocytosis

Principle

Capsules composed of mucoid polysaccharides or

polypeptides

The capsule stain technique takes advantage of this

phenomenon by staining around the cells


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CAPSULE STAIN An acidic stain such as congo red or nigrosin that stain

the background Basic stain that colorizes the cell

The capsule remains unstained and appears as a white

halo between the cells and the colored background

CAPSULE STAINMetode pengecatan kapsul

• Metode Burry

Cat yg dipakai Safranin / Methylene Blue

• Metode Stitt Hiss

Cat yg dipakai Basic Fuchsin dgn Alkohol

• Metode Welch

Cat yg dipakai Carbol Fuchsin dgn NaCl• Metode Anthony

Kristal Violet dgn CuSO4


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CAPSULE STAIN (metode burry)

FLAGELL STAIN

Purpose:

The flagella stain allows direct observation of flagella

Presense and arrangement of flagella may be useful in

identifying bacterial species

Principle:

mordant akan meningkatkan afinitas flagel untuk

menyerap zat warna. Koloidal garam asam tanat menyebabkan presipitat pada dinding sel dan flagel,

sehingga tampak lebih besar dan terwarna dengan

karbolfuhksin


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Pembuatan Preparat

1. Objek glass dibersihkan

dan disterilkan dengan

melewatkan di atas api

sebanyak 3x

2. Sebanyak 1 tetes sampel

bakteri diteteskan di tepi

objek glass menggunakan

pipet tetes steril

3. Objek glass dimiringkan

hingga tetesan tadimengalir ke ujung yang lain

4. Objek glass dikeringkan diincubator pada suhu 560C

FLAGELL STAIN

Pengecatan

1. Preparat digenangi dengan

larutan Mordant dan

didiamkan selama 10 menit

2. Preparat dicuci dengan air

mengalir

3. Preparat digenangi dengan

cat Carbol Fuchsin selama 5

menit

4. Preparat dicuci dengan air

mengalir 5. Periksa dibawah mikroskop

dengan perbesaran 1000X

dengan imersi oil

FLAGELL STAIN

Mordant yang terdiri dari :

5 cc Larutan jenuh Kalium aluin dalam

aquadest 2 cc Larutan Asam Tanin 20% dalam aquadest

2 cc Larutan HgCl2 jenuh dalam aquadest

0,4 cc Larutan Basich Fuchsin jenuh dalam

Alkohol 96%

Fungsi Mordantmengintensifkan pengecatan

dengan memperbesar bentuk dan diameter flagell


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TERIMAKASIH

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6 PEWARNAAN BAKTERI