6 PEWARNAAN BAKTERI

17/03/2016BACTERIAL STAINING RENITA YULIANA Kenapa bakteri harus diwarnai? STAINING? Cara mewarnainya ? 1 sehingga sulit untuk dilihat dengan mikroskop tanpa pewarnaan/ pengecatan  1 pengecatan dapat menentukan/ membedakan  morfologi sel.17/03/2016 PEWARNAAN BAKTERI  Key  Magnification (perbesaran) & Resolution Contrast  Untuk melihat bentuk sel bakteri  (warna) spesimen harus kontras dengan latar belakang dari lapang pandang mikroskop   sitoplasma pada dasarnya transparan. ukuran. struktur dan sifat bakteri 2 . dan susunan PEWARNAAN 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  Prinsip : zat warna akan bergabung secara kimiawi dengan protoplasma bakteri  Fungsi : mengamati morfologi. 17/03/2016 STAIN CATEGORIES Morphological – size. dan susunan sel bakteri Prinsip: Surface of most bacterial cell Hanya menggunakan 1 macam cat pewarna  Methylene Blue  Safranin  Crystal violet 3 . ukuran. 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. 5. 2.   6. 3.   4.   Clean slide LABEL Smear in circle Broth Solid + H20 Air dry first Heat fix (usually) Kill organism Adhere to slide Problems Too thick Wash off specimen 4 .17/03/2016 Slide Preparation 1. sedangkan sel tidak akan terwarnai NEGATIVE STAIN  Prinsip: Chromogen  acid  membawa muatan negatif Muatan negatif di permukaan sel bakteri menolak muatan negatif chromogen  sel tidak terwarnai. background yang terwarnai 5 . 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).17/03/2016 NEGATIVE STAIN  Teknik untuk menentukan morfologi dan sususan sel bakteri yang tidak tahan panas (fiksasi). congo red  Prinsip : pewarna asam akan mewarnai latar belakang. 17/03/2016 6 . 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 7 .17/03/2016 GRAM STAIN  Untuk membedakan antara bakteri Gram positif dan Gram negatif  Untuk menentukan morfologi sel. 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 8 .17/03/2016 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. 17/03/2016 Time Frame 1) 1 minute 2) 1 minute 3) 15 seconds 4) 1 minute 9 . 17/03/2016 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 10 . 17/03/2016 ACID FAST STAIN ZN Method:  Phenolic compound  carbolfuchsin (as the primary stain  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 11 . 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 12 .17/03/2016 ACID FAST STAIN Other Staining  Fluorescent dyes. 17/03/2016 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 dan memudahkan masuknya zat warna. impermeabilitas dari spora akan melindungi zat yang mewarnai spora dari perlakuan pelunturan atau penambahan zat pewarna sel vegetatif 13 . 17/03/2016 ENDOSPORE STAIN Schaeffer Fulton method  Primary stain  malachite 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) 14 . 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 15 .17/03/2016 CAPSULE STAIN Purpose:  To detect cells capable of producing an extracelluar capsule  Capsule production increase virulence in some bacteria. 17/03/2016 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 STAIN Metode 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 16 . 17/03/2016 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. sehingga tampak lebih besar dan terwarna dengan karbolfuhksin 17 . Koloidal garam asam tanat menyebabkan presipitat pada dinding sel dan flagel. Preparat digenangi dengan larutan Mordant dan didiamkan selama 10 menit 2. Objek glass dikeringkan di incubator pada suhu 560C Pengecatan 1. Objek glass dimiringkan hingga tetesan tadi mengalir ke ujung yang lain 4. Periksa dibawah mikroskop dengan perbesaran 1000X dengan imersi oil FLAGELL STAIN Fungsi Mordant  mengintensifkan pengecatan dengan memperbesar bentuk dan diameter flagell 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% 18 . Preparat dicuci dengan air mengalir 3. Preparat digenangi dengan cat Carbol Fuchsin selama 5 menit 4. Sebanyak 1 tetes sampel bakteri diteteskan di tepi objek glass menggunakan pipet tetes steril 3. Objek glass dibersihkan dan disterilkan dengan melewatkan di atas api sebanyak 3x 2. Preparat dicuci dengan air mengalir 5.17/03/2016 FLAGELL STAIN Pembuatan Preparat 1. 17/03/2016 TERIMAKASIH 19 .