PEWARNAAN GRAM

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Pewarnaan Gram ini pertama kali dikembangkan oleh seorang ahli histologik Christian Gram (1884). Dengan pewarnaan Gram, bakteri-bakteri dapat dibagi atas 2 golongan yaitu Gram positif dan Gram negatif. Gram positif warnanya violet (ungu) karena mengikat zat warna utama ―kristal violet‖. Sedangkan Gram negatif berwarna merah jambu karena melepaskan zat warna utama dan menangkap zat warna penutup ‖fuchsin‖. Prinsip atau pokok-pokok pewarnaan Gram meliputi 4 tingkatan yaitu : Pewarnaan dengan zat warna utama (kristal gentian violet yang warnanya violet). Merekatkan (mengintensifkan) dengan suatu larutan mordant, yaitu larutan lugol (J-KJ). Menambahkan zat decolorisasi (bahan peluntur) misalnya alkohol atau alkohol-asam. Pemberian zat penutup (counter stain), misalnya : larutan fuchsin, safranin, dll.

Pulasan menurut Gram mempunyai banyak modifikasi, sebaiknya pakailah salah satu cara saja diantara yang banyak. Kesalahan biasanya terdapat pada ‖overstaining‖ dan ‖overdecolozing‖, yaitu terlalu lama memberikan zat-zat warna atau pancucian dengan alkohol. Akibatnya Gram-positif dapat menjadi Gram negatif. Teknik mewarnai hendaknya dikontrol juga dengan melakukan pemulasan terhadap bakteri yang telah diketahui Gramnya. Larutanlarutan zat warna yang digunakan senantiasa diperiksa, apakah sudah terdapat kristal-kristal atau kotoran-kotoran lainnya. Gunakanlah selalu larutan-larutan zat warna yang disaring dengan kertas saring. Perlu kita ketahui bahwa perbedaan sifat antara kedua golongan bakteri tadi, tidaklah absolut tegas dan spesifik, melainkan tergantung juga pada beberapa faktor, antara lain: a) Bakteri-bakteri Gram positif sering kali tidak dapat menyerap dan mengikat zat warna kristal violet, terutama apabila dibuat preparat dari bakteri-bakteri biakan murni yang telah tua (rough). b) Ada bakteri-bakteri tertentu yang sangat peka terhadap cara-cara yang mengalami sedikit perubahan. c) Selain daripada itu ada juga bakteri-bakteri yang bersifat ‖gram variable‖, dll. Gentian violet dapat diganti dengan crystalviolet atau methylviolet, jika gentian violet tidak ada. · Larutan Standard Gentian Violet. Bubuk (kristal) gentian violet : 5 gram. Alkohol 95-96% : 95 ml. Larutan Pakai. Larutan standard gentian violet : 10 ml. Karbol (phenol) 5% : 90 ml. Larutan Standard Fuchsin.

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Cipto Mangunkusumo di Jakarta. Gentian violet 1 gram. Lugol dibuang dan preparat dicelupkan ke dalam alkohol 96%. langsung dipakai tanpa ditipiskan lagi. · A. Dapat juga kita sediakan larutan lugol dalam 5x atau 10x kuat. Alkohol 95-96% : 95 ml. keringkan dalam temperatur kamar. biarkan 24 jam kemudian disaring. Larutan Pakai. 4. Bubuk digerus dalam mortir dengan alkohol sedikit demi sedikit. 5. biarkan kira-kira 1-2 menit. biarkan selama 5 menit. Buat sediaan pada objek gelas. Phenol kristal 2 gram. . diamkan selama kira-kira 1-3 menit. lihat dengan mikroskop memakai lensa rendam minyak. kemudian rekatkan (fiksasi) 3x di atas api Bunsen. B. Larutan-larutan zat warna : 1. 2. Gram positif = ungu. Larutan standard fuchsin : 10 ml. Gentian violet digerus dengan alkohol dalam mortir tambahkan phenol dan campur. 3. Larutan Lugol (J-KJ). Aquadest : 90 ml. Bila perlu disaring. Zat warna dibuang dan bubuhi dengan larutan mordant (lugol). Kemudian tambah air sedikit demi sedikit dengan mengaduk terus. Mula-mula Jodium + KJ dalam kira-kira 10 ml aquadest. Genapkan volume alkohol sampai volume yang diperlukan. Pada waktu pemakaian encerkan dengan aquadest. Filtrat ini dipakai untuk pewarnaan. kemudian bubuhi dengan cat-penutup (counter stain) larutan water-fuchsin. sampai warna gentian violet lepas (sampai gentian violet tidak ada luntur lagi). Cuci dengan air kran. Jodium kristal : 1 gram Kalium jodida (KJ) : 2 gram Aquadest : 300 ml. genapkan volumenya menjadi 300 ml. Sesudah jodium larut. Carbol Gentian Violet. Biarkan 24 jam baru dapat dipakai. Tuangi dengan larutan karbol-gentian-violet (sesudah sediaan dingin).· Bubuk (kristal) fuchsin : 5 gram. Jika hendak dipakai larutan stam ini harus diencerkan dahulu 10x dan disaring. VARIASI PEWARNAAN GRAM yang digunakan di Bagian Pathologi Klinik Fakultas Kedokteran dan Rumah Sakit Dr. setelah larut masukkan ke dalam botol (untuk gentian violet harus botol yang cokelat atau sawo matang). 100 ml. Cuci dengan air kran sampai bersih. 6. Larutan lugol ini disimpan dalam botol yang sawo matang. Gram negatif = merah. Cara pewarnaan Gram : 1. Larutan lugol yang dibuat seperti di atas. Alkohol 95 % 10 ml dan aquadest ad. keringkan.

Larutan ini gunanya sebagai mordant. biarkan kering dalam temperatur kamar dan periksa dengan mikroskop memakai lensa rendam minyak. biarkan selama 1 menit. Sediaan sesudah difiksasi 3x di atas api. kemudian segera ditambah dengan larutan Natrium-Bikarbonat 5%. Bersihkan dengan air. Bubuhi larutan decolorisasi tetes demi tetes (bahan peluntur) yang terdiri dari : 1 bagian ether + 3 bagian aceton. Gram positif : biru-ungu. Bersihkan dengan air. Larutan Lugol (J-KJ) Jodium 1 gram. biarkan 24 jam. ~ ~ ~ ~ ~ ~ Cara pewarnaan : Buat sediaan pada objek gelas. dituangi dengan larutan 1% gentian violet (crystal violet) dalam alkohol. Bact. yaitu Karbol Fuchsin Z. Boleh juga dicuci lagi dengan aquadest. Safranin 1 gram. Pewarnaan Gram modifikasi BURKE. Geruslah safranin dengan alkohol. 3. Hasil pewarnaan Gram : Bakteri Gram positif : Biru-ungu (ungu kebiru-biruan). Larutan safranin (counter stain).5 % beberapa detik lamanya. 2. Catatan : Sebagai counter-stain dapat juga digunakan karbol fuchsin encer. Cuci dengan aquadest. Segera dicuci dengan air. lihat dengan mikroskop memakai lensa rendam minyak.2. keringkan pada temperatur kamar. Neelsen 1 ml + 9 ml aquadest (pengenceran 10x). mortir berkali-kali dicuci dengan air. biarkan 24 jam. . bubuhi dengan larutan mordant yaitu larutan lugol ( 1 gram J + 2 gram KJ + 200 ml aquadest). Cuci dengan aquadest. Cuci dengan aquadest. tambah air sedikit demi sedikit. alkohol 95% sebanyak 4 ml dan aquadest 360 ml. (Burke’s modifikation : J. Saring dan disimpan dalam botol yang cokelat. 4. Jika karbol fuchsin ini dipakai waktunya C. buang zat warna dengan alkohol (decolorisasi) sampai tidak ada lagi warna yang dilepaskan dari sediaan. bubuhi lugol selama 30 detik. 7: 159. Geruslah Jodium bersama KJ hingga homogen. kemudian bubuhi dengan larutan counter stain (cat penutup) safranin 0. rekatkan di atas api Bunsen 3x. 1922). Saring dan siap untuk digunakan. 1. Cuci degan air. Bakteri Gram negatif : Merah kekuning-kuningan. 5. biarkan selama kira-kira 1 menit. tunggu sampai dingin. sampai zat warna utama terlepas atau bahan peluntur tidak berwarna lagi. Pulas dengan larutan safranin (counter stain) kira-kira 30 detik. KJ 2 gram dan aquadest 300 ml. tuangkan ke dalam botol. Gram negatif : merah kekuning-kuningan. 3. Pulas dengan karbol gentian violet selama 60 detik.

dipersingkat. Karbol fuchsin encer ini sering digunakan untuk memulas vibrio. Susunan pulasan karbol fuchsin lihat pada pewarnaan tahan asam. . paling lama 1 menit. Vibrio berwarna merah jambu dan bentuk koma. waktunya kira-kira 5-10 detik.

not all bacteria can be definitively classified by this technique. Contents     1 History 2 Uses o 2. search A Gram stain of mixed Staphylococcus aureus (Gram positive cocci) and Escherichia coli (Gram negative bacilli). The Gram stain is almost always the first step in the identification of a bacterial organism. The word Gram is always spelled with a capital.Gram staining From Wikipedia. the inventor of Gram staining.[1] A Gram positive results in a purple/blue color while a Gram negative results in a pink/red color. which is present in a thick layer in Gram positive bacteria. the free encyclopedia Jump to: navigation. It is based on the chemical and physical properties of their cell walls. While Gram staining is a valuable diagnostic tool in both clinical and research settings.2 Staining mechanism 3 Examples o 3.1 Medical o 2. referring to Hans Christian Gram. the most common Gram stain reference bacteria Gram staining (or Gram's method) is a method of differentiating bacterial species into two large groups (Gram-positive and Gram-negative). Primarily. thus forming Gram-variable and Gramindeterminate groups as well. it detects peptidoglycan.1 Gram-positive bacteria o 3.2 Gram-negative bacteria o 3. and is the default stain performed by laboratories over a sample when no specific culture is referred.3 Gram-indeterminate bacteria 4 See also .

and is especially important when infection would make an important difference in the patient's treatment and prognosis. which are far more specific and informative than differential staining. some organisms are not susceptible to either stain used by the Gram technique. Staining mechanism .[4][8] .[7] Medical See also: Gram-negative bacterial infection and Gram-positive bacterial infection Gram stains are performed on body fluid or biopsy when infection is suspected.[6] The Gram stain is not an infallible tool for diagnosis. It has been largely superseded by molecular techniques even in the medical microbiology lab. or phylogeny. since these microorganisms yield widely varying responses that do not follow their phylogenetic groups. examples are cerebrospinal fluid for meningitis and synovial fluid for septic arthritis.The similarity of the results of both Gram stain and PCR for diagnosis of gonorrhea was 99. formerly archaeabacteria. particularly with regards to gonorrhoea diagnosis in Kuwait. In a modern environmental or molecular microbiology lab.[3] Uses Gram staining is a bacteriological laboratory technique[4] used to differentiate bacterial species into two large groups (Gram-positive and Gram-negative) based on the physical properties of their cell walls.  5 References 6 External links History The method is named after its inventor. they may stain either negative or positive). Gram devised his technique not for the purpose of distinguishing one type of bacterium from another but to enable bacteria to be seen more readily in stained sections of lung tissue.[2] He published his method in 1884. Gram stains yield results much more quickly than culture. most identification is done using genetic sequences and other molecular techniques. and it is of extremely limited use in environmental microbiology. who developed the technique while working with Carl Friedländer in the morgue of the city hospital in Berlin. Gram-staining has proven to be as effective a diagnostic tool as PCR. Some organisms are Gram-variable (that means. and included in his short report the observation that the Typhus bacillus did not retain the stain. identification.[5] Gram staining is not used to classify archaea.4% in Kuwait. the Danish scientist Hans Christian Gram (1850–1938).

the crystal violet stain will be removed from both Gram-positive and negative cells if the decolorizing agent is left on too long (a matter of seconds). resulting in Gram-negative staining of these Gram-positive cells.Gram-positive bacteria have a thick mesh-like cell wall made of peptidoglycan (50-90% of cell envelope).[10] Crystal violet (CV) dissociates in aqueous solutions into CV+ and chloride (Cl−) ions. the age of the culture may influence the results of the stain. Iodine (I− or I− + 3) interacts with CV and forms large complexes of crystal violet and iodine (CV–I) within the inner and outer layers of the cell. the Gram-positive cell remains purple and the Gram-negative cell loses its purple color. . There are four basic steps of the Gram stain:     Applying a primary stain (crystal violet) to a heat-fixed smear of a bacterial culture. Heat fixing kills some bacteria but is mostly used to affix the bacteria to the slide so that they don't rinse out during the staining procedure. which is usually positively charged safranin or basic fuchsin. is applied last to give decolorized Gram-negative bacteria a pink or red color. which are stained pink by the counter-stain. after staining with the Gram stain. Iodine is often referred to as a mordant. a Gram-positive cell becomes dehydrated from an ethanol treatment. color the cell. but is a trapping agent that prevents the removal of the CV–I complex and.[14] In addition. The large CV–I complexes become trapped within the Gram-positive cell due to the multilayered nature of its peptidoglycan. and Propionibacterium have cell walls particularly sensitive to breakage during cell division. The CV+ ion interacts with negatively charged components of bacterial cells and stains the cells purple. in all bacteria stained using the Gram stain. yield a Gram-variable pattern: a mix of pink and purple cells are seen. Butyrivibrio. Arthobacter. a decrease in peptidoglycan thickness during growth coincides with an increase in the number of cells that stain Gram-negative. In contrast. The genera Actinomyces.[11] When a decolorizer such as alcohol or acetone is added. Corynebacterium. The addition of a mordant. which are stained purple by crystal violet.[9] Carbol fuchsin is sometimes substituted for safranin since it will more intensely stain anaerobic bacteria but it is much less commonly employed as a counterstain. Counterstain. After decolorization. and Clostridium.[12][13] Some bacteria. and the inner peptidoglycan layer is left exposed. These ions penetrate through the cell wall and cell membrane of both Gram-positive and Gram-negative cells. In cultures of Bacillus. The CV–I complexes are washed from the Gram-negative cell along with the outer membrane. Mycobacterium. A Gram-negative cell will lose its outer lipopolysaccharide membrane. therefore. which binds to crystal violet and traps it in the cell (Gram's iodine) Rapid decolorization with alcohol or acetone. it interacts with the lipids of the cell membrane. and Counterstaining with safranin. The decolorization step is critical and must be timed correctly. whereas Gram-negative bacteria have a thinner layer (10% of cell envelope).

It has also been expanded to include the Mollicutes. This rule is followed by two phyla — Firmicutes (except for the classes Mollicutes and Negativicutes) and the Actinobacteria. but are derived from such forms. positive or indeterminate. It includes many well-known genera such as Bacillus. . Enterococcus.[5][15] Historically. and Clostridium.[5][15] Members of the Deinococcus-Thermus group.Examples Gram-positive bacteria Main article: Gram-positive bacteria Gram-positive bacteria have generally a single membrane (monoderm) surrounded by a thick peptidoglycan. members of the Chloroflexi (green non-sulfur bacteria) are monoderms but possess a thin or absent (class Dehalococcoidetes) peptidoglycan and can stain negative.[5][15] Gram-indeterminate bacteria Gram-indeterminate bacteria do not respond to Gram staining and. spirochaetes and green sulfur bacteria. but not limited to. Streptococcus. cannot be determined as either Gram-positive or Gram-negative. the Gram-positive forms made up the phylum Firmicutes. Examples of them. and most Proteobacteria (exceptions being some members of the Rickettsiales and the insect-endosymbionts of the Enterobacteriales).[5][15] In contrast. Staphylococcus. are Gram-variable and acid fast bacteria. Most bacterial phyla are Gram-negative. Listeria. Gram-negative bacteria Main article: Gram-negative bacteria Gram-negative bacteria generally possess a thin layer of peptidoglycan between two membranes (diderms). a name now used for the largest group. therefore. including the cyanobacteria. stain positive but are diderms with a thick peptidoglycan. bacteria like Mycoplasma that lack cell walls and so cannot be stained by Gram.

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