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PEWARNAAN GRAM 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 : 1. Pewarnaan dengan zat warna utama (kristal gentian violet yang warnanya violet). 2. Merekatkan (mengintensifkan) dengan suatu larutan mordant, yaitu larutan lugol (J-KJ). 3. Menambahkan zat decolorisasi (bahan peluntur) misalnya alkohol atau alkohol-asam. 4. 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. Larutan- larutan 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.

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Page 1: pewarnaan gram

PEWARNAAN GRAM

  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 :

1.    Pewarnaan dengan zat warna utama (kristal gentian violet yang warnanya violet).2.   Merekatkan (mengintensifkan) dengan suatu larutan mordant, yaitu larutan lugol (J-KJ).3.    Menambahkan zat decolorisasi (bahan peluntur) misalnya alkohol atau alkohol-asam. 4.   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. Larutan-larutan 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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Bubuk (kristal) fuchsin         : 5 gram.Alkohol 95-96%                   : 95 ml.

·        Larutan Pakai. Larutan standard fuchsin      : 10 ml. Aquadest                            : 90 ml.

Bubuk digerus dalam mortir dengan alkohol sedikit demi sedikit, setelah larut masukkan ke dalam botol (untuk gentian violet harus botol yang cokelat atau sawo matang). Genapkan volume alkohol sampai volume yang diperlukan. Biarkan 24 jam baru dapat dipakai. Jika hendak dipakai larutan stam ini harus diencerkan dahulu 10x dan disaring. Filtrat ini dipakai untuk pewarnaan.

·        Larutan Lugol (J-KJ).Jodium kristal                     : 1 gramKalium jodida (KJ)               : 2 gramAquadest                            : 300 ml.Mula-mula Jodium + KJ dalam kira-kira 10 ml aquadest. Sesudah jodium larut, genapkan volumenya menjadi 300 ml. Larutan lugol ini disimpan dalam botol yang sawo matang. Dapat juga kita sediakan larutan lugol dalam 5x atau 10x kuat. Pada waktu pemakaian encerkan dengan aquadest. Bila perlu disaring. Larutan lugol yang dibuat seperti di atas, langsung dipakai tanpa ditipiskan lagi.

A.    Cara pewarnaan Gram :

1. Buat sediaan pada objek gelas, keringkan, kemudian rekatkan (fiksasi) 3x di atas api Bunsen.

2. Tuangi dengan larutan karbol-gentian-violet (sesudah sediaan dingin), biarkan selama 5 menit.

3. Zat warna dibuang dan bubuhi dengan larutan mordant (lugol), diamkan selama kira-kira 1-3 menit.

4. Lugol dibuang dan preparat dicelupkan ke dalam alkohol 96%, sampai warna gentian violet lepas (sampai gentian violet tidak ada luntur lagi).

5. Cuci dengan air kran sampai bersih, kemudian bubuhi dengan cat-penutup (counter stain) larutan water-fuchsin, biarkan kira-kira 1-2 menit.

6. Cuci dengan air kran, keringkan dalam temperatur kamar, lihat dengan mikroskop memakai lensa rendam minyak. Gram positif  = ungu. Gram negatif = merah.

B.    VARIASI PEWARNAAN GRAM yang digunakan di Bagian Pathologi Klinik Fakultas Kedokteran dan Rumah Sakit Dr. Cipto Mangunkusumo di Jakarta.

Larutan-larutan zat warna :

1.  Carbol Gentian Violet.

Gentian violet 1 gram; Phenol kristal 2 gram; Alkohol 95 % 10 ml dan aquadest ad. 100 ml. Gentian violet digerus dengan alkohol dalam mortir tambahkan phenol dan campur. Kemudian tambah air sedikit demi sedikit dengan mengaduk terus, biarkan 24 jam kemudian disaring.

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2.  Larutan Lugol (J-KJ)

Jodium 1 gram, KJ 2 gram dan aquadest 300 ml. Geruslah Jodium bersama KJ hingga homogen, tambah air sedikit demi sedikit, biarkan 24 jam. Saring dan disimpan dalam botol yang cokelat. Larutan ini gunanya sebagai mordant.

3.  Larutan safranin (counter stain).

Safranin 1 gram, alkohol 95% sebanyak 4 ml dan aquadest 360 ml. Geruslah safranin dengan alkohol, tuangkan ke dalam botol, mortir berkali-kali dicuci dengan air, biarkan 24 jam. Saring dan siap untuk digunakan.

Cara pewarnaan :~     Buat sediaan pada objek gelas, rekatkan di atas api Bunsen 3x, tunggu sampai dingin.~     Pulas dengan karbol gentian violet selama 60 detik.~     Cuci dengan aquadest, bubuhi lugol selama 30 detik.~   Cuci dengan aquadest, buang zat warna dengan alkohol (decolorisasi) sampai tidak ada lagi

warna yang dilepaskan dari sediaan. Boleh juga dicuci lagi dengan aquadest.~     Pulas dengan larutan safranin (counter stain) kira-kira 30 detik.~      Cuci dengan aquadest, biarkan kering dalam temperatur kamar dan periksa dengan mikroskop

memakai lensa rendam minyak.Hasil pewarnaan Gram :Bakteri Gram positif   : Biru-ungu (ungu kebiru-biruan).Bakteri Gram negatif   : Merah kekuning-kuningan.

C.    Pewarnaan Gram modifikasi BURKE.(Burke’s modifikation : J. Bact. 7: 159, 1922).

1.    Sediaan sesudah difiksasi 3x di atas api, dituangi dengan larutan 1% gentian violet (crystal violet) dalam alkohol, kemudian segera ditambah dengan larutan Natrium-Bikarbonat 5%, biarkan selama kira-kira 1 menit.

2.   Cuci degan air, bubuhi dengan larutan mordant yaitu larutan lugol ( 1 gram J + 2 gram KJ + 200 ml aquadest), biarkan selama 1 menit.

3.   Segera dicuci dengan air. Bubuhi larutan decolorisasi tetes demi tetes (bahan peluntur) yang terdiri dari : 1 bagian ether + 3 bagian aceton, sampai zat warna utama terlepas atau bahan peluntur tidak berwarna lagi.

4.   Bersihkan dengan air, kemudian bubuhi dengan larutan counter stain (cat penutup) safranin 0,5 % beberapa detik lamanya.

5.   Bersihkan dengan air, keringkan pada temperatur kamar, lihat dengan mikroskop memakai lensa rendam minyak.Gram positif : biru-ungu.Gram negatif : merah kekuning-kuningan.

Catatan :Sebagai counter-stain dapat juga digunakan karbol fuchsin encer, yaitu Karbol Fuchsin Z. Neelsen 1 ml + 9 ml aquadest (pengenceran 10x). Jika karbol fuchsin ini dipakai waktunya

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

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Gram stainingFrom Wikipedia, the free encyclopediaJump to: navigation, search

A Gram stain of mixed Staphylococcus aureus (Gram positive cocci) and Escherichia coli (Gram negative bacilli), 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).

It is based on the chemical and physical properties of their cell walls. Primarily, it detects peptidoglycan, which is present in a thick layer in Gram positive bacteria.[1] A Gram positive results in a purple/blue color while a Gram negative results in a pink/red color.

The Gram stain is almost always the first step in the identification of a bacterial organism, and is the default stain performed by laboratories over a sample when no specific culture is referred.

While Gram staining is a valuable diagnostic tool in both clinical and research settings, not all bacteria can be definitively classified by this technique, thus forming Gram-variable and Gram-indeterminate groups as well.

The word Gram is always spelled with a capital, referring to Hans Christian Gram, the inventor of Gram staining.

Contents

1 History 2 Uses

o 2.1 Medical o 2.2 Staining mechanism

3 Examples o 3.1 Gram-positive bacteria o 3.2 Gram-negative bacteria o 3.3 Gram-indeterminate bacteria

4 See also

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5 References 6 External links

History

The method is named after its inventor, the Danish scientist Hans Christian Gram (1850–1938), who developed the technique while working with Carl Friedländer in the morgue of the city hospital in Berlin. 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, and included in his short report the observation that the Typhus bacillus did not retain the stain.[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] Gram staining is not used to classify archaea, formerly archaeabacteria, since these microorganisms yield widely varying responses that do not follow their phylogenetic groups.[6]

The Gram stain is not an infallible tool for diagnosis, identification, or phylogeny, and it is of extremely limited use in environmental microbiology. It has been largely superseded by molecular techniques even in the medical microbiology lab. Some organisms are Gram-variable (that means, they may stain either negative or positive); some organisms are not susceptible to either stain used by the Gram technique. In a modern environmental or molecular microbiology lab, most identification is done using genetic sequences and other molecular techniques, which are far more specific and informative than differential staining.

Gram-staining has proven to be as effective a diagnostic tool as PCR, particularly with regards to gonorrhoea diagnosis in Kuwait.The similarity of the results of both Gram stain and PCR for diagnosis of gonorrhea was 99.4% in Kuwait.[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. Gram stains yield results much more quickly than culture, and is especially important when infection would make an important difference in the patient's treatment and prognosis; examples are cerebrospinal fluid for meningitis and synovial fluid for septic arthritis.[4][8] .

Staining mechanism

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Gram-positive bacteria have a thick mesh-like cell wall made of peptidoglycan (50-90% of cell envelope), which are stained purple by crystal violet, whereas Gram-negative bacteria have a thinner layer (10% of cell envelope), which are stained pink by the counter-stain. 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.

The addition of a mordant, which binds to crystal violet and traps it in the cell (Gram's iodine)

Rapid decolorization with alcohol or acetone, and Counterstaining with safranin.[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.[10]

Crystal violet (CV) dissociates in aqueous solutions into CV+ and chloride (Cl−) ions. These ions penetrate through the cell wall and cell membrane of both Gram-positive and Gram-negative cells. The CV+ ion interacts with negatively charged components of bacterial cells and stains the cells purple.

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. Iodine is often referred to as a mordant, but is a trapping agent that prevents the removal of the CV–I complex and, therefore, color the cell.[11]

When a decolorizer such as alcohol or acetone is added, it interacts with the lipids of the cell membrane. A Gram-negative cell will lose its outer lipopolysaccharide membrane, and the inner peptidoglycan layer is left exposed. The CV–I complexes are washed from the Gram-negative cell along with the outer membrane. In contrast, a Gram-positive cell becomes dehydrated from an ethanol treatment. The large CV–I complexes become trapped within the Gram-positive cell due to the multilayered nature of its peptidoglycan. The decolorization step is critical and must be timed correctly; 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).

After decolorization, the Gram-positive cell remains purple and the Gram-negative cell loses its purple color. Counterstain, which is usually positively charged safranin or basic fuchsin, is applied last to give decolorized Gram-negative bacteria a pink or red color.[12][13]

Some bacteria, after staining with the Gram stain, yield a Gram-variable pattern: a mix of pink and purple cells are seen. The genera Actinomyces, Arthobacter, Corynebacterium, Mycobacterium, and Propionibacterium have cell walls particularly sensitive to breakage during cell division, resulting in Gram-negative staining of these Gram-positive cells. In cultures of Bacillus, Butyrivibrio, and Clostridium, a decrease in peptidoglycan thickness during growth coincides with an increase in the number of cells that stain Gram-negative.[14] In addition, in all bacteria stained using the Gram stain, the age of the culture may influence the results of the stain.

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Examples

Gram-positive bacteria

Main article: Gram-positive bacteria

Gram-positive bacteria have generally a single membrane (monoderm) surrounded by a thick peptidoglycan. This rule is followed by two phyla — Firmicutes (except for the classes Mollicutes and Negativicutes) and the Actinobacteria.[5][15] In contrast, members of the Chloroflexi (green non-sulfur bacteria) are monoderms but possess a thin or absent (class Dehalococcoidetes) peptidoglycan and can stain negative, positive or indeterminate.[5][15] Members of the Deinococcus-Thermus group, stain positive but are diderms with a thick peptidoglycan.[5][15]

Historically, the Gram-positive forms made up the phylum Firmicutes, a name now used for the largest group. It includes many well-known genera such as Bacillus, Listeria, Staphylococcus, Streptococcus, Enterococcus, and Clostridium. It has also been expanded to include the Mollicutes, bacteria like Mycoplasma that lack cell walls and so cannot be stained by Gram, but are derived from such forms.

Gram-negative bacteria

Main article: Gram-negative bacteria

Gram-negative bacteria generally possess a thin layer of peptidoglycan between two membranes (diderms). Most bacterial phyla are Gram-negative, including the cyanobacteria, spirochaetes and green sulfur bacteria, and most Proteobacteria (exceptions being some members of the Rickettsiales and the insect-endosymbionts of the Enterobacteriales).[5][15]

Gram-indeterminate bacteria

Gram-indeterminate bacteria do not respond to Gram staining and, therefore, cannot be determined as either Gram-positive or Gram-negative. Examples of them, but not limited to, are Gram-variable and acid fast bacteria.