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Microorganisms that exist in nature has a morphology, structure and unique properties, as well as bacteria.

Bacteria that live almost colorless and the contrast with water, in which bacterial cells are suspended. One way to observe the bacterial cell shape is so easy to identify with the method of painting or staining. It also serves to determine the nature of the physiological reactions that determine the bacterial cell wall through a series of coloring Microorganisms are hard to see with the light microscope, because it does not adsorb or refract light. The reason is what causes the dyes used to color or background microorganisms. The dye adsorbs and refract light so the contrast enhanced disekelilingya microorganisms. The use of dyes allowed the observation of cell structures such as spores and infectious materials containing starch and phosphate granules. Staining is used to look at one specific cell structures called coloring. While staining is used to differentiate the differential staining of microorganisms called bacteria differentiate into groups of gram positive and gram negative. Another is the differential staining Ziehl Neelsen who choose bacteria become resistant to acid groups and acid resistant (Dwidjoseputro.1998). Introduction of microbial forms (morphology), except for microalgae to be done first so that staining can be observed clearly (Hadiutomo. 1990). In general, bacteria are translucent, this is because a lot of the bacteria that has no dyes (Waluyo, 2004). The aim of staining is to facilitate the observation of bacterial cell shape, expand the size of jazad, observe the structure inside and outside the bacterial cell, and see the reaction to the dye jazad given that the physical or chemical properties of a substance can be determined (Hadiutomo. 1990). Staining method was first discovered by Christian Gram in 1884. With this method. Bacteria can be grouped into two yatu, gram-positive and gram-negative bacteria. Its based off of the reaction or the nature of the bacteria to the paint. Or the nature of the reaction is determined by the composition of the bacterial cell wall that does not bias gram staining done on microorganisms that do not have cell walls as Mycoplasma sp (Waluyo, 2004). Success or failure of a coloring is determined by the timing of the color and culture of the colored age (age of culture that is both 24 hours). Commonly used dyes are salts that are built by the ions that positively and negatively charged ions which one is colored. The dye grouped into two, namely the acidic dyes and bases. If the ion containing the color is a positive ion dye called dye bases. And if the color is ion-containing negative ions then dye called negative dye (Hadiutomo. 1990). The dye used in coloring is alkaline and acidic. In alkaline dye part role in providing color called called chromophores and have a positive charge. Conversely, the acid dyes instrumental sections provide dyes having negatively charge dye base more widely used because of the negative charge is found on the walls of cells, and cell membrane staining process sitoplasmasewaktu positive charge on the dye bases will be associated with a negative charge inside the cell, thus microorganisms more clearly visible (Dwidjoseputro.1998).

The dye is negatively charged acid is not normally used for coloring microorganisms, but is usually used to stain microorganisms, but is usually used to color the background coloring preparations. The dye is negatively charged acid can not be associated with a negative charge contained in the cell structure. Sometimes negative dyes used to color the cell which is positively charged, it should be noted that the charge and power tie dye to the cell structure may change depending on the pH of the surrounding area during the dyeing process (Dwidjoseputro.1998). Staining procedure that produces staining microorganisms called positive staining in staining procedure can be used dyes are positively charged alkali and acid dyes are negatively charged. In contrast to the negative staining of microorganisms around the colored background to enhance contrast with colorless microorganisms. Staining includes preparation of microorganisms by preparations pillowcase (Dwidjoseputro.1998) Before coloring made reviews of bacteria on the glass object. This review then fixed. The number of bacteria contained in the review should be enough so that it can be seen as forms and penataanya observed. The mistake often made is to use bacterial suspension is too dense, especially when the suspension comes adari not solid media. In contrast to a bacterial suspension if too thin, you will get trouble when looking bacterial preparations (Sutedjo.1991). To observe the morphology of cells staining microorganisms so often after making preparations pillowcase made fixation followed by staining. Fixation can be done by passing over the fire or soaking preparations with methanol. Fixation is used for: 1. Observing bacteria because bacteria cells more clearly visible after the colored 2. Bacteria attach to the object glass 3. Killed bacteria In simple staining is only one kind of dye is used to enhance the contrast between microorganisms and their surroundings. Commonly, this staining procedure using alkaline dyes such as crystal violet, methylene blue, basic fuchsin carbolic acid, safranin or green malakit. Sometimes negative dyes used for coloring is simple: acid dyes are frequently used nigrosin and red kongo (Lay.1994). Staining procedure is simple, easy and fast, so the coloring is often used to look at the shape the size and arrangement of the bacteria in the bacteria known mikoorganisme bentu round (coccus), rod (bacillus), and spiral. With a simple staining of bacteria can also be seen arrangement. In coccus can be seen coloring like chain (stertococcus), grapes (stafilococcus), pairs (diplococcus), cube shape consisting of 4 or 8 (saranae) (Lay.1994). Some microbes difficult colored with dyes that are alkaline, but easily visible with negative staining, the method of microbial mixed with Indian ink or nigrosin, then rubbed on glass objek.Zat color will not stain the bacteria, but bacteria color the surrounding environment. With microbes microscope will appear colorless with black background (Lay.1994).

Staining method was first discovered by a biotechnology expert from Denmark named Christian Gram in 1884. Finding the staining method by accident. With this method. Bacteria can be grouped into two yatu, gram-positive and gram-negative bacteria. Its based off of the reaction or the nature of the bacteria to the paint. Or the nature of the reaction is determined by the composition of the bacterial cell wall so that the painting can not be done on a gram microorganisms that do not have cell walls as Mycoplasma sp. Gram stain is a differential staining is very useful and most widely used in the microbiology laboratory. Staining is an important step in the characterization and identification of bacteria (Lay, 1994) Gram stain gives good results, when used fresh culture aged 24-48 hours. When used old culture, there is the possibility of storage of gram staining results. In old cultures, many cells were damaged on the walls of his cell. Damage to the walls of these cells causes the dye to come out when washed with lartan pemucat. This means that gram-positive bacteria with damaged cell walls can no longer be preserved so it looks as crystal violet gram-negative bacteria (Lay, 1994) The characteristics of gram negative characteristic: - The structure of the cell wall thin, about 10-45mm, three-or multi-layer coated - The walls of his cell contained more fat (11-22%), peptidoglycan layer found in rigid, next to the small amount of 10% of the dry weight, not lactic acid. - Less susceptible to penicillin compounds. - Not resistant to physical disturbance The characteristics of gram-positive bacteria: - Thick walls - The walls of his cell containing a normal lipid - Equity more susceptible to penicillin compounds - Growth is inhibited significantly by dyes such as crystal violet - Composition required more complex - More resistant to physical disturbance. Painting gram carried out in 4 stages: a. Provision of primary colored paint (liquid crystal violet) color purple b. Pengintensifan paint color with the addition of a solution of Mordant c. Laundering (dekolarisasi) with acid alcohol solution d. Giving your opponent the paint color paint safranin Many organic seenyawa color (dyes) used to color microorganisms for microscopic examination and gram staining procedures have been developed for: - Observing the morphology of microorganisms both in rough - Identify the parts of structural cells of microorganisms - Help identify or distinguish similar organisms

Tools and Materials Tools - Microscope - Needle ose - Grail petri - Bunsen - Cotton - Pipette drops - Bottle - Beaker glass - The glass trophies - Cover glass - Paper filter - Tweezers - Attraction glass - Scissors Ingredients - Alcohol 95% - aquades - Carbol fuchsin - Crystal Violet - Nigrosin - Chinese Ink - Malachite green - Lugol's iodide - safranin - Tissue - Alcohol 70% Pure culture of bacteria

The Step Simple staining 1. Preparations glass cleaned with 70% alcohol then fixed on the Bunsen 2. Burned needle ose then dipped into distilled water and given too little distilled water on glass using a needle ose preparations 3. Burned again and grab the needle loop of bacteria from the media and then spread over a glass preparations 4. Dried and aerate preparations 5. Dropped into crystal violet dye solution as much as 1 or 2 drops 6. Dried and aerate for 1 minute 7. Washed with running water 8. Dried preparations to aerate, and 9. Observed under the microscope and the characteristic form of the bacteria Negative staining 1. Preparations glass cleaned with 70% alcohol then fixed on the Bunsen 2. Burned needle ose then dipped into distilled water and also a little distilled water on glass using a needle ose preparations

3. Burned again ose needle and bacteria taken from the media over the past diratakkan glass preparations 4. Dried and aerate preparations 5. Dripped dye solution 1 drop of Indian ink 6. Dried and aerate preparations 7. Washed with running water 8. Dried preparations to aerate, and 9. Observed under a microscope Gram staining 1. Preparations glass cleaned with 70% alcohol then fixed on the Bunsen 2. Burned preparations using a glass needle ose 3. Dbakar longer needle ose and bacteria taken from the media over the past diratakkan glass preparations 4. Dried and aerate preparations 5. Dropwise a solution of the dye crystal violet 2-3 drops and settling for 1 minute 6. Dried and aerate preparations 7. Washed with water and dried 8. Dripped with Lugol solution and allowed to stand for 1 minute and then washed with running water and pat dry diangin 9. Then washed with 95% alcohol for 30 seconds, then washed with running water and wind dried 10. Given a solution of basic fuchsin or safranin for 2 minutes 11. Washed with running water and wind dried 12. Observed under a microscope Spore staining 1. Preparations glass cleaned with 70% alcohol then fixed on the Bunsen 2. Burned needle ose then dipped into distilled water and given too little distilled water on glass using a needle ose preparations 3. Burned again ose needle and bacteria taken from the media over the past diratakkan glass preparations 4. Closed with filter paper 5. Dripped malachite green then fixed 6. Allowed to stand for 5 minutes and discarded filter paper 7. Rinsed with running water and then aerate 8. Safranin dripped and dried without fixation

9. Observed under a microscope