Extract Ethyl Acetate Red Fruit (Pandanus Conoideus Lam.) as a Counterstain in Gram Staining Technique

Objective: to test the extract of ethyl acetate Red Fruit (Pandanus Conoideus Lam.) As dye substitution in Gram staining technique. Material and Methods: Made Preparations from the suspension Escherichia coli ATCC 25922 and Streptococcus sanguine ATCC 10556 counted 30 preparation, and then done Gram staining process to this preparation. 1 preparation using counterstain basic fuchsin as a control and 29 other preparations using ethyl acetate extract of Red Fruit as a counterstain, the result of staining compared to using standard degradation of RHS color chart. Result: From 29 sample preparations used the counterstain extract of ethyl acetate Red Fruit (Pandanus Conoideus Lam.), 12 preparations produced a color close to color code 62B on the standard degradation of RHS color chart that is moderate purplish pink. Conclusions: From this preliminary study that examined the extract of ethyl acetate Red Fruit as a counterstain on Gram staining resulted in a color contrast between Gram + and Gram bacteria though not as good as if using compressed/counterstain basic fuchsin.


Introduction
Microbiology is one of the basic branches of medical science (Basic Medical Science) who studied the life of microorganisms are bacteria that can cause disease in humans and animals. Bacteria have unique characteristics both of shape, size, and formation. This characteristic can be seen through microscope examination, but the size of bacteria is very small that is about 0,2 μm -0,5 μm and transparant, so to be able to see bacteria under microscope need to be done the process of putting dye into the body of bacteria called by the process of staining Bacterial. Bacteria do not absorb or refract light, this is why bacteria are difficult to see with a direct light microscope. Therefore, it is necessary to dye to color the microorganisms, this dye is able to absorb and refract light. The combination of microscopic lighting and coloring processes became one of the most important procedures in the field of microbiology to study the nature of bacteria and group them into more specific groups. 1,2,3 Bacteria staining process can be done by using one kind of dye which is referred to as Simple Staining and two kinds of dyes called Differential staining as an example is the Gram stain. 1,2 Through microscopic examination of bacteria that have been colored can be seen morphology of shape, size, formation and its nature. Is it Gram + (purple) or Gram -(pink / red). 3 Microscopic examination is an important step in the process of a bacteriological diagnosis which is a step / initial step for diagnosing the disease in humans and animals.
Bacterial staining methods used until now still use dyes synthetic chemicals that are quite expensive as carbolic Gentian Violet, Basic Fuchsin, safranin, Methylene Blue, Malachite Green and others. 1,2 Indonesia as an agricultural country is very rich in its natural resources including its agricultural products. Eastern part of Indonesia (Papua) there are quite abundant agricultural products namely Red Fruit (Pandanus conoideus Lam.) Were utilized and consumed by the people of Papua as a source of food as well as food coloring agents. The content of beta carotene from this fruit causes the red color, carotenoids contained therein have been utilized by the people of Papua as a natural food coloring good. 4.5 On the basis of this reason, the authors are interested in conducting preliminary research on Red Fruit to be used as a substitute of natural dyes from herbs to the process of bacterial staining.

Materials and Methods
The materials used to make this preliminary study are synthetic ingredients or chemical staining for Gram as carbolic Gentian Violet, Basic Fuchsin, Iodine, Alcohol 96% and ethyl acetate extract of red fruit in the form of a standard solution. For the test microorganisms used in this preliminary study is Escherichia coli ATCC 25922 and Streptococcus sanguis ATCC 10556.
The method of staining bacteria used is the Gram staining method. The procedure of making red fruit extract with the solvent ethyl acetate is as follows:

Results
Results ethyl acetate extracts were prepared by maceration for 24 hours at room temperature after filtration and evaporation using a rotary vacuum evaporator make the solution viscous and red like the picture below.

Figure 2. Red Fruit Extract Ethyl Acetate
The resulting color of ethyl acetate extract of red fruit than the color of basic fuchsin looks like the image below.

Figure 3. Color Comparison (A) Of Ethyl Acetate Extract Red Fruit (B) Basic Fuchsin
Results Gram of the 30 preparations were stained with basic fuchsin as a control and the other 29 were stained with counterstain ethyl acetate extract of red fruit will be compared with the degradation table by the RHS Color Chart color as seen in the table below.  Gram staining results that use basic fuchsin counterstain and used as a control in this preliminary study can be seen in the picture below.

Discussion
Red fruit in addition to containing nutrients is also active compounds in high enough levels, among others tocopherol, alpha-tocopherol, oleic acid, linoleic acid, and beta-carotene carotenoid. Betacarotene is what causes the red color of the red fruit. This substance has long been exploited by the people of Papua as a good natural food coloring. 4.5 In this study, the extract of ethyl acetate red fruit, as a counterstain could staining bacteria on Gram staining preparations although the resulting color is not as good as the dye synthetic basic fuchsin, but the purpose of the Gram stain to get an idea of the contrast between Gram + (purple) and Gram-(which is red or pink) is relatively visible.