Bacterial Cell Staining

BACTERIAL CELL Staining 6.1. Introduction Recognition of microbial forms (morphology), except for microalgae, must be stained first so that they can be observed clearly. The purpose of coloring is: 1. Makes it easier to observe the shape of microorganism cells (especially bacteria). 2. Clarify the size of the body 3. Can observe the external structure and internal structure of microbial cells. 4. See the reaction of the body to the dye given, so that the physical and chemical properties of the body can be known. Thus, we can use staining as a way to classify bacteria. Whether the coloring is successful or not is largely determined by the time the color is applied and the age of the culture being colored (a good culture age is 24 hours). 6.2 Various coloring methods 6.2.1 Direct staining with basic dyes Generally, dyes are salts formed by positively or negatively charged ions, where one of the ions is colored. If a bacterial cell whose cell wall has a relatively negative charge combines with positively charged ions from a dye, it causes the bacterial cell to be colored.Coloring substances can be grouped into two groups, namely dyes that are basic and dyes that are acidic. If the positive ion of the dye contains the color, then the dye is a basic dye. And if the color is in a negatively charged ion, then the dye is an acid dye. Tools and materials a. Bacterial cultures isolated in previous practicums and some pure cultures. b. Methylene blue dye c. Crystal violet dye. d. Carbol fuhsin e. Clean slide and cover glass. Ways of working . a. Place a grease-free glass slide on the work table. b. Place a drop of water in the middle of the object glass. c. Using a flared Ose needle, take a small amount of the bacterial culture that you isolated in the previous practicum or from the pure culture of bacteria provided by your assistant. d. Make a bacterial smear in water placed on a glass slide by rubbing a loop needle containing the bacterial culture, so that you get a thin and even mixture. e. Fixation over a Bunsen flame with a distance of about 30 cm. From the flame. When fixing this, don't overheat it because it can damage the shape of the cells. f. Drop one of the dye solutions provided by the assistant onto the specimen that has been fixed. Methylene blu dye will color cells in 30 - 60 seconds, Kristak violet in 10 seconds, and Carbol fuhsin in 5 seconds. g. Wash with running water. h. Dry the preparation by placing it between filter paper. i. Observe with a microscope using an objective lens with 100 times magnification, and immersion oil. j. Note and draw the cell shape and cell color of the microorganisms you see. Question : 1. Is the culture you are isolating a pure culture, why? 2. What is the meaning of fixing preparations? 6.2.2. Negative staining or indirect staining One of the acid dyes is nigrosin or china ink. Because the coloring power of this substance is in negative ions and does not react with other negative ions from bacterial cells, this dye does not color cells. In this case, what is colored is the environment around the cells. In this way, you can observe the shapes of bacterial cells and their sizes clearly. Tools and materials a. Bacterial culture results from isolation or culture provided by the assistant. b. Nigrosin liquid or Chinese ink c. Grease free slide. Ways of working a. Drip one drop of nigosin liquid or Chinese ink on the edge of the slide. b. Using a lighted Ose needle, take a small amount of bacterial culture and suspend it in a drop of nigrosin on the surface of a glass slide. c. Spread the bacterial suspension in nigrosin on the surface of this slide using another slide. d. Let it dry at room temperature, and observe with a microscope (100 x objective lens magnification), and use immersion oil. e. Draw the shape of the bacteria that you see. The following staining methods are differential staining techniques, as one way of classifying bacteria. 6.2.3. Gram stain Gram stain is a very common stain in the field of bacteriology. With this staining, bacterial groups can be divided into two,namely the groups of Gram-positive bacteria and Gramnegative bacteria. There are many modifications of this coloring technique, but all of these techniques are based on the same principles, namely: 1. Color the microorganisms with basic dyes, namely crystal violet or gentian violet. 2. Color fixation, namely to strengthen the attachment of the base color, for example using iodine salt (modification of Lugol's solution). 3. Washing or removing the base color with alcohol, acetone, or a mixture of alcohol and acetone. 4. Re-staining with a comparison or contrast dye that is different from the basic dye, namely to color cells whose color has been lost by color removal. For example, in this case safranin or carbolic fuhsin are used. Bakery which after being colored with basic dye does not have its color removed by alcohol will be violet because it is colored by crystal violet, and no longer absorbs the contrast dye. The group of bacteria that have this characteristic is grouped into Gram-positive bacteria. Meanwhile, groups of bacteria that have been colored with basic dyes and the color is erased after being treated with alcohol, will absorb the safranin or carbolic fuhsin dye which is used as a contrast dye, so that the preparation will appear red (the color of safranin or carbolic fuhsin). This group of bacteria is called Gram negative bacteria. Tools and materials a. Young bacterial cultures isolated or provided by an assistant. b. Crystal violet solution c. Iodine salt solution. d. 95% alcohol. e. Safranin solution f. Object glass Ways of working a. Make a bacterial smear on a dry and clean glass slide. b. Fixation over a Bunsen flame or in air. c. Stain with crystal violet solution for 1 - 1.5 minutes. d. Wash with distilled water. e. Drizzle with iodine salt solution, and leave for 1 minute. f. Wash with 95% alcohol solution until the color is removed, usually for 0.5 minutes (30 seconds). g. Wash with water. h. Stain with safranin or carbolic fuhsin for 2 minutes.i. Wash with water, and remove excess water using blotting paper, without rubbing the preparation. j. Dry in air or over a Bunsen flame. k. Observe under a microscope with 1000 times magnification. (remember to use immersion oil). l. Record your observations.