INTRODUCTION Gram staining is the most essential and universally used staining technique in bacteriology laboratory. Gram-staining was firstly introduced by Cristian Gram in 1883.This method is used to distinguish between gram positive and gram-negative bacteria which have consistent differences in their cell walls. gram-positive bacteria stain blue-purple and Gram-negative bacteria stain pinkred. There are several objective why we should do this experiments such as knowledge of the differences between gram positive and gram negative bacteria, develop the lab skill, and be more familiar with gram staining procedure. There are also several material that we use in this experiment such as Hucker’s crystal violet, Gram’s iodine, 95 % alcohol, safranin and many others. Before that, we absolutely must know about the proper procedures during this experiment such as prepare smears from culture of microorganisms, heat the smears, place the slide on a staining rack and so on. OBJECTIVE 1. Knowledge of how distinguish the gram-positive and gram-negative bacteria properly. 2. Develop the laboratory skill in gram staining in proper ways. 3. Be more familiar with gram staining procedure. 4. To understand how the gram stain reaction affects Gram positive and Gram negative bacteria based on the biochemical and structural differences of their cell walls.
MATERIAL Gram stain reagents: Hucker’s crystal violet Gram’s iodine 95 % ethyl alcohol Safranin Microscope slides Staining rack Bibulous paper
PROCEDURE 1. Smears from cultures of at least two microorganisms was prepared, one from each of the two groups. 2. The smears was air dried and heated. 3. The slides on a staining rack and flood each smear were placed with crystal violet. 4. The crystal violet from each slide with tap water and drain off excess water were wash after staining for 1 minute. 5. Each smear was flooded with iodine. 6. The iodine from each slide with tap water and lightly blot were washed with bibulous paper to remove excess water after 1 minute, but the slide was not dried completely. 7. Each slide was tilted and decolorized with 95 % ethanol until the alcohol draining from the slide appears colorless about 15 seconds. 8. The slide was briefly washed with tap water and the excess was drained off. 9. The smear was counterstained with safranin for about 20-30 seconds. 10. The smear was briefly washed with tap water and blot dry. 11. The slides were examined under the oil-immersion objective. Before that, the slides were given the oil in order to refract light rays towards the center of the lenses.
DISCUSSION In this experiment, we focused on gram staining and the mechanisms in gram staining. As usual, before we started the experiment, it is crucial for us to perform aseptic technique In order to minimize contamination. Firstly, we prepared smears of two microorganisms which are Bacillus anthracis and Escherichia coli. In theory, both of this microorganisms are from two different groups, and we expected to observe two different result in this gram staining experiment. After preparing the smears of the microorganisms, the smears were air dried and heat fixed. Then we stain both smears using crystal violet dyes. Crystal violet dyes are basic dyes that have positively charged particle that helps them to bind to negatively charged molecule like teichoic acid at the cell wall of bacteria. This crystal violet dye can dissociates into cv+ and cv- ions. These ion can penetrate deeps into the cell wall of bacteria and interacts with the negatively component on the bacterial cell wall. 1 minute later, the crystal violet was washed with tap water and then the slides are dried. The next step is to add iodine onto each smears. Iodine was being added as a mordent to form crystal violet-iodine complex, CVI complex. This complex enables the dyes to not be easily being removed. Next, we washed the iodine with tap water and dried off the excess water. After that, 95% of ethyl alcohol was being added to acts as a decolorizing agent. It interacts with the lipid membrane of both positive and negative bacteria, and this would cause the gram-negative bacteria to lost their outer membrane and exposing the peptidoglycan. The CVI complex are being washed from the outer membrane of gram-negative bacteria and cause the purple color to decolorize. Meanwhile, for gram-positive bacteria, the addition of alcohol dehydrated the layer of peptidoglycan which in turn would trap the CVI complex. This cause the gram positive bacteria appeared to be purple color as the CVI complex are being retained. The addition of alcohol is not be more than 15 seconds as this would break the cell wall of the bacteria, thus resulting in no stain to be observed. The slides are washed with water and dried off. In the next step, safranin was used to counterstain both smears. This is to enables the gram negative bacteria to be visualize easily as it can be stain in pink color. The gram-positive bacteria does not being stained pink when safranin was being introduced because the peptidoglycan layer already have have CVI complex. Then, the slides were washed using tap water and dried off. Finally, we observed our
specimen using microscope under oil-immersion objective lenses. This particular lens have more mirrors inside and it requires the use of oil to refract light rays towards the center of the lenses. QUESTION 1. If the iodine step were omitted what color would you expect a Gram- negative microorganism to be ? A Gram-positive ? If the iodine step were omitted in Gram’s stain, the crystal violet which enters the cell will not form the crystal violet iodine ( CVI ) complex. The CVI complex molecule has larger size than that crystal violet molecule and hence Gram positive bacteria are able to retain the stain. 2. Could other dyes be substituted for crystal violet in gram stain procedure? For safranin? Explain your
answers.
Crystal violet dye can be replaced with Methylene blue. This is because both are basic dye that have a positive charge that able to bind to negatively charged particle like teichoic acid at the bacterial cell wall. Therefore Methylene blue can be used to replace Crystal violet dye. Meanwhile, for safranin, it can be replaced with basic fuchsin. Both dyes are able to stain gram-negative bacteria a pink colour, but based on the information that we have searched, basic fuchsin gives much brighter color in order for the bacteria to be seen clearly. 3. What role does the mordant play in the Gram-stain procedure? The mordant is the substance that set dyes the iodine in gram stain fixed the crystal violet to bacterial cell wall of the gram positive bacteria. When gram-positive bacteria are treated with ethanol , the alcohol is thought to shrink the pores of thick peptidoglogycan. Thus, the dye-iodine complex is retained during this short decolorization step and the bacteria remains purple. 4. What correlation is there between cell wall composition and Gram reaction ? is gram reaction correlated with sensitivity to various antibacterial agents? Crystal violet (CV) dissociate into Cl- ions and CV+ which interact with negatively charged components of the cells and stain it purple. Iodide ion is then react with CV+, forming CVI complex. Alcohol interacts with the lipid of cell membrane, causing gram negative cell lose its outer lipopolysaccharides (LPS) membrane, exposing the peptidoglycan layer. Therefore, CV-I and the outer membrane is washed away. However, in the gram positive cell, the alcohol dehydrated the cell, trapping the CV-I because of the thick peptidoglycan. The gram reaction is somehow correlated with sensitivity to various antibacterial agents.
Gram positive cells are sensitive to the action of certain antibacterial agents like penicillin due to lack of outer membrane which acts as barrier to certain antibacterial agents, while gram negative cells are more susceptible to mechanical breakage as the amount of peptidoglycan is small. However, the gram reaction does not ensure that the same antibacterial agent is able to destroy the 2 different species or strain of bacteria even though they showed the same result in the gram reaction. This is because the antibacterial agents have different types of action such as injury to the plasma membrane, inhibition of cell wall synthesis and protein synthesis, does not only depend on the gram reaction.
CONCLUSION From the experiment that we have done, finally, we can conclude that gram staining is the method of distinguishing between gram positive and gram negative bacteria. In this experiment, we were provided some material to help us for reaching the aim of this experiment such as Crystal violet, Gram’s iodine, 95 % ethyl alcohol, safranin, and microscope slide. However, before doing the experiment, we absolutely need to pay attention on the precautions. There are several procedures that we have to do in order to avoid the error in this experiment, such as prepare smear from cultures of microorganism, heat fix the smears, place the slides on a staining rack, and so on.
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