Bacteria are uniquely different and while some of the bacteria are harmful to human beings and cause various health complications, some of them are harmless and provide various benefits to the environment and the human body. On the other hand, some groups of bacteria are capable of causing both, meaning that they can be beneficial to human beings or harmful at a given quantity. Escherichia coli , which is commonly abbreviated as E. Coli is an example of a bacterium that is found in different environments. E. Coli is found in places such as animal intestines, soil, food, and many surfaces that are dump. This bacterium is diverse and belongs to the Escherichia genus . Most of the E. Coli bacteria are not harmful to human beings and this is the reason why we leave with most of them within our environments. Some of the E. Coli bacteria are even found within the human digestive system where they survive in the intestinal tract helping with the digestion and absorption of some nutrients into the body. However, some E. Coli bacteria are very harmful to human beings. These are E. Coli bacteria such as E. Coli 0157:H7 that can cause serious health complications. It is very important to get to identify these bacteria that cause infection so that they can be controlled. When people get sick, most are taken to the hospital for check-up and depending on the procedures used by medical lab experts, harmful E. Coli bacteria may be detected. Gram stain is one of the methods that can be used to test for the presence of a given bacteria such as E. Coli .
Methods
Various methods can be used in performing bacteria experiments such as five stains or biochemical tests and all these can help to identify bacteria. Depending on various factors such as availability of the testing resources, the expertise of the person conducting the test, and the environment, one can choose any test method available. Examples of the five stain tests are, fermentation, capsule strain, litmus milk, TSI, and gram stain.
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The method that is going to be used in performing a test for the E. Coli bacteria is a gram stain. Compared to the other five stain tests, the gram stain method appears to be more complex because it entails the use of two dyes. In most cases, simple stains such as methylene blue may be used but for the gram stain technique, the complexity is brought by the differential procedures involved. In this case, the gram stain method is appropriate because it involves the use of more than one stain to differentiate the cellular components of our specimen. In the experiment, three different stains going to be used are crystal violet, iodine, and safranin.
The Theory Under Which the Gram Stain Works
In an aqueous solution, the crystal violet component will dissociate into two ions which are the CV+ and CV- ions. The two ions can penetrate the membrane and walls or the gram-negative and gram-positive cells. The positive ions CV+ will react with the negative charge components of the cells of bacteria. When this interaction occurs, CV+ will take up the purple color. In addition to iodine, the CV+ will interact with the iodine cations leading to the formation of large complexes of CV1. All these will be formed on the outer layers and cytoplasm of the cell.
When the decolorizing agent is added such as ethanol, they will interact with the membrane lipids of the gram-negative and gram-positive, and gram-negative positive. The peptidoglycan layer which is found on the cell walls of the bacteria has different thicknesses and this is what causes the color difference when de-colorizing agents are used. For example, due to the difference in this layer, the gram-positive bacteria will retain the crystal violet stain even after using acetone or alcohol de-colorizing agent. The decolorizing agent will achieve its purpose by dehydrating the peptidoglycan which is done through tightening and shrinking motions. In gram-negative cells, the outer membrane will not be able to retain the color of crystal violet iodine and this will cause the change of color.
Under this mechanism, the outer membrane is lost causing the peptidoglycan layer to be left exposed. In the gram-negative cells, the decolorizing agent will further cause the walls to be leaky. When the walls are leaky, they will not be able to hold the large complexes of CV-L when doing de-colorization.
Requirements for The Gram Stain Experiment
Sample: in this case, the E. Coli bacteria will be used as the sample.
Microscope glass slide.
Distilled water
Burner (a heater)
Alcohol
Gram stain: the components chosen for the experiment are Iodine, Crystal violet.
A collecting wire loop
How the Problem Is Going to Be Studied
Smear Preparation
Heating of the wire loop before scooping any sample.
Allow cooling of the heated wire loop (heating and cooling is done in order to make a sterile.)
Scooping and placing the broth culture on to a sterile slide. Scooping done by the wire loop.
Gently spread the sample on the slide.
A sterile wire loop used again to drop saline solution to the plate cultures containing the bacteria.
Use of another sterile wire loop to scoop the sample followed by stirring on the drop of water to make an emulsion.
Gram Staining
The following are some of the steps that are going to be taken when starting gram staining experiment,
Flooding the smear with crystal violet for a period of 60 seconds. Done when the slide is placed on a staining rack.
Tilting the slide slightly and rinsing with water. This can either be tap or distilled water. Caution should be taken not to roughly wash the glass slide.
Flooding the sample with iodine. Allowing the slide to stand approximately 40 to 60 seconds.
Tilt the slide again slightly and then rinsing by the use of distilled or tap water.
Tilting the slide and performing de-colorization by the use of either acetone or ethanol. De-colorization is done by application of ethanol in drops for about 9 seconds.
Rinsing gently with water.
Blotting the slide dry
Placing the slide under the microscope to view the sample.
Results
The table below shows the summary of the results that were obtained.
| Escherichia coli bacteria | Color shown |
| E. Coli gram-negative | Absence of purple color |
| E. Coli gram-negative | Pink |
When the slide was viewed under the microscope, the specimen was observed to have a pink color. The purple color was absent in the specimen observed on the slide. The fact that the purple color was absent indicated that the specimen was a gram-positive bacterium. In other words, the absence of the purple color indicated the absence of gram-negative E. Coli
Conclusion
In conclusion, gram stain is one of the five stain tests that can be used to identify bacteria. Currently, the gram stain method has remained to be one of the most common procedures applied in microbiology. As opposed to the simple stain that uses only one dye, the gram stain method is considered to be complex because it entails differentiation. The staining technique distinguishes between two types of bacteria, gram-negative and gram-positive. In our problem, the gram stain method was used to characterize Escherichia coli as gram-negative bacteria because the color turned pink in color. It is the absence of the purple color that will indicate a gram-positive Escherichia coli When carrying out this experiment, one has to make sure that the smear is not too thick and this may make the cells not to be de-colorized or the dyes not to penetrate the cell. Lastly, one has to ensure that the de-colorization step is properly timed. The Escherichia coli bacteria are medically significant because they can be used to make certain medications. For example, the Escherichia coli bacteria are used in making cancer-fighting drugs, antibiotics, and also manufacturing insulin.
