Introduction
Gram staining is a form of differential staining used to determine the form of infection that an organism has. That is, whether the infection has been caused by either a gram positive or gram negative bacteria. It involves both a primary and a counter stain that help in categorizing the Eubacteria strains. Occasionally, the presence of white blood cells in the area attacked by bacteria shows the presence of an infection. This gives a clinician more information on diagnosis.
This kind of information can enable a doctor or clinician to quickly prescribe particular antibiotics that would help in treat the infection. This form of test is quick and acts as an alternative test to culture diagnostic tests. Culture evaluation may be coupled with gram staining test and endospore tests. This can help evaluate the type of bacteria strain and its removal methodology. Hence, it is possible to perform other tests that would give more information on progress of any infection. Some diseases are related to the spikes or spores in bacteria. These spikes do cause wound development on organisms.
Hypothesis
Gram stain test gives much information on color of bacteria. That is either pink or purple color. In addition, the shape of the bacteria can also be analyzed. That is whether the bacteria are round shaped or rod shaped. This would help give much information on origin of most infectious diseases.
On the other hand, there has been a formed view that some bacteria cause wound by use of their spikes. For this to be proved, certain tests like endospore staining can be done to categorize the bacteria. This test is normally done for the purpose of determining certain forms of bacteria that have spores which are resistant to stain and other harsh conditions. In addition, the test is used in studying certain bacterial structures such as the vegetative cells. The experiment would show whether the spores produced are due to nutrient limitation or resistance to the dye component.
Procedure for Gram Staining
A heat fixed bacteria smear was taken. The smear was then flooded with crystal violet for one minute and it was rinsed. The crystal violet was the primary stain. The next step included a smear by use of iodine for a minute and the stain was washed with water. The smear was then flooded with Acetone which was quickly rinsed with water. A counter stain was added on the smear and left for a minute. This was again washed with water. Lastly, the smear was air dried and observed under the microscope.
Results
Observation of the slides under the microscope showed the following results:
- Gram positive bacteria stained purple while gram negative bacteria did not stain at all.
Endospore Staining
Procedure for Endospore Staining
Half a drop size of distilled water was placed on a slide using a dropper. A small portion of agar was taken aseptically and mixed with water on slide to form a cloudy sample. The remaining bacterium was then burned off the loop. The suspension was then spread to form a thin film using the loop. Afterwards, the suspension on the thin film was allowed to dry. A sample of the organism was then heat fixed onto a slide by passing it through a Bunsen burner. The slide was then placed over a steam bath and was covered with Malachite green. The stain was left over the bath for three minutes. The Malachite green was then damped off to allow the stain to cool. Thereafter, the slide was rinsed to remove excess stain. The smear was then covered with Safranin for two minutes and then the slide was rinsed to remove excess stain. Lastly, the stain was blot dried and viewed under the microscope.
Results
Observation of the endospore experiment resulted in the following:
Clostiridium tetani
- Careful observation of the endospore was to show blue coloration under the microscope.
- While careful observation of the vegetative material would result into a colorless form.
Clostiridium botulinum
- Careful observation of the endospore would show green coloration under the microscope.
- While careful observation of the vegetative material would result into a red coloration.
Bacillus anthracis
- Careful observation of the endospore was to show green coloration under the microscope.
- While careful observation of the vegetative material would result into a red coloration.
Discussion
Gram Staining
The cell wall is the main contributor of the bacterial stain in gram staining experiments. The gram positive bacteria have got some additional components on their cell wall that is different from the cell wall of the gram negative bacteria. One major component is the peptidoglycan layer. This layer is thought to have a high retaining power of the dye used in gram staining experiment. Moreover, sensitivity to the antibiotics is likened to the gram positive bacteria. This is because the antibiotics predominantly interfere with the cell wall layer hence compromising the fundamental functions of the cell wall. This can automatically leads to the death or destruction of the gram positive bacteria.
In addition, the gram positive bacteria are sensitive to the enzyme lysozyme. There are times that one might find that the gram negative bacteria are stained. This could majorly occur in occasions where an old culture has been used, when there has been improper washing between steps, when a very thick smear has been applied and when there was use of a contaminated culture. This therefore calls for careful check up of steps when the experiment is being performed.
Endospore Staining
It is very hard to stain an endospore. This resistance to staining could be due to the characteristic nature of the endospore. Other factors that can contribute to the resistance are like, endospore DNA stabilizer such as Calcium Dipicolinate, DNA binding proteins that are heat resistant and lastly the enzymes that repair DNA. These do offer recovery to a damaged bacterial endospore. The staining therefore requires heat that would catalyze the reaction and strong dyes that are able to produce coloration when they come into contact with the endospore. This test is mostly done for the bacterial strains such as Clostridium and Bacillus.
Conclusion
The test can only be accurate when the right procedure has been employed. The endospore test is interesting in that the spores do not take dye easily. But, when the dye is absorbed, it is not easily decolorized. During the test, the bacteria are to be grown on Agar. The nutrient agar should be removed or the agar should be deprived of any nutrient to allow for the formation of spores. These staining tests give more elaboration on the species of bacteria as color is the main differentiating factor. Many bacterial strains do posses different components that enable them to act out the disease causing effect. For this reason, they are able to cause different forms of diseases. Therefore, the different strains should be noted by their color stains specific parts of the bacterial cell components.