Bacilli and Cocci Bacteria Classification

In this study of prokaryotic organisms, an experiment was performed in order to understand the differences between eukaryotic cells and bacteria. The purpose of the study was to distinguish different types of bacteria based upon their morphology and the properties of the molecules on their exterior. We examined the observable differences in eukaryotic cells of Bacilli and Cocci bacteria and classified them based upon the way they orient themselves around other bacteria. To perform this, we set out a clean, sterilized glass slide and placed it on a staining tray covered with bacterial proteins and smeared with crystal violet and gram’s iodine. We then held the slide at a slight incline and rinsed the crystal violet with water after which we dropped ethanol on the slanted slide until a faint violet color was seen in the alcohol rinse. We proceeded by covering the smear with safranin for 1-2 minutes and later rinsed the safranin to completely dry the slide with bibulous paper. On examining the slide under oil immersion, our results showed a subset of cocci bacteria having a very thick peptidoglycan cell wall composed of carbohydrates crossed linked to proteins. Another subset of bacteria had two cell walls: a thin inner wall composed of peptidoglycans plus an outer wall composed of carbohydrates, proteins and lipids.

Introduction

The classification of microorganism bacteria takes into consideration two heterogeneous groups of prokaryotes and eukaryotes. Based on the differences that exist in the cellular organism of prokaryotes and eukaryotes, these bacteria’s that belongs to the kingdom of protista are classified based upon their morphology. Furthermore, the bacteria can be classified based upon the way in which they orient themselves around other bacteria. This is to imply that bacteria can occur in single structures or in chain. For instance, Bacilli

bacteria mostly appear in elongated cigar or basically in rectangular shaped microbes, staphylo are categorized by their grape-like clusters whereas Cocci bacteria are known by their round, spherical or circular shaped microbes.

However, some bacteria can perform the role of decomposers, some can live at extremely high temperatures, and some live in the gastro intestinal tract of human beings and some are the source of human diseases. For eukaryotes in this case, we have the algae, fungi and protozoa bacteria’s with most of them possessing peptidoglycan cell wall composed of carbohydrates crossed linked to proteins. The synthesis of proteins in these structures takes place in cytosol with structurally different ribosomes where gram staining is smeared.

Materials and Methods

First, we set out a clean glass slide onto which we placed a small drop of deionized water. We used a an inoculating loop and placed it on the incinerator to help transfer a small amount of bacterial colony to the deionized water drop to sterilize it. We further mixed them well so that the water and bacterial mixture was about the size of the nickel. With the dried mixture of bacterial colony and water, we heat-fixed the glass slide by passing it over the exterior of the incinerator for about 15 times. The purpose of heat-fix process was to coagulate the bacterial proteins to the slide so that the cells would not be washed off during the staining period. We then sterilized the glass slide and placed it on a staining tray covered with bacterial proteins and smeared with crystal violet and gram’s iodine.

Thereafter we then held the slide at a slight incline and rinsed the crystal violet with water after which we dropped ethanol on the slanted slide until a faint violet color was seen in the alcohol rinse. We proceeded by covering the smear with safranin for 1-2 minutes and later rinsed the safranin together with the Gram’s iodine for one minute to completely dry the slide with bibulous paper. While de-staining the smear by dropping ethanol down the slanted slide one drop at a time, we continued drop-wise until only a faint violet color was seen in the alcohol rinse. We then concluded by rinsing the slide with water and then covering the smear with safari for 1-2 minutes until all the water had blotted dry on the slide. By so doing we were trying to distinguish different types of bacteria based upon their morphology and the properties of the molecules on their exterior.

Results

Results showed a subset of cocci bacteria having a very thick peptidoglycan cell wall composed of carbohydrates crossed linked to proteins. Such a cell wall will retain a purple color when stained with a substance known as crystal violet. Another subset of bacteria had two cell walls: a thin inner wall composed of peptidoglycans plus an outer wall composed of carbohydrates, proteins and lipids. These bacteria do not stain purple when using crystal violet, and instead maintain a red stain from the staining component safaranin.

The variation for staining for these two particular types of bacteria allows them to be categorized into two physiologically different groups: gram (+) and gram (-). The gram staining technique was developed by Hans Christian Grams in 1884 and is still being used widely today to assist in the identification of various types of bacteria. More importantly, some treatments will only work on gram (+) and gram (-) bacteria.

Result Chart for Gram Staining

Reaction and Appearance of Bacteria

Solution in order Applied

Grams Positive

Grams Negative

1. Crystal-Violet (C-V) -Primary Stain

Cells stain violet

Cells stain violet

2. Gram’s Iodine (I) - Mordant

CV-I complex formed within cells: Cells remain violet

CV-I complex formed within cells: Cells remain violet

3. Alcohol- Delororizer

Cell wall dehydrated, pores shrink, permeability decreases, CV-I complex retained; cells remain violet

Lipid extracted form cell walls; porosity increases, CV-I removed from cells; cells colorless

4. Safranin-Counter Stain

Cells not affected, remain violet

Cells takes up the stain; becomes red

Figure 1: Effects of Different Materials used in gram staining on gram (+) and gram (-) cells

Results for Comparison of Bacteria to Eukarya

Shown below were some of the observable differences between the prokaryotic cells of the bacteria and the eukaryotic cells of the bacteria.

Figure 2: Schematic of a Bacterium and its various cellular components

Bacteria lack the membrane-bound nucleus of eukaryotes, and their DNA forms a tangle known as nucleoid and is found in the cytoplasm of the cell. Additionally, while the proteins are bound to the DNA they do not keep it in a structured shape and bacteria chromosomes are cellular rather than linear. Also, additional DNA may be found within bacteria that exist in loops known as plasmids. These plasmids can be ejected from the cytoplasm of the cell into any extracellular space and can also be transferred out of one cell and into another. This ability of bacteria to trade genes is what can eventually lead certain strains of bacteria to become resistant to particular drugs or treatments.

Classification Grain Staining Bacteria

Based on the results shown above, it may seem as though all bacteria have a peptidoglycan cell wall and that the characteristic of this cell wall are the same in all types of the bacteria. However, this is not the case and it turns out this particular cell wall is showing different results when a grape-like cluster of cocci bacteria is observed. In the figure below shows the three images, one of each gram stain. Give bacteria name, shape and gram status in description.

Figure 4: Drawings and labels showing the three types of bacteria based upon their morphology

Tabular Results

Figure 4: Determination of shape and gram stain of unknown samples

Results chart for Bacteria vs Eukarya

Shown below is the results chart for Bacteria vs Eukarya for the study

Characteristics

Bacteria

Eukarya

Nucleus

Chromosomes

Organelles

Unicellular or Multicellular

Sexual Reproduction

Plasma Membrane

Cell Wall Material

Simple or Complex RNA polymerases

Figure: Identification of the Difference that exist between bacteria and Eukarya

Medical Microbiology

External and internal organs of the body are covered by over one hundred trillion bacteria, and these bacteria are required for healthy living. While we provide these bacteria with food and space to live, they protect us from more pathogenic strains of bacteria. Additionally, some of these bacteria can produce the nutrients needed by the body to maintain homeostasis. However, the introduction of virulence factors can result in alterations in these bacteria that lead them to cause disease in human. This delicate balance implies that it is extremely important for certain regions of the body to remain as sterile s possible. Such sites are not typically exposed to the external environment. Outlined below are the four sterile sites for the human body that should not contain bacteria;

Ø Head

Ø Brain

Ø Liver

Ø Pancreas

Discussion

Based on the results shown above, it may seem as though all bacteria have a peptidoglycan cell wall and that the characteristic of this cell wall are the same in all types of the bacteria. However, this is not the case and it turns out this particular cell wall is showing different results when a grape-like cluster of cocci bacteria is observed. These results are important because they help us identify which type of  bacteria are capable of performing the role of decomposers as well as those that can live at extremely high temperatures, and some live in the gastro intestinal tract of human beings and also classify which bacteria are the source of human diseases.

In the medical field, the study of bacteria and gram staining is important because it helps in the identification of various types of bacteria in human beings. Additionally, clinicians use the results of bacteria and gram staining to differentiate between gram positive organisms and gram negative organisms thus helping one to know which gram cells take up the crystal violet, and which is then fixed in the cell with the iodine mordant.

References

Gupta, R. S. (2018). What are archaebacteria: life's third domain or monoderm prokaryotes related to Gram‐positive bacteria? A new proposal for the classification of prokaryotic organisms. Molecular microbiology, 29(3), 695-707.

Woese, C. R., Kandler, O., & Wheelis, M. L. (2017). Towards a natural system of organisms: proposal for the domains Archaea, Bacteria, and Eucarya. Proceedings of the National Academy of Sciences, 87(12), 4576-4579.