Literature Review (Minh Tri)

Chosen Research Topic: Investigation on effects of various beverages on probiotics(Lactobacilius)

Since our topic is about different antibiotics and Lactobacillus. Lactobacillus, the probiotic, plays a significant part in our research. Since we are using Lactobacillus Casei as a representative for probiotics, we would like to make L. Casei most similar to the other probiotics to make the research accurate.

There are many kinds of classifications, groupings of bacteria. One common one is the Gram Stain. How does Gram Stain work? As Monica(2012) said, Gram Staining involves 3 processes.

The 3 Processes:

Staining

• The bacteria, cells, tested are stained with a crystal violet dye. Then, Gram's Iodine solution(iodine and potassium) is added to form a complex with the crystal violet dye. This complex is then becomes insoluble in water.

 Decolorization

• A decolorizer such as ethyl alcohol or acetone is added to the cells, which dehydrates the          peptidoglycan layer, shrinking and tightening it. The crystal violet-iodine complex is not able to penetrate this tightened and shrunk peptidoglycan layer, and is thus trapped in the cell in Gram Positive Bacteria. On the other hand, because the peptidoglycan layer of the Gram Negative Bacteria is on the inside, under the outer-membrane and the periplasmic space, the decolorizer degrades the outer-membrane. Due to the thinner peptidoglycan layer of the Gram Negative bacteria, without the outer-membrane, the layer cannot retain the crystal violet-iodine complex and loses it's violet stain. 

CounterStaining

• Counterstaining uses a counterstain. An example of a counterstain would be the water soluble safranin. This counterstain, safranin, is added to the cells, bacteria tested, which would stain them(Gram Negative) red. This is because the Gram Negative Bacteria was emptied of the Crystal Violet-iodine complex and just got stained red. Since safranin is lighter than crystal violet, it does not disrupt the purple coloration in Gram Positive Cells. In other words, this safranin does not overwrite the already violet stained Gram positive cells. 

Due to the differences in thickness of the peptidoglycan layer in the cell membrane between the Gram negative and Gram Positive Bacteria, Gram Positive Bacteria(With thicker peptidoglycan layer) retain the Crystal Violet during the decolorization process, while the Gram negative bacteria(With thinner peptidoglycan layer) will lose the Crystal Violet Stain and instead get stained by the safranin during the counterstain process. Eventually, at the end of the experiment, the Gram Positive Bacteria, under a microscope, will look dark violet or violet, indicating that they are Gram Positive. While the gram negative bacteria, will look red, like pink rods under the microscope.

So the question to ask is, what is the difference between the Gram positive and negative bacteria? 

The following characteristics are shown in Gram Positive Bacteria: (Wikipedia)

• Cytoplasmic Lipid membrane.
• Thick peptidoglycan layer. (multilayered)
• Flagellum (Only in some species. If present, it contains two rings for support as opposed to four in Gram-negative bacteria because Gram-positive bacteria have only one membrane layer.)
• Mainly exotoxins produced.

The following characteristics are shown in Gram Negative Bacteria:

• Cytoplasmic membrane
• Thin peptidoglycan layer. (single layered)
• Outer membrane containing lipopolysaccharide (LPS). 
• Porins exist in the outer membrane.
• Presence of periplasmic space.
• Mainly endotoxins produced.

Comparing the 2 characteristics above, we can see that the differences between Gram Positive and Gram Negative Bacteria are:

• Presence of Outer membrane.
• Presence of periplasmic space.
• Thickness of peptidoglycan layer.
• Presence of lipopolysaccharide.(LPS)
• Toxins produced.

From the differences, I can safely say the Gram Negative Bacteria is more resistant to antibiotics, as well as more "irritating" to Scientists. Let me explain.

Melissa(2000) said, "the distinctive feature of gram-negative bacteria is the presence of a double membrane surrounding each bacterial cell. Although all bacteria have an inner cell membrane, gram-negative bacteria have a unique outer membrane. This outer membrane excludes certain drugs and antibiotics from penetrating the cell, partially accounting for why gram-negative bacteria are generally more resistant to antibiotics than are gram-positive bacteria."

She also said that Gram negative bacteria have a great facility for exchanging genetic material (DNA) among strains of the same species and even among different species. This means that if Gram negative bacterium undergoes mutation, or acquires genetic material that confers resistant to antibiotic, the bacterium may later share its DNA with another strain of bacteria and that this another strain of bacteria can become resistant as well. This causes great pain to Scientists as Gram negative bacteria might get resistant to antibiotics faster, as well as unexpectedly, and is very "irritating" as these scientists have to make a new antibiotic to fight against these mutated bacterium.

Coming back to our research, all the above is precisely why we use Lactobacillus Casei as the test subject of our research/experiment.

LPS is known as an endotoxin, found in the outer membrane of the Gram negative bacteria. Injections of small amount of endotoxins have shown produce fever, a decrease and blood pressure and activation of inflammation.
With LPS in their outer membrane, most Gram negative bacteria are either harmful or harmless, little, or none beneficial.
We use L. Casei to represent good bacteria due to the fact that it is found in our body, as well as it being a probiotic. Of course, it is a Gram positive Bacteria. 

References:

Monica Z.B (2012, May 29). Gram Staining. Microbial Life. Retrieved July 9, 2013, from http://serc.carleton.edu/microbelife/research_methods/microscopy/gramstain.html

Gram-Positive Bacteria. (n.d). In Wikipedia. Retrieved July 9, 2013, from http://en.wikipedia.org/wiki/Gram-positive_bacteria

Gram-Negative Bacteria. (n.d). In Wikipedia. Retrieved July 9, 2013, from https://en.wikipedia.org/wiki/Gram-negative_bacteria

Melissa K. (2000). The problem with gram-negative bacteria. Retrieved July 9, 2013, from http://www.anapsid.org/gramnegative.html