Sunday, September 1, 2013


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

Background Research
Chosen Research Topic: Investigation on effects of various beverages on probiotics (Lactobacilius).
Inside our bodies, antibodies are constantly created and churned out, used by our immune system to detect and eliminate or neutralize foreign threats found in our body. (Mandal, 2013). These antibodies either tag or eliminate the said foreign threats.

Those foreign threats range from then bacterium causing illnesses such as syphilis, tuberculosis, salmonella, and some forms of meningitis. (Nordqvist, 2007)

Sometimes, however, especially during serious illnesses, antibodies naturally created by the body are not enough to successfully defend the body form external threats and thus external intervention may be required to the external threats. In this case, the external intervention comes in the form of antibiotics.

According to Nordqvist(2007), “Antibiotics, also known as antibacterials, are types of medications that destroy or slow down the growth of bacteria. The Greek word anti means "against", and the Greek word bios means "life" (bacteria are life forms).”

He also wrote (2007), “Before bacteria can multiply and cause symptoms, the body's immune system can usually destroy them. We have special white blood cells that attack harmful bacteria. Even if symptoms do occur, our immune system can usually cope and fight off the infection. There are occasions, however, when it is all too much and some help is needed.....from antibiotics.” (Nordqvist, 2007)

As shown, our bodies are generally well equipped to fight off infections, however, when the body fails to eliminate the threats, antibiotics might be needed to turn the tides and aid the body’s immune system. Antibiotics also come in different forms, such as penicillin and tetracyclines.
Penicillin is one of the most effective, safest, cheapest, and most common form of antibiotic in our modern world.  Penicillin was originally discovered by Ernest Duchesne, a medical student from the late 19th Century, when he, in a study, saw a sample of Staphylococcus being contaminated by mold (Penicillium fungi), and that all of the Staphylococcus bacteria cells close to the mold were observed to be dying. (Paddock, 2011)

However, penicillin only truly came into global spotlight in 1928, when Alexander Fleming accidentally discovered it’s antibacterial properties.

Paddock wrote (2011), “Bacteria are constantly rebuilding their cell walls, which is how they protect themselves and maintain their structure. Penicillins work by damaging and penetrating these cell walls, thus killing the bacteria cells.”(Paddock, 2011)

Simply, penicillin works in killing bacteria as it penetrates and damages the cell walls of various bacterias, in a process known as 'peptidoglycan synthesis’. Penicillin is known as a bacteriocidal.

Another common antibiotic available would be Tetracycline. Tetracycline also is used to treat many different bacterial infections. When they were first discovered, tetracyclines were found to have had the broadest antimicrobial spectrum among the antibiotics mankind had to offer. They were found to have been effective against both the Gram Positive and Gram Negative strains of bacteria, as well as large viruses, such as members of the lymphogranuloma group. (Klajn, n.d.) 

Klajn said (n.d.), “Tetracyclines inhibit a lot of enzyme reactions essential for the vital processes of bacterial cells. The most sensitive biochemical reaction that is inhibited is the synthesis of proteins. Tetracycline works by binding specifically to the 30S ribosome of the bacteria, preventing attachment of the aminoacyl tRNA to the RNA-ribosome complex. It simultaneously inhibits other steps of the protein biosynthesis. Tetracycline can also alter the cytoplasmic membrane and this in turn causes leakage of nucleotides and other compounds out of the cell. This does not directly kill the bacteria but instead inhibit it.”.

As such, tetracycline are known as a bacteriostatic drug, which simply stops bacteria replication, as opposed to penicillin, a bacteriocidal one, which kills off bacteria.

From the above mentioned antibiotics, it might seem on the surface that antibiotics might do far more good than harm in protecting the body. However, there are actually many less known side effects which are damaging to the body as well.

In fact, antibiotics often affect the digestive system whenever they are taken, occurring in around 1 in 10 people, bringing side effects such as diarrhoea, bloating, indigestion, abdominal pain and loss of appetite. These side effects usually disappear, however, when a course of probiotics is taken. (NHS Choices, 2012)

They also wrote that approximately around 1 in 15 people have an allergic reactions to antibiotics, especially penicillin and cephalosporin. In most cases, the allergic reaction is mild to moderate and can be display symptoms such as a raised itchy skin rash, known as urticaria or ‘hives’, coughing, wheezing, and a tightness of breath, which often causes breathing difficulties.

In very rare cases, which are estimated to be somewhere between 1 to 5 in 10,000, antibiotics can cause a severe and potentially life-threatening allergic reaction known as anaphylaxis. Initial symptoms of anaphylaxis are often the same as above, with the potential to eventually lead to a rapid heartbeat, increasing breathing difficulties due to swelling and tightening of the neck and chest, a sudden intense feeling of apprehension and fear, a sharp and sudden drop in your blood pressure, which can make one feel light-headed and confused, or even unconsciousness. (NHS Choices, 2012)

As such, antibiotics are shown to have a vast number of negative side effects, with the reasons as shown below:

There are thousands of species of bacteria, yeast and micro-organisms live in and on the human body. Balance in the stomach flora helps keep our body healthy. For example, the bacteria in our gut helps in the digestion of stomach content. However, antibiotics make the bacteria composition in our stomach flora imbalanced as a side effect as antibiotics do not discriminate in the killing of bacteria. These results in the symptoms and conditions stated above. (Lewine, 2012)

Despite there being effects negative to one’s well being, there are solutions to potentially negate or reduce them, with the simplest and often most effective solution being the taking of probiotics.

The idea behind using probiotics to counter the effects of antibiotics is that probiotics tend help populations of good bacteria recover more quickly and restore order to the intestines as they often contain the bacteria essential for our bodies, reversing the and reducing the damage of the antibiotics and returning the bacterial compositions in the body to healthier levels. (Lewine, 2012)

Chen said (2012), “I had an older female patient who came to me for irritable bowel syndrome with a propensity toward constipation and gastroesophageal reflux. After our initial discussion as to what might be beneficial for her work up, she decided she wanted food sensitivity testing. When the results came back, we explored the categories of foods that caused inflammation in her system and she consequently removed those foods from her diet. Her symptoms improved about 60 percent, but she wanted to know if there was anything else she can do to help the rest of her symptoms.
When she first started seeing me, she wasn't sure she wanted any more supplements, since she felt that she takes enough and felt certain that removal of food triggers alone would rid her of all her gastrointestinal issues. After our discussion once she had removed food triggers and symptoms improved but plateaued, she wanted to give probiotics a try. We started her on a gluten-free, dairy-free probiotic and after about four weeks on it, she noticed that her symptoms were mostly gone. We discussed the idea that she may have had some small bowel dysregulation in flora after many years of irritation and inflammation of the gastrointestinal tract from food triggers and that she needed to rebalance the flora after the removal of food triggers to get her bowel to fully heal. She is now on probiotics daily and recently told me that she had fewer colds last winter season, which she attributes to the probiotic's role on a healthy immune system.”

In the case of that older patient, it is quite evident that the probiotics helped with the gastrointestinal issues as well as helping the patient achieve and maintain a healthy immune system for fighting against colds. As such, this is an example of how probiotics positively aided in creating a healthy body.

It is undeniable that we take in more than one liquid per day. Hence, when drinking probiotics, it is inevitable that they would consume another liquid. Hence, we had to choose to pick the common drinks, out of the many from WebMD (2012), Coffee, Tea, Cola, Energy Drinks and Milk. From these drinks, we would want to find out its effects Lactobacillus. Casei,(representing probiotics) since it is commonly found in many probiotic drinks.

As a result, we decided to choose Lactobacillus casei and various commonly consumed beverages as the basis of the project. Lactobacillus, a probiotic, plays a significant part in our research. Due to its easy access, we decided to use L.casei as the main basis in conducting this experiment, which simultaneously also serve as a representative for the wide range of probiotics.

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:

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.

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 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 existing in the outer membrane, Presence of periplasmic space, Mainly endotoxins(harmful) 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.

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

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. While on the other hand, Gram Positive bacteria are mostly harmless, or have some benefits (which later can be used to represent probiotics), and some harmful ones. (Little)

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.

The results would tell us the effects of the various beverages on lactobacillus and thus will inform us about which beverage is good for probiotics and warn the people to avoid which beverages when drinking probiotic drinks.

Research Question 
Inside the human body, there are naturally occurring antibodies manufactured by the immune system. By function, these antibodies detect and eliminate or neutralize the foreign threats in our body. These foreign threats ranging from harmful bacteria to viruses. However, they have limits and antibiotics for medical use are often brought in to help face the more dominant threats. With antibiotics introduced, there are positive benefits, but also negative impacts. These disadvantages are also the antibiotic’s side effects as stated in the prescription of these antibiotics. These side effects are caused when the balanced composition of intestinal flora is disrupted, due to the beneficial bacteria being caught in friendly fire in the process when antibiotics eliminates the harmful bacteria in the body, which becomes a hazard to human health. As a result, we take probiotic drinks to counter this imbalance in stomach flora. However, probiotic drinks are frequently taken with common beverages. Therefore, we wanted to find out whether these beverages taken have any effect on the probiotics in the gut flora which leads to our study, Investigation on effects of Lactobacillus on various beverages.

Question addressed
As mentioned in 1.2.1, we want to find out whether the consumption of commonly consumed beverages affect probiotic growth, particularly lactobacillus in our body. So, the research question is, “Do the beverages we consume affect probiotics (lactobacillus) or it’s growth in our body?”.

All of beverages tested would affect Lactobacillus. casei, by at most 30% negatively compared to Sterilized Water. (Amount of Colonies grown after being put with beverages is 30% less than amount of colonies grown after being put with Sterilized Water.)

Why is this so? Sterilized Water is our control. It represents the normal flora. The most favorable situation we would want is all the beverages affecting the Lactobacillus as though they were in the normal flora. In this case, sterilized water represents our normal flora. Why 30%? 30% is a neutral number as anything more than 30% would be considered quite harmful to the human body and would not be that possible. Killing above 30% of bacteria would make the beverage like an antibiotic, where it will even make the bacterial composition in flora worse. 30% because 40% is too much as it is almost half of the whole bacterial population and would be harmful. 30% is the ideal percentage as it is the line between harmful and harmless.
We expect that most of the beverages would affect the Lactobacillus colonies positively, and little actually inhibiting the growth. However, there might be just some chemicals in the beverages that Lactobacillus that might be sensitive to and thus, we set the cap of the beverages affecting the lactobacillus colonies  negatively at 30%.

Independent variable(s)
Type of beverages used.

Dependent variable
Amount of Lactobacillus grown on MRS plates. (CFU)

1. Location of testing environment.
2. Testing Equipment (sizes of sterile filter dish and petri dishes)
3. Type of lactobacillus. (Lactobacillus. Casei Strain Shirota)
4. Amount of lactobacillus (CFU)
5. Type of beverage used for each test (e.g. If a bottle of coke is used for trial 1, same bottle of coke should be used for trial 2)
6. Amount of beverage (ml)

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