If I use Lysol Toilet Bowl Cleaner, then the most bacteria will be reduced, because Lysol contains a higher concentration of hydrochloric acid, thus increasing chemical reactions and promoting particle collisions.

Background Research

 

What are Bacteria?

 

Bacteria are microscopic single-celled prokaryotes of the kingdom of eubacteria that possess a peptidoglycan cell wall and a cytoplasmic membrane, ranging from 0.1 to 5 micrometers in diameter, causing them to be considerably smaller than most eukaryotic cells. They live everywhere, including inside of you, with roughly 10 times more bacteria cells than human cells within the human body. That may sound scary, but only a small fraction of them are pathogenic, while the majority of bacteria are actually beneficial for us. The majority help you digest food, with at least 30 trillion bacteria within your gut, up to 300 trillion, but there are many other bacteria throughout the rest of your body, including your reproductive system and urinary tract. 

 

How Might You Classify Bacteria?

 

Bacteria are classified in many ways, but some of the most common methods are shape, cell wall, motility, taxonomic ranks. These allow scientists to understand and classify a new bacterium or narrow a disease down. Additionally, it can allow for more precise findings during testing, where a potentially life-threatening disease could be mistaken for harmless bacteria, as in the case of nontuberculous mycobacteria. These bacteria are found in our soil and are inhaled constantly every day, only affecting a few individuals as a lung infection, but are in the same genus as tuberculous mycobacteria, or, as it is more commonly known as, tuberculosis. If a scientist were to mistake these two, it could have potentially life-threatening consequences for the public.

 

Shape of Bacteria

 

Arguably the most common method is the shape of the bacteria, as it is the simplest, and requires only a microscope. Bacteria have three main shapes, sphere-shaped, rod-shaped, and spiral-shaped, called cocci, bacilli, and spirochetes respectively. These are the most common, as there are a vast number of unusually shaped bacteria, such as Bdellovibrio and filamentous bacteria, but these appear very little when compared to the vast number of cocci, bacilli and spirochetes. Additionally, there are coccobacilli, which are oval-shaped and appear commonly throughout nature. 

 

Taxonomic Hierarchy

 

Additionally, you can sort bacteria by their taxonomic hierarchy. This is used to classify all living things, from mammals to plants to everything in between. Because bacteria are living things, they are also placed on the hierarchy. There are three main parts of taxonomy, classification, nomenclature, and identification, but for this portion, we will be talking about identification. For this example, we will use the bacterium Aureus anaerobius, which is in the bacteria domain. Domains are used to separate Eucarya, Archaea and Bacteria, and afterwards separate them additionally. At the highest level after domain, which is called the kingdom, Prokaryotes, there are the most living organisms, but it is the least specified, being used for further classification for eukaryotes. Afterwards, the phylum is slightly more specific, in splitting into Bacteria and away from other prokaryotes. The division Firmicutes consists mostly of cells with a Gram-positive cell wall. The class Bacilli are based on the shape of bacteria, while Bacillales is an order containing the most productive order in the Firmicutes division. Family and genus begin to get more specific, with Staphylococcus and Staphylococcaceae containing many medically significant bacteria. Finally, species and subspecies are extraordinarily precise and are more focused on the species itself. 

 

Bacterial Motility

 

Bacterial motility is the ability of a bacterium to actively move through the environment, typically as a response to various stimuli such as chemotaxis, phototaxis and magnetotaxis. This is essential for survival, enabling a bacterium to find nutrients, evade harmful substances, and take over an environment. There are 5 primary methods of motility, being through flagella, twitching, gliding, swarming and spirochete motility. The most common method of motility is flagellar motility, which is through the use of flagella, whip like appendages that rotate like propellers to push or pull the cell forward. This method has three variations, being monotrichous, lophotrichous and peritrichous, which are based around the number of flagella a cell has. Monotrichous cells have a single flagellum, such as Vibrio cholerae. Lophotrichous cells have a cluster of flagella at one end, such as Pseudomonas. Finally, peritrichous cells have flagella distributed all over the surface of the cell, as in the case of Escherichia coli. Swarming motility is similar, as it is the coordinated movement of a bacterial population using flagella, generally across a moist surface, such as with Proteus mirabilis.

Another type of motility is twitching motility, which is when bacteria utilize retractable type 4 pili to attach to the environment and pull themselves forward, as shown with the bacteria Neisseria gonorrhoeae. Additionally, gliding motility is a slower, smoother movement of bacteria occurring without flagella, often involving the secretion of polysaccharides or surface proteins, which is utilized by Cyanobacteria.

Spirochetes have their own method of motility, through the use of internal flagella, or axial filaments. These filaments cause corkscrewing of the cell body through viscous environments, such as in the case of Borrelia burgdorferi.

 

Gram Staining

 

Bacteria cell walls consist of complex, multilayered cell envelopes that help protect them from the often hostile environment around them. The bacteria cell envelope falls into 2 main categories, Gram-negative and Gram-positive. 

Since as early as the 1830s, scientists have understood that all living organisms are composed of cells. These cells have a definite boundary called the plasma membrane. That means that the living matter must be contained within the membrane, and everything outside the membrane is non-living. In almost all cells, this structure is a phospholipid bilayer that surrounds and contains the cytoplasm, which helps keep the structure of the cell. Additionally, there are proteins interspersed throughout the cell wall, which makes each cell unique as there are trillions of possible combinations.

In 1884 a scientist by the name Christian Gram found out the main way to differentiate the types of bacteria, called gram staining. Many things differentiate them, but the main differences are based on the cell wall. Gram-negative bacteria contain a peptidoglycan (PG) cell wall surrounded by a membrane containing lipopolysaccharides (LPS), which help prevent osmotic pressure. Gram-positive bacteria lack an outer membrane, succumbing to osmotic pressure more easily, but have a peptidoglycan cell wall that is several times thicker than that of Gram-negative bacteria. Threading through these layers are anionic polymers called teichoic acids, which are involved in interactions with the host's immune system, inflammation, and even septic shock.

Different cell walls can additionally impact the uses of common antibiotics such as penicillin, which works by inhibiting cell wall synthesis by targeting penicillin-binding proteins in the cell wall, working extraordinarily well against gram-positive bacteria. However, the outer membrane of gram-negative bacteria acts as a barrier against penicillin. This makes it so that gram-negative bacteria are more antibiotic-resistant, needing different antibiotics to break down the cell membrane.

Moreover, these cell envelopes allow specific nutrients through while preventing other more dangerous substances into the cell. This is called selective nutrient transfer, and one of the main ways that some cleaners operate is by having a similar chemical structure to these nutrients. These chemicals are then absorbed into the cell and start to deform the cell, whether by coagulating proteins or by disabling the cell wall and allowing outside sources to harm and kill the bacterium.

 

 

Key Differences Between Gram-Positive and Gram-Negative Bacteria:

 

Types of Cleaners and the Effects of Them on Bacteria

 

Various cleaners can have vastly different effects on bacteria, as they break down bacteria differently, but the majority of cleaners target the cell wall, which is the main portion of the cell that separates it from the environment. Therefore, a cleaner can either pierce the cell wall and damage the inside of the cell or break apart the cell wall and let exposure damage the cell beyond repair.

One of the ways that some cleaners work is by disrupting the cell wall, behaving like proteins in the cell and replacing them, called chemical antagonists. Additionally, they can behave by damaging the amino acids within the cell wall, such as some aldehydes like formaldehyde. Furthermore, ethyl alcohol works in a process called denaturation, which is the unfolding of proteins within a cell. These mainly work on bacteria, and have little to no effect on spores and viruses. Moreover, some cleaners coagulate enzymatic proteins, killing the bacteria, whilst some acids such as acetic acid are absorbed through the cell membrane. 

 

DEFINITIONS

 

Amphipathic 

— Of a molecule, especially a protein, with both hydrophilic and hydrophobic parts.

 

Anionic Polymer

— A chain of millions of carbon atoms on which negatively charged spots are attached along the chain, acting like molecular hooks. 

 

Chemotaxis

— Directed cellular movement due to chemical energy

 

Flagella

 

— Strands of hair-like structures on bacteria that allow movement through various env

 

Glycoconjugates 

— Compounds in biology consisting of carbohydrates linked to non-sugar molecules like proteins, peptides, and lipids.

Lipopolysaccharides

— Large amphipathic glycoconjugates consisting of a hydrophobic lipid domain attached to a core oligosaccharide and a distal polysaccharide.

 

Gram-staining

— The act of staining bacteria to differentiate the cell wall of bacteria, Gram positive bacteria stain

 

Magnetotaxis

— Directed cellular movement due to magnetic energy

 

Monosaccharides, Oligosaccharides, and Polysaccharides 

— Monosaccharides are the simplest carbohydrates, chemically being aldehydes or ketones possessing 2 or more hydroxyl (-OH) groups. Oligosaccharides contain 2–10 monosaccharides, and polysaccharides contain over 10.

 

Peptidoglycan

— A carbohydrate that borders the bacterial cytoplasmic membrane and helps preserve the structure of the cell from environmental pressure.

 

Phototaxis

— Directed cellular movement due to light energy

 

Sulfhydryl

— A sulfur atom, with two lone pairs bonded with a hydrogen atom, contributes to protein structure and amino acids such as cysteine.

 

Teichoic Acids

— Anionic polymers found in Gram-positive bacteria cell walls, made up of polyglycerol phosphate units (approximately 20–30 repeats). They are involved in the regulation of cell morphology and division.

 

Manipulated: 

• Type of cleaner used (Mr. Clean, Method, Lysol Toilet Bowl Cleaner, or vinegar)

Responding:

• Amount of bacteria left in the agar plate at the end of the experiment

Controlled:

• Location the bacteria is grown in.
• Amount of cleaner used in experiment. (10ml)
• Brand of petri dishes.
• Amount of bacteria applied onto petri dish.
• Application of bacteria onto petri dish.
• Application of cleaners on petri dish.
• Amount of time for the bacteria to grow.
• Location the bacteria is taken from. (Kitchen sink drain)
• Amount of time the cleaner contacts the bacteria.
• Same temperature of cleaner.
• Same age of cleaner.
• No contamination. (Oils, food, etc.)

​​​​​​Materials:

•  Premade agar plates from Agar Plates - Amazon.ca 
•  Latex gloves
•  Face mask
•  Lysol toilet bowl cleaner (20ml, divided into two 10ml containers)
•  Vinegar (20ml, divided into two 10ml containers)
•  Mr. Clean (20ml, divided into two 10ml containers)
•  Method bathroom cleaner (20ml, divided into two 10ml containers)
•  Kitchen sink
•  Safe location to keep bacteria
•  Safe way to dispose of bacteria

 

Procedure:

1.  Put on protective gear such as gloves and face mask.
2.  Prepare petri dishes as per the instructions on the package.
3.  Swab kitchen sink drain with a cotton swab.
4.  Smear the swab onto the petri dish in a zigzag pattern to cover the most area.
5.  Seal petri dish by taping it shut with duct tape.
6.  Let bacteria grow for 72 hours.
7.  Set 2 petri dishes aside for control petri dishes.
8.  Open petri dish by breaking tape seal.
9.  Apply 10ml of cleaner onto petri dish.
10.  Wait 24 hours for cleaner to work.
11.  Repeat steps 3–9 for all cleaners twice.
12.  After gathering data, dispose of bacteria in a safe location.

 

Observations:

 

Analysis:

 

The most effective cleaner was Mr. Clean, being consistently above a 70% reduction rate, except for one of the trials, where it didn’t eliminate any large colonies, as no large colonies grew in that sample. Lysol was moderately effective on colonies of a middling size, but surprisingly wasn’t effective on larger colonies. Vinegar was the least effective, eliminating under 40% of the bacterial colonies in the majority of trials, even allowing growth of medium colonies in one circumstance. The only occurrence of a reduction of over 50% with vinegar was when only one large colony was grown in one of the vinegar trials, which was eliminated. Finally, Method was the overall least consistent cleaner, not reducing any of the larger colonies, and in one trial didn’t reduce any medium colonies either, but in the second trial, reduced 20 of the 21 medium colonies, leading to a decrease of over 95%. 

These surprisingly low total decreases across all trials may be attributed to the cell wall of the bacteria used in this experiment, which I believe may have primarily been Gram-positive. There are two types of cell walls, Gram-positive and Gram-negative. These have two fundamental differences, being the thickness of the cell wall, and the possession of a cell membrane. A Gram-positive cell has a thicker peptidoglycan wall, with up to 40 layers of peptidoglycan, although lacking a cell membrane. This allows for a stronger protective barrier against the cell wall being pierced, but allows more absorption of debris or proteins, as well as chemicals. Meanwhile, Gram-negative bacteria have only two or three peptidoglycan layers, but have an outer cell membrane, which helps to prevent unnecessary or harmful debris from being absorbed, including some chemical cleaners, at the lack of protection from piercing or otherwise damaging the cell wall. This is important, as some cleaners work by being absorbed through the cell wall, and others break down the cell wall.

One of the cleaners that are absorbed through the cell wall, and thus was more effective, was Mr. Clean, with a rate of reduction over 65%. Mr. Clean is a sodium hypochlorite (bleach) based cleaner, which is absorbed through the cell wall and coagulates proteins within the cell, rendering the cell inoperative. This type of cleaner is most effective on Gram-positive bacteria, as the outer membrane of Gram-negative bacteria restricts the ability for the cleaner to be absorbed into the cell. Furthermore, Lysol is hydrochloric acid based, which works by breaking down the cell membrane, something that is considerably easier to do to Gram-negative bacteria, due to their substantially thinner cell walls, requiring less effort to break apart. As the Lysol did poorly, this furthers the theory that the bacteria were Gram-positive, as Lysol had a rate of reduction under 40%. This is further supported by the fact that Mr. Clean is 6% sodium hypochlorite, whilst Lysol is almost 10% hydrochloric acid. A higher concentration of cleaner leads to a higher amount of collisions with the particles, allowing for more cleaning in less time. 

Finally, the more natural cleaners did relatively poorly, as they have lower chemical concentrations, with vinegar being 5% acetic acid, and Method having potassium hydroxide, a chemical used in conjunction with water to make eco-friendly soap. Acetic acid is most effective on Gram-positive cells, as its main antimicrobial effect is through passive diffusion of the cell wall, something that is exceedingly more difficult to do to Gram-negative bacteria. This is in stark contrast to the poor reduction of bacteria, which may have been considerably worse had the bacteria been mostly gram negative. Furthermore, Although potassium hydroxide is used in high concentrations, normally with a 25% concentration, the chemical itself is not very effective, being gentle enough on skin cells to not elicit reactions. Potassium hydroxide is ineffective to use on Gram-positive bacteria, as it behaves through a process called alkaline lysis, which is the degrading of the cell wall. This is most effective to utilize on Gram-negative bacteria, as they have considerably thinner cell walls. In addition, the gentleness of the cleaner may have been an additional component in the ineffectiveness of Method. To conclude, both of these cleaners are relatively gentle cleaners, which could lead to them being more ineffectual on bacteria, as they are made by either having a lower concentration, as in the case of vinegar, or by using more impotent chemicals, although at a higher ratio, which is most likely why these cleaners both had average reductions of under 35%, with Method having an average reduction of less than 25%. 

 

Trends and Outliers:

 

The cleaners worked less effectively than anticipated, as there was a relatively low average reduction across all trials. Significant differences were observed between the highest reduction and the average reduction for some cleaners. For instance, Method had an average reduction of 24%, but in one trial had over 95% reduction of medium colonies. Similarly, vinegar did poorly overall, with a 31% reduction, but had a 100% reduction of large colonies in one trial. Mr. Clean appeared to be the most consistent, with its only anomaly occurring when no large colonies grew. In severe contrast, Lysol performed considerably worse than expected. Although it achieved high reductions of medium colonies in both trials, over 70%, the average reduction was only 40%, as it was ineffective on large colonies. 

Additionally, another outlier was in one of the vinegar trials. In this trial, there were 7 more medium colonies after the cleaner was applied (69), than when before the cleaner was applied (62). This implies that some bacteria within that petri dish were able to not only survive in acetic acid, but grow and thrive within it.

 

Conclusions:

 

In conclusion, my hypothesis was incorrect, as Lysol was over 30% less effective than Mr. Clean, working poorly on larger colonies. I believe that this was most likely due to the fact that most of the bacteria were Gram-positive, inhibiting the potential effectiveness of the hydrochloric acid within the Lysol, due to the considerably thicker cell wall. This is further evidenced by the fact that Mr. Clean, the most effective cleaner, is bleach based, working most effectively on Gram-positive bacteria, as bleach is absorbed through the cell wall and coagulates proteins within the bacteria, something that is substantially more difficult to do to Gram-negative bacteria, due to the cell membrane.

 

Real World Applications:

The potential real world applications are vast for this science fair project. Firstly, knowing which cleaners to use to clean your sinks and countertops are important, especially so in a kitchen, where, without proper disinfection, you run the risk of getting salmonella or a similar foodborne illness. In addition to kitchens, this information could be utilized for cleaning healthcare facilities, where disinfecting medical equipment, operating rooms, and patient rooms can be integral to immunocompromised patients. Schools and daycare centres have shared surfaces such as desks, which can have a host of harmful bacteria, similarly to public areas such as transportation and gyms, where there are shared spaces and equipment. Finally, water treatment plants require proper disinfection of the water to make it potable. To conclude, there are many real world applications, allowing for substantial research and study on this topic.

 

Although the margins of error were relatively small, there were a few areas where human error could have played a role in problems that occurred. For instance, although I had protective equipment, there was potential for contamination of the agar plates, exposing the experiment to external sources of bacteria. This would have expanded the bacterial spectrum, heavily impacting the effectiveness of various cleaners. Moreover, I could have incorrectly sealed the containers, exposing the bacteria to extraneous environments, severely impacting the scope of bacteria, increasing it to unreasonable levels for this experiment.

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I would like to acknowledge the traditional territories and oral practices of the Treaty Seven nations, including the Siksika, the Piikani, the Kainai, the Stoney Nakoda, consisting of the Chiniki, Bearspaw, and Good Stoney, and the Tsuut'ina first nations. I would also like to acknowledge the Metis of the treaty 7 region of southern Alberta. Likewise, I appreciate the opportunity to call this land my home, alongside the First Nations peoples who have lived here for millennia. Additionally, I would like to thank my family and friends for all the support they have given me, over the past several months.