Disinfectant Investigation

Science

Aim: 

I want to investigate how different disinfectant concentrations can affect the growth and reproduction of micro-organisms, more specifically bacteria.

Hypothesis:
I predict that the least concentrated disinfectant will kill the least amount of bacteria compared to a highly concentrated sample, therefore affecting growth and reproduction in bacterial communities.

Variables:

Independent Variable;

Because this is a fair-test investigation, the variable I will be changing is the concentration of the disinfectant. In order to change the concentration, the amount of Dettol will be the same for all samples, but the amounts of water added to the disinfectant will vary among all samples.

For this variable, the equipment I will use is:

• Full-strength Dettol
• Manufacturers instructions
• No Dettol - i.e water
• Diluted Dettol

Dependent Variable;
The variable being measured is the size of the clear-zone around the Dettol disc. Using a ruler, I will measure the diameter of the clear-zone. Measuring in millimeters in this case is more suitable and accurate compared to centimeters.

Other Variables;
Other variables that can change the outcome of the experiment:

• Growing temperature - needs to have the same temperature for all samples
• Growing medium - needs to have the same medium for all samples
• Growing time - needs to have the same amount of time for all samples
• Size of disc - the size of the disc needs to have the same size for all samples

Reliability:

To ensure reliability, I will repeat the experiment three times to be consistent in this investigation.

Equipment:

• Agar plate
• Dettol
• Vivid
• Filter paper
• Hole punch
• Water
• Sellotape
• A solution of bacteria
• Pipette
• Tweezers 
• Measuring cylinder 
• Ruler

Method:

1. Gather equipment

2. Using the vivid, evenly mark out the agar plate into quadrants. Then at the bottom of the agar plate, label the four quadrants appropriately (Full-Strength, Manufactured Instructions, Diluted, Water) Then label name on the rim of lid.

               

"F" stands for Full-strength

"M" stands for Manufacturers Instructions

"W" stands for Water

"D" stands for Diluted

3. Using the hole punch, punch out four disc of filter paper

4. Using a pipette, transfer a few drops of pepper solution onto the agar plate. Swirl. Tip out the excess solution from the agar plate.



5. Using the tweezers, soak an unused disc into water and shake out any excess liquid. Place the disc into the quadrant that is labelled "Water". (0% concentrate of Dettol)

               

6. Using the tweezers, soak a disc in full-strength Dettol. Shake out any excess liquid and place the disc into the quadrant that is labelled "Full-Strength". (100% concentrate of Dettol)

     

7. Pour 200mL of Dettol into measuring cylinder and then add 400mL of water. Using the tweezers, soak an unused disc into the mixture and shake out any excess liquid. Place the disc into the quadrant that is labelled "Manufacturers Instructions". (1:20 diluted/5% concentrate of Dettol)

              

8. Add a further 400mL of water to the manufacturers instructions mixture, and using the tweezers, soak an unused disc into the diluted mixture and shake out any excess liquid. Place the disc into the quadrant that is labelled "Diluted". (2.5% concentrate of Dettol)

                

9. Using sellotape, fasten the lid onto the container. Then incubate the agar plate at 20 degrees for 24 hours upside down so that any remaining moisture will fall to the bottom.

             

10. Using a ruler, measure the diameter of the clear zone for each individual disc.

11. Repeat the experiment two more times for reliable results and analyze results.

Notes From Trial:

The results from the trial aren't as successful as we hoped it would be. They are not uniform and the clear zones are indistinguishable. There are many contributing factors as to why these results are unreliable. For instance:

The results from the trial may not be reliable because variables that should've been regulated changed the outcome of the experiment. The bacteria was meant to be incubated over 24 hours at 20 degrees, but since the agar plate was left out for three days before being incubated for 24 hours (variable affecting the experiment would be time) and was left at changing room temperature for those three days (variable affecting the experiment would be temperature), this would've indefinitely affected the growth and reproduction of bacteria since bacteria grows every 20 minutes.

Another factor that might've affected the outcome of the experiment is that it could've been Claudia and I not sealing the lid to the container properly. Perhaps if we had taped it along the rim, no excess air would be able to enter the agar plate. Although, I will admit this theory is quite unlikely since the sealing was sufficient.

Changes to the experiment:

• Incubate the agar plate once the plate is sealed properly and upside down.
• Disinfect the tweezers in between discs

Results:

Hypothetically, the more concentrated the solution, the bigger the clear zone. On all results (exempting trial), I came to the realization that the labels were incorrect and misplaced since it didn't follow the outline. As a result of this, I've taken the liberty of swapping the labels to their more practical places. 

         

         

Analysis:

Plate 1's results and measurements are consistent on paper, but not so easily distinguishable visually.

Plate 2's results is the most successful out of the four. The clear zones are more preferable as they are more distinguishable. 

Plate 3's results are smaller compared to the other results and may affect the average, but Plate 3's results are correct in terms of proportion.

Plate 4 is unreliable since water ended up killing a great number of bacteria. This is improbable because the conditions the water was incubated at (20°C) was in the danger zone (4.4°C-60°C). The danger zone is where bacteria grows most rapidly. Temperatures extremely above and below this range will not allow bacteria to grow and multiply, so I can only conclude that somehow excess Dettol got onto the water disc. It is also plausible that because Christchurch's water supply is chlorinated, perhaps the chlorine killed bacteria. According to the online news media, Stuff: "The council has since recorded an average of 0.64ppm (parts per million) over 1395 tests" in areas all around Christchurch. But from what I can conclude, in all of the other plates, the results are consistent in being 0mm, indicating that the low concentration of chlorine in the water haven't killed this bacteria solution (microflora of black-pepper), so I am discounting Plate 4's results, as it would affect the average.

Conclusion:

To conclude, I predicted that the higher the concentrate of the disinfectant was, the more the amount of bacteria would be killed compared to a lower concentrated sample, therefore affecting the growth and reproduction of micro-organisms. From my graph, the trend of the line increases. As the Dettol concentration increases, so does the size of the clear zone around the filter paper.

Explanation:
 

Structure of Bacteria
Bacteria are singular-celled micro-organisms that exist both inside and outside other organisms, living in communities of millions. Some are quite helpful to humans, whilst others can make humans sick... or in extreme cases, some bacteria can be lethal. There are five basic types of bacteria: cocci (spherically-shaped), spirilla (spiral-shaped), vibrios (comma-shaped), spirochaetes (corkscrew-shaped), and the one we're focusing on: bacilli (rod-shaped). Unlike protista, fungi, plant, and animal cells which are eukaryotic, bacteria are prokaryotes and do not contain a nucleus. Instead, circular DNA are found in an area called the nucleoid region which contain the genetic information. The nucleiod is a region of cytoplasm which is not in fact bound by a nuclear membrane. Bacteria are cellular structures and the gel-like cytoplasm which fills up the cell consist of water, nutrients, enzymes and gases. Cytoplasm is where all chemical reactions happen as the functions for cell growth, metabolism, and replication are performed here as the cytoplasm contains cell structures like plasmids, chromosomes, and ribosomes. From the above mentioned, plasmids are small, extra-chromosomal genetic material which codes for non-essential characteristics. In no shape or form are they involved in  the primary means of reproduction in bacteria... only the DNA contains the genetic instructions for both initiating and carrying out binary fission (where the cell divides to make exact copies of themselves, which I will get into more depth later). Also free-floating in the cytoplasm are ribosomes. Ribosomes are where RNA is translated into protein (protein synthesis). Proteins are crucial for fundamental cell functions like repairing damage or directing chemical processes. There are two to three layers that encase the cell called the cell envelope. The first layer is made up of phospholipids and proteins, and is called the cell membrane. Enclosing the interior of the bacteria, the cell membrane both regulates what materials enter and exit the cell, and is adaptive to different living conditions. The second layer is made up of peptidoglycan (a polymer), and is called the cell wall. The cell wall is rigid and gives the cell its structure whilst protecting the cell membrane from the environment. Depending on the strength of the wall, the cell wall is responsible for keeping the cell from bursting. And in some species of bacteria, a third protective layer is present and is called the capsule. Made up of polysaccharides (complex carbohydrates), the capsule's responsibilities are to keep the cell from drying out and protect the bacteria from being engulfed by larger micro-organisms. The capsule is also a vital factor in causing disease. For locomotion, bacteria have a lash-like appendage which originates from the cytoplasmic membrane, which is anchored by the cell wall and protrudes out from the capsule called the flagella. The flagella is a tail and acts like a propeller so that the cell can move towards nutrients and away from toxic chemicals. Like the flagella, pili also originate from the cytoplasmic membrane, are anchored by the cell wall and project from the capsule. Some types of plasmids form pili, which are small and hair-like and their purpose is to allow the cell to attach to other cells and surfaces and transfer copies of the plasmid/genetic material to other bacteria (conjugation). Conjugation is beneficial in the respects of sharing advantageous genetic material with other bacteria, such as antibiotic resistance.

https://simple.wikipedia.org/wiki/Binary_fission

Bacterial Reproduction

Most common in prokryates such as bacteria, binary fission -- a kind of asexual reproduction -- is where the cell divides to produce two new exact copies of itself. For most plasmid types, copies in the cytoplasm will also be passed on to daughter cells during binary fission. First, the parent cell elongates as it builds up more cell wall. Secondly, the bacterial genome (the complete set of genes) replicates whilst remaining attached to the cell membrane. During this step, any remaining copies of plasmids in the cytoplasm are replicated. Duplicated DNA then separate, moving towards the poles as the cell elongates further. The fourth and fifth step is where the cell wall forms a furrow. Breaking free from each other, one cell finally becomes two as two identical daughter cells are produced. Typically, Escherichia coli cells (a bacteria found in black peppercorn which is in our bacterial solution) divide about every 20 minutes at 37 °C. The purpose of binary fission is to reproduce and grow bacterial communities.

http://lifeofplant.blogspot.com/2011/12/active-transport.html

Nutrition
In order for bacteria to grow and reproduce, bacteria use a process called direct transport. Through direct transport, bacteria secrete enzymes onto their food to break it down into simpler forms. And depending on the circumstances, bacteria can either absorb nutrients through the cell membrane by passive diffusion or active transport. Through passive diffusion, nutrients move from a high concentration (outside of the bacteria) to a low concentration (in the bacteria) -- the act similar to the function of a sponge. Unlike passive diffusion, active diffusion goes against the concentration gradient (from low to high), and because of this, requires the use of an energy/protein carrier (e.g ATP). Pump-mediated, the direct active transport method is where the walls of the bacteria act as a vacuum and absorb the surrounding nutrients.

Bacteria Found in the Bacterial Solution
The bacterial solution we used for this experiment was made out of black peppercorn and water. Black peppercorn is especially rich in bacteria and consists of many colonies of fungi/microflora such as Escherichia coli (gram-negative), E. freudii, Serratia sp. (gram negative), Klebsiella sp. (gram-negative), Bacillus sp. (gram-positive), Staphylococcus sp. (gram-positive), and Streptococcus sp. (gram-positive). The thickness of the bacterial cell wall can determine if the bacteria is either gram-positive or gram-negative. A technique known as gram staining is used to differentiate these two groups. Gram-positive cells have a thick layer of peptidoglycan in their cell walls and stain violet; Dettol is most effective against gram-positive cells. Gram-negative cells have a thinner peptidoglycan wall and stain red; Dettol is generally more resistant to gram-negative cells.

Chloroxylenol and it's Effects on Bacteria
80 years ago, Dettol Antiseptic solution was first used in hospitals to disenfect the skin before procedures... today, Dettol can be found in most family homes with the sole purpose to kill germs and create a clean hygienic environment. Chloroxylenol (C₈H₉OCl) is an antiseptic and a chemical used in Dettol. This chemical is most commonly used for disinfecting skin, surgical equipment, households, and wounds to prevent infection. From this experiment, the Dettol from the disc diffuses into the bacterial cells from the bacterial solution and the agent breaks the cell wall and deactivates enzymes in the bacteria. Enzymes help accelerate the rate of chemical reactions in bacteria and ultimately help accelerate growth. In this case, once the enzyme in the bacteria is deactivated, the ability in the bacteria to consume waste and turn it into simple waste is limited because the enzyme can't convert the more complex waste into simpler waste. Chloroxylenol acts as a proton gradient disrupter. Using proton gradients, bacteria produce Adenosine triphosphate (ATP) -- which is an energy system/store in biological systems. By disrupting the cell membrane potentials, this act blocks ATP, therefore starving the bacterial cells to death. Ultimately, chloroxylenol at higher concentrations will rapidly kill bacterial cells since the proteins and nucleic acids in the cell are suffocated and cease to function. Without cells to reproduce, the population decreases rather than increases as evident from the experiment. In everyday life, using full-strength dettol would be impractical since it's a waste of resource. Yes, it kills the highest abundance of bacteria... but this experiment has proved that a lower concentration of Dettol is still efficient in killing bacteria. On the manufacturers instruction's disc samples, the clear-zone around the discs was an average of 18.33mm. This is why manufacturers instructions recommend a 1:20 dilution of Dettol since it's both efficient in killing bacteria and saving money.

Evaluation:
Overall, the results of this experiment are successful since the aim of the investigation: "to investigate how different disinfectant concentrations can affect the growth and reproduction of bacteria," is evident from the experiment. The higher the concentration of Dettol, the more bacteria killed, therefore affecting the growth and reproduction of bacteria in a community. The results from the experiment are reliable since the experiment has been repeated three times to prove consistency, exempting the trial and the fourth plate. As mentioned before, the trial is unreliable since variables that should've been regulated to be equal with the other plates (time and temperature) changed the outcome of the experiment. To remedy the trial, we repeated it an extra three times, regulating every variable to be the same. Although, one anomaly in the experiment that didn't fit the pattern was plate 4's water clear-zone. Plate 4's water clear-zone was 12mm and all the other results are consistent in being 0mm. From this information, I have concluded that somehow excess Dettol got onto that disc. For future experiments, extra precautions should be put into sterilizing equipment in between every disc to prevent future anomalies. One question that has now arisen from this investigation is the possibility of bacteria growing a resistance to Dettol. Can the bacteria present in the solution gain a mutation to resist chloroxylenol if lower concentrations of the chemical is used?

Bibliography:
https://www.stuff.co.nz/the-press/news/103631494/chlorine-levels-in-some-christchurch-water-might-be-enough-for-swimming-pool

https://micro.magnet.fsu.edu/cells/bacteriacell.html

https://www.medicalnewstoday.com/articles/157973.php

https://www.quora.com/How-do-bacteria-consume-food

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC546988/

http://www.dettol.co.nz/about-us/our-history/our-history/

https://en.wikipedia.org/wiki/Chloroxylenol

https://sites.google.com/site/ksnc14scienceproject/how-does-chloroxylenol-kill-bacteria