Bacteria and disinfectants

Title: Bacteria and disinfectants
Purpose: How concentration can affect the performance rate of disinfectants in killing micro-organisms.

Part 1

Aim: Our aim is to find out how concentration can affect the performance rate of disinfectants in killing micro-organisms.

Hypothesis: I think the higher the concentration of savlon, the bigger the clear zone will be.

Independent Variable: The independent variable that we will be changing is the concentration of the savlon.
Dependent Variable: The dependent variable that we will be measuring is size of the clear zone around the piece of filter paper soaked in a concentration of savlon.
We will be measuring the size of the clear zone using a ruler and the units we'll be using are in millimeters.

Quadrant 1 will be controlled, this means there will not be any dettol on a piece of filter paper.'
Quadrant 2 will have 10% dettol on a piece of filter paper.
Quadrant 3 will have 0.1% dettol on a piece of filter paper.
Quadrant 4 will have 0.01% dettol on a piece of filter paper.
We will not be using 100% dettol because it is too strong and it will clear all the bacteria. This means there won't be any bacteria to see and the experiment would be a fail.

To ensure the data is accurate we will be using the same type of yoghurt, we will be keeping the temperature of the room the same, and we will be using the same type of disinfectant.
To ensure our data is reliable and that we have enough data we will be doing the experiment 3 times.

Variables that need to be kept the same	Why they need to be kept the same
Type of Yoghurt	By using the same type of yoghurt we know the test will be fair because all the ingredients to grow bacteria are the same.
Temperature	By growing the bacteria in the same temperature we know the test will be fair because high temperature increases bacteria growth and a cold temperature slows down bacteria growth. So by growing them in the same temperature they are growing at the same rate.
Type of disinfectant	We have to make sure we are using the same disinfectant in all our experiments because not all disinfectants are the same, and others may have different chemicals in them that could impact the experiment.

Equipment:

• Agar Plate
• Hole Punch
• Filter paper
• water
• beaker with dettol
• beaker with water
• Yogurt
• Permanent marker
• pipette
• spotting tile
• tweezers
• bunsen burner
• ethanol
• sellotape

Method:

1. Use the permanent marker to draw an equal sized quadrant on the bottom side of the agar plate. Number them 1-4.
2. Using a cotton bud, spread a small amount of yogurt inside the agar plate careful not to disrupt the agar jelly, immediately place the lid on after to prevent contamination. 
3. Place a small amount of savlon into 1 beaker, and some water in another beaker. 
4. Using the pipette, place 1 drop of savlon into a spotting tile, dilute the savlon with 9 drops of water, swirl the diluted savlon with the pipette to ensure it is combined.
5. Take 1 drop of the diluted savlon and put it in another tile, add 9 more drops of water to the diluted savlon. Being sure to mix using the pipette.
6. Lastly take 1 drop of the dilute diluted savlon and add another 9 drops of water, also mixing with the pipette.  
7.  Cut out 4 pieces of filter paper using a hole punch. 
8. Dip the tweezers in ethanol and swipe it through the flame 3 times, repeat after picking up each piece of filter paper. 
9. Dip one piece of filter paper into a concentration of savlon, do the same with 2 more pieces of filter paper but dip them into different concentrations of savlon. Leave 1 piece of filter paper without any savlon on it.
10. Before putting the pieces of filter paper that have been soaked in a concentrate of savlon in the agar plate, Be sure to put them on a piece of paper towel to remove the excess liquid. 
11. Put each piece of filter paper in the center of a separate quadrant in the agar plate.
12. Put the lid on, and tape it up so it doesn't come loose, then place it in a warm place overnight. 

Change in method:

Instead of using a cotton bud to spread the yoghurt onto the agar plate in step 2, we decided to water the yoghurt down and apply a thin layer all over the agar plate. We altered this step to decrease the chances of contamination. 

Part 2

Data

	Concentration of the savlon	Clear Zone Plate 1	Clear zone Plate 2	Average
Concentration 1	Controlled ( 0% )	0 mm	0 mm	0 mm
Concentration 2	0.1%	5 mm	6 mm	5.5 mm
Concentration 3	1%	9  mm	10 mm	9.5 mm
Concentration 4	10%	13 mm	20 mm	16.5 mm

Observations:
Quadrant 1 - There is no clear zone surrounding the piece of filter paper. The bacteria growing look like little white dots, they are covering almost the whole quadrant.
Quadrant 2 - Quadrant 2 has the largest area of clear zone surrounding the filter paper, outside of the clear zone there are once again little white dots of bacteria that have formed.
Quadrant 3 - Quadrant 3 has the second largest area of clear zone surrounding the filter paper. Outside the clear zone are white areas of bacteria.
Quadrant 4 - Quadrant 4 has the smallest clear zone surrounding the filter paper. There is quite a large area of white-looking bacteria growing.

Conclusion 
In conclusion, based on the data we collected, and from what we can see in the graph, we found that the higher the concentration of savlon, the bigger the clear zone,  In relation to the data my hypothesis was correct. In the graph, from what we can see, as the concentration of the savlon increases, the line also increases. The trend like shows us if the line is increasing, stationary, or decreasing. The purpose of this investigation was how concentration can affect the performance rate of disinfectants in killing micro-organisms. The concentrations of savlon we were using created various clear zones, in which bacteria could not grow or reproduce.

Discussion
Bacteria are unicellular microorganism. Bacteria reproduces asexually which means they are creating a clone of themselves, although they are genetically identical a mutation can occur that creates variation in the bacteria. Mutation can occur in bacteria when a bacteria cell transfer data through pili to another bacteria cell. If a bacteria has a certain resistance to an antibiotic the bacteria can transfer the resistance to another bacteria cell. They use the process of binary fission to replicate and grow colonies. The process of binary fission begins when a bacteria cell replicates their DNA, after which it elongates and eventually spits off into two daughter cells. Bacteria reproduce every 20 minutes. Bacteria colonies are thousands, millions, or even billions of bacteria cells that are piled up which then become visible to the naked eye. They belong to a group all to themselves, this is because they are neither plant, nor animal.  There are 4 phases for the growth of Bacteria which is shown in a table below.
Savlon is an antiseptic that contains two antiseptics, cetrimide and chlorhexidine gluconate. Cetrimide is the antiseptic in savlon that kills bacteria, it kills bacteria by weakening the bacteria's membranes and it then enters the cell. It takes 15 seconds for cetrimide to enter the bacteria cell. Methyl hydroxybenzoate and propyl hydroxybenzoate are the preservatives in savlon that prevent bacteria from growing. There are two ways that the savlon can enter the cell wall, diffusion and active transport. Active transport occurs when an enzyme in bacteria allows the chemical to pass through, much like a gate keeper. Chlorhexidine is a positively charged molecule that is attracted to negatively charged phospholipids in bacteria's cell wall. The chlorhexidine bind themselves to the cell wall that causes it to burst. Because the cell wall bursts, fluid leaks out which causes death of the bacteria cell. The cell structure of bacteria are made up of 8 main things, the capsule is the outer layer which protects the cell and aids in defence against immune system. The cell wall is the strong layer inside the capsule that protects and provides the shape of the cell. The cell membrane is the third layer, it is a flexible barrier that controls what enters and exits the cell. The cytoplasm is a gel-like material inside the cell that holds the cells contents such as ribosomes, plasmids, and the nucleoid. Protein synthesis occur in the ribosomes, plasmids are small circular pieces of DNA that code for non-essential processes. The nucleoid cells genetic information that code for essential life processes. The flagellum looks like a tail, it aids in the movement of the cell. Located on the outside of the bacteria cell there are small things named pili, pili are used to connect to other bacteria cells like a bridge and transfer genetic information. Below is an image of the process of binary fission, and a diagram of the 4 phases of bacteria growth. Bacteria is living thing, we know this because of the anagram MRS GREN. MRS GREN stands for:
Movement - all living things move
Respiration - Getting energy from food
Sensitivity - detecting changes in the surrounding
Growth - all living things grow
Reproduction - making more living things of the same type
Excretion - getting rid of waste
Nutrition - taking in and using food

Phase #	Explanation
Phase 1: Lag Phase	Bacteria initially placed in the area suitable for growth begin to take in nutrients, synthesize their RNA and their proteins. They are not ready to replicate yet.
Phase 2: Exponential or Log Phase	Bacteria replicate quickly, using the readily available nutrients in the broth.
Phase 3: Stationary Phase	Essential nutrients begin to decrease which cause the growth rate and death rate of bacteria to be the same.
Phase 4: Death Phase	As there are no more nutrients the bacteria die.

EVALUATION
I believe that the our experiment was not reliable. I believe this because we originally had 3 agar plates and only 2 had measurable data. We decided not to include the 3rd agar plate in our assessment because the data couldn't be measured. We changed step 2 for our method to reduce the risk of contamination. Instead of using a cotton bud to apply the yoghurt directly to the agar plate, we watered down the yoghurt with water and swished the liquid around the agar plate until all of the base was covered, then we tipped out the remaining liquid. If we were to do the experiment again I would increase the concentration of the savlon. I would do this because the higher concentration of savlon would create larger clear zones which would be easier to measure. We grew our bacteria on a type of agar plate called a nutrient agar. Nutrient agar is made of 0.5% peptone, 0.3% beef extract/yeast extract, 1.5% agar, 0.5% sodium chloride, and distilled water. The bacteria in the yoghurt we used is called streptococcus thermophilus and lactobacillus bulgaricus. Streptococcus thermophilus is a probiotic (good bacteria) that breaks down sugar in milk. For our experiment the agar plates were put in an incubator at 25.C, if the temperature was set higher the bacteria would grow a lot faster.

Bibliography
https://en.wikipedia.org/wiki/Savlon 
http://www.wired.co.uk/article/whats-inside-savlon-antiseptic-cream
http://microbiologyonline.org/about-microbiology/introducing-microbes/bacteria
https://en.wikipedia.org/wiki/Nutrient_agar