Investigation for the effect of disinfection on bacterial growth

Francesca Tate 10D

Investigating the effect of disinfectant on bacterial growth:

There are various ways on destroying micro organisms. Antiseptics, disinfectants, heat sterilisation, heat treatment, radiation and antibiotics are all ways of killings micro organisms outside the body.

I am going to investigate the effect of disinfectant on bacterial growth. The definition of a disinfectant is a chemical that kills micro organisms. There are many different types of disinfectant; Milton’s, tcp, dettol, demestos, alcohol and bleach. I am going to investigate the effect of one particular disinfectant. Ethanol. Ethanol is used in medical wipes and as an antiseptic. Ethanol kills organisms by denaturing their proteins and dissolving their lipids (fats). These fats are found in the cell membrane.

Bacteria are small, single cell organisms that are big enough to be seen under the light of a microscope. There are many different types and can be located almost anywhere. A bacteria cells consists of a cell membrane, inside this membrane is a fluid called cytoplasm. At the centre of the cell is a string of DNA. Attached to the cell are sometimes flagella, these help the cell to move. Bacteria divide by mitosis; bacteria can roughly double their population in just 20 minutes. This leads to colonies forming and these colonies are visible. These visible colonies are the things I will be counting.

The bacteria I am going to use is E coli (Escherichia coli). A common type of bacteria, which normally lives in the body in your intestines where it helps your body break down and digest food. When the E coli moves from your intestines into other parts of your body you can get sick. Symptoms of E coli poisoning include fevers, chills, bad cramping, stomach pains, vomiting and diarrhoea.

I intend to vary the concentration of ethanol and use this to determine the optimum concentration to prevent the growth of E coli on agar jelly.

So this leaves me with an improved investigation title:

Investigate the effect of ethanol concentration on the growth of E coli.

Aim: To find the optimum concentration needed to destroy E coli living on agar jelly.

Prediction: As the concentration increases the number of colonies formed will decrease.

I predict that as the concentration (x axis) goes up the

Number of colonies counted (y axis) will go down. This

Is because there will be more ethanol particles in the

Disinfectant and therefore more particles to denature the

Proteins and lipids on the cell membrane of the bacterium

cells, therefore denaturing the bacteria cells altogether.

The cell membrane, which now can no-longer function

Correctly causes the cell to denature. The purpose

Of a cell membrane is to control what is passing in and

Out of the cell such as water or nutrients. The cell membrane being unable to function correctly means that the cell will not be able to absorb what is “truly” needs to multiple and/or survive.

I selected equipment that I could use to carry out my experiment.

Bunsen burner
6 sterile test-tubes and bungs
1 bottle of sterile water
1x1cm³ syringe
2x10cm³ syringes
Beaker of disinfectant (swabbing)
Bottle of disinfectant – 70% alcohol
1 nutrient broth culture of E coli bacteria
1 inoculating loop
1 piece of tissue
1 stop clock
11 agar plates
1 marker pen
Sellotape and scissors

I carried out a preliminary experiment.

This experiment was done initially to determine the type of disinfectant I was going to use for this experiment and the sort of ...

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I selected equipment that I could use to carry out my experiment.

Bunsen burner
6 sterile test-tubes and bungs
1 bottle of sterile water
1x1cm³ syringe
2x10cm³ syringes
Beaker of disinfectant (swabbing)
Bottle of disinfectant – 70% alcohol
1 nutrient broth culture of E coli bacteria
1 inoculating loop
1 piece of tissue
1 stop clock
11 agar plates
1 marker pen
Sellotape and scissors

I carried out a preliminary experiment.

This experiment was done initially to determine the type of disinfectant I was going to use for this experiment and the sort of range of concentrations I would be looking at.

Initially when glancing at the table of results, you think that this was a useless experiment. Infact this first experiment gave me a lot of information I needed for my final experiment. These agar plates were incubated at 20-25°C – this was useful to me as it shows that incubating the agar plates at this temperature produces growth. 0.1cm³ of E coli was placed in 10cm³ of disinfectant this proved to be too small. I left the E coli in the disinfectant for 2 minutes, this seemed like a good time.

As a class we pooled our results and decided that the optimum time for leaving E coli in the disinfectants was between 3 and 4 minutes. Also 1cm³ of E coli in 10cm³ of disinfectant seemed like a more reasonable amount. We also decided that ethanol would be the disinfectant to use from this point on (hence the background information on E coli and ethanol/alcohol stated previously). We felt that 70% would be the absolute maximum concentration and to produce a suitable set of results choose 5 or more concentrations between 0% and 70%.

I then produced this method:

After putting on labcoat and safety goggles, take the piece of tissue and the beaker of disinfectant and disinfected the work surface.
Throw away the used tissue, as this is now very flammable.
Make up the concentrations of ethanol from the bottle of disinfectant (i.e. 70% alcohol) and place into the 6 sterile test tubes using the 10cm³ syringes.
Label each test-tube.
Light the Bunsen burner to create an updraft (this causes any microbes in the air to be lifted up away from the experiment being performed on the work surface).
Place 1cm³ of E coli in the first test-tube using the 1cm³ syringe.
Shake the test-tube 6 times to ensure mixing.
Start stop-clock
Leave for 3 minutes.
In the time here waiting you can label the agar plates with the date the solution was placed on the jelly and with concentration and your initials.
Place inoculating loop in the flame till it glows red hot.
Wait briefly for the loop to cool before removing the bung from the relevant test tube.
Usually we could “Surface sterilise this (by swiping it through the flame)” but as ethanol is being used and ethanol is extremely flammable it is advised not to do this.
Dip the inoculating loop into the solution and moving quickly to avoid contamination swipe the inoculating loop in a zigzag pattern across the jelly. The lift of the agar plate should be lifted at the smallest angle as possible to decrease the risk of contamination from airborne bacterium.
Stop the clock
Place 2 or 3 strips of sellotape across the agar plate just to hold it in together, not to seal it completely as the bacteria need to breathe.
Do this for all the concentrations.
Repeat each concentration twice. And/or further if unsure of results.
Leave the agar plates in a warm place with a thermometer or incubate them at approximately 24°C i.e. room temperature.
Check on plates after 3 days.

I used the method above and used the table below to make up the following concentrations:

When I attempted this experiment using the above method I encountered the following problems:

My results from this experiment after 3 days were:

I can see from these results that these are not suffice to conclude my experiment. I have therefore decided to increase the time in which I record my results to 4 days and add one more concentration on to my list to make up the whole 11 agar plates. I will also take into mind the problems with my original method and repeat my experiment.

Obtaining Evidence

*I had to repeat this agar plate, as there was contamination. The number above is the number after being repeated.

**I have rounded this number to 1 as it is impossible for there to only be half a colony.

Analysis

From the results I can see that straightaway that there is trend that as the concentration increases the number of colonies decreases. I have decided to draw a graph to see what the actual relationship between the ethanol and the E coli bacteria is.

I recorded these results by counting up the number of colonies that I could see on the surface of the agar jelly. The graph is a gentle curve which means that there is a steady change to begin with, with the lower concentrations (which I did not perform, but only predict using my trendline) and then slopes off at the bottom to eventually hit a 0.

The evidence I have here clearly matches up with my original prediction of

“Prediction: As the concentration increases the number of colonies formed will decrease.”

As we can tell this by the negative gradient. Unfortunately I was not totally successful in predicting my experiment. My prediction graph and my actual graph are two different things but both giving the general hint of the experiment. My prediction graph was a straight down line, whereas my actual graph is a gentle sloping curve as I said earlier. The graph has a negative gradient because the greater the concentration of ethanol the greater the number of ethanol particles in the disinfectant’s volume. Therefore more particles in the disinfectant to destroy the bacteria.

Evaluation

I have divided my evaluation up into 3 different parts. Method, Apparatus and Results.

Method

Naturally when performing an experiment like this there is a high risk of contamination. Contamination can be recorded when a different colour colony is identified on the plate or a slightly larger colony which is a mouldy colour and usually has spores on it. In a school science laboratory there are only certain ways we can over come this risk. As I said earlier in my method, you should use sterilised equipment and disinfect the work surface using tissues. Another way to prevent contamination would be to wear a surgical mask to stop us breathing over the experiment (i.e. the agar jelly). As expected you cannot be sure of your aseptic technique till after a few days where you find out you results.

Another problem encountered with this method was what I mentioned earlier with the inoculating loop being too hot when being placed in the disinfectant. This was overcome in final experiment as I identified this in my second experiment as a problem. It was overcome by letting the inoculating loop cool for 45 seconds close to the flame after glowing red-hot. A good thing about my method is that I used repeats. I decided against repeating my control, as this would require 12 plates in total when I only had 11.

The concentrations I used in my opinion were a good choice because they used both the maximum and the minimum concentrations. In order to increase the accuracy of my experiment I could do more concentrations between 0% and 30% (i.e. 10% and 20%). On my graph I can see that there is a curve. It would have been interesting to see how critical it was between 40% and 50%, between 50% and 60% etc. I would of done this by doing the concentrations every 3% or 5%.

Something that could have affected my results was the time the E coli was left in the ethanol (disinfectant). The longer the E coli is left in disinfectant the more likely it is that the results have a lower number of colonies. I settled on 3 minutes, as this is minute longer than I used in my preliminary work.

Unsurprisingly a way to improve my results from my method would be to do more repeats and to use more concentrations. Also to be tighter about touching my hair/face during the experiment. I should also wash my hands after the experiment as E coli when in other parts of the body causes (as I said on page 1) fevers, chills, cramping, stomach pains, vomiting and diarrhoea.

Apparatus

Apart from trying to create the impossible of a sterile environment in the school laboratories there was the task of trying to keep the agar plates at a constant temperature. As we did not watch the plates twenty-four hours of the day we cannot be 100% sure that they remained at a constant temperature but we can be reasonably sure that they were. In order to make my experiment more accurate I could of used glass syringes rather than plastic syringes.

The Bunsen burner lifts the microbes away from the experiment in a circular motion, meaning that they rise and fall continuously. This technically means that microbes could contaminate the plates in this motion. The apparatus could have been better if I had performed this in a special laboratory, which is specifically designed for this type of experiment therefore reducing the chances of contamination dramatically.

Results

Judging by my results I can say that overall my technique was good. The only mishap was in the 40% concentration trial number 1; I had some contamination so I decided to repeat that particular plate in order to gain my average. As I can see from my graph that the points are all very close the trendline, which shows that they are reasonably accurate. Microbes are incredibly small as I have already said (the E coli bacterium is approx. 3 microns), which means there is a huge room for error.

When looking at my results I can safely say that I have produced results that are useful to my initial title and prediction. My prediction was

As the concentration increases the number of colonies formed will decrease.

I can say this was decreased by the fact that the graph has a negative gradient (meaning it slopes downwards) and this can be supported by the table which shows the numbers decreasing in these two columns. My prediction has thus been proved. I could of improved my results by measuring to another degree of accuracy such as mm² covered etc.

I could of measured my colonies differently. I chose to just count them, whereas I could of drawn four streaks and counted the colonies on the forth streak or draw a shape calculated the area covered by the microbes. Any of these ways would have been ways of counting the number of colonies. These are not necessarily more accurate than the way I have chosen. Possibly the microbes on the final streak might have been more accurate. It would be interesting to find out if it was or not.

In conclusion I can say that judging my situation (i.e. a school laboratory) that my results have produced suitable results for my experiment. Naturally there are ways I could have improved my method, apparatus and results I have taken these into account and shall remember them if I decide to repeat my experiment in the future.

Mrs. Dolan