1. Project overview and assessment

The overall aim of this practical is for you to plan, develop and perform your own experiment to test a scientific hypothesis. You will have two weeks (four sessions) to obtain data on variables affecting the contamination of food that has been in contact with a surface. After the practical sessions, you will write up your findings as a lab report, which will be worth 20% of the final grade for the [30-credit Level 1 Skills Module]. More information on the lab report will be provided in session 4.

Aims of the practicals

1. To formulate a scientific hypothesis.
2. To work as a team to design and perform an experiment to test your hypothesis.
3. To apply your results to make public health recommendations.
4. To write up your data in a lab report.

Specific objectives

• To design and perform your own experiment.
• To define the variables of your experiment.
• To use controls appropriately.
• To interpret your data appropriately.
• To present data in a clear and coherent way.
• This practical gives you the chance to build on many module learning objectives, but the key one is “investigate scientific questions by formulating hypotheses and designing experiments to test them, including appropriate controls”.

What to do before you attend the lab

1. Read through the entire schedule (15 – 20 minutes).
2. After you have read the schedule, answer the pre-lab questions below (10 minutes).
3. Read the [material on group work from our academic skills centre]

Pre-lab questions

1. What is a positive control? What does it tell you?
2. What is a negative control? What does it tell you?
3. Is it possible to have more than one negative/positive control? Why?
4. Why do we need to include replicates within a scientific experiment?
5. What is the difference between a technical replicate and a biological replicate?

2. Background information

In 2017, CBBC’s Newsround defined the five second rule as:

“If you drop something on the floor and pick it up in less than five seconds it’s ok to eat”

Is this an urban myth or a reliable description of reality? Does the length of time food is on the floor determine its level of contamination? You’re going to help us to find out…

The challenges of food safety

In the UK each year as many as 2.4 million people suffer from food-related illness (Food Standards Agency, 2022). However, food contaminated with pathogenic organisms rarely looks, smells or tastes bad so it is impossible to know whether or not it is safe to eat. There are two ways that foodborne microorganisms can make you ill:

1. Infection (by consuming pathogenic microorganisms).
2. Foodborne intoxication (by consuming toxins derived from microorganisms).

It is worth noting that not all microorganisms found on food are harmful. Some are neutral; some are beneficial; and some cause food spoilage but are not harmful to humans.

Scientists have used various methods to study food poisoning and to assess the transfer of different bacterial species from contaminated surfaces onto food (Dawson et al., 2007, Miranda et al., 2016). In this project, we will use a bacteria called Serratia marcescens to analyse the transfer of food contamination. This strain was chosen because S. marcescens is non-pathogenic, and because it is easy to identify because of the red pigment it produces.

References

CBBC (2017) Is it safe to eat food you’ve dropped on the floor? [Available here].

Dawson, P., Han, I., Cox, M., Black, C. and Simmons, L. (2007) ‘Residence time and food contact time effects on transfer of Salmonella Typhimurium from tile, wood and carpet: testing the five-second rule’, J Appl Microbiol, 102(4), pp. 945-53. [Available here].

Food Standards Agency (2022) ‘FSA research suggests new higher estimates for the role of food in UK illness’. [Available here].

Miranda, R. C. and Schaffner, D. W. (2016) ‘Longer Contact Times Increase Cross-Contamination of Enterobacter aerogenes from Surfaces to Food’, Applied and Environmental Microbiology, 82(21), pp. 6490-6496. [Available here].

3. Practical schedule – what is happening each day?

Day 1: Protocol Development: which sampling method?

You need to quantify contamination using the equipment available. There are many possible ways of doing this – which ones do you think are best? How have other scientists measured this?

To do today:

• • Set up some simple experiments to test methods of sampling.
  • Start simple and try several methods (suitable for low and high levels of bacterial load)
  • Replicates are not important at this stage.

Day 2: Protocol Development – which variables?

Today, you will explore the different variables that you might want to test. What variables are you interested in exploring in your experiments (time / contamination level / food type)? How can these be tested with the equipment and reagents you have available?

To do today:

1. • Analyse your data from Day 1. Which of your sampling methods worked best? What will you change/keep in your experiments today?
   • Design some simple experiments to test which variables are suitable for analysis.
   • These are still pilot experiments so replicates are not important yet.

Day 3: Your final experiment

Today, you will use the data from Days 1 and 2 to plan and set up your experiment, making sure to incorporate appropriate experimental controls and replicates.

To do today:

• • Analyse your data from Day 2. Did any of your variables give better results than others? Which variable(s) will you choose for your final experiment?
  • Formulate a hypothesis that you wish to test.
  • Set up your final experiment. You need to consider a number of factors:
    • Technical repeats – how many?
    • Biological repeats – how many?
    • Do you have appropriate positive and negative controls?
    • How will you analyse the data?

Day 4: Data analysis

Today, you will analyse the data from your final experiment.

To do today:

• • Collect your results from Day 3.
  • Analyse the data and draw suitable conclusions from it.
  • Carry out any appropriate statistical analysis.

4. Experimental planning

Working in teams, you will design an experiment to test the five-second rule. Successfully planning an experiment is a skill that you are just beginning to learn. Here are some steps to get you started (you will not necessarily carry them out in exactly this order and you may have to repeat steps):

1. Analyse preliminary data – did it work as intended? What do you need to change?
2. Adjust your experimental approach in light of your preliminary data.
3. Choose variables (independent and dependent).
4. Generate a testable hypothesis.
5. Design controls (positive and negative). Are they the right controls?
6. Repeat your experiments (including appropriate controls and replicates).
7. Present and analyse your data.
8. Draw some valid conclusions from your analysis.

If you are at all unsure about any stage of this (experimental plan, controls, replicates, etc.), then discuss your ideas with a demonstrator before proceeding. Even if you are sure about your ideas, we still recommend discussing your plans with a demonstrator!

Below is an explanation of some of the scientific terms you will come across during these practicals.

Hypothesis

In science, a hypothesis is distinct from a theory. A hypothesis is an educated guess as to what you think is the explanation for an observation. You do not need any data or evidence to formulate a hypothesis, although a hypothesis is usually based on existing knowledge. A key feature of a hypothesis is that it should be testable – it should make a prediction that you can experimentally test. Importantly, it doesn’t matter whether your hypothesis is correct or incorrect! When writing a hypothesis, you should express it as a statement, not a question.

A theory, in contrast, is a well-established and widely-accepted explanation that is based on a large body of experimental evidence.

Variables

The independent variable is the thing that you will change in your experiment. There are many variables that you could test, but we would recommend focusing on one or two. Here are some suggestions of variables that you may wish to consider (this is not an exhaustive list and you are more than welcome to choose your own):

• Transfer time
• Type of food
• Type of surface
• Amount of contamination
• Type of contamination (residual contamination / artificially contaminated surface)

The dependent variable is the thing that you will measure, after manipulating the independent variable. For you this is bacterial contamination and one of the first things you need to do is work out a good way of doing this.

Controls

You need both positive and negative controls. A negative control is something that you would expect to display no effect. A positive control is something that you would expect to produce the desired outcome. Good controls can be hard to design, and there is often more than one control that you could choose. Discuss your ideas with each other and a demonstrator.

Replicates

Replicates are important but not all replicates are the same – there are technical replicates and biological replicates. The difference between the two is summarised here and there are two examples below to help you understand the difference.

Technical replicates. This is when you take multiple measurements from the same sample. It ensures that your measuring technique is robust and reproducible and that your equipment is functioning normally. You do not perform statistical analysis on technical replicates – you would normally take an average of them all and call that the measurement of that sample.

Biological replicates. This is when you measure multiple samples to check for biological variation. This is what you are most interested in and biological replicates are what you perform your statistical analysis on.

Replicates example 1: How long is a mouse’s tail?

Obviously, not all mice will have exactly the same length of tail. In order to answer this question properly, you will need to measure the tail of more than one mouse and then average the results. These are biological replicates because you are performing the same test on multiple samples (mice).

However, as a thorough scientist (which of course you are) you will also want to make sure that you are measuring accurately, and so you would measure each mouse’s tail more than once. This is a technical replicate because you are measuring the same sample (mouse’s tail) multiple times.

Here are the results from this experiment:

You now have three measurements of mouse tail length from three different mice. These are your biological replicates and this data can be used to answer your question, “how long is a mouse’s tail?” When analysing data like this, it is important to remember that this is a sample (you obviously can’t measure the tails of the entire mouse population). You would usually express your data as an average (the mean) of the mouse tail lengths:

Mean tail length from your sample = (104 mm + 116 mm + 95 mm) / 3 = 105 mm.

You would also usually include some descriptive statistics (such as standard deviation / standard error) that provide some information about the variation in your sample.

Replicates example 2: How many bacteria (colony forming units) are there in a culture of E. coli?

Imagine that you have a flask containing 50 ml of bacterial culture. You wish to measure the number of viable bacteria (Colony Forming Units, CFUs) in this culture. To do this, you will take a 100 μl sample from the flask, spread this on an agar plate, and wait for the bacteria to grow and form colonies. Counting the number of colonies that grow will tell you how many viable bacteria were in the sample you took.

Technical replicates would entail taking more than one sample from the bacterial culture. This ensures you have taken a representative sample of the culture and that your sampling technique is consistent.

Biological replicates would involve growing more than one flask of culture, to control for any variability between cultures.

You carry out this experiment and set up three cultures of bacteria. You take two 100 μl samples of each culture and count the number of viable bacteria as above. Here are your results:

These values are the biological repeats and can be used to calculate a mean value for CFUs in the bacterial culture:

Mean CFU = (104 + 90 + 108) / 3 = 101 CFU per ml

5. Reagents and equipment

This is a list of all the things you will have available for your experiments. If you would like to use anything which is not listed below, please check ASAP with a member of the technical team whether it is available. We can source things (within reason), but we need warning!

Foodstuffs

• Apples
• Grapes
• Cheese
• Substitute food:
  • Sterilised 1 cm³ wooden cubes
  • Sterilised marbles

Reagents and media

• LB agar plates
• Sterile LB media and universal tubes for aliquotting
• 100% ethanol (see safety information below)
• Serratia marcescens liquid cultures (will form pink-red colonies)

Equipment and consumables:

• Sterile tubes in various sizes (1.5 ml, 5 ml, 15 ml, 25 ml, and 50 ml)
• Empty sterile petri dishes
• Glassware; cylinders, beakers, conical flasks, Duran bottles, etc.
• Chopping boards and knives
• Forceps
• Ethanol baths and spreaders
• Ceramic tiles

Practical laboratory tips

Making a spread plate. The ideal volume of bacterial culture to spread on an agar plate is between 50 – 200 μl. If you have too small a volume, you won’t get an even spread. If the volume is too large, it will take forever to dry. If your plates are taking a long time to dry, you can leave the lids ajar to help speed this up (make sure open plates are close to a blue bunsen flame, to keep them sterile).

Sterile technique. Good aseptic technique is essential – don’t forget to use it when appropriate. It applies when you are handling bacteria and also when you are opening any tubes / plates / media that you will use for bacterial cultures. [Link to internal videos showing various elements of sterile technique]

Other lab equipment. You are probably familiar with most equipment by now. Here are some videos of equipment that you might have had less experience with: [Link to internal videos showing how to use the spectrophotometer and centrifuges]

6. Lab safety

Risk Assessment Information

[see appendix]

General safety information

Please don’t contaminate the lab!

Please do not artificially contaminate permanent surfaces (for example, the bench or the floor). If you are using these surfaces as your source of contamination, use only the residual contamination found on these surfaces. If you wish to artificially contaminate a surface, use something disposable/washable (for example, a tile or a petri dish).

Hungry?

Do not eat the food!