Summary

The aim of this experimental challenge is to provide you with direct experience of planning, designing, organising and implementing a drug discovery project. You will work with a small team of colleagues to extract, purify and characterise the anti-microbial properties of a natural product from a number of fruits, roots or vegetables: you can choose.

Background

Natural products form the bedrock of the pharmaceutical industry. We know that from very early times extracts from animals and plants have been used to alleviate pain, treat fever and generally provide some form of respite from illness and disease. It wasn’t until the turn of the last century that such “herbal remedies” became properly formulated and characterised. In fact, the drug industry we know today primarily grew out of the two World Wars, when public and political pressure to treat the victims of warfare intensified, as the casualties mounted. The development of the first synthetic drugs (such as Salvarsan) by pioneers like Paul Ehrlich, alongside the more serendipitous experiments of the pioneer of antibiotics, Alexander Fleming, and the subsequent translation of Penicillin into daily use by the Australian clinician, Howard Florey (the former head of Pathology at Sheffield) and the German Biochemist Ernst Chain, are amongst the greatest scientific achievements of the last century. The challenges that face us today include eliminating the threat of Malaria and developing a new generation of antibiotics to head off the AMR (antimicrobial resistance) threat.

What are Natural Products?

Natural Products are simply molecules produced by microbes and plants (mainly), some of which over many years have been shown to have therapeutic value. The many attempts to rationally design drugs over the last century have met with poor success rates. In fact, many synthetic drugs were first isolated from living organisms, as Natural Products, and have then been chemically modified in order to enhance their efficacy. Examples include the pain killers paracetamol and aspirin, and antibiotics that are modified versions of Fleming’s original beta lactam antibiotic from Penicillium notatum. It seems that microbes and plants have evolved secondary metabolic pathways for the synthesis of compounds that higher organisms cannot synthesise, and it is this broad spectrum of molecules that has fed the pipeline of the pharmaceutical sector. Today, most pharmaceutical companies have field laboratories searching and screening for Natural Products and the search for new antimicrobials is currently being extended to microbes that are traditionally difficult to grow on agar plates (the so-called unculturable or uncultivable microbes).

The project

You are given the challenge of discovering antimicrobial compounds in plant tissue from a range on offer (see table below); it could be coconut, citrus fruit or ginger!

The aim is simple: you should design an experimental strategy for extracting bioactive compounds and a method for screening the antimicrobial activity (a range of safe bacterial strains that mimic common pathogens will be provided).

You must keep a detailed set of experimental records and you must prepare detailed plans for each stage of the project e.g. choice of source material, extraction methods, assay development, quantitative measurements, and dose response evaluation. We are also looking for evidence of your investigation of the biology of the source material, previous attempts that are published, companies competing with you, etc. There will be no schedule for you to follow; you must research and plan all experimental protocols yourself. You will be given advice at all stages of the project together with an introductory analysis session.

Reagents and Equipment

IMPORTANT INFORMATION!

Please read the following before beginning your project

General procedures

A general safety information sheet has also been provided. Any additional hazard information related to items you have requested can also be found in the box. Please read all safety information carefully before starting; it is your responsibility to work safely

• Please note that if you give us anything for autoclaving it will not be returned to you until the following session, as the autoclave takes a long time to complete a cycle and cool down. That’s why we are providing you with media on the first day, but after that you will be expected to make your own; you will therefore need to plan accordingly!
• Not all items can withstand autoclaving (e.g. pestle and mortars) – please check before planning to autoclave anything!
• Ingredients have been provided for you to make your own media as required. Media (including agar) should be prepared for autoclaving in the BLUE-TOPPED DURAN BOTTLES ONLY.
• When labelling items for autoclaving, write on the autoclave tape itself, as writing on other surfaces can disappear during the autoclaving procedure. It really helps us get the labels off again if you fold over one end – thank you!
• Don’t forget to choose a STERILE container for growing cultures
• If helping yourselves to chemicals from the communal area, please decant some rather than taking the whole container back to your station – other groups might also need to use it.
• You should send your used laboratory glassware through the usual washing routes, but you will need to WASH ALL FOOD-RELATED EQUIPMENT YOURSELF (graters, pestle & mortars, chopping boards, juicers, zesters etc etc). Washing-up brushes and draining racks are provided. THE ONLY EXCEPTION TO THIS IS KNIVES which should be left in your bay please.
• You should label every item you prepare with your GROUP (a.m. or p.m.), BAY NUMBER and GROUP NAME, otherwise we will struggle to return the items to you.
• There are several trays at the end of your bay, for different procedures or incubating at various temperatures. Please check carefully that you are placing your items in the correct tray(s)!

Safety guidelines

Please carefully read these guidelines prior to starting your experimental work, and if in doubt about the safety of a particular chemical or procedure then ask a demonstrator or a member of the technical team before proceeding.

Always read the safety labels on any chemicals you use, and if ordering specialist chemicals for your experiment, always read the MSDS (Material Safety Data Sheet) that is supplied.

If you are pregnant or suspect you may be then please let a member of staff know so that we can advise you on which chemicals to avoid using.

Solvents (e.g. ethanol or isopropanol)

• Do not heat these chemicals or expose them to an open flame (except for when using an alcohol bath and glass spreader)
• Do not flame plastic universal tubes containing flammable solvents
• Do not dispose of solvents down the drains, pour them into the solvent waste bottle provided (on one of the sinks).
• In addition to being flammable, isopropanol is an irritant, so you should wear gloves and goggles when handling to prevent exposure.

Antibiotics

• Always wear gloves when handling antibiotic solutions
• Kanamycin is toxic to the reproductive system at the concentration provided, and may cause damage to an unborn child or damage fertility.

Acids (e.g. hydrochloric acid, sulfuric acid)

• Always wear gloves and goggles when using concentrated acid solutions as they are corrosive.
• When diluting acids you should always add acid to water and not the other way around. Doing this the wrong way will cause the acid to fizz and possibly spatter up out of the beaker.

Base solutions (e.g. sodium hydroxide)

• Always wear gloves and goggles when using concentrated alkaline solutions and these are corrosive and can cause serious damage to your skin and eyes.

Other

• When carrying out the coconut/fruit extractions be careful with the knives as they are very sharp. Do not walk around the lab carrying knives; leave them where they are placed within the bays.
• When breaking open the coconuts, first place them within a plastic bag before smashing it with the hammer, as this will prevent shards of the shell from flying everywhere and causing injury. Wear goggles.
• Please be aware that sand baths can become very hot and so always wear gloves, and where possible use tongs when placing and removing items. When hot, the sand may stick to your skin and cause serious burns.
• Do not overfill alcohol baths. Do not fill to more than a third full, otherwise you risk splashing burning alcohol onto the bench.

[see appendix for more safety information]

Tips for planning your experiments

• Remember to include controls
• Don’t try to test every parameter e.g. extracts from every kind of fruit, prepared in several different ways, on both bacterial strains. Choose just one or two things to focus on so you can do them thoroughly
• Talk to demonstrators about your plans before ordering anything or doing any experimental work – it might be that something you plan to do isn’t possible or won’t work
• Refresh your memory on biological and technical replicates – you will need to incorporate these towards the end of your project (below)
• Don’t get frustrated if clear-cut positive results seem elusive – remember that most research projects last several years because experimental work is a very slow process. The most important thing is that you are gaining experience of the process of experimental design (see Learning Objectives)

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