Biology A level revision resource: Testing antimicrobial agents

Antibiotics often have a specific mode of action that enables them to kill bacteria, for example, penicillin inhibits the proteins that cross link the peptidoglycan layer in bacterial cell wall. Penicillin is most effective against gram-positive bacteria, as their cell walls are mostly peptidoglycan, rather than gram-negative bacteria who also have a lipopolysaccharide and protein layer. As the cell tries to divide it is unable to rebuild this peptidoglycan cell wall, it then cannot maintain turgor pressure and the cell will 'pop' or lyse. To combat this, bacteria have developed a number of defences including 'penicillinase' enzymes which render penicillin ineffective, modifying the proteins that penicillin acts on, or even developing membrane pumps that actively remove penicillin from within the bacterial cell. 

Other antimicrobial agents tend to be more broad-acting than antibiotics, which can make them less vulnerable to the development of resistance. Bleach, for example is very good at killing bacteria as hypochlorous acid has a similar effect on enzymes as heat does. The antimicrobial agent in garlic, allicin, inhibits the action of a broad range of enzymes. However, just as bleach is highly toxic to humans and other animals, garlic is also toxic to much more than just bacteria It has anti-fungal, anti-parasitic and antiviral activity; it can even be toxic to animals that are less able to digest it than humans. 

How do we test antimicrobial agents?

How do I master aseptic technique?

Good aseptic technique is a foundation of safe and effective working in the laboratory. Cross contamination of samples can ruin hours of painstaking work in the lab, and, if working with potentially hazardous organisms, cause harm to human health. Although in a school setting you would be unlikely to ever work with especially harmful strains of bacteria, most bacteria have the potential to do harm if they get can access areas of the body where they should not be. 

There are a few basic tenets to developing effective aseptic technique. Firstly, ensure that you always wear the correct personal protective equipment. You should be advised by your teacher about suitable safety equipment but remember that it is not just there to protect you - it also protects your experiment from contamination. Secondly, remember that time is always a factor and the key to good aseptic technique is to work efficiently rather than quickly. If you do not have a vital piece of equipment close to hand you increase the risk of an open petri-dish becoming contaminated, so arrange everything beforehand to avoid delays. Finally, prepare your work area and equipment properly. Close windows to avoid drafts, and always use a Bunsen burner to draw air currents up and away from your work area. Ensure that you disinfect every surface before and after, and each piece of equipment with every use. A combination of Bunsen flame and alcohol is an effective way of sterilising metal equipment like wire loops or tweezers. If your bench area is difficult to disinfect, then you might consider covering it with a more easily disinfected material on which to work.

You can see Chris using the Bunsen flame in the image above to make sure that air currents are drawn up and away from the petri-dish that he is working on. For the purpose of taking the picture he is holding the petri-dish, but better aseptic technique would be to have the dish flat on the bench with the lid easily accessible nearby; at least he is wearing correct personal protective equipment! He should also use alcohol and the Bunsen flame to sterilise the metal tweezers after each use. 

What does inhibited growth look like?

If you have performed the experiment correctly, with good aseptic technique and accurate serial dilutions, you should see clear areas, or 'zones of inhibition', around discs of antimicrobial agent. The size of this zone of inhibition shows how effective the antimicrobial agent is at killing bacteria or inhibiting their growth, and you would expect that the larger zones would be found around the stronger concentrations of antimicrobial agent. Hopefully your control disc, containing only water, should show no zone of inhibition.

Depending on the antimicrobial agent that you have used, you would expect different levels of growth inhibition. Going back to the example of penicillin, we would expect a much larger inhibition on the growth of S. aureus than on E. coli, as S. aureus is gram-positive and E. coli is gram-negative. However, using a wide ranging antimicrobial such as bleach, we would expect the growth of both bacteria to be similarly inhibited.

Bacteria can find ways around even broad-acting antimicrobials such as bleach. There are a special class of proteins called heat shock proteins (HSP) that are produced in response to stressful conditions including heat and exposure to toxins. These proteins 'chaperone' other cellular proteins and help to protect them against damage, and one called HSP33 was recently shown to protect bacteria against concentrations of bleach that would usually kill those without the protein. Being able to survive an environmental condition that kills other microorganisms is a huge selective advantage, and so the rapid evolution of resistance in microorganisms will always be a challenge for human populations.