The Garlic Center is most grateful to Gareth Browning for sharing his work and allowing us to publish it on the internet. This is one novel approach to determining the excellent antibiotic properties of garlic. If you have work of a similar nature – please email it to us and we will place here in our research center.

Garlic, or Allium sativum, has been used for hundreds of years to treat a variety of illnesses. As it has been used continually over many generations, it is likely that garlic actually has an effect against these illnesses, so I am going to investigate in a laboratory situation the effects of garlic against the growth of bacteria.

If it is found that garlic has such an effect that it is easily testable, I will then go on to investigate the effect of concentration of the garlic on the growth of bacteria.

A preliminary experiment was performed first to find suitable testing conditions.

Discs were cut from filter paper using a hole-punch, and soaked in 3g of crushed garlic mixed with 5ml of water. Single discs were placed on aseptic agar plates which had 0.2ml of E.coli in broth poured on them. The plates were incubated for 24 hours and the growth was then looked at.

Each disc on the agar plates gave an inhibition zone where no growth of the bacteria occurred, between 11 and 14mm in diameter.

Now that I can be sure the effects of garlic are testable, I will move on to looking at concentration.

Five agar plates were poured using aseptic methods. 0.2ml of broth culture of E.coli, which had been incubated overnight, was spread evenly over each plate using a right- angled spreader made from a glass pipette.

Small discs were cut from filter paper using a hole-punch previously washed in ethanol (to ensure it was sterile).

3g of garlic bought from a local supermarket was crushed using a pestle and mortar, with 5ml of water to act as the solvent. The small discs were then soaked in the supernatant formed and then allowed to dry in a sterile petri dish.

One disc was placed in the centre of the first agar plate, two discs on top of each other in the centre of the second agar plate, three on top of each other in the next and so on. The duplicated discs gave the effect of increasing concentration of the garlic.

The plates were again incubated for 24 hours and the zones of inhibition were looked at.

This gave good results but I will know repeat the experiment to ensure they are correct, and to account for any anomalous results. I will also extend the experiment by increasing the number of duplicated discs tested.

It was also noticed in both experiments that as the number of discs got higher the definition of the inhibition zone got stronger. With the lower numbers of discs the border between growth and no growth was sometimes quite vague, but with the higher numbers the inhibition zones were very clear.

From my experiment I have noticed two main trends:

1. Garlic has an inhibitory effect on the growth of the bacteria Escherichia coli.
2. Increasing the number of discs impregnated with garlic increases the effect of its inhibitory actions.

The concentration of whatever was causing the growth of the bacteria to be stopped was obviously increased as the number of impregnated discs got higher. The increased number of discs gave a higher concentration, so there was a higher level of garlic that could act on the bacteria cells trying to grow near the discs.

This is shown clearly by the graphs from my experiment. The curves showing the relationships between number of discs and diameter of inhibition zone give standard curves with decreasing gradients. (This is what should be expected, as the increase in concentration increase decreases as disc number gets higher. For example adding one disc to one disc doubles the concentration, but adding one disc to 5 discs only increases the concentration by 20%.) With disc number and area of inhibition zone, a more direct relationship was found and this is shown especially so in experiment two. A clear straight-line graph was obtained meaning the area of inhibition is proportional to the number of discs.

The observation made where the clarity of the zone increased with the number of discs, also shows that increased concentration gives increased effect by the garlic. The bacteria having different tolerances to the effect of garlic could explain this effect. At the low concentrations some bacteria may be able to survive and grow, but at the higher concentrations nearly all will be affected meaning the clarity of the zone will be strong.

Two anomalous results were obtained during the course of the whole experiment. These were both obtained at 4 discs, in experiment one and two, and there are a few reasons for them not giving the expected results. Firstly, as the inhibition zone size in both cases was very similar to the previous number of discs, it could have been that the number of discs was counted incorrectly in one or both of these cases. The discs were very small, and hard to separate and count after soaking so it is possible this mistake could have occurred. However it is quite unlikely that this happened, as the number of discs for each plate were counted a number of times. As, in both cases, the size of inhibition zone was smaller than could have been expected, it might have been that the discs used had not been soaking for quite as long as the others used on other plates, and so were not quite as effective.

Garlic contains many sulphur-containing compounds the most important of which in this case is alliin. When garlic is cut, damaged or crushed this alliin comes into contact with an enzyme present called alliinase. The alliinase converts the alliin to allicin, which is responsible for the distinctive smell of garlic and its actions against the growth of micro-organisms. The allicin interferes with RNA production and lipid synthesis. If RNA cannot be produced, or only produced in less than satisfactory amounts then protein synthesis will be severely hindered. It would be stopped at every stage due to the absence of messenger RNA, ribosomal RNA and transfer RNA. If amino acids and proteins cannot be produced then growth and development of the organism will not occur as they are essential for all parts of cell structure. Also, as lipid synthesis is affected, other parts of the cell are interfered with. The main effect being that the phospholipid biolayer of the cell wall cannot form correctly in both Gram positive and Gram negative bacteria. All these things contribute to the bacteria not being able to grow in the presence of allicin.

It is likely that because of these very reasons garlic is also effective against the growth of fungi and even viruses. In a separate experiment performed by another student the effect of garlic was shown against yeast cells using the same method as in this experiment. Zones of inhibition of similar or greater size were obtained, proving this theory (against fungi).

These results help towards the biological significance of my results, of which there is really only one thing. The actions of garlic allow it to protect itself against parasites and fungi present in the soil. If the species can have a resistance to certain illnesses then it is far more likely to survive and go on to grow and reproduce, and will be more competitive against other species living in the same area. It is obviously a great advantage to be able to kill, or inhibit the growth, of potentially threatening micro-organisms near where growth will occur.

Tests by scientists have shown garlic to be the equivalent of 1% penicillin, and it is said to be more effective than 1% penicillin against the illness typhus. It is also good for cholera, dysentery and enteritis. With all these illnesses the allicin blocks two groups of enzymes; cysteine proteinases and alcohol dehydrogenases.

Cysteine proteinases are among the main culprits in infection, and they provide infectious organisms with the means to damage and invade tissues.

Alcohol dehydrogenases play the major role in the harmful organisms metabolism and survival.

The allicin reacts with the important component of the enzymes; the sulphydryl group (SH), meaning the enzymes cannot perform their roles, so garlic is also important in defence against actual illnesses. Because of the method of which garlic is not susceptible to these illnesses, it is very unlikely that the disease causing micro-organisms will develop a resistance to the effects of garlic, as this would require modifying the very enzymes that make their activity possible.

Garlic may have a role in reducing the build-up of cholesterol in humans. The sulphydryl group is also a crucial component of some enzymes that participate in the synthesis of this potentially lethal lipid. It may therefore prevent the production of cholesterol, by stopping these enzymes, reducing the chances of arteries becoming clogged.

As with all experiments there were certain limitations to the techniques used. As described earlier, it was often quite hard to separate the individual discs, so there was always the risk of having the incorrect number of discs on a plate.

It would have been hard to increase the number of discs past eight, as above this the stack becomes quite large. During the 24-hour incubation period, where the plates obviously have to be stored upside down to prevent condensation, it is likely that the discs in a large stack would fall off from the agar.

With the lower number of discs, as the clarity of the inhibition zone was not as good as the higher number of discs, it was quite hard to distinguish between growth and no growth. It was therefore harder to measure these zones, and small inaccuracies could have occurred during measurements.

There are many ways that this investigation could be furthered. First of all, the area below one disc could be investigated. This could be done by decreasing the concentration of the original supernatant by, for example, using 10ml of water instead of 5ml to effectively halve the concentration. One disc could be used again but it would have half the strength of the original one.

Different bacteria and also fungi could be studied. Genuses such as Staphylococcus and Lactobacillus, and also Saccharomyces would be easily testable. The effect of garlic could be investigated further by looking at what happens in differing conditions. For example the temperature could be changed, or the pH varied to see if garlic is just as effective in extreme conditions. Different species of garlic could also be investigated. Allium ursinum could be compared to Allium sativum. Completely different plant species could be investigated such as onions, leeks and shallots and even others such as oregano and thyme to discover if these have any similar properties.

By Gareth Browning, 2000