The effect of different antibiotics on two bacterias, the gram-negative Escherichia Coli and the gram-positive Bacillus Subtilis through disc diffusion
This experiment is to investigate the rate of inhibition and effectiveness of antibiotics, by measuring the inhibition zones on the incubated agar plates. (1)Antibiotics as a start, are best known to prevent growth of microorganisms, in this case being the bacteria. Penicillin was the first antibiotic that was derived naturally from other fungi and bacteria and was the first step to synthesizing different antibiotics. Each antibiotic works sufficient to certain infections or bacterias and in order to determine what antibiotic is best to use, we can use the Kirby-Bauer disk diffusion method. This method is a way of seeing which antibiotic works best for a certain bacteria, by swabbing a thin layer of bacteria across a agar plate and then add a filter disc soaked in a particular antibiotic creating a inhibition zone with time. The effectiveness of the antibiotics concentration decreases the further away you are from the disc and so in that particular area, if the antibiotic compound is effective against bacteria no colonies will grow where the concentration is greater or equal to that of a effective concentration. This particular circular region where no bacteria grows is known as the “Zone of inhibition” and so the size of its diameter is the measure of the antibiotics effectiveness.
Also, the two bacterias used are the gram-positive Bacillus Subtilis and the gram-negative Escherichia Coli. The gram-positive bacteria are easier to kill considering their thick peptidoglycan outer layer absorbs antibiotics easily. However, the gram-negative bacteria has a thin peptidoglycan as well as multilayered membrane structure making it more resistant to getting affected by antibiotics. Thus, the additional membranes act as controllers to what reaches the inner core enabling them to get rid of threatening antibiotics before it reaches the core also making the gram-negative more dangerous in contrast to the gram-positive.
How effective are the six different antibiotics on two bacterias, the gram-negative Escherichia Coli and the gram-positive Bacillus Subtilis through disc diffusion?
The aim of this procedure, was to investigate how the six different types of antibiotics effect/prevent the growth of the gram-negative Escherichia Coli and the gram-positive Bacillus Subtilis.
The gram-negative Escherichia Coli and the gram-positive Bacillus Subtilis are not that dangerous, however still bacterias that could cause diseases, thus we had to be careful to not have skin contact with them as well as not open the agar plates ones we placed a layer of bacteria on it so that it would not get in contact with the laboratory area to keep the environment clean.
6 agar plates
The gram-negative Escherichia Coli and the gram-positive Bacillus Subtilis
24 Antibiotic discs of 6 different types :
Cotton tipped swabs
Sterilizing alcohol 90%
All the equipments were sterilized inside a autoclave with high pressure before use.
Our hands were sterilized before starting with 90% alcohol.
Six agar plates were placed on the table as well as the bacteria Escherichia Coli and Bacillus Subtilis.
With the use of a cotton swab, three of the agar plates were rubbed with a thin even layer of Escherichia Coli and the three others with Bacillus Subtilis.
All 6 plates had 4 different types of antibiotics with a forcep and then placed in an incubator with high pressure and temperature for 48 hours
The next day, the agar plates were taken out and the diameter of the inhibitions of the antibiotics were measured with a ruler.
If we compare Graph1. containing the Escherichia Coli with Graph2. containing Bacillus Subtilis Bacillus Subtilis, we can see that the antibiotic Ampicillin had zero inhibition on Escherichia Coli bacterium, but it did inhibit Bacillus Subtilis Bacillus Subtilis with an average diameter of 1.55 cm ±0.01. However, Erythromycin had an overall higher average diameter of inhibition on Bacillus Subtilis Bacillus Subtilis than on Escherichia Coli. Trimethoprim, Tetracycline and Sulfonamide all had higher average diameter inhibition zones on Bacillus Subtilis Bacillus Subtilis than on Escherichia Coli. Observing from the Standard Deviation calculated for both the Escherichia Coli and Bacillus Subtilis Bacillus Subtilis values on both the graphs, we can see that Escherichia Coli has a higher STDEV than Bacillus Subtilis Bacillus Subtilis, which is for the most part because of the gap caused by Ampicillin zero inhibition on the bacterium making the values more separated and spread out.
Looking at the qualitative data collected seen on table 5&6 , we can that certain antibiotics such as Trimethoprim did not inhibit at all, which shows that some of the discs were too old and probably expired, making it a big limitation in values we did not obtain. So for future experiments, buying fresh antibiotics would be a necessity.
Also, another issue we had was that several of the inhibition zones were not perfect circles, thus making it harder for us to find the most reliable diameter to measure and depend on. This might also be because of the discs not being fresh and some of them expired as well. However, another reason for this might also be that the bacterias were not completely evenly layered on top of the agar plates with the cotton swabs. Another, more efficient application of the bacterias on the agar plates would have been better, such as using a disc the same size as the agar plates size and dip it in the bacteria and then taking it out with a forcep letting it stand until it does not drip anymore and then place it on top of the agar plates and then removing them to place the antibiotic discs on later. Maybe some sort of a machinery would be a good idea as well or just dipping the agar plates in the bacteria and letting it stand upside down with the top on until it does not drip anymore and then remove the top and pour out the excess liquid bacteria solution for further placement of antibiotics on top. Otherwise, measuring all the diameters from all the sides and then taking the average could also be a good idea.
As a conclusion from the data collected as well as the discussion section above, the investigation shows that the antibiotics in general were more efficient on the gram-positive Bacillus Subtilis Bacillus Subtilis than on the gram-negative Escherichia Coli. This can be explained from with the help of the background information, considering that even though the gram-positive bacterium has a thicker layer of peptidoglycan protecting it, the gram-negative Escherichia Coli has multilayer membrane structure and even if the peptidoglycan is thin layer, the antibiotic would have to absorb through each layer of membrane to reach the core in order to kill the bacteria.
Science online labs. Lab 3 antibiotics. (Available online): http://labs.7bscience.com/lab-3---antibiotics.html (Accessed 2/17-18)
Eoscu. Gram Positive vs Gram Negative Bacteria and the Fight Against HAIs. (Available online): http://blog.eoscu.com/blog/gram-positive-vs-gram-positive (Accessed 2/17-18)
IB biology book. Andrew Alliot (Accessed 2/17-18)