Biology SNAB Core Practical - Gram Staining Bacteria

AS and A Level Science

Core Practical – Gram Staining Bacteria

Introduction

When a bacterial infection is diagnosed antibiotics may be

prescribed. Different antibiotics are not equally effective

against all bacteria, so the correct antibiotic must be selected

for a particular bacterial infection. In some cases the most

effective antibiotic is known, but in other cases tests need to

be carried out by a pathology department. In this activity I

will be testing the effectiveness of several types of antibiotics

on bacteria

Hypothesis

The aim of the experiment is to test the effectiveness of different types of antibiotic on bacteria. The bacteria that we will be using will be Escherichia Coli and Bacillus Subtilis. I predict that the antibiotics will be more effective on B. Subtilis.

Variables

The Independent variable in this investigation is the presence of the different types of antibiotics. This will be changed by using a mast ring which has all the different types of antibiotics around it. The Dependant variable in this investigation is the zone of inhibition around the disc. This will be measured by measuring the zone around the disc, using a ruler, after the Petri dish had been left for an adequate amount of time. The control variable was the length of time that the antibiotics had to kill the bacteria. This was controlled by everyone leaving the dishes for the same amount of time, 24 hours. Another variable is type of bacteria, which is altered by putting E. Coli in one Petri dish and B. Subtilis in the other. Temperature will be controlled by leaving both dishes at the same temperature (30 degrees) so that this cannot affect the results.

Equipment

● Agar plate seeded with known bacteria

● Sterile Pasteur pipette

● Bunsen burner

● Beaker of disinfectant, 1% Virkon

● Bench spray of disinfectant, 1% Virkon

● Bactericidal soap

● Paper towels

● Marker pen

● Forceps

● Mast ring or antibioticimpregnated paper discs

● Adhesive tape

● Incubator set at 30 °

Method

Label two Petri dishes with the names of the bacteria and pour in the agar jelly, which was incubated to 30 degrees.
Add in the bacteria to the agar plate, making sure to flame the neck of the bottle of bacteria, and not letting the lid touch the surface.
Sterilise the forceps and use them to pick up the mast ring, ...

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Equipment

● Agar plate seeded with known bacteria

● Sterile Pasteur pipette

● Bunsen burner

● Beaker of disinfectant, 1% Virkon

● Bench spray of disinfectant, 1% Virkon

● Bactericidal soap

● Paper towels

● Marker pen

● Forceps

● Mast ring or antibioticimpregnated paper discs

● Adhesive tape

● Incubator set at 30 °

Method

Label two Petri dishes with the names of the bacteria and pour in the agar jelly, which was incubated to 30 degrees.
Add in the bacteria to the agar plate, making sure to flame the neck of the bottle of bacteria, and not letting the lid touch the surface.
Sterilise the forceps and use them to pick up the mast ring, and place it in the agar plate containing the bacteria. Don’t open the lid for too long as contamination could take place.
Tape the lid to the base using adhesive tape and place in the incubator for 24 hours.

After incubation, observe and measure the clear zones around the antibiotic – the zone of inhibition.

Risk Assessment

Likelihood and Severity is on a scale of 1-5, one being low, five being high. Risk is calculated by multiplying likelihood and severity, so is out of 25. Generally, if the risk is over 10 then the experiment should not take place.

Validity

The results will be valid because the experiment is designed to test the hypothesis, so the experiment will test what it is intended to. The results may not be that reliable as we are not doing any repetitions. The results should be accurate as the equipment is all functioning correctly currently.

Results

Zone = Zone of Inhibition (clear area around antibiotic)

PG = Penicillin

NO = Novobiocin

OX = Oxacillin

FC = Fucidic acid

T = Tetracyline

E = Ethylronyin

C = Chlorophenicol

S = Streptomycin

S. Albus was used as a reference bacteria, as we did not carry out the experiment on this bacteria.

Conclusion

The patterns in the results is that generally the results support the hypothesis which is that the B. Subtilis has greater zones of inhibition in more of the antibiotics due to the antibiotics being more effective against this bacteria.

It is hard to know if there are any anomalies, as we did not repeat the experiment, so we cannot see if there are any results that do not fit in with the general trend.

The basic trend on the graph is that E. Coli is the most resistant to antibiotics, as there are no zones of inhibition for penicillin, oxacillin, or fucidic acid. Whereas B. Subtilis has Zones on all of the antibiotics except for penicillin.

E. Coli is gram negative and B. Subtilis is gram positive, which could explain the results. The fact that E. Coli is gram negative means that it has an outer membrane. This outer membrane protects the bacteria from several antibiotics, such as penicillin. Whereas B. Subtilis does not have this protection of the outer membrane so has decreased resistance.

Evaluation

During the experiment, I think that a few systematic and random errors may have taken place.

Firstly, a systematic error was that whilst measuring the zone of inhibition, there was some confusion about whether or not to include the disc of antibiotic. Some did, and some didn’t, which could affect the results. If I was to do the experiment again, I would either have the same person measuring all the zones, or say for sure that you didn’t count the disc containing the antibiotic.

Next, some of the zones were not circular in shape, and therefore had sides that were longer than others. This could create different results depending on the angle at which you measure the zone of inhibition. To get around this I would make it clear at the start to measure the longest diameter of the zone, so as to get consistent results.

A random error may have occurred for E. Coli at the antibiotic Ethylronyin as the reading is significantly lower

Different amounts of agar jelly may also have been used, which could produce different results, as this is the medium for the bacteria. Therefore in future I would say that we shouls use a set amound to agar. The same could be said for the bacteria as the amount added was just ‘a pipette’.

I think that the results were valid, as they tested the hypothesis. I don’t think that the results are very reliable, as we did not do any repetitions, however I think that the results were accurate due to the simplicity of the method and equipment.

If I was to take this investigation further, I would test more bacteria against more types of antibiotics.