Documenti di Didattica
Documenti di Professioni
Documenti di Cultura
Antibiotics have varied modes of action and are usually categorized by their
chemical class. Beta-lactam is the chemical name for antibiotics that have a
chemical/lactam ring. This extensive family includes the penicillins4
Gram positive bacteria appear bluish purple under a light microscope as they
take up the crystal violet stain and remain unaffected by the rest of the
process12. Examples include staphylococcus and bacillus bacteria;
characteristics common to these include two basic layers, a plasma
membrane and a thick layer of peptidoglycan. Typically these bacteria are
particularly sensitive to antibiotics such as penicillin and sulphonamides.
6) http://helios.bto.ed.ac.uk/bto/microbes/penicill.html
7) Same as above
8) http://print.factmonster.com/ce6/sci/A0846951.html
9)http://www.bmb.leeds.ac.uk/mbiology/ugteach/dental/antibiotics/dantibiobact/p
rotag.html
10) Pg. 394, Bacteria in Biology, Biotechnology & Medicine, Paul Singleton
11) Pg. 476, Microbiology, 4th Edition, M.J.Pelczcar, R.D.Reid and E.C.S Chan
12)Pg. 592, Biology, 2nd Edition, Ann Fullick, Heinemann Educational Publishers
2000
Gram negative bacteria such as salmonella, haemophilus and escherichia
coli, appear to red under a light microscope, as any crystal violet which does
bind to the bacterial smear is readily decolorised by the alcohol and replaced
with red safranin dye. Gram negative bacteria have thinner walls containing
less peptidoglycan, and are usually sensitive to broad-spectrum antibiotics
such as streptomycin.
13) http://people.ku.edu/~jbrown/ecoli.html
14) Pg. 448, Bacteria in Biology, Biotechnology & Medicine, 5th Edition, Paul
Singleton
15) http://www.bact.wise.edu/Bact330/lectureecoli
16) Same as above
17) http://www.bact.wise.edu/Bact330/lectureecoli
18) Pg. 369, Bacteria in Biology, Biotechnology & Medicine, 5th Edition, Paul
Singleton
19)Pg. 376, Microbes in Action, A Laboratory Manual of Microbiology, 4th Edition,
Seeley, Vandemark, Lee
20) Pg. 376, Bacteria in Biology, Biotechnology & Medicine, 5th Edition, Paul
Singleton
inoculum is pipetted into a sterile petri dish via aseptic techniques. The
melted warm agar is then poured into the plate and the dish is gently swirled,
three or four times in each of the three directions. On incubation colonies
develop within as well as on the medium20.
13) http://people.ku.edu/~jbrown/ecoli.html
14) Pg. 448, Bacteria in Biology, Biotechnology & Medicine, 5th Edition, Paul
Singleton
15) http://www.bact.wise.edu/Bact330/lectureecoli
16) Same as above
17) http://www.bact.wise.edu/Bact330/lectureecoli
18) Pg. 369, Bacteria in Biology, Biotechnology & Medicine, 5th Edition, Paul
Singleton
19)Pg. 376, Microbes in Action, A Laboratory Manual of Microbiology, 4th Edition,
Seeley, Vandemark, Lee
20) Pg. 376, Bacteria in Biology, Biotechnology & Medicine, 5th Edition, Paul
Singleton
Hypothesis
It has been noted that spread plating the bacteria will ensure an even
distribution of confluent growth across the plate. Hence the hypothesis to be
tested is that the spread plating technique will aid the antibiotic’s
effectiveness by exerting a noticeable effect on the diameter of the zone of
inhibition
Independent Variable
Dependent Variable
Controlled Variables
• Antibiotic used
After the sensitivity test in the pilot study is performed, the type of
antibiotic used (either penicillin or streptomycin) will be confirmed for use
in the experiment itself. This antibiotic will remain consistent in its type
and concentration for every sample it is included in.
Please note
An aseptic technique will be used for the pilot study and the experiment.
Pipettes will be wrapped in foil prior to the trials to increase their sterility,
petri dishes will be kept in a sealed plastic bag and any instrument which is
to be involved in transfers and is less likely to catch fire will be flamed (e.g.
bottle necks, spreaders, forceps).
Outline Method
Three lawn plates were created by first flaming the bottlenecks of the
McCartney bottles containing the nutrient agar. This was then poured into
each of the petri dishes and left to cool and solidify, 0.2cm3 of the culture was
then transferred into each petri dish. The spreader was then dipped in
ethanol and flamed to increase its sterility and left to cool. Once the spreader
had thoroughly cooled, the sample of bacteria in each petri dish was spread
as evenly as possible by revolving each plate slowly on the bench. Forceps
were dipped in ethanol, flamed and left to cool. The antibiotic discs of
penicillin and streptomycin were placed in two of the plates. A disc of sterile
filter paper was placed in the third (to act as a control group).
Three pour plates were created by first flaming the neck of the McCartney
bottle containing the culture, 0.2cm3 of this was transferred into each petri
dish. The bottlenecks of the McCartney bottles containing the nutrient agar
were flamed. These were then poured into each petri dish and left to solidify.
The forceps were then dipped in ethanol and flamed. To increase sterility and
left to thoroughly cool. Then antibiotic discs of streptomycin and penicillin
were added to two of the petri dishes, to the third a sterile filter paper disc
was added as a control.
Equipment
− 6 Sterile Petri dishes, Sterile filter paper discs, Sterile 1cm3 pipettes and
pipette filler
− Verkon Disinfectant
− Ethanol
− Autoclave bag
− Ruler (30cm)
Risk Precaution
A basic sensitivity test was done as the pilot study, to gain practice in the
microbiology techniques, discover which antibiotic is the most effective
(creates the larger zones of inhibition) and to also establish where there is
overall room for improvement before the real experiment is executed.
Please note that control groups were made for each culture technique but
could not be included in this table as they were designed to display the
absence of a ZoI.
* In this result the growth of colonies was slightly skewed, as there was no
even distribution of bacteria. Hence there was no growth around the penicillin
disc for it to inhibit.
− Verkon Disinfectant
− Ethanol
− Autoclave bag
− Ruler (30cm)
The investigation was performed in the biology laboratory of Bedford College. All
practical work was supervised and took approximately 1hour and 30 minutes to complete.
All petri dishes were dated and labeled before the experiment.
1) The bench was wiped down with a disinfectant and a Bunsen Burner was
lit on the surface.
2) A McCartney bottle containing nutrient agar was taken from the water
bath thoroughly dried with a paper towel, to prevent contamination.
3) The lid of the McCartney bottle was removed and the neck of the bottle
was flamed to increase sterility.
4) The lid of the petri dish was opened was opened as little as possible whist
the agar was poured into it.
5) With the plate on the table the agar was gently swirled three or four times
in each of the three directions to ensure even distribution of the agar
6) The steps (2) to (5) were repeated until 3 plates were created.
7) The McCartney bottle of the culture in nutrient broth was shaken well, to
abate any solid cultures that may have been present. The lid was then removed
and the bottleneck was flamed.
8) Once the agar had solidified, 0.2cm3 of the culture was transferred using
aseptic techniques, into a petri dish containing a solid medium of nutrient agar.
11) The steps (8) to (9) was repeated for each of the 3 plates.
12) The forceps were dipped in ethanol, flamed then allowed to burn and
thoroughly cool.
13) An antibiotic disc of streptomycin was added to each of the two plates. A
sterile filter paper disc was added to the third to act as a control group.
(Method cont’d)
Preparation of Pour Plates
2) The lid of the McCartney bottle containing the culture in nutrient broth was removed
and the neck was flamed, 0.2cm3 of this was then transferred using aseptic techniques,
into a petri dish.
3) A McCartney bottle containing nutrient agar was removed from the water bath and
dried thoroughly to prevent any contamination.
4) The lid of the bottle was removed and the neck was flamed to increase sterility.
5) The lid of the petri dish was opened as little as possible whilst the nutrient agar was
poured into it.
6) With the plate on the table the nutrient agar was gently swirled three or four times to
ensure an even distribution of agar.
7) The steps (2) to (6) were repeated until 3 plates were created. The agar plates were
then left to solidify.
8) The forceps were dipped in ethanol, flamed and then allowed to burn and thoroughly
cool.
9) A disc of streptomycin was added to each of the two plates. To the third a disc of
sterile filter paper was added, to arise as the control group.
10) The entire experimental procedure was repeated to obtain averages and accuracy.
12) After incubation, the diameter if the inhibition zones were measured using a 30cm
ruler on the base of the petri dish.
13) Results were then tabulated and all samples were disposed of in an autoclave bag.
The Experiment
These are the results after 96 hours of incubation.
Round I
Spread 2 26mm
Round II
Table of Averages
Taking into account all the values produced regardless of which round
they belonged to, the Range (26-22) was found to be 4mm.
The Statistical Test
(Mann Whitney U-Test)
Spread (NA) 23 24 25 26
RANKA 1 2 3 4
Pour (NB) 22 22 22 23
RANKB 2 2 2 4
ΣRA = 10
ΣRB = 10
NANB= 4 X 4
=16
Analysis
Table of Averages
The average of the results obtained in Round I was taken and then the same
was done for the results obtained in Round II. Hence these two averages were
tabulated in a Table of Averages. The difference between these two averages
is 2mm. This displays some supporting evidence for the hypothesis.
Graph # 1
This is a composite bar chart, which displays the results obtained from Round
I and Round II of the experiment. This chart displays some evidence of
supporting the hypothesis as it proves that spread plating had a slightly
larger effect on the effect of the diameter of the inhibition zone. The Range
(which can be found below the table of results) was calculated to be 4mm,
this displays some variability between the two techniques.
Graph #2
This is a standard bar chart, which only displays the results obtained in Round
I of the experiment. Again this contributes some supporting evidence toward
the hypothesis as the mean difference between the two techniques was
found to be 3mm. This was calculated by finding the difference in the squares
of the bar chart between 1 and 2, and 3 and 4. The mean of these values was
then taken.
Graph #3
This is also a standard bar chart, but it only displays the results obtained in
Round II of the experiment. This also displays supporting evidence towards
the hypothesis. The mean difference was also calculated for this data and
was found to be 1.5mm. This indicates a slight difference between the two
techniques.
The Mann Whitney U-Test was the statistical test used in this experiment. The
U-Test was used to compare the median values between the two sets of data.
It also helped to identify whether there was any difference between them.
The U-Test is a non-parametric test, which allows us to use it when the
sample is relatively small: this is a relevant and important advantage to this
experiment.
After performing the U-test from the results obtained, it can be concluded
that:
Further Work
After performing the experiment I feel there is still room for improvement. In
the event of a future investigation, I would:
• Make more replicates of both the spread and pour plating technique, as
well as other culturing techniques (e.g. streak plating) to produce a clear
picture of the overall results.