1AO3
Biology
Lab Biology Three:
Microbiology and Efficacy of Different Antibiotics
Date of Lab: 4 November 2010
Date of Submission: 11 November 2010
Abstract
In this laboratory lab, the treatment of water samples is tested on by antimicrobial agents to analyze
the effectiveness of treatments. In part A, through serial dilutions of the untreated water sample and the
treated water sample, the colonies of bacteria left can be observed in agar plates, the calculation of the
concentration of the bacteria per milliliter of a sample in both, control and treatment, can be compared. It has
been observed that bacterial colonies in the least concentrated treatment are significantly less than the
bacterial colonies in control. In Part B of the lab, four different antibiotic discs are placed in each of the
plates of E. coli, M. luteus and untreated water sample. After 24 hours of incubation, the amount of clearing
was exhibited an thus, the gram positive and gram negative nature of the lawns were observed. Gram +ve
and gram ve are differ in which antimicrobial treatment is more effective when the zone of inhibition is
measured.
Introduction:
Untreated water can turn into a deadly case, and measures must be taken to treat it. The issue is to
measure the approximate number of bacteria contaminating a water sample, and suggest an experiment that
contributes to the reduction of microbial contamination in the water. Using the technique of serial dilution,
the bacteria can be counted in a contaminated solution with the naked eye. The untreated water is mixed with
a large amount of saline, called a diluent. Then, a series of 10-fold dilutions of the original mixed fluid is
carried out a number of times. Serial dilution is important because the experimenter can use the culture plates
that produce countable numbers from the different dilutions to calculate colony-forming units per millileter
of the sample, as well as how many bacteria in total were present. The treated water sample with the least
concentration is expected to exhibit the least amount of bacterial colonies and opposed to the lesser diluted
samples.
In this laboratory experiment, the treatment used in part A is sodium hypochlorite or bleach. Sodium
hypochlorite has a lot of uses and is an excellent antimicrobial agent.. Since it is strong oxidizer, it works by
denaturing a bacterias proteins causing insoluble aggregations, and clumping of proteins. This leads to loss
of proper function of essential bacterial growth, and the cell eventually dies. This will show how many cells
of the colonies are still alive in the treated water samples and compared to the untreated water samples.
Antibiotics can be selective; some are non-toxic to plants, animals and unstimulated bacterial cells,
but are toxic to bacteria that are being reproduced. There are four ways that antibiotics can inhibit bacterial
reproduction; since bacterial cells have a cell wall are composed of murein that is important for structural
support and bacterial growth, antibiotics can inhibit murein formation; antibiotics can damage cell
membrane; they can interfere with protein synthesis; and they can inhibit nucleic acid metabolism.
How effective an antibiotic is against bacteria can be dependent on whether the bacteria is Gram +ve
or Gram -ve. In Part B, four different antibiotic discs were placed in each Petri dish of a Gram ve culture
(E.coli), a Gram +ve cultutre (M.luteus), and an untreated water sample lawn., The antibiotics used in this
experiment include penicillin, streptomycin, tetracycline, and chloramphenicol. These antibiotics kill
bacteria by inhibiting protein synthesis which prevents bacterial reproduction. After a day of incubation, the
amount of clearing is analyzed.
Purpose
The purpose of this laboratory experiment is to create serial dilutions to accurately count the
population number of bacteria within liquid sample. Also, effects of antibiotics on E.coli, M. Luteus, and
untreated water are observed so that an effective decontamination method can be determined. Qualitative and
quantitative results are obtained for analysis.
Hypothesis
For part A, the untreated water sample is treated with bleach (sodium hypochlorite). It is
hypothesized that this treatment would result in a lower amount of colonies in comparison to the untreated
water sample due to the strength of the bleach as a decontaminator. This result can be drawn from the control
and treated serial dilutions. The number of colonies of the bacteria can be compared to the concentration
calculated: the higher the concentration, the increase in the amount of the colonies of bacteria. For part B, it
is hypothesized that the effects of the antibiotics would differ depending on whether the bacteria is gram
positive or gram negative based on their chemical composition of the cell wall.
Materials and Methods
Part A:
Materials:
5l of bleach (treatment)
A bunsen burner
8 sterile 2 ml snap-cap micro-centrifuge tubes
A P1000 pipette
A P200 pipette
Sterile saline
Numerous sterile blue tips
Numerous sterile yellow tips
2 Petri plates
A Bunsen burner
A wire loop
Methods:
1) Have your gloves on all the time during the procedure to avoid contamination.
2) Clean your bench with Dettol and start the Bunsen burner to keep the bench reasonably sterilized
throughout the procedure.
3) Label the 8 sterile microfuge tubes as followed:
The control serial dilutions: C10-1,C10-2,C10-3,C10-4
The treatment serial dilutions: T10-1, T10-2, T10-3, T10-4
4) With P1000 and a blue tip, place 900l (0.9ml) of sterile saline into each labeled microfuge tube.
5) With P200 and a fresh sterile tip for each time
The control serial dilutions:
o
Withdraw 100l of untreated water sample, drop it into the C10-1 tube and mix it well.
o
Withdraw 100l of solution in the C10-1 tube, drop it into the C10-2 tube and mix it well.
o
Withdraw 100l of solution in the C10-2 tube, drop it into the C10-3 tube and mix it well.
o
Withdraw 100l of solution in the C10-3 tube, drop it into the C10-4 tube and mix it well.
6) For the treatment serial dilutions, add 5l bleach together with the untreated water sample, wait for 5
mins, and then follow closely to Step 5).
7) Draw 4 quadrants at the bottom of each Petri plate and name each quadrant: C10 -1, C10-2, C10-3, C10-4
and T10-1, T10-2, T10-3, T10-4.
8) Sterilize the wire loop on the Bunsen burner, leave it to lower the temperature
The control serial dilutions:
o
Obtain 1l of solution from the C10 -1 tube and cover the C10-1 quadrant with it evenly.
Make sure to recap the test tube before moving on to the next dilution.
o
Obtain 1l of solution from the C10 -2 tube and cover the C10-2 quadrant with it evenly.
Make sure to recap the test tube before moving on to the next dilution.
o
Obtain 1l of solution from the C10 -3 tube and cover the C10-3 quadrant with it evenly.
Make sure to recap the test tube before moving on to the next dilution.
o
9)
Obtain 1l of solution from the C10-4 tube and cover the C10-4 quadrant with it evenly.
For the treatment serial dilutions, follow closely to Step 2)
10) Independent Variable: Dilution of the untreated water and treatment samples.
Dependent Variable: Number/Density of the colonies
11) Do not discard the untreated water sample, it is to be expected for Part B.
Part B:
Materials:
3 Petri plates
E. coli lawn
M. luteus lawn
Untreated water sample from Part A
A P200 pipette
3 sterile blue cell spreaders
4 different antibiotic discs for each Petri plate (Penicillin, Streptomycin, Tetracycline & Chloramphenicol)
Numerous sterile yellow tips
Methods:
1) Independent Variable (s): Types of bacteria and antibiotics
Dependent Variable: Diameters of the clear-areas surrounding each antibiotic disc
2) Label the bottom of each Petri plate as: E. coli lawn, M. luteus lawn and Untreated water sample lawn.
3) Withdraw 100l of E. coli using the P200 pipette. Empty the sterile tip on the E. coli plate and smooth it
out with the blue cell spreader. Make sure the liquid culture is spread out evenly on the agar and that it
has been fully absorbed.
4) Using a fresh sterile tip and blue cell spreader each time, repeat Step 3) for M. luteus and untreated water
sample.
5) Place one of each antibiotic disc (Penicillin, Streptomycin, Tetracycline & Chloramphenicol) on every
Petri plate.
6) Bundle all 5 Petri plates from the entire experiment with masking tape. With the Part A Petri plates
placed upside-down.
7) Come back 24 hours later to view the results.
8) Quantitative and qualitative observations of the visible bacteria were taken. The concentration of bacteria
was calculated which acted as the dependent variable.
Results
Part A
In Table 1.1 of Appendix B, the total dilution is calculated of each individual micro-centrifuge tube.
The dilutions of 10-1, 10-2 , 10-3 , 10-4 resulted from the process of serial dilution which allows for four separate
concentrations for both the control and treated water solutions.
Referring to Appendix B Table 1.3, there is a distinct trend of reduction in the number of colonies
as solution the becomes more diluted. Colonies in Quadrant C10-1 and C10-2 are much to concentrated to be
counted. Quadrant C10-3 was less condensed than the first 2 quadrants, but there was much more colonies in
it than there was in Quadrant C10-4. The average concentration of bacteria was 5.4 *107, it was calculated as
the mean value of the concentrations of C10-3 and C10-4.
Referring to Appendix B Table 1.4, the results of treatment serial dilutions are shown. A small
amount of bleach (5l) is added to the treatment group. Quadrant T10 -1 contains 3 colonies, and Quadrant
T10-2 contains only 1 colony left. Both Quadrants T10-3and T10-4 contain no colony at all. The average
concentration of bacteria, 3.25 * 104 cfu/ml, is much lower than the control serial dilution. The quadrants in
the treatment Petri dish are a lot more clear or have fewer spots of bacteria compared to the untreated water
sample Petri dish
Part B
In the E. coli agar plate, the highest clearing is by the antibiotic chloramphenicol (25 mm), then
penicillin (12 mm).The other two bacteria, streptomycin and tetracycline, resulted in a nonexistent diameter.
In the M. luteus agar plate, penicillin is of the highest diameter (26 mm) followed by
chloramphenicol (17 mm). The other two antibiotics displayed minicule uneven diameter that could be
recorded properly.
The untreated water sample in the agar plate displayed diameter of 30 mm due to chloramphenicol,
15 mm due to penicillin, and none due to others.
As shown in Appendix B - Table 1.3, both, penicillin and chloramphenicol seemed, to work well
with all 3 types of bacteria, while Streptomycin and Tetracycline had no effect on the bacteria.
Chloramphenicol was most effective with the E.coli lawn and the untreated water sample lawn. Penicillin
worked best for the M. luteus lawn.
Discussion
Part A
A 9 mL of untreated water sample was treated with 5 L of the independent variable bleach in a
sterile microfuge tube and acted as a treatment for five minutes. We needed to do the serial dilutions because
otherwise, the untreated water sample would be too concentrated and the colonies would be too numerous to
count no matter how tiny the amount of volume we have plated, giving us inaccurate results. With an optimal
dilution, the density of the bacteria can be accurately counted. Since we dont know how many colonies are
present until we have incubated the plates for 24 hours, we need to plate many different dilutions and see
which produce (s) countable numbers.
The hypothesis is that there would a decrease in the number of colonies is due to the
treatment of bleach. This is hypothesized because bleach is an antibacterial agent and would prevent
bacterial reproduction. It is observed that there is a decrease in the bacterial population due to the
bleach added to the untreated water sample as predicted. The results match the hypothesis. The
purpose of including a control series is so that we can see the difference between the two Petri
dishes and analyze results according to that. We can figure out how effective our treatment against
the bacteria. We determined how many bacteria were present in the whole sample by calculating the
concentration (cfu/ml sample = number of colonies per/(amount plated (ml)*dilution)) and
multiplying it with the volume of the volume of the sample.
However, there are factors that could have affected the concentrations in the control and treatment
solutions. These factors include whether one of the tubes in the serial dilution was heavily contaminated with
bacteria before the dilution series was made, a fresh pipette was not used at all steps of serial dilution, an
agar plate could have been contaminated before dilution was spread on it, the wire loop did not cool and
killed all the bacteria when plating a quadrant, or mixing of each dilution tube before taking a sample was
not performed properly. The unexpected result was how effective the treatment had been against the
untreated water sample, resulting in a virtually clear Petri dish. If this was true, then bleach is quite powerful
when interfering with the bacterias RNA transcription and protein manufacture. The error can be that to the
bacteria was let to sit with the treatment for too long.
An extension of this laboratory experiment may be testing more different treatment methods with
bacteria to analyze growth in different situations. There can be different temperatures, concentrations, or
amount set. If we can test different concentration of bleach in contaminated water, we can determine when it
is most efficient without harming or being toxic to other living things, such as humans. By using different
temperatures and treatments, we can further determine when and which antibacterial agent to use in order to
maximize efficiency. Also, the development of bacterial resistance to antibacterial agents can be tested at
different dilutions of treatments.
Part B
All the antimicrobial agents used work to inhibit protein synthesis, resulting in prevention of
bacterial growth. Penicillin works by destroying the cell wall, inactivating an enzyme necessary for the cross
linking of bacterial cell wall, and the bacteria soon dies. Streptomycin blocks protein synthesis by binding to
the bacterial ribosome. Tetracyclines inhibit synthesis of protein important for survival of cells.
Chloramphenicol also interferes with protein synthesis.
For part B, it was hypothesized that specific antibiotics work more or less effectively depending on
whether the bacteria is gram +ve or gram ve, and its chemical composItion due to that. The cell
composition differ by the fact that the cell wall of gram-negative bacteria is a thin layer peptidoglycan
between the cytoplasmic membrane and the lipid-containing outer cell envelope, whereas the gram-positive
cell wall is of a thicker diameter of peptidoglycan that encircles the cell., lacks the cell envelope, and
contains additional substances, such as proteins. If the zone of clearing or inhibition is of larger amount, then
the bacteria is more susceptible to that specific antimicrobial agent than the others.
E. coli (a gram ve culture) is shown to be more resistant towards streptomycin (0 mm), tetracycline
(0 mm), and penicillin (12 mm). It is more susceptible to chloramphenicol because it exhibited the greatest
diameter of the zone of clearing (25 mm). This can be due to the fact that E. coli is composed more of lipids
and carbohydrates than proteins. In the agar plate of M. luteus, the gram +ve culture shows greater
susceptibility to penicillin (26 mm) than chloramphenicol (17 mm). In the case of the untreated water sample
Petri dish, the diameters are very similar when compared to the E.coli Petri dish results. This may show that
untreated water sample in our experiment contained more of gram ve bacteria. It is possible that penicillin
works better with gram +ve bacteria because its primary goal is to destroy the cell walls of bacteria, can the
cell walls of M. luteus are thicker and are more sensitive to lysosomes that digest it. Gram ve bacteria has to
2 cell layers, rich in lipids and protieins and is less sensitive to lysozome which may explain why
chlorapmphenicol works better.
However, there may have been errors as the quantitative observations might have inaccuracies: the
aseptic technique may not have been present throught the lab, the correct amount of solutions may not be
micropipetted, or the diameters may have been wrongly measured as some of them were of uneven distance.
For future extended experiments, it would be interesting to increase the number of bacterial strains to test.
For example, we could also test disinfectants on a gram-positive bacterium, such as Stapylococcus epidermis
and figure out whether S. epidermis susceptible to the same disinfectants as E. coli?
Literature Cited
1. "Serial Dilutions." UMass Amherst: The College of Natural Sciences: Departments. N.p., n.d. Web. 5
Nov. 2009. <http://www.bio.umass.edu/micro.
2. Johnson, T. and C. Case, 1995. "Chemical Methods of Control," adapted from Laboratory
Experiments in Microbiology, Brief Edition, 4th ed. Redwood City, CA: Benjamin/Cummings
Publishing Co., available online from The National Health Museum, Access Excellence Activities
Exchange [accessed September 11, 2006]
http://www.accessexcellence.org/AE/AEC/CC/chance_activity.html .
3. Van Hoeck, Baker and Tokuhisa, 2004. "What is a zone of inhibition on an agar plate?" Ask A
Scientist, Molecular Biology Archive, University of Chicago and Argonne National Laboratories,
Newton Bulletin Board System [accessed September 11, 2006]
http://www.newton.dep.anl.gov/askasci/mole00/mole00531.htm.
4. Rollins, D.M. and S.W. Joseph, 2000. "Antibiotic Disk Susceptibilities," Department of Cell Biology
and Molecular Genetics, University of Maryland, College Park [accessed September 11, 2006]
http://www.life.umd.edu/classroom/bsci424/LabMaterialsMethods/AntibioticDisk.htm .
5. Bio1A03 Lab Manual, 2009. Microscopy and Efficacy of Different Antibiotics. Department of
Biology.