Recycled Liquid Cloth Wash Bacteria Biology Essay

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This project was devised to investigate the effect of different concentration of recycled liquid cloth wash on bacterial growth and the resistance of bacteria against the specific concentration of recycled cloth wash. The effect was investigated by using a serial ten-fold dilution technique to dilute the recycled liquid cloth wash and observing its effect on the growth of bacterial colonies of Echerichia coli sp. and Bacillus subtilis sp. with a control group of sterilized distilled water over successive periods of 24 hours. The results showed that the increase in dilution results in the increase in number of colonies appearing, and that the increase did not occur by chance, agreeing with the hypothesis. Also, it appeared that the difference between the median of the groups in treatment of 100% concentration of recycled liquid wash and 1% concentration was significant, thus 100% concentration of recycled liquid wash was still effective against microorganisms.

Experimental Hypothesis

The greater the dilution of recycled liquid cloth wash, the higher the bacterial growth.

Null Hypothesis

There is no relationship between the dilutions of recycled liquid clothes wash and the growth of bacteria.

Research and Rationale

An investigation is conducted to investigate the effect of different concentrations of recycled liquid cloth wash on bacterial growth and how this could be applied to the efficiency of recycling used liquid cloth wash in our daily lives.

Bacteria are prokaryotic enzymes that may be pathogenic and cause harm to the human body. Although some are beneficial to living organisms, others invade organisms and secrete endotoxins or exotoxins that harm the host's life. Endotoxins are lipopolysaccharides that are part of the outer layer of Gram negative bacteria which cause symptoms of a disease while exotoxins are soluble proteins that are produced and released into the body by bacteria which cause symptoms of disease1. Echerichia coli sp. (E. coli) is a diverse species of bacteria found in abundance in the environment around us. E.coli sp. infects the digestive and respiratory systems of human and often causes diarrhoea. It is a Gram negative bacteria, characterized by staining red in Gram staining1 and having a peptidoglycan layer thinner than Gram negative bacteria, which are bacteria that stain blue in Gram staining. E. coli sp. was chosen as the model organism in this experiment due to its simple structure and is easily grown in the laboratory7. Bacillus subtilis sp. is the equivalence of E. coli sp. of Gram positive bacteria. Thus, both species were used and compared in this experiment.

The number of bacterial colonies that appear on the Petri dishes signify the resistance exhibited by the particular strain of bacteria against a specific concentration of recycled liquid wash over a period of time.

The answers obtained may shed some light on the potential of reusing available sources of disinfectant instead of spending more resources, money and energy to manufacture new products to be used domestically. The 100% concentration of recycled liquid wash that effectively inhibited the growth of bacterial colonies is a clear proof that even after its primary usage of killing bacteria in clothes, the used liquid wash can still be used to disinfect surfaces such as house pavements and stairwells of public places where the utility of a strong disinfectant may be spared.

Planning

A trial experiment was conducted to obtain the best concentration of recycled liquid wash that would be effective for the longest time on both bacterial species. To prepare the recycled cloth wash liquid, a solution of liquid wash (10 mℓ of liquid Dynamo in 200 mℓ of distilled water) was used to soak a piece of dirty cloth for 12 hours. The bacterial lawns were prepared in a sterilised condition, adhering to the procedure below. The independent variable in this experiment was the concentration of disinfectant used and it was varied by using concentrations of 100% and diluting other samples to concentrations 10%, and 1%. The dependent variable in this experiment was the resistance of bacterial cultures against the different concentrations of disinfectant and it was assessed by counting the number of bacterial colonies over a period of time. Conditions such as the temperature and amount of bacterial inoculums were set at 30°C and 200 μℓ, respectively. The trial experiment was repeated to compare the precision of data.

The techniques used were aseptic and ten-fold serial dilution techniques. Aseptic technique was carried out by sterilising the table top where the experiment was conducted, heating the head of the glass containing the bacteria and the nutrient agar in a Bunsen flame, and sterilising the apparatus used in an oven. Ten-fold serial dilution was conducted by adding a 100% concentration of recycled liquid wash into a test tube and transferring a small amount of content from the first test tube into a second test tube before adding sterilized distilled water into the test tube to dilute it. The ratio of recycled liquid wash to sterilized distilled water is 1 : 9, forming a 10% concentration. Similarly, the content in the second test tube was further diluted into a third test tube. The test tubes were centrifuged to allow even mixing of solutions.

Procedure

The surface of the table on which the experiment was conducted was sterilized by spraying 70% of alcohol and wiping the area with a piece of tissue paper.

A ten-fold serial dilution was carried out on the recycled liquid wash, forming solutions of 100%, 10%, and 1% concentration. An extra control group was set up using sterilized distilled water.

100 μℓ of each the solutions was placed into a sterile Petri dish.

200 μℓ of bacterial inoculum of Echerichia coli sp. was placed into a Petri dish using 200 μℓ micropipette and teat.

Steps 3 and 4 were repeated for bacterial inoculum of Bacillus subtilis sp. and a control group of sterilized distilled water.

The Petri dishes were placed in an incubator at temperature 30.0°C for 24 hours.

Observations made were recorded.

The data obtained were analysed by employing the Kruskal-Wallis Test2 which is a non-parametric test to compare the medians of four independent groups of sampled data.

Trial Experiment Results

The results for the resistance of bacteria against different concentrations are as follows.

Dilutions

Number of Bacterial Colonies

Temperature (°C)

Escherichia coli sp.

Bacillus sp.

Distilled Water

100%

0

0

1

30.0 ± 0.1

10%

6

6

3

30.0 ± 0.1

1%

15

21

6

30.0 ± 0.1

0% (dH2O)

1

0

2

30.0 ± 0.1

After 24 Hours - Trials

From the table above, it was observed that the number of bacterial colonies increases with the dilution of recycled liquid wash. However, the variation between the number of colonies at different dilutions was not clearly seen, which suggests that the data should be amended. Also, very little number of colonies was seen in the set containing sterilized distilled water. The bacterial species used were maintained for the real experiment as the effect of the dilutions was clearly seen on them.

The temperature of 30.0°C was retained for the real experiment as it is suitable for the growth of both E. coli sp. and Bacillus sp. since they are mesophiles6 - bacteria whose optimal growth occurs at temperature between 20-40°C.

From the results obtained, the procedure was amended for the main experiment by using 400 μℓ of bacterial inoculum instead (using a 1000 μℓ micropipette) as the effect of recycled liquid wash concentrations on the bacterial colonies was subtle. This is because, after 24 hours, the results obtained for Bacillus sp. too was not encouraging as the difference between the numbers of colonies observed was too small to note a significant difference. This could probably be due to the high amount of liquid wash as compared to the amount of bacterial inoculum, the former being too effective in inhibiting the growth of bacteria.

Risk Assessment

E. coli sp. and Bacillus subtilis sp. were considered a risk factor in this experiment. This is a potentially harmful experiment as bacterial infection is possible should the inoculums be not handled properly. Therefore, the hands were washed with a disinfectant thoroughly and dried at a distance from the Bunsen burner flame before and after the experiment. To prevent complications from exposure to microorganisms, the procedure was designed such that the bacterial cultures were not in direct contact with the hands by using micropipette and teats to handle the inoculums.

Observing and Recording

The Effect of Dilutions of Recycled Cloth Wash on the Growth of Bacteria

Results:

Petri dishes

Number of Bacterial Colonies

Temperature (°C)

Escherichia coli sp.

Bacillus sp.

Distilled Water

1

0

0

3

30.0 ± 0.1

2

12

14

7

30.0 ± 0.1

3

55

62

10

30.0 ± 0.1

4

5

6

5

30.0 ± 0.1

After 24 Hours - Real Experiment

Key: 1 - 100% concentration (Tube 1)

2 - 10% concentration (Tube 2)

3 - 1% concentration (Tube 3)

4 - Distilled water

It is important to note that despite keeping the temperature of the incubator constant at 30.0°C, the temperature in the incubator did fluctuate within the range of ± 0.1-0.3°C and this was noted as a limitation. However, the fluctuations did not affect the rate8 of enzyme activity of the bacteria significantly.

Interpreting and Evaluation

Investigating the Effect of Dilutions of Recycled Cloth Wash on the Growth of Bacteria

Statistics

The statistical demonstration of data was done by using the Kruskal-Wallis Test4.

Ranks

Concentration

N

Mean Rank, Ri

1 (100%)

3

2.00

2 (10%)

3

8.67

3 (1%)

3

10.33

4 (dH2O)

3

5.00

Total

12

Where N = Number of groups, dH2O = Distilled water

Experimental Hypothesis

The greater the dilution of recycled liquid cloth wash, the higher the bacterial growth.

(HA = There is a difference between the medians of at least one pair of the samples)

Null Hypothesis

There is no relationship between the dilutions of recycled liquid clothes wash and the growth of bacteria.

(H0 = There are no differences between the medians of the pairs in the samples)

Kruskal-Wallis Test

The Kruskal-Wallis test was conducted11 using the following data:

100% Conc.

10% Conc.

1% Conc.

dH2O

E. coli sp.

0

12

55

5

Bacillus sp.

0

14

62

6

dH2O

3

7

10

5

Table 1 - Number of Colonies According To Dilutions

Using the values from the mean ranks calculated, the H-test equation was used to derive the H-value.

100% Conc.

(Group 1)

10% Conc.

(Group 2)

1% Conc.

(Group 3)

dH2O

(Group 4)

1.5

9

11

4.5

1.5

10

12

6

3

7

8

4.5

Σ Ri

6.00

26.00

31.00

15.00

2.00

8.67

10.33

5.00

Table 2 - Ranked Hypothetical Increases in Range of Number of Colonies (N = 12) After Each Treatment, With R Being The Sum of the Ranks in the i-th Sample

The H-Test Equation

H = - 3(N+1)

H = - 3(12+1)

= 0.0769 (12.00 + 676.00 + 961.00 + 225.00) - 39

= 9.666667

Correction Factor for Ties

1 - = 1- = 0.993

Corrected Value of the H Statistic for Ties

H =

=

= 9.735

Tabled Value of the H Statistic as Compared With Computed Value of the H Statistic

Reference to the χ2 table10 indicates that a value of the H statistic of ≥ 7.82 with df = 4-1 = 3 has a probability of occurrence when H0 is true of P < 0.05. Thus, because the observed value of the H statistic (9.735) exceeds the tabled value of the H statistic (9.735), the null hypothesis is rejected.

Conclusion

It may be concluded that there are differences among the four dilution groups relative to the changes in the dependent variable (number of colonies) that did not occur by chance.

Computation of Multiple Comparisons among Dilutions

The hypothesis: For groups u and v (number of groups),

H0 : θu = θv

HA : θu ≠ θv

The number of comparisons possible in this case is computed by:

Number of comparisons = = = 6

Equality used to test the significance of individual pairs of differences:

≥ za/i(i-1)

The critical difference for comparison is:

Using the table of normal distribution:

za/i(i-1) = z0.05/4(4-1) = z0.0042 ≈ 2.64

The critical difference is then calculated:

za/i(i-1) = 2.64 = 7.77

The differences are obtained for the four pairs of groups:

= = 6.67

= = 8.33

= = 3.00

= = 1.66

= = 3.67

= = 5.33

The comparison:

The critical difference is compared with the differences among the average rankings for the four groups of dilutions. As only the difference between groups 1 and 3 (8.33) exceeds the critical value of 7.77, that difference is considered to be significant. It may be concluded that the medians between the groups 1 and 3 are different.

Conclusion:

Test Statisticsab

Number of Colonies

Chi-square

9.735

d.f. (Degree of Freedom)

3

P-value

0.021

a. Kruskal-Wallis Test

b. Grouping variable: Concentration

The results show that there was a significant difference in the medians, χ2 (3, N = 12) = 9.735, p = 0.021. As the right tail probability was less than 0.05, the null hypothesis, H0, was thus rejected and the alternative hypothesis, HA, accepted. Therefore, the dilution in group 1 (100% concentration) results in a different outcome from that of group 3 (1% concentration). This suggested that the probability of the differences between the pairs of data did not occur by chance and the median of at least one pair of groups (100% Concentration vs. 1% Concentration) differed.

Assumptions:

The dependent variable (dilution of recycled liquid wash) had an underlying continuous distribution to avoid the problem of ties and it was measured on at least an ordinal scale.

The numbers of bacterial colonies in one dilution were independent of the numbers of bacterial colonies in the other dilutions.

The number of bacterial colonies of a bacteria species in each dilution was not influenced by the number of colonies of other bacterial species within the same dilution.

Data Analysis

Graph 1

From Graph 1, it is clear that the increase in dilutions results in the number of bacterial colonies to increase. The 100% concentration of recycled liquid cloth wash was the most effective in preventing the growth of bacteria, followed by the 10% concentration and 1% respectively, while distilled water, the control group in this experiment reported minimal number of colonies after a period of 24 hours. Despite being recycled, the active ingredient of pentasodium triphosphate (10-30%)12 (inorganic chelating agents) in Dynamoâ„¢ was still effective in inactivating13 the proteases of the bacteria. The enzymes contained in the recycled liquid wash were not saturated and were very effective against the bacteria. The liquid wash, being ionic in nature, dissolved14 the membrane protein of the bacterial inner membrane. As the dilutions increase, the number of moles of recycled liquid cloth wash per volume of solvent (sterilized distilled water) decreases. This results in smaller amount of enzymes available to inhibit the growth of bacteria, hence the number of bacterial colonies increases with the dilutions as shown in Graph 1. Sterilised distilled water contains no minerals vital to the growth of bacteria, thus it is apparent that the number of colonies appearing in both the species remained low, varying slightly with the dilutions used. In the control groups (where the bacterial inoculums were replaced with sterilized distilled water), growth of bacteria still occurred at a low rate (between 5-6 colonies) despite the absence of bacterial inoculums. It was also noted that the shape of bacterial colonies was irregular in the control group, unlike the spherical shape of colonies in other bacterial lawns. Possibly, the bacteria in the control groups originated from the dirty cloth, developed resistance against the liquid wash and reproduced in the nutrient agar.

Graph 2

From Graph 2, it was observed that both bacterial species of E. coli sp. and Bacillus sp. exhibited similar trend in growth with each dilution. The effect of different dilutions of recycled liquid wash was similar to both the Gram-negative E. coli sp. and Gram-positive Bacillus sp. As the dilutions increase, the number of colonies increased too, with zero growth at 100% concentration and maximum growth at 1% concentration. The relatively thick15 (20-80 nm) and continuous cell wall of the Gram-positive Bacillus sp., which is made of peptidoglycan, may be better at resisting the enzymatic actions of the recycled liquid wash than the Gram-negative E. coli sp. which has a thinner cell wall (5-10 nm). Although it is not clearly seen in the 100% treatment group, the enzymatic effect of the recycled liquid cloth wash on the bacterial cell wall of different thickness was seen in the 10% and 1% groups, with more of Bacillus sp. colonies to E. coli sp. The growth of both the bacterial inoculums in the agar containing sterilized distilled water was very low due to the inhibitory effects of sterilized distilled water against the growth of bacteria.

Limitation

The main source of limitation in this investigation was the presence of foreign bacteria in the solution of recycled liquid cloth wash which was inevitable as the liquid cloth wash used has to be recycled, thus used to immerse a dirty cloth first. The dirty cloth already contained microbes on its surface, which was treated with the liquid cloth wash. However, there is always a chance of bacteria surviving the treatment, developing resistance and contaminating the bacterial lawn upon the introduction of recycled liquid wash into the Petri dishes. The effects of the limitation were minimized by leaving the immersion of dirty cloth for no longer than 12 hours to allow as much microbes to be killed and simultaneously preventing the development of resistance of the microbes against the liquid cloth wash. The temperature of the incubator, which was set to 30.0°C but fluctuated within the range of ± 0.1-0.3°C, was a source of limitation. To minimize the effects from the small changes of the temperature, a piece of cloth could be placed over and around the Petri dishes. The reduced rate of heat loss from the cloth when the temperature of the incubator fluctuates would also reduce the effects of the relatively high temperature differences onto the bacterial growth. Also, when the lid of the Petri dish was opened slightly to introduce the inoculums, the entry of airborne microbes is possible, and may cause interspecific competition between the bacterial inoculums and the microbes. The risk was minimized by transferring the agar and inoculums quickly and disinfecting the head of the conical flask (containing the molten agar) by placing it in a Bunsen flame.

A range of source was chosen for use in this investigation. Sources 4 and 10 were taken from the website of the University of Leicester and the University of North Carolina at Chapel Hill, that are well-established universities in the UK and the USA. Source 1 is the official Biology textbook endorsed by Edexcel International which was written by an expert and reviewed for error before publishing. Sources 6 and 7 are books written by experts in the field of Biology whose educational backgrounds are clearly mentioned. Source 8 is a research paper printed in a journal released by an authority in Microbiology in the USA, with good citation to literature references, in which the facts presented are still acceptable to date.

Appendices

Books

1 Ann Fullick, 2009, Edexcel A2 Biology, Pearson Education Limited.

6 Solomon, Berg, Martin, 2004, Biology 7th Edition, Thomson Learning.

7 Karp, Gerald, 2005, Cell Biology 6th Edition, John Wiley & Sons.

9 Lehmann EL, Nonparametrics: Statistical Methods Based on Ranks, New York, NY: McGraw-Hill Inc. 1975.

Web source

2 Richard Lowry, The Kruskal-Wallis Test, http://faculty.vassar.edu/lowry/ch14a.html, 20th April 2010

3 Kruskal Wallis Test, http://www.statsdirect.com/help/nonparametric_methods/krusk.htm, 21st April 2010

4 Kruskal Wallis Non-Parametric ANOVA, University of Leicester, http://www.le.ac.uk/bl/gat/virtualfc/Stats/kruskal.html, 25th April 2010

10 On-Line Chi-Squared Table, http://www.unc.edu/~farkouh/usefull/chi.html, 25th April 2010

12 Colgate-Palmolive Company., Material Safety Data Sheet, Colgate-Palmolive, http://www.colgate.com.au/Colgate/AU/Corp/MSDS/pdfs/dynamo_2xultra_liquid_Regular.pdf, 25th April 2010

15 Cell Wall and Gram-Negative Cell Envelope, http://www.ncbi.nlm.nih.gov/bookshelf/br.fcgi?book=mmed&part=A289, 26th April 2010

Journal

8 D.A. Ratkowsky and colleagues, Relationship Between Temperatures and Growth Rate of Bacterial Cultures, American Society of Microbiology, Journal of Bacteriology, 1982, 149:1-5.

11 Chan Y, Walmsley RP, Learning and Understanding the Kruskal-Wallis One-Way Analysis-of-Variance-by-Ranks Test for Differences Among Three or More Independent Groups, Journal of Physical Therapy, 1997, 77:12; 1755-1761.

13 W. Schreiber, Inactivation of an Alkaline Bacillopeptidase by Pentasodium Triphosphate, Biochemische Laboratorien, Henkel and Cie GmBH, Düsseldorf, Germany.

14 Filip C. et al., Solubilisation of the Cytoplasmic Membrane of Escherichia coli by the Ionic Detergent Sodium-Lauryl Sarcosinate, American Society of Microbiology, Journal of Bacteriology, 1973, 115:3; 717-722.

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