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Topic A

Compare the effects of the antibiotic streptomycin (S) and oreganum essential oil on the bacteria Escherichia coli or Pseudomonas aeruginosa

Contents:

1. What type of investigation will this be?

2. Background information

3. Aim and objectives

4. Population and Sampling

5. Controls

6. Variables

7. Replication

8. Statistics

9. Influencing outcomes

10. Assumptions and Bias

11. Repeatability

12. Back to the beginning

1. What type of investigation will this be?

1

Is this an observational investigation or an experiment?

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We are starting out with a question or hypothesis so this is an experiment.Check your answer

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2. Background information

The undergraduate reviewed the literature on this topic. She was interested in examining the effectiveness as bactericides of substances other than antibiotics.

i. The justification

Oregano essential oil has been found to inhibit food-borne pathogens and spoilage micro-organisms (e.g. Friedman et al 2002) and is of interest to the food industry.

ii. Information about practicalities

The student contacted suppliers of antibiotics and arranged to use antibiotics on pre-prepared and standardised paper discs. She carried out two pilot studies: firstly, to determine how best to apply the oregano oil, and, secondly, to ensure an appropriate bacterial lawn was obtained on her agar plates she used a dilution series on the bacterial species.

Reading and the pilot study enabled the student to decide how to assess the effect of the potential bactericides. When the discs of treated paper were placed on the agar plate and this was then incubated, a clear region - the zone of inhibition - was evident. The student decided the measure the diameter of this zone of inhibition.

iii. The context

The literature review provided general background information but no additional specific data was required to carry out this investigation.

References

FRIEDMAN, M., HENIKA, P.R., MANDRELL, R.E. (2002). Bactericidal activities of plant essential oils and some of their isolated constituents against Campylobacter jejuni, Escherichia coli, Listeria monocytogenes and Salmonella enterica. Journal of Food Protection 65 (10): 1545 - 1560.

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3. Aim and objectives

2

Draft an aim and objective(s).

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As is common in small undergraduate studies the aim is very similar to the objective. Only in large studies where you have several experiments are the objectives likely to be very different in phrasing from the objective.

Draft Aim: The effectiveness of oregano essential oil as a bactericide.

Draft Objective: A comparison of the zones of inhibition resulting from treatment with either streptomycin or oregano essential oil on plates inoculated with either Escherichia coli or Pseudomonas aeruginosa.

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4. Population and Sampling

3

What is the statistical population? Will you need to sample? By what method(s)? Reflect on whether your sampling method will generate a representative sample. Indicate roughly the number of samples and/or observations you intend to collect and your rationale.

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  1. What is the statistical population?

There are four elements to this investigation, two species of bacteria (Escherichia coli or Pseudomonas aeruginosa) and two potential bactericides (streptomycin and oregano essential oil). There are therefore in essence four statistical populations: E. coli treated with streptomycin, E. coli treated with oregano oil, P. aeruginosa treated with streptomycin and P. aeruginosa treated with oregano oil.

  1. Will you need to sample? By what method(s). Reflect on whether your sampling method will generate a representative sample.

The sampling, therefore, relates to the samples of Escherichia coli or Pseudomonas aeruginosa,which you can assume are representative of their species or variant, and the batches of antibiotic and oregano oil, which you will assume are representative of the antibiotic/oil in general.

  1. Indicate roughly the number of samples and/or observations you intend to collect and your rationale.

For this investigation there will be four samples determined by the four combinations of treatments:

    • E. coli treated with streptomycin
    • E. coli treated with oregano oil
    • P. aeruginosa treated with streptomycin
    • P. aeruginosa treated with oregano oil

Step 8 (statistics) may lead to a revision in the number of samples chosen for the experiment and, therefore, must always be carried out as part of the planning process.

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5. Controls

4

Do you need a control(s). If yes, what? If no, why not?

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Yes. Two technical controls are needed:

  1. Inoculated plates without the discs of bactericides should be cultured to ensure that the inoculation has been successful and to provide a further visual record of the appearance of an inoculated plate to compare it to treated plates.
  2. Agar plates made from the original agar but not inoculated with bacteria to ensure that the original agar is sterile.
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6. Variables

5

What is (are) your treatment(s)? What may cause non-treatment variation?

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What is (are) your treatment(s)?

There are two treatment variables: species of bacteria (Escherichia coli or Pseudomonas aeruginosa) and potential bactericides (streptomycin and oregano essential oil).

What may cause non-treatment variation?

  • Variants within the bacterial species
  • Batch by batch variation in cultures of bacteria
  • Batch by batch variation in agar and therefore agar plates
  • Batch by batch variation in antibiotic discs and commercial supplies of oregano essential oil
  • Variation between the antibiotic impregnated paper discs
  • Variation between the oregano oil impregnated discs
  • Micro-environmental variation within the incubator
  • Asymmetrical diameter of the zone of inhibition
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6

For each possible cause of non-treatment variation how might the effect be minimised?

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Potential source of non-treatment variation

How to minimise impact on hypothesis testing

Variants within the bacterial species.

Use one specified supplier and known identified variants. Acknowledge that findings may not apply to all variants of any one species of bacteria.

Batch-by-batch variation in cultures of bacteria.

Use a single batch of each species throughout the investigation.

Batch-by-batch variation in agar and therefore agar plates.

Make up one initial volume of agar; divide into two for inoculation with one or other species of bacteria. Use only these two batches to pour all the plates used in the investigation. (Reserve some of the initial volume of agar to pour as un-inoculated control plates).

Batch-by-batch variation in antibiotic discs and commercial supplies of oregano essential oil.

Use one specified supplier and with clearly specified concentration, date of production etc.

Variation between the antibiotic impregnated paper discs.

Use replicates to gain some estimate of the inherent technical variation within the experiment.

Variation between the oregano oil-impregnated discs

Use replicates to gain some estimate of the inherent technical variation within the experiment.

Micro-environmental variation within the incubator.

Randomise the location of the plates for the four treatments within the incubator.

Asymmetrical diameter of the zone of inhibition.

Take two measurements at right angles to each other in a consistent direction on all plates. These two measures can be averaged. (Note: usually you should not summarise your data before analysing it as you loose information about the variation within your experimental system. In this case we judge that an average or mean measure of the diameter is more useful in the investigation and so have decided that in this instance an average is acceptable).



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7. Replication

7

Will you use replicates? Why? Indicate how many and explain why this number.

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The student decided to use 8 plates for each combination of treatments. This replication will allow the undergraduate to obtain some indication of the technical variation inherent in her experimental system and her analysis and hypothesis testing will allow her to distinguish between the variation due to non-treatment variation (sampling error) and the effects of her treatments. A larger number of replicates would enable more sensitive hypothesis testing, but would also use more time, materials and incubator space.

Step 8 may lead to a revision in the number of replicates chosen for the experiment and, therefore, must always be carried out as part of the planning process.

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8. Statistics

8

Have a go at choosing what might be the correct test to analyse the data from this experiment. Explain your choice.

This is invariably the step that students find the hardest and, therefore, we return to this topic in the interactive exercises for chapters 4 - 8.

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Using the information in appendix b of the text: which statistical test should I choose?

B.1. What type of investigation am I designing?

In this investigation we are starting out with a question so this is an experiment.

B.2. Which type of hypotheses am I testing?

There are three types of hypotheses that you need to choose between. If you are not sure which type of hypotheses you will be testing read the information in B.2.1 - B.2.3 before deciding. For more information about hypotheses and hypothesis testing read Chapter 4.

  1. Does the data match an expected ratio?

or

  1. Is there an association between two or more variables?

or

  1. Do samples come from the same or different populations?

It is not always easy to decide which type of hypothesis you are testing. In this case the student wanted to compare the effects of treatments on samples. She had no expectation and did not want to examine the association between the bacteria and bactericides. She did want to compare samples and see if they were the same or statistically different. We are therefore testing the third type of hypotheses. Most undergraduate work examines this type of hypothesis.

B.2.3. Do samples come from the same or different populations?

To test this type of hypothesis you need to decide if the data are likely to be parametric. To tell if your data are likely to be parametric (Normally distributed) you should refer to Box 3.2 in the book and in the Statistical Software section of the Online Resource Centre. When designing an investigation you can only use criterion 'a' to decide whether your data may be parametric. The remaining criteria should be used when the investigation has been carried out to confirm your decision.

Criterion a: Are the data measured on an interval scale which is therefore quantitative and continuous, such as mm and grams?

In this example the answer is YES, the scale of measurement is mm which is an interval scale. Therefore, we will assume at present that the data may be parametric and can now choose the statistical test.

B.2.3.1. Parametric tests

These are largely selected on the basis of the experimental design - how many variables, how many categories in each variable, how many replicates in each category. In this investigation there are two treatment variables (species of bacteria and bactericides), each with 8 replicates. Each category for variable 1 is combined with each category from variable 2. Examining the table you can see that we should therefore consider using the parametric two-way ANOVA.

Experimental design

Test

You have one treatment variable. You are going to compare two samples. The data is unmatched.

t or z test for unmatched data (7.1 or 7.2).

You have one treatment variable. You are going to compare two samples. The data is matched.

t or z test for matched data (7.3)

You have one treatment variable. You are going to compare two or more samples. You wish to test general and specific hypotheses.

One-way parametric ANOVA and Tukey's test (7.5 and 7.6)

You have two treatment variables. Each variable has at least two categories or classes and all categories from one variable are combined with all categories from the second variable. You wish to test general and specific hypotheses.

Two-way parametric ANOVA and Tukey's test (7.7. and 7.8.)

You have two treatment variables. Each variable has at least two categories. One variable is randomised or nested with respect to the second variable. You wish to test general hypotheses.

Two-way nested ANOVA (7.9.)

You have three treatment variables. Each variable has at least two categories and all categories from each variable are combined with all other categories from the other variables. You wish to test general and specific hypotheses.

Three-way parametric ANOVA (7.10.)

None of the above.

Chapter 8 and Sokal & Rohlf, 1981.



The criteria for using the two-way parametric ANOVA are found in 7.7.1. You:

  1. Wish to test for differences in population means
  2. Have two treatment variables
  3. Have parametric data
  4. Allocate all items at random to each sample
  5. Have variances that are similar (homogeneous)
  6. Have the same number of replicates (observations) in each sample
  7. Have an orthogonal design

Criteria 1, 2, 4, 6 and 7 are met by the design of the investigation. We are assuming at present that as the scale of measurement will be mm the data may be parametric but this will need to be confirmed. We cannot test criterion 5 until we have the data. Therefore, at present there is no reason for believing that this test will not be suitable and our current design needs no revision to ensure that the data should be analysable.

We return to this example in the interactive exercise 2 in chapter 7 and interactive exercise 2 in chapter 8 where we analyse the data from this experiment.

Check your answer

9

Finalise your aim and objectives.

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Aim: The effectiveness of oregano essential oil as a bactericide.

Objective: A comparison of the mean diameter (mm) of the zone of inhibition resulting from treatment with either streptomycin or oregano essential oil on plates inoculated with either Escherichia coli or Pseudomonas aeruginosa.

Check your answer

10

Read section 4.1.2 of the text and draft your hypotheses.

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If you use a parametric two-way ANOVA there are three pairs of hypotheses. The first relates to the first variable, the second pair of hypotheses relate to the second variable and the third pair of hypotheses related to the possible interaction between the two variables.

(Columns)

H0: There is no difference between the mean diameter of the zone of inhibition (mm) between Escherichia coli and Pseudomonas aeruginosa when treated with possible bactericides.

H1: There is a difference between the mean diameter of the zone of inhibition (mm) between Escherichia coli and Pseudomonas aeruginosa when treated with possible bactericides.

(Rows)

H0: There is no difference between the mean diameter of the zone of inhibition (mm) resulting from treatment by oregano oil compared to streptomycin on plates inoculated with bacteria.

H1: There is a difference between the mean diameter of the zone of inhibition (mm) resulting from treatment by oregano oil compared to streptomycin on plates inoculated with bacteria.

(Interaction)

H0: There is no difference between the mean diameter of the zone of inhibition (mm) due to an interaction between bacteria species and putative bactericide.

H1: There is a difference between the mean diameter of the zone of inhibition (mm) due to an interaction between bacteria species and putative bactericide.

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9. Influencing outcomes

11

Is this relevant to your experiment? In what way?

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It is unlikely (other than the student having a poor sterile technique) that she will influence the outcome of the investigation.

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10. Assumptions and Bias

12

List all the assumptions and possible causes of bias in your design.

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The response of these bacteria reflect the response of all bacteria in this variant of each species.

The effect of this batch of streptomycin and the batch of oregano essential oil is typical of all streptomycin and oregano oil treatments of similar concentration.

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11. Repeatability

13

Could this experiment be repeated?

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a) Yes
b) No
Correct. There is no reason why this experiment could not be repeated. If the results from a repeat of this experiment confirm the findings the more confidence will be attached to these results.Incorrect. There is no reason why this experiment could not be repeated. If the results from a repeat of this experiment confirm the findings the more confidence will be attached to these results.
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12. Back to the beginning

14

Provide a complete review of your experimental design written in such a way that another person could use this review to carry out your experiment.

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Aim: The effectiveness of oregano essential oil as a bactericide.

Objective: A comparison of the mean diameter (mm) of the zone of inhibition resulting from treatment with either streptomycin or oregano essential oil on plates inoculated with either Escherichia coli or Pseudomona aeruginosa.

Experimental Design:

The following is a summary of our experimental design. Clearly there are some further details that need to be considered such as the volume of agar required for the experiment, before this is a finalised method.

Paper discs impregnated with a known concentration of streptomycin were obtained from a known supplier. Similarly oregano essential oil was obtained from a known supplier at a given concentration. A pilot study was carried out and it was determined that a small paper disc dipped in the oregano oil consistently soaked up a given volume of the oil. These oregano discs and the streptomycin discs were then used in the following experiment. A volume of agar was divided into two flasks and autoclaved. On cooling, two plates from each flask of agar were poured under sterile conditions and sealed. These plates were one of the two controls. The two flasks of warm agar were inoculated with either Escherichia coli or Pseudomonas aeruginosa and the inoculated agar poured into 20 petri dishes each. When the agar had solidified the dishes were marked with the points of a compass (north, south, east, and west) and the centre spot identified. Two plates for each bacterium were set aside as the second technical control. A disc (either streptomycin or oregano oil) was placed in the centre of the plate. Eight plates inoculated with E. coli were selected at random and allocated to the streptomycin treatment. The remaining 8 plates were treated with the oregano oil discs. Similarly the 16 plates inoculated with P. aeruginosa were also allocated at random, 8 were treated with streptomycin and 8 with oregano oil. All plates were incubated at 37°C for 24 hours after which time the plates were examined. Two measures (east - west) and (north-south) of the diameter of the zone of inhibition (mm) were made and averaged.

It is expected that the data will be parametric and if so may be analyzed using a two-way ANOVA. As far as it is possible to check, the criteria for using this test are met by the experimental design. The criteria that cannot be checked at present will be checked when the data are available.

Assuming the criteria are met then the hypotheses to be tested are:

H0: There is no difference between the mean diameter of the zone of inhibition (mm) between Escherichia coli and Pseudomonas aeruginosa when treated with possible bactericides.

H1: There is a difference between the mean diameter of the zone of inhibition (mm) between Escherichia coli and Pseudomonas aeruginosa when treated with possible bactericides.

H0: There is no difference between the mean diameter of the zone of inhibition (mm) resulting from treatment by oregano oil compared to streptomycin on plates inoculated with bacteria.

H1: There is a difference between the mean diameter of the zone of inhibition (mm) resulting from treatment by oregano oil compared to streptomycin on plates inoculated with bacteria.

H0: There is no difference between the mean diameter of the zone of inhibition (mm) due to an interaction between bacteria species and putative bactericide.

H1: There is a difference between the mean diameter of the zone of inhibition (mm) due to an interaction between bacteria species and putative bactericide.

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Reflection

15

Describe how you felt at the various stages of planning this experiment. Identify one step that you found difficult and need further practice. How does our answer differ from yours? In what ways were your ideas better?

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Well done! You have now completed this interactive exercise. You may now wish to print this page for your reference, and also the Word documents if you wish to keep a permanent record of your answers.Check your answer