Posted: July 1st, 2015

# Evaluate the outcomes from D and z calculations and compare your D and z values with published data within Journal papers (this might be hard to find for D121)

D and z values

Introduction

As you are aware, or should be aware, the D value of a microorganism is the time in minutes at a particular temperature or dose of radiation that it takes for the population to be decreased by 90% (1 log10 reduction) i.e. from 1 x 107 cells to 1 x 106 cells.

Obviously the value depends on the organism, the medium it is treated in and the degree of treatment (temperature or dose of radio activity) used.

For any given organism the D value is reproducible provided all the other conditions are constant.

So if the D value for Escherichia coli in water at 80OC is 0.05 minutes, it will always be 0.05 minutes for that strain in water at 80OC. Change the temperature of the suspending medium and then the D value will change. This is why you see D values with a number subscript. So for the example above the D value would be expressed as D80 = 0.05 min

In relation to pharmaceutical products sterilised by moist heat at 121OC then the D value of most interest is D121.

D values are easily determined experimentally by plotting the log10 survivors when a suspension of cells is held at a high temperature. This is only easy at relatively modest high temperatures but at 121OC would require high temperature oil baths and samples sealed in heating tubes.

To find the D121 of an organism therefore, use can be made of the z value. Again you should be aware that if you know the D value of an organism at one temperature and you know the value of z you can calculate the D value at another temperature. The z value is the change in temperature required to bring about a 90% (1 log10 reduction) change in the D value and can be calculated from a plot of log10D value against temperature.

In this practical exercise you will measure the D value of Salmonella enterica serovar Typhimurium at four different temperatures, determine the z value for this organism and from your results calculate the D121 for Salmonella enterica serovar Typhimurium.

HAZARD WARNING

Microbiological hazard: Category 2 organism,

Bunsen burners, Hot (≥ 50OC) water baths

Method

You will be working in pairs and each pair will be responsible for determining the D value of Salmonella enterica serovar Typhimurium at a particular temperature.

You are provided with

Finn pipette for 500 μl volume plus sterile tips (blue)

Finn pipette for 20μl volume plus sterile tips (yellow)

Stop watch

32 test tubes containing 4.5 ml of sterile Ringer

10 sterile TSA plates

5 ml of Salmonella enterica serovar Typhimurium broth culture

Access to a water bath at 48OC, 52OC, 54OC, 56OC, or 58OC containing a flask with 200 ml of sterile TSB.

CONTROL

For ALL pairs you need to determine the cfu/ml in your flask at time zero. You could add the inoculum, mix and sample immediately but this will give you an inaccurate result especially at the higher temperatures.

To get an accurate time zero count you need to know how many viable cells there are per ml of your Salmonella enterica serovar Typhimurium broth culture.

Label eight test tubes each containing 4.5 ml of sterile Ringer 10-1 to 10-8

Mix the contents of the Salmonella enterica serovar Typhimurium broth culture and aseptically remove 500 μl of the culture into your 10-1 tube, discard the tip. Mix the contents of the 10-1 and with a fresh tip remove 500 μl from the 10-1 tube into the 10-2 tube. Carry on with the rest of the tubes to complete the 10-8 dilution.

Mark out two sterile TSA plates as shown in the diagram below and inoculate the plates as described.

Miles and MISRA METHOD FOR Control

1. Mark duplicate agar plates provided into 6 sectors on the base, labelled

10-3 to 10-8 (see diagram). Also label with your name, date, sample time and sample temperature

 -8
 -7
 -6
 -5
 -4
 -3

1. Starting with the most dilute (10-8) and using a sterile Finn tip, place 20ml of the suspension on the sector labelled 10-8 on each of the two plates.   Hold the tip 3cm above the agar sector and allow the drop of the dilution to fall gently into the centre of the sector. Replace the Petri-dish lids.
2. Similarly take a sample from 10-7 dilution using the same Finn tip, and apply that to the appropriate sector (10-7) on the agar plates.
3. Proceed until all dilutions to 10-3 are assayed onto the agar plates and DO NOT MOVE PLATES UNTIL THE DROPLETS HAVE SOAKED INTO THE AGAR.
4. Incubate inverted at 37oC for 24 hours.

To determine the survival of Salmonella enterica serovar Typhimurium at your temperature you will have to perform a viable count on samples taken at 5, 10, 20, and 30 minutes after adding the culture. The viable count method you will be using is the Miles and Misra method (see below).

TEST PROCEDURE

.

Add 2ml of the culture to your flask with swirling to mix and IMMEDIATELY start your stop watch. You can now dispose of the stock S. enterica Typhimurium culture (wire basket on trolley) as it will not be needed again.

For each timed sample you will need six test tubes labelled 10-1 to 10-6 each containing 4.5 ml of sterile Ringer and two sterile TSA plates marked as in the diagram below and labelled appropriately

At the appropriate sample time gently swirl the flask (DO NOT TAKE THE FLASK OUT OF THE WATER BATH) and aseptically remove 500 μl of the culture into your 10-1 tube, discard the tip.

Mix the contents of the 10-1 and with a fresh tip remove 500 μl from the 10-1 tube into the 10-2 tube.

Carry on with the rest of the tubes to complete the 10-6 dilution.

Repeat at each sample time with fresh tubes, tips and plates.

Miles and MISRA METHOD FOR TEST

1. For each sample time mark duplicate agar plates provided into 6 sectors on the base, labelled

10-1 to 10-6 (see diagram). Also label with your name, date, sample time and sample temperature

1. Starting with the most dilute (10-6) and using a sterile Finn tip, place 20ml of the suspension on the sector labelled 10-6 on each of the two plates.   Hold the tip 3cm above the agar sector and allow the drop of the dilution to fall gently into the centre of the sector. Replace the Petri-dish lids.
2. Similarly take a sample from 10-5 dilution using the same Finn tip, and apply that to the appropriate sector (10-5) on the agar plates.
3. Proceed until all dilutions are assayed onto the agar plate and DO NOT MOVE PLATES UNTIL THE DROPLETS HAVE SOAKED INTO THE AGAR.
4. Incubate inverted at 37oC for 24 hours.

Results

Individual temperature = …………..OC

Individual pairs: raw plate counts

 Time Zero control Zero control Zero control Dilution Count one Count two average 10-3 10-4 10-5 10-6 10-7 10-8

 Time Dilution Count one Count two average 10-1 10-2 10-3 10-4 10-5 10-6

 Time Dilution Count one Count two average 10-1 10-2 10-3 10-4 10-5 10-6

 Time Dilution Count one Count two average 10-1 10-2 10-3 10-4 10-5 10-6

 Time Dilution Count one Count two average 10-1 10-2 10-3 10-4 10-5 10-6

Individual pair viable counts

 Temperature ………OC Sample Time min Count one Count two Mean count Dilution used Cfu/ml original Log10 cfu/ml 0 *

* You will need to divide this number (control only) by the dilution resulting from adding 2.0 ml of inoculum to 200 ml TSB

Cfu/ml of original = mean count x 1/volume (ml) x 1/dilution used

Plot log10 cfu/ml against time in minutes and from your graph determine the D value for Salmonella enterica serovar Typhimurium at your temperature.

Class Data

 Temperature OC D value D value D value D value Average D value Log10 Average D value 48 52 54 56 58

Plot log10 D value against temperature and determine the value of z from your graph.

Calculate the value of D121 for Salmonella enterica serovar Typhimurium.

In your practical report you will be expected to compare your value those presented in the literature and discuss any differences.

D and z Report.

Preamble.

The D & z practical will have a strict maximum word limit of 2,000 words.

This limit will exclude class data tables, graphs and the list of references but will include tables or figures imported from the literature into the Introduction / Discussion. Reports that exceed the word limit will be penalised.

Results should contain the “individual pair viable counts” table from the top of page 6 in your schedule. Show your calculations. This table was the one you used to draw your graph in class to determine the D value for your allocated temperature.

You are NOT required to submit this graph used to determine D, nor draw a new one, of your individual pair data for this report.

You ARE required to include the table of class data below, draw and submit the graph from which you calculate the z value and of course show your calculation of the z value and the D121 value.

Class data for D values at various temperatures

You are advised to use a spreadsheet such as Excel to perform the calculations, (equation STDEV.P was used above for Standard Deviation, and then copy and paste the complete table into your report, as above). Then plot log10 of the mean of D against the temperature. Your Graph should be plotted by hand, contain y-axis error bars of log10 ( Mean of D, plus and minus standard error) and the axes correctly labelled.

Actual values for 48 degrees are included above as an example.

According to theory, the graph should give a linear negative slope through all the data points.   Clearly a best straight line of fit (by eye is acceptable) through as many data points as possible is appropriate.

From your straight line you will be able to calculate the z value – refer to lecture notes for explanation of how to do this. (You should get a value in the range around -5 to -10).

Use this z value to calculate the D121 value (refer to lecture notes for explanation of how to do this) for Salmonella enterica serovar Typhimurium

Your graph should be plotted by hand, contain error bars and be correctly titled and the axes correctly labelled.

The following is what we will be looking for in your reports:

Completed Declaration – typed “signature” is acceptable – do not scan and paste.

Save Word file before electronic submission as:

“Surname” “Given name” “Student Number” “Report 3”

Abstract

Concise, clear and accurate

Intro: This ought to BRIEFLY cover following points

• D value & z value
• Definition of the relevant type of sterilisation
• Holding/heating up times
• Relevant properties of “Salmonella typhimurium
• Effects of heat on bacterial cells

Methods

Refer to schedule: note and explain changes

Results

Table for Individual pair data

Correct calculation

Class data for D at all temperatures

Correct calculations of data as noted above

Graph for class means of log10D vs temp

Title

correct orientation of axes

sensible scale

labelled axes

accurate plot of points

sensible line

Correct calculation of z

Correct calculation of D121

Discussion

Accuracy of cfu/ml especially at higher temp

Accuracy of class data (why such high standard error at low temps?)

Evaluate the outcomes from D and z calculations and compare your D and z values with published data within Journal papers (this might be hard to find for D121)

Eg

Thermal Inactivation of Salmonella spp. in Chicken Broth, Beef, Pork, Turkey, and Chicken: Determination of D‐and Z‐values.

Juneja, VK, Eblen, BS and Ransom GM – Journal of Food Science, 66 (1), 146–152.

http://onlinelibrary.wiley.com/doi/10.1111/j.1365-2621.2001.tb15597.x/pdf

The European Commission Scientific Committee on Emerging and Newly Identified Health Risks (SCENIHR) reported on “The Safety of Reprocessed Medical Devices Marketed for Single-Use” in July 2010 and quoted a D121 value for Salmonella spp as 0.1 sec.

Conclusion

Correct citation and listing of refs using Harvard system

Grammar, spelling, Latin names, italics

How to write Practical Reports

1 INTRODUCTION

Practicals form an essential and valuable part of Sciences courses, and this is especially true for Pharmacy.   Firms employing sandwich students and graduates comment favorably on the practical skills of students from this institution. Ideally these practicals should illustrate the theory presented in lectures as well as allowing you to acquire specific practical skills. In addition they are ideal for developing other skills such as;

Initiative & planning

Team work

Data collection

Data manipulation

Numeracy

Data evaluation

Presentation of data and conclusions drawn from the evaluation of the data.

Communication skills (relates to team work and to presentation of results).

Scientific writing

To get the most from practicals therefore you should NOT leave the laboratory UNTIL you have completed all your own work and have CONTRIBUTED to and RECORDED class results.

Do not waste time on idle gossip.

2 PRACTICAL REPORTS

1. i) You should develop the habit of keeping a ‘lab. book’ in which you record all essential information; weightings, absorbance values, dilutions, samples assayed etc. in a clear and orderly form. These lab books should be considered as your source of raw data when you come to write up the practical. Recording such vital information on scraps of paper, even in loose leaf files, is not a good idea as they are too easily lost.

1. ii) Reports should be written in the style of papers published in the scientific press. The Society for General Microbiology publishes a journal called Microbiology which has an acceptable style. A copy of the Journal’s instructions to authors is available here http://mic.sgmjournals.org/misc/ifora.pdf and you should adopt the style they recommend. The major points are as follows;

Abstract: This should state clearly and concisely what was done and the result. It should NOT contain any details of methods or explanation of results.

Introduction: DO NOT copy the introduction from the schedule. Present additional information that is useful and relevant to the practical and indicates the aim and context. Remember to reference additional information

Methods: These are usually presented in some detail on the schedule. Again DO NOT COPY OUT the methods. It is sufficient to refer to the schedule (which you MUST hand in with your practical report- see later).

If you have used a modification of the published method you must describe it and explain how the modification may affect the results.

If you are only performing some of the method as part of a team practical, identify the part you performed, record the identity of your laboratory partners and clearly indicate the data you have provided.

Results: These should be clearly presented in tables or as fully labeled HAND DRAWN graphs. On most schedules you will find blank tables for you to record the data.

Where calculations are required on the data show clearly ALL STAGES of your calculations. If there are a lot of repetitive calculations show full stages of a couple as examples. Calculations will be checked and assessed.

A results section should be used to present and to describe your data but NOT‚ to discuss it.

Graphs should be neatly drawn with appropriate scales. Where more than one line is drawn on the same axes use different symbols for each line. Acceptable symbols are circles, squares, triangles, diamonds, inverted triangles and these can be open (○) or closed (●), thus allowing up to ten lines per graph. If you have more lines then you can use ‘+’ or ‘x’ as well but only as a last resort. NEVER use small dots.

Using all of these you could get 12 lines on one graph.

Discussion: The discussion should be a critical evaluation and interpretation of your results and NOT just a repetition of the Results section.

The quality and reliability of your data should be made clear.   The source of errors should be identified and their effect on the data explained. NB vague statements about ‘experimental error’ or ‘student error’ are not acceptable. Therefore the discussion allows you to show that you have understood the aims and principles of the practical and that you can assess your results in the light of previously published or expected results. Material from published sources or text-books should be clearly identified and the Harvard system used for references.

Questions: In some schedules you will be asked specific questions or set specific tasks related to the practical. These should be answered on separate pages as part of your report. Such questions are NOT a substitute for a report.

References: These should be in the Harvard style – see papers in the journal Microbiology for correct citation and listing.

There are no set number required, although the extremes of absence (or meagre) and excessive referencing should be avoided, and will be detrimental to your mark.

Guidance on Harvard referencing can be found at:

http://www.wlv.ac.uk/lib/skills-for-learning/referencing/harvard-referencing/

Use peer reviewed journals, electronic journals or academic websites. Wikipedia is not acceptable.

• PRESENTATION OF REPORT

The reports will usually be handed in 1-2 weeks after the session (refer to deadlines on WOLF) in which results were collected. Reports handed in after the deadline will not be assessed. They should be submitted by the deadline.

The word limit is 2,000 words excluding references. There will be severe penalties imposed if you go over the word limit and/or submit after the deadline.

The report must be clearly written and follow a logical order.

The report may be written using a word processor but Graphs must be hand drawn.

You must hand in the Declaration with your work. Type your name into the “Signature” and copy and paste the whole declaration into the beginning of your report.

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