5PY017 Pharmaceutical Microbiology

Evaluation of antimicrobial effectiveness of disinfectants

There are a number of methods available to assess the effectiveness of antimicrobial products such as disinfectants and antiseptics. The European and British Standard methods consist of a range of methods to cover medical, veterinary and food/industrial area:
Phase 1 – simple methods to screen for antimicrobial activity against standard organisms in suspension test.
Phase 2 – methods designed to simulate usage in suspension (Phase 2 step 1) or surface tests (Phase 2 step 2)
Phase 3 – Factory trials

This practical is based on BS EN1276:1997 ‘Chemical disinfectants and antiseptics – Quantitative suspension test for the evaluation of bactericidal activity of chemical disinfectants and antiseptics used in food, industrial, domestic and institutional areas – test method and requirements (phase 2, step1)’. The disinfectant is tested at three concentrations (labelled T1, T2 and T3 in the practical which are <in-use, in-use and >in-use respectively) against Gram positive (Staphylococcus aureus and Enterococcus hirae) and Gram negative (Pseudomonas aeruginosa and Escherichia coli) bacteria in conditions to simulate usage (presence of organic load and hard water). To pass the test a disinfectant must reduce the count of the four organisms by five log orders within the five minute contact time.

Students working in pairs to test one organism and one disinfectant. Aseptic microbiological techniques must be used throughout the practical.

Method

1. Record which bacterial suspension and disinfectant are to be tested.
2. Label duplicate Petri dishes with student names, name of the organism, and either N10-6, N10-7, A, B, C, T1, T2, T3.

3. Take the bacterial suspension labelled ‘N’ (Neat) and perform a decimal dilution by removing 0.5ml of the suspension and adding to 4.5ml Ringers to give a 10-1 dilution. Continue the dilution series to 10-7 and pour plate in duplicate 1ml aliquots from the 10-6 and 10-7 dilutions into the plates labelled N10-6 and N10-7
Keep the 10-5 dilution and label ‘D’ (Diluted) as this will be used to inoculate controls in the test.
Controls
4. Label 3 sterile test tubes ‘A’, ‘B’ and ‘C’. A and B are controls to ensure that the medium and neutraliser do not affect the microorganisms. Control C is to check that the neutraliser is effective in inactivating the disinfectant at the end of the contact time.
5. To test tube A, add 1ml Bovine serum albumin (BSA), 1ml of bacterial suspension D and 8ml Sterile distilled water (SDW) and then vortex. After 5 min, vortex and remove duplicate 1.0 ml for duplicate pour plates using the plates labelled A.
6. To test tube B, add 8ml neutraliser, 1ml sterile distilled water (SDW) and 1ml of bacterial suspension D and vortex. After 5 min, vortex and remove duplicate 1.0 ml aliquots for duplicate pour plating using the plates labelled B.
7. For Control C, add 1ml BSA, 1ml water and 8ml disinfectant (T3, the most concentrated sample) and vortex. After 5 min, remove 1ml to a tube containing 8ml neutraliser and vortex. After 5 min, add 1ml bacterial suspension D and vortex. After 5 min, remove duplicate 1 ml aliquots for pour plating using plates labelled C.

Test
8. The disinfectant is assessed at three concentrations T1, T2 and T3 which are lower than the in-use, the in-use and higher than the in-use concentrations respectively. For the disinfectant test, label 6 sterile test tubes T1, T2, T3 (Test), T1N, T2N, T3N (Test neutralised). To T1N, T2N and T3N aseptically transfer 8ml neutraliser and 1ml sterile water (SDW)
9. To the test tube labelled T1, add 1ml BSA and 1ml bacterial suspension N (NEAT) and vortex. Add 8ml disinfectant T1, vortex and after exactly 5 min, transfer 1ml to the tube labelled T1N and vortex. After 5 min, remove duplicate 1.0 ml for pour plating using plates labelled T1.
10. Repeat as 9 above for disinfectant concentration T2 and T3.
11. Incubate all plates at 37oC for 24 hr.
Summary
Pair Results

Count the cfu/plate and record in the tables below.

Bacterial Culture…………………………..

Table 1 Calculation of concentration of bacterial suspension N
10-6 10-7
Duplicate 1
Duplicate 2
Mean cfu ml-1
Mean cfu ml-1 in undiluted* N
* Use the dilution that gives 30-300 cfu ml-1 for calculation

Table 2 Controls
A B C
Duplicate 1
Duplicate 2
Mean cfu ml-1
For results that are too numerous too count, record as >300

Table 3 Test
T1 T2 T3
Duplicate 1
Duplicate 2
Mean cfu ml-1
Mean x10 cfu ml-1 in test mixture T
Calculation of Reduction Factor (RF) for each concentration of disinfectant

RF= NX10-1
T
Where N is the number of cfu ml-1 in the bacterial suspension
(from the shaded box in Table 1 above)
T is the number of cfu ml-1 in the test mixture for T1, T2 or T3 (from the shaded boxes in Table 3 above)

To pass the disinfectant test, the in-use concentration (T2) must give a reduction factor of>105 for all four organisms within the 5 minute contact time.

Class results
Collect class data of the reduction factors for all four organisms and three concentrations of the disinfectant benzalkonium chloride

Reduction Factor (RF)
T1
%
BAC T2
%
BAC T3
% BAC
S.aureus
E.coli
P.aeruginosa
E.hirae

The drug I have used is E.coli the tem is 54
Results
Disinfectant Practical report

See the notes on WOLF for general practical write-up requirements.

The report must contain:
Abstract – a concise and detailed summary of the experimental findings
Introduction – focused background to set the context of the experiment supported by references cited in the Harvard style in the text.
Methods – refer to the practical schedule but note any changes
Results – Present numbered tables with detailed titles, describe the results and refer to the tables
Discussion – explain the results obtained and in particular
Pair results – comment on the controls, the results obtained for the organisms tested and validity of the test.
Class results – discuss the results obtained with respect to repeatability, concentrations tested and the susceptibilities of the organism used.
References – listed in the Harvard format alphabetically
Table 1. Class data showing reduction factors of benzalkonium chloride (BAC)

BAC Conc (%)
Organism T1 – 0.005% T2 – 0.1% T3 – 0.5%

S.aureus
3.07 x107
1.31×106
1.33×106
1.83×102
7.5×106
2.35×105
1.4×105
E.coli
1.2×105
1.28×103
5.97×103
1.38×107
6.25×105
P.aeruginosa
4.7×103
3.57×102
1.3×106
E.hirae
1.25×104
2.01×103
7.65×106
3.25×104
1.0×107
1.0×101
4×106

Table 1 Calculation of concentration of bacterial suspension N
10-6 10-7
Duplicate 1 150 24
Duplicate 2 180 28
Mean cfu ml-1 165 26
Mean cfu ml-1 in undiluted* N 1.6×108 2.6 x108
* Use the dilution that gives 30-300 cfu ml-1 for calculation

Table 2 Controls
A B C
Duplicate 1 210 190 206
Duplicate 2 252 180 189
Mean cfu ml-1 231 185 197.5
For results that are too numerous too count, record as >300

Table 3 Test
T1 T2 T3
Duplicate 1 1 7 1
Duplicate 2 3 5 1
Mean cfu ml-1 2 6 1
Mean x10 cfu ml-1 in test mixture T 20 60 10

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

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.
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.
Use peer reviewed journals, electronic journals or academic websites. Wikipedia is not acceptable.
3 PRESENTATION OF REPORT

The reports will usually be handed in one week after the session in which results were collected. Reports handed in after the deadline will not be assessed. They should be submitted by 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.

.

5PY017 Pharmaceutical Microbiology

Evaluation of antimicrobial effectiveness of disinfectants

There are a number of methods available to assess the effectiveness of antimicrobial products such as disinfectants and antiseptics. The European and British Standard methods consist of a range of methods to cover medical, veterinary and food/industrial area:
Phase 1 – simple methods to screen for antimicrobial activity against standard organisms in suspension test.
Phase 2 – methods designed to simulate usage in suspension (Phase 2 step 1) or surface tests (Phase 2 step 2)
Phase 3 – Factory trials

This practical is based on BS EN1276:1997 ‘Chemical disinfectants and antiseptics – Quantitative suspension test for the evaluation of bactericidal activity of chemical disinfectants and antiseptics used in food, industrial, domestic and institutional areas – test method and requirements (phase 2, step1)’. The disinfectant is tested at three concentrations (labelled T1, T2 and T3 in the practical which are <in-use, in-use and >in-use respectively) against Gram positive (Staphylococcus aureus and Enterococcus hirae) and Gram negative (Pseudomonas aeruginosa and Escherichia coli) bacteria in conditions to simulate usage (presence of organic load and hard water). To pass the test a disinfectant must reduce the count of the four organisms by five log orders within the five minute contact time.

Students working in pairs to test one organism and one disinfectant. Aseptic microbiological techniques must be used throughout the practical.

Method

1. Record which bacterial suspension and disinfectant are to be tested.
2. Label duplicate Petri dishes with student names, name of the organism, and either N10-6, N10-7, A, B, C, T1, T2, T3.

3. Take the bacterial suspension labelled ‘N’ (Neat) and perform a decimal dilution by removing 0.5ml of the suspension and adding to 4.5ml Ringers to give a 10-1 dilution. Continue the dilution series to 10-7 and pour plate in duplicate 1ml aliquots from the 10-6 and 10-7 dilutions into the plates labelled N10-6 and N10-7
Keep the 10-5 dilution and label ‘D’ (Diluted) as this will be used to inoculate controls in the test.
Controls
4. Label 3 sterile test tubes ‘A’, ‘B’ and ‘C’. A and B are controls to ensure that the medium and neutraliser do not affect the microorganisms. Control C is to check that the neutraliser is effective in inactivating the disinfectant at the end of the contact time.
5. To test tube A, add 1ml Bovine serum albumin (BSA), 1ml of bacterial suspension D and 8ml Sterile distilled water (SDW) and then vortex. After 5 min, vortex and remove duplicate 1.0 ml for duplicate pour plates using the plates labelled A.
6. To test tube B, add 8ml neutraliser, 1ml sterile distilled water (SDW) and 1ml of bacterial suspension D and vortex. After 5 min, vortex and remove duplicate 1.0 ml aliquots for duplicate pour plating using the plates labelled B.
7. For Control C, add 1ml BSA, 1ml water and 8ml disinfectant (T3, the most concentrated sample) and vortex. After 5 min, remove 1ml to a tube containing 8ml neutraliser and vortex. After 5 min, add 1ml bacterial suspension D and vortex. After 5 min, remove duplicate 1 ml aliquots for pour plating using plates labelled C.

Test
8. The disinfectant is assessed at three concentrations T1, T2 and T3 which are lower than the in-use, the in-use and higher than the in-use concentrations respectively. For the disinfectant test, label 6 sterile test tubes T1, T2, T3 (Test), T1N, T2N, T3N (Test neutralised). To T1N, T2N and T3N aseptically transfer 8ml neutraliser and 1ml sterile water (SDW)
9. To the test tube labelled T1, add 1ml BSA and 1ml bacterial suspension N (NEAT) and vortex. Add 8ml disinfectant T1, vortex and after exactly 5 min, transfer 1ml to the tube labelled T1N and vortex. After 5 min, remove duplicate 1.0 ml for pour plating using plates labelled T1.
10. Repeat as 9 above for disinfectant concentration T2 and T3.
11. Incubate all plates at 37oC for 24 hr.
Summary
Pair Results

Count the cfu/plate and record in the tables below.

Bacterial Culture…………………………..

Table 1 Calculation of concentration of bacterial suspension N
10-6 10-7
Duplicate 1
Duplicate 2
Mean cfu ml-1
Mean cfu ml-1 in undiluted* N
* Use the dilution that gives 30-300 cfu ml-1 for calculation

Table 2 Controls
A B C
Duplicate 1
Duplicate 2
Mean cfu ml-1
For results that are too numerous too count, record as >300

Table 3 Test
T1 T2 T3
Duplicate 1
Duplicate 2
Mean cfu ml-1
Mean x10 cfu ml-1 in test mixture T
Calculation of Reduction Factor (RF) for each concentration of disinfectant

RF= NX10-1
T
Where N is the number of cfu ml-1 in the bacterial suspension
(from the shaded box in Table 1 above)
T is the number of cfu ml-1 in the test mixture for T1, T2 or T3 (from the shaded boxes in Table 3 above)

To pass the disinfectant test, the in-use concentration (T2) must give a reduction factor of>105 for all four organisms within the 5 minute contact time.

Class results
Collect class data of the reduction factors for all four organisms and three concentrations of the disinfectant benzalkonium chloride

Reduction Factor (RF)
T1
%
BAC T2
%
BAC T3
% BAC
S.aureus
E.coli
P.aeruginosa
E.hirae

The drug I have used is E.coli the tem is 54
Results
Disinfectant Practical report

See the notes on WOLF for general practical write-up requirements.

The report must contain:
Abstract – a concise and detailed summary of the experimental findings
Introduction – focused background to set the context of the experiment supported by references cited in the Harvard style in the text.
Methods – refer to the practical schedule but note any changes
Results – Present numbered tables with detailed titles, describe the results and refer to the tables
Discussion – explain the results obtained and in particular
Pair results – comment on the controls, the results obtained for the organisms tested and validity of the test.
Class results – discuss the results obtained with respect to repeatability, concentrations tested and the susceptibilities of the organism used.
References – listed in the Harvard format alphabetically
Table 1. Class data showing reduction factors of benzalkonium chloride (BAC)

BAC Conc (%)
Organism T1 – 0.005% T2 – 0.1% T3 – 0.5%

S.aureus
3.07 x107
1.31×106
1.33×106
1.83×102
7.5×106
2.35×105
1.4×105
E.coli
1.2×105
1.28×103
5.97×103
1.38×107
6.25×105
P.aeruginosa
4.7×103
3.57×102
1.3×106
E.hirae
1.25×104
2.01×103
7.65×106
3.25×104
1.0×107
1.0×101
4×106

Table 1 Calculation of concentration of bacterial suspension N
10-6 10-7
Duplicate 1 150 24
Duplicate 2 180 28
Mean cfu ml-1 165 26
Mean cfu ml-1 in undiluted* N 1.6×108 2.6 x108
* Use the dilution that gives 30-300 cfu ml-1 for calculation

Table 2 Controls
A B C
Duplicate 1 210 190 206
Duplicate 2 252 180 189
Mean cfu ml-1 231 185 197.5
For results that are too numerous too count, record as >300

Table 3 Test
T1 T2 T3
Duplicate 1 1 7 1
Duplicate 2 3 5 1
Mean cfu ml-1 2 6 1
Mean x10 cfu ml-1 in test mixture T 20 60 10

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

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.
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.
Use peer reviewed journals, electronic journals or academic websites. Wikipedia is not acceptable.
3 PRESENTATION OF REPORT

The reports will usually be handed in one week after the session in which results were collected. Reports handed in after the deadline will not be assessed. They should be submitted by 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.

.