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MEDICAL MICROBIOLOGY MB2020 PRACTICAL REPORT 2021 REASSESSMENT   IMPORTANT NOTES CONCERNING PLAGIARISM AND COLLUSION By submitting your work, you are declaring: “I confirm that I understand the University’s regulations regarding plagiarism and that this is my own work

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MEDICAL MICROBIOLOGY MB2020

PRACTICAL REPORT

2021 REASSESSMENT

 

IMPORTANT NOTES CONCERNING PLAGIARISM AND COLLUSION

By submitting your work, you are declaring: “I confirm that I understand the University’s regulations regarding plagiarism and that this is my own work. It has not been copied from any person’s work (published or unpublished), and has not previously been submitted for assessment”.

If you actively co-operate with other students to jointly produce work where there is a requirement that it is produced independently (collusion) the School will investigate your work as evidence of academic misconduct under Senate Regulation 11: Regulations governing student conduct and discipline.

INSTRUCTIONS

The report has four sections:

1. Determination of bacterial susceptibility to an antimicrobial agent (20 marks)
2. Isolation of bacteria from sputum (10 marks)
3. Application of growth assays for analysis of bacterial populations (20 marks)
4. Main characteristics of the identified pathogen (100 marks)

Complete tables, provide calculations, answer questions and write a 600-word essay in Section 4. Marks for each section are indicated in brackets. The final mark for the report will be calculated as the percentage of the maximum mark.

 

 

Section 1. determination of bacterial susceptibility to an antimicrobial agent (20 marks)

Write the name of tested antimicrobial: isoniazid

Results of disk assay

Table 1. Diameter of inhibition zones – model data

Culture	Diameter of inhibition zone, mm
	Water control	Replicate 1	Replicate 2	Replicate 3	Mean±STDV
Culture 1	0	15	17	14
Culture 2	0	21	25	28

Results of microdilution assay

Table 2. Growth of M. smegmatis strain 1 in microplate – model data

Antimicrobial concentration (µg/ml)	Wells	Number of pink wells	Number of blue wells
100	A1-D1	0	4
50	A2-D2	0	4
25	A3-D3	0	4
12.5	A4-D4	0	4
6.3	A5-D5	0	4
3.2	A6-D6	0	4
1.6	A7-D7	3	1
0.8	A8-D8	4	0
0.4	A9-D9	4	0
0.2	A10-D10	4	0
0.1	A11-D11	4	0
0	A12-D12	4	0

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Determine the MIC for strain 1 and write the value:

 

 

 

 

 

Table 3. Growth of M. smegmatis strain 2 in microplate – model data

Antimicrobial concentration  (µg/ml)	Wells	Number of pink wells	Number of blue wells
100	E1-H1	0	4
50	E2-H2	0	4
25	E3-H3	0	4
12.5	E4-H4	0	4
6.3	E5-H5	0	4
3.2	E6-H6	0	4
1.6	E7-H7	0	4
0.8	E8-H8	0	4
0.4	E9-H9	3	1
0.2	E10-H10	4	0
0.1	E11-H11	4	0
0	E12-H12	4	0

 

Determine the MIC for strain 2 and write the value:

 

 

 

 

 

Discuss results of both disk and microdilution assays and make a conclusion about antimicrobial susceptibility patterns of strain 1 and strain 2.

 

 

 

 

 

Explain the difference between Minimum Inhibitory Concentrations (MIC) and Minimum Bactericidal Concentrations (MBC). Briefly describe how you would set an experiment for determination of MBC.

 

 

 

 

 

SECTION 2. ISOLATION OF BACTERIA FROM SPUTUM (10 MARKS)

Table 4. Number of colonies observed on agar plates 1 and 2

Dilution	Sputum: colony numbers in spots	Mean, sputum	Decontaminated sputum: colony numbers in spots	Mean, decontaminated sputum
Undiluted sputum	confluent	N/A	confluent
10-1	confluent	N/A	confluent
10-2	confluent	N/A	confluent
10-3	21, 22, 28		24, 20, 19
10-4	2,0,1		0,3, 0
10-5	0, 0, 0		0, 0, 0
10-6	0, 0, 0		0, 0, 0
10-7	0, 0, 0		0, 0, 0

 

2.       Calculate CFU numbers using the following formula:

 

CFU ml-1=Mean number of colonies in spot x dilution factor x 100

 

 

 

Write your calculations below:

Sputum

 

 

 

 

 

Decontaminated sputum

 

 

 

 

 

Questions

1. Compare the results obtained with sputum and decontaminated sputum and suggest possible explanations for any differences. Discuss how these results may influence experiments with clinical sputum samples?
2. Provide two examples of molecular tests made with sputum samples. Briefly describe these tests.

 

 

 

 

Section 3. Application of growth assays for analysis of bacterial populations (20 marks)

Determining percentage of viable in mixed live/dead bacterial suspension

A mixture of live and dead Micrococcus luteus bacteria with a total count of 1x10⁷ cells ml^-1 was serially diluted and three 10 µl drops from each dilution were spotted on agar plates. Drops were allowed to dry; plates were sealed and incubated at 37°C for 3 days. Colonies in each spot were counted and colony counts were recorded in Table 5.

Table 5. Numbers of colonies on agar plates

Dilution	Number of colonies in individual spots	Mean number of colonies in spot
Undiluted culture	Confluent growth	N/A
10-1	Confluent growth	N/A
10-2	Confluent growth	N/A
10-3	8, 21, 22
10-4	1, 0, 0
10-5	0, 1, 1
10-6	0, 0, 0
10-7	0, 0, 0

Your tasks

1.  To calculate mean numbers of colonies and complete relevant sections of Table

2. Based on values from Table 1 calculate the CFU number per 1 millilitre using the following formula:

CFU ml^-1=Mean number of colonies in spot x dilution factor x 100

 

 

 

 

In the same experiment, 10 µl from each dilution was inoculated in each of 8 replicate wells containing LB medium. Plates were sealed and incubated at 37°C for 3 days. Growth was assessed by visual inspection and by measurement of optical density (OD) at 600 nm. The results of these assessments were recorded in Table 6.

Table 6. Growth of M. luteus in MPN microplate

Dilution	Number of positive wells by visual inspection	Number of positive wells by OD measurement
Undiluted culture	8	8
10-1	8	8
10-2	8	8
10-3	8	8
10-4	8	8
10-5	0	2
10-6	0	0
10-7	0	0
Negative control	0	0

 

Your tasks

1. To work out the MPN count using values obtained by OD measurement. Select three successive dilutions, so the last dilution has fewer than 8 positive wells.

2. Workout the MPN using Supplementary Table 1. See explanations below in “EXAMPLE” section.

3. Determine the MPN bacterial count per 1 millilitre in the initial cell suspension using the following formula:

 

MPN ml^-1= Value from table X dilution factor X 100

 

 

EXAMPLE: if the observed growth pattern is the following: in neat, 10^-1, 10^-2, 10^-3 samples all 8 wells are positive; in 10^-4 dilution 6 wells are positive and 2 wells are negative; in 10^-5 - 2 positive and 6 negative wells; no growth in 10^-6 and 10^-7 dilutions. For estimation of MPN you should use 8-6-2 combination (the numbers of positive wells in 10^-3, 10^-4 and 10^-5 dilutions). The value from Supplementary table 1 is 16.6 cells; it corresponds to the probable number of cells in 10^-3 dilution. In this case the dilution factor is 1000 or 10³. In each well only 10 µl from each dilution was inoculated; to calculate the viable count per 1 ml you have to multiply your value by 100. The MPN count is 1.66x10⁶ viable cells ml^-1.

MPN ml^-1=16.6X1000X100=1.66x10⁶ viable cells ml^-1

4. Calculate the percentage of viable cells obtained by the two methods using the following formulae: 

% Viable bacteria CFU= (CFU ml^-1 /1x10⁷) X 100

% Viable bacteria MPN= (MPN ml^-1 / 1x10⁷) X 100

 

Remember that the total count of bacterial suspensions provided originally was 1x10⁷ cells ml^-1.

 

 

 

Discuss these results.

 

 

 

 

Describe two advantages and two disadvantages of each method.

 

 

 

 

Provide three practical recommendations for conducting reliable and robust CFU and MPN assays.

 

 

 

 

 

 

 

Section 4. main characteristics of THE identified pathogen (100 marks)

Mycobacterium abscessus was isolated from in the sputum experiments described above. Complete Table 7

Table 7. Main characteristics of the identified pathogen

N	Characteristic	Description (10 words maximum per 1 answer)
1.	Staining pattern
2.	Cultivation media
3.	Diseases caused
4.	Symptoms
5.	Populations affected
6.	Diagnostic methods
7.	Treatment
8.	Prevention
9.	Safety precautions for handling
10.	Antimicrobial resistance

Write a 600-word essay to describe three major virulence factors of the identified pathogen and provide three references.

 

 

Supplementary information

Supplementary Table 1. Values of the M.P.N. for 8 tubes (wells) inoculated from each of three successive 10-fold dilutions (Norman&Kempe, 1960)

Number of turbid tubes observed at three successive dilutions			M.P.N. (per inoculum of the first dilution)	Number of turbid tubes observed at three successive dilutions			M.P.N. (per inoculum of the first dilution)
8	8	7	208	8	3	2	7.18
8	8	6	139	8	3	1	5.82
8	8	5	98.2	8	3	0	4.67
8	8	4	70.2	8	2	4	8.07
8	8	3	51.0	8	2	3	6.72
8	8	2	38.5	8	2	2	5.50
8	8	1	30.1	8	2	1	4.45
8	8	0	24	8	2	0	3.62
8	7	8	59.6	8	1	3	5.22
8	7	7	50.8	8	1	2	4.27
8	7	6	43.3	8	1	1	3.50
8	7	5	36.9	8	1	0	2.87
8	7	4	31.4	8	0	2	3.38
8	7	3	26.7	8	0	1	2.80
8	7	2	22.6	8	0	0	2.31
8	7	1	19.1	7	7	1	5.47
8	7	0	15.9	7	7	0	4.84
8	6	6	28.4	7	6	2	5.30
8	6	5	25.0	7	6	1	4.71
8	6	4	21.8	7	6	0	4.15
8	6	3	18.9	7	5	2	4.58
8	6	2	16.6	7	5	1	4.04
8	6	1	13.8	7	5	0	3.55
8	6	0	11.5	7	4	3	4.46
8	5	6	21.3	7	4	2	3.95
8	5	5	18.9	7	4	1	3.47
8	5	4	16.6	7	4	0	3.04
8	5	3	14.4	7	3	3	3.86
8	5	2	12.3	7	3	2	3.40
8	5	1	10.30	7	3	1	2.98
8	5	0	8.42	7	3	0	2.59
8	4	5	14.8	7	2	3	3.33
8	4	4	13.0	7	2	2	2.92
8	4	3	11.1	7	2	1	2.55
8	4	2	9.40	7	2	0	2.20
8	4	1	7.74	7	1	3	2.87
8	4	0	6.22	7	1	2	2.51
8	3	5	11.8	7	1	1	2.17
8	3	4	10.2	7	1	0	1.86
8	3	3	8.67	7	0	2	2.14

 

 

 

Number of turbid tubes observed at three successive dilutions			M.P.N. (per inoculum of the first dilution)	Number of turbid tubes observed at three successive dilutions			M.P.N. (per inoculum of the first dilution)
7	0	1	1.83	4	2	1	1.09
7	0	0	1.55	4	2	0	0.93
6	6	1	3.08	4	1	2	1.08
6	6	0	2.77	4	1	1	0.92
6	5	1	2.73	4	1	0	0.76
6	5	0	2.44	4	0	2	0.91
6	4	2	2.69	4	0	1	0.75
6	4	1	2.41	4	0	0	0.60
6	4	0	2.14	3	4	0	1.01
6	3	2	2.38	3	3	1	1.00
6	3	1	2.11	3	3	0	0.85
6	3	0	1.86	3	2	1	0.85
6	2	2	2.09	3	2	0	0.70
6	2	1	1.84	3	1	2	0.84
6	2	0	1.60	3	1	1	0.70
6	1	2	1.82	3	1	0	0.56
6	1	1	1.58	3	0	2	0.69
6	1	0	1.35	3	0	1	0.55
6	0	2	1.56	3	0	0	0.41
6	0	1	1.34	2	4	0	0.79
6	0	0	1.13	2	3	1	0.79
5	5	1	2.07	2	3	0	0.66
5	5	0	1.85	2	2	1	0.65
5	4	1	1.84	2	2	0	0.52
5	4	0	1.63	2	1	1	0.52
5	3	2	1.82	2	1	0	0.39
5	3	1	1.61	2	0	2	0.51
5	3	0	1.41	2	0	1	0.38
5	2	2	1.60	2	0	0	0.26
5	2	1	1.40	1	3	0	0.49
5	2	0	1.21	1	2	1	0.36
5	1	2	1.39	1	2	0	0.36
5	1	1	1.20	1	1	1	0.24
5	1	0	1.01	1	1	0	0.36
5	0	2	1.19	1	0	1	0.24
5	0	1	1.01	1	0	0	0.12
5	0	0	0.83	0	2	0	0.23
4	4	0	1.28	0	1	1	0.23
4	3	1	1.27	0	1	0	0.11
4	3	0	1.10	0	0	1	0.11

 

MEDICAL MICROBIOLOGY MB2020

PRACTICAL REPORT

2021 REASSESSMENT

 

IMPORTANT NOTES CONCERNING PLAGIARISM AND COLLUSION

By submitting your work, you are declaring: “I confirm that I understand the University’s regulations regarding plagiarism and that this is my own work. It has not been copied from any person’s work (published or unpublished), and has not previously been submitted for assessment”.

If you actively co-operate with other students to jointly produce work where there is a requirement that it is produced independently (collusion) the School will investigate your work as evidence of academic misconduct under Senate Regulation 11: Regulations governing student conduct and discipline.

INSTRUCTIONS

The report has four sections:

1. Determination of bacterial susceptibility to an antimicrobial agent (20 marks)
2. Isolation of bacteria from sputum (10 marks)
3. Application of growth assays for analysis of bacterial populations (20 marks)
4. Main characteristics of the identified pathogen (100 marks)

Complete tables, provide calculations, answer questions and write a 600-word essay in Section 4. Marks for each section are indicated in brackets. The final mark for the report will be calculated as the percentage of the maximum mark.

 

 

Section 1. determination of bacterial susceptibility to an antimicrobial agent (20 marks)

Write the name of tested antimicrobial: isoniazid

Results of disk assay

Table 1. Diameter of inhibition zones – model data

Culture	Diameter of inhibition zone, mm
	Water control	Replicate 1	Replicate 2	Replicate 3	Mean±STDV
Culture 1	0	15	17	14
Culture 2	0	21	25	28

Results of microdilution assay

Table 2. Growth of M. smegmatis strain 1 in microplate – model data

Antimicrobial concentration (µg/ml)	Wells	Number of pink wells	Number of blue wells
100	A1-D1	0	4
50	A2-D2	0	4
25	A3-D3	0	4
12.5	A4-D4	0	4
6.3	A5-D5	0	4
3.2	A6-D6	0	4
1.6	A7-D7	3	1
0.8	A8-D8	4	0
0.4	A9-D9	4	0
0.2	A10-D10	4	0
0.1	A11-D11	4	0
0	A12-D12	4	0

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Determine the MIC for strain 1 and write the value:

 

 

 

 

 

Table 3. Growth of M. smegmatis strain 2 in microplate – model data

Antimicrobial concentration  (µg/ml)	Wells	Number of pink wells	Number of blue wells
100	E1-H1	0	4
50	E2-H2	0	4
25	E3-H3	0	4
12.5	E4-H4	0	4
6.3	E5-H5	0	4
3.2	E6-H6	0	4
1.6	E7-H7	0	4
0.8	E8-H8	0	4
0.4	E9-H9	3	1
0.2	E10-H10	4	0
0.1	E11-H11	4	0
0	E12-H12	4	0

 

Determine the MIC for strain 2 and write the value:

 

 

 

 

 

Discuss results of both disk and microdilution assays and make a conclusion about antimicrobial susceptibility patterns of strain 1 and strain 2.

 

 

 

 

 

Explain the difference between Minimum Inhibitory Concentrations (MIC) and Minimum Bactericidal Concentrations (MBC). Briefly describe how you would set an experiment for determination of MBC.

 

 

 

 

 

SECTION 2. ISOLATION OF BACTERIA FROM SPUTUM (10 MARKS)

Table 4. Number of colonies observed on agar plates 1 and 2

Dilution	Sputum: colony numbers in spots	Mean, sputum	Decontaminated sputum: colony numbers in spots	Mean, decontaminated sputum
Undiluted sputum	confluent	N/A	confluent
10-1	confluent	N/A	confluent
10-2	confluent	N/A	confluent
10-3	21, 22, 28		24, 20, 19
10-4	2,0,1		0,3, 0
10-5	0, 0, 0		0, 0, 0
10-6	0, 0, 0		0, 0, 0
10-7	0, 0, 0		0, 0, 0

 

2.       Calculate CFU numbers using the following formula:

 

CFU ml-1=Mean number of colonies in spot x dilution factor x 100

 

 

 

Write your calculations below:

Sputum

 

 

 

 

 

Decontaminated sputum

 

 

 

 

 

Questions

1. Compare the results obtained with sputum and decontaminated sputum and suggest possible explanations for any differences. Discuss how these results may influence experiments with clinical sputum samples?
2. Provide two examples of molecular tests made with sputum samples. Briefly describe these tests.

 

 

 

 

Section 3. Application of growth assays for analysis of bacterial populations (20 marks)

Determining percentage of viable in mixed live/dead bacterial suspension

A mixture of live and dead Micrococcus luteus bacteria with a total count of 1x10⁷ cells ml^-1 was serially diluted and three 10 µl drops from each dilution were spotted on agar plates. Drops were allowed to dry; plates were sealed and incubated at 37°C for 3 days. Colonies in each spot were counted and colony counts were recorded in Table 5.

Table 5. Numbers of colonies on agar plates

Dilution	Number of colonies in individual spots	Mean number of colonies in spot
Undiluted culture	Confluent growth	N/A
10-1	Confluent growth	N/A
10-2	Confluent growth	N/A
10-3	8, 21, 22
10-4	1, 0, 0
10-5	0, 1, 1
10-6	0, 0, 0
10-7	0, 0, 0

Your tasks

1.  To calculate mean numbers of colonies and complete relevant sections of Table

2. Based on values from Table 1 calculate the CFU number per 1 millilitre using the following formula:

CFU ml^-1=Mean number of colonies in spot x dilution factor x 100

 

 

 

 

In the same experiment, 10 µl from each dilution was inoculated in each of 8 replicate wells containing LB medium. Plates were sealed and incubated at 37°C for 3 days. Growth was assessed by visual inspection and by measurement of optical density (OD) at 600 nm. The results of these assessments were recorded in Table 6.

Table 6. Growth of M. luteus in MPN microplate

Dilution	Number of positive wells by visual inspection	Number of positive wells by OD measurement
Undiluted culture	8	8
10-1	8	8
10-2	8	8
10-3	8	8
10-4	8	8
10-5	0	2
10-6	0	0
10-7	0	0
Negative control	0	0

 

Your tasks

1. To work out the MPN count using values obtained by OD measurement. Select three successive dilutions, so the last dilution has fewer than 8 positive wells.

2. Workout the MPN using Supplementary Table 1. See explanations below in “EXAMPLE” section.

3. Determine the MPN bacterial count per 1 millilitre in the initial cell suspension using the following formula:

 

MPN ml^-1= Value from table X dilution factor X 100

 

 

EXAMPLE: if the observed growth pattern is the following: in neat, 10^-1, 10^-2, 10^-3 samples all 8 wells are positive; in 10^-4 dilution 6 wells are positive and 2 wells are negative; in 10^-5 - 2 positive and 6 negative wells; no growth in 10^-6 and 10^-7 dilutions. For estimation of MPN you should use 8-6-2 combination (the numbers of positive wells in 10^-3, 10^-4 and 10^-5 dilutions). The value from Supplementary table 1 is 16.6 cells; it corresponds to the probable number of cells in 10^-3 dilution. In this case the dilution factor is 1000 or 10³. In each well only 10 µl from each dilution was inoculated; to calculate the viable count per 1 ml you have to multiply your value by 100. The MPN count is 1.66x10⁶ viable cells ml^-1.

MPN ml^-1=16.6X1000X100=1.66x10⁶ viable cells ml^-1

4. Calculate the percentage of viable cells obtained by the two methods using the following formulae: 

% Viable bacteria CFU= (CFU ml^-1 /1x10⁷) X 100

% Viable bacteria MPN= (MPN ml^-1 / 1x10⁷) X 100

 

Remember that the total count of bacterial suspensions provided originally was 1x10⁷ cells ml^-1.

 

 

 

Discuss these results.

 

 

 

 

Describe two advantages and two disadvantages of each method.

 

 

 

 

Provide three practical recommendations for conducting reliable and robust CFU and MPN assays.

 

 

 

 

 

 

 

Section 4. main characteristics of THE identified pathogen (100 marks)

Mycobacterium abscessus was isolated from in the sputum experiments described above. Complete Table 7

Table 7. Main characteristics of the identified pathogen

N	Characteristic	Description (10 words maximum per 1 answer)
1.	Staining pattern
2.	Cultivation media
3.	Diseases caused
4.	Symptoms
5.	Populations affected
6.	Diagnostic methods
7.	Treatment
8.	Prevention
9.	Safety precautions for handling
10.	Antimicrobial resistance

Write a 600-word essay to describe three major virulence factors of the identified pathogen and provide three references.

 

 

Supplementary information

Supplementary Table 1. Values of the M.P.N. for 8 tubes (wells) inoculated from each of three successive 10-fold dilutions (Norman&Kempe, 1960)

Number of turbid tubes observed at three successive dilutions			M.P.N. (per inoculum of the first dilution)	Number of turbid tubes observed at three successive dilutions			M.P.N. (per inoculum of the first dilution)
8	8	7	208	8	3	2	7.18
8	8	6	139	8	3	1	5.82
8	8	5	98.2	8	3	0	4.67
8	8	4	70.2	8	2	4	8.07
8	8	3	51.0	8	2	3	6.72
8	8	2	38.5	8	2	2	5.50
8	8	1	30.1	8	2	1	4.45
8	8	0	24	8	2	0	3.62
8	7	8	59.6	8	1	3	5.22
8	7	7	50.8	8	1	2	4.27
8	7	6	43.3	8	1	1	3.50
8	7	5	36.9	8	1	0	2.87
8	7	4	31.4	8	0	2	3.38
8	7	3	26.7	8	0	1	2.80
8	7	2	22.6	8	0	0	2.31
8	7	1	19.1	7	7	1	5.47
8	7	0	15.9	7	7	0	4.84
8	6	6	28.4	7	6	2	5.30
8	6	5	25.0	7	6	1	4.71
8	6	4	21.8	7	6	0	4.15
8	6	3	18.9	7	5	2	4.58
8	6	2	16.6	7	5	1	4.04
8	6	1	13.8	7	5	0	3.55
8	6	0	11.5	7	4	3	4.46
8	5	6	21.3	7	4	2	3.95
8	5	5	18.9	7	4	1	3.47
8	5	4	16.6	7	4	0	3.04
8	5	3	14.4	7	3	3	3.86
8	5	2	12.3	7	3	2	3.40
8	5	1	10.30	7	3	1	2.98
8	5	0	8.42	7	3	0	2.59
8	4	5	14.8	7	2	3	3.33
8	4	4	13.0	7	2	2	2.92
8	4	3	11.1	7	2	1	2.55
8	4	2	9.40	7	2	0	2.20
8	4	1	7.74	7	1	3	2.87
8	4	0	6.22	7	1	2	2.51
8	3	5	11.8	7	1	1	2.17
8	3	4	10.2	7	1	0	1.86
8	3	3	8.67	7	0	2	2.14

 

 

 

Number of turbid tubes observed at three successive dilutions			M.P.N. (per inoculum of the first dilution)	Number of turbid tubes observed at three successive dilutions			M.P.N. (per inoculum of the first dilution)
7	0	1	1.83	4	2	1	1.09
7	0	0	1.55	4	2	0	0.93
6	6	1	3.08	4	1	2	1.08
6	6	0	2.77	4	1	1	0.92
6	5	1	2.73	4	1	0	0.76
6	5	0	2.44	4	0	2	0.91
6	4	2	2.69	4	0	1	0.75
6	4	1	2.41	4	0	0	0.60
6	4	0	2.14	3	4	0	1.01
6	3	2	2.38	3	3	1	1.00
6	3	1	2.11	3	3	0	0.85
6	3	0	1.86	3	2	1	0.85
6	2	2	2.09	3	2	0	0.70
6	2	1	1.84	3	1	2	0.84
6	2	0	1.60	3	1	1	0.70
6	1	2	1.82	3	1	0	0.56
6	1	1	1.58	3	0	2	0.69
6	1	0	1.35	3	0	1	0.55
6	0	2	1.56	3	0	0	0.41
6	0	1	1.34	2	4	0	0.79
6	0	0	1.13	2	3	1	0.79
5	5	1	2.07	2	3	0	0.66
5	5	0	1.85	2	2	1	0.65
5	4	1	1.84	2	2	0	0.52
5	4	0	1.63	2	1	1	0.52
5	3	2	1.82	2	1	0	0.39
5	3	1	1.61	2	0	2	0.51
5	3	0	1.41	2	0	1	0.38
5	2	2	1.60	2	0	0	0.26
5	2	1	1.40	1	3	0	0.49
5	2	0	1.21	1	2	1	0.36
5	1	2	1.39	1	2	0	0.36
5	1	1	1.20	1	1	1	0.24
5	1	0	1.01	1	1	0	0.36
5	0	2	1.19	1	0	1	0.24
5	0	1	1.01	1	0	0	0.12
5	0	0	0.83	0	2	0	0.23
4	4	0	1.28	0	1	1	0.23
4	3	1	1.27	0	1	0	0.11
4	3	0	1.10	0	0	1	0.11