4th year thesis microbiological assessment of a cleanroom

Microbiological assessment of a cleanroom

Shane Kearney

X00069160

Experimental Review Document

For Research to be carried out under the Supervision of

Sylvia Healy

Department of Science

Institute of Technology Tallaght

Submitted in Part Fulfilment of the Award of Bachelor of Science Honours in Bioanalysis (Course Code TA-SBIOL_D)

Semester 8

April 2015

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ABSTRACT

A standardised sixteen day monitoring program was developed for the validation of a class A and

class C clean room with the identification of at least six potentially hazardous organisms. The method

was used to monitor the C.F.U count using micriobiological and particle tests. Both sections of the

cleanroom were mapped out and given a number for the type of test to be carried out. Settle plates ,

contact plates and swab tests were carried out to gain C.F.U counts along surfaces where the bio-test

was used to count C.F.U in the air . Lastly a particle counter was used to count particles that formed in

the air. The results showed that both the class A and class C sections broke E.U guidance limits set in

the annex 1 2008 for the maximium permitted C.F.U allowed within a cleanroom. Limits were broken

in each of the microbiological tests as C.F.U counts were high but cumulative particle counts fell

within the limits for both the Grade A and grade C. A selection of six microorganisms were able to be

identified ranging from the Bacillus family to the Staphylococcus family all with the potential of

contaminating medical products. In conclusion the following results mean the Institute of technology

Tallaght cleanroom falls out of specification in many areas and corrective action is needed

immediately in order to bring the standards back into the levels permitted by the E.U guidelines.

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ContentsABSTRACT................................................................................................................................................2

Chapter 1

Key objectives........................................................................................................................................4

1.1 Introduction.....................................................................................................................................4

Chapter 3

Method:.................................................................................................................................................8

3.1 Preparation of agar.....................................................................................................................8

3.2 Settle/contact plate.....................................................................................................................8

3.3 biotest..........................................................................................................................................8

3.4 Particle counter...........................................................................................................................8

3,5 Swab Test.....................................................................................................................................9

3.6 Oxidase test.................................................................................................................................9

3.7 API Test........................................................................................................................................9

3.8 0.2 µm filtration kit for swabs.....................................................................................................9

3.9 Incubation / storrage of plates....................................................................................................9

Chapter 4

Results.................................................................................................................................................10

Chapter 5

Discussion............................................................................................................................................18

Conclusion...........................................................................................................................................22

Chapter 6

References...........................................................................................................................................23

Map of clean room..............................................................................................................................26

Appendix.............................................................................................................................................27

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Chapter 1

Key objectives Development of a standardised monitoring plan Comparison of the class A section to the class C section Identification of a selection of microbes isolated from the cleanroom and the treat they pose Recommendation on future management and maintenance of the clean room To determine whether ITT Dublin clean room meets design specifications

1.1 Introduction Creating a good monitoring program (1)

The setting up of a program for monitoring contamination is vital if a cleanroom is to be kept up to

the standards Required. A Monitoring program should consist of the following

Selection of an area within the room to be tested and deciding the type of test suitable for the

area

The frequency your tests should be taken. If the room being tested has a constant flow of

operators moving in and out a trend can be developed on the room when it is in operation and

when it is idle.

Using methods that have been FDA approved and guaranteed to give the best possible results

Monitoring the C.F.U counts and making sure they do not pass the E.U action limits

Frequency of sampling is based on the type of hazard that is found in the room. as well as the

consequences that can be caused by that contaminated product. So the higher the risk the more

frequent sampling should take place. If an area is deemed to be a critical zone then this zone must be

monitored more frequently than other area in the cleanroom. This means that surface monitoring

would be less frequent outside of the critical zone. Transfer areas would need a high amount of

monitoring due to the fact that products are being moved outside of their sterile environment. This

frequency of testing must be carried out in a controlled manner and this is where standardisation

comes in. The importance of standardization is vital as a means of recording consistent and important

results. Tests must be carried out in the same manner in the same position under the same parameters

Eg. Settle plates must be placed in the same position at near enough the same time so that all plates

can be exposed for an equal amount of time and that one plate does not get less or more time then

another plate the in the following set of tests . It creates a consistent day by day set of results . The

fact that these methods are being carried out in the same manner over and over again means

contamination can be spotted easier.(2)

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The complexity of the design of the cleanroom always comes down to the type of products that the

clean room is being used for. This determines the microbial counts allowed and not allowed in the

room. Grade A clean room would have particularly stringent dressing in conjunction with highly

frequent monitoring where a Grade C would be less strict and less frequent monitoring protocals. This

means that each section would carry a higher or lower action limit then one another. If these levels

where to be passed then a corrective action will have to be taken. This means an immediate solution

for the root of the problem must be found which is documented for future reference in case a re-

occurrence is ever to happen. The corrective action should review working procedure , (staff re-

training) review of gowning and de-gowning procedures and the overall maintance of that particular

section of the room and equipment and this can all help towards a good GMP . (3)

When it comes to clean room technology (GMP) Good manufacturing practice of the environment in

the clean room is key in which a medical device or manufacturing of a medical product is being

monitored. An employee who works within the room must be able to specify the critical areas in what

the clean room must meet before manufacturing of any product can take place. This means a

program , SOP(standard operating procedure) must be created and followed like a routine( weekly ,

monthly ) for these critical areas of manufacture which is standardisation.. The data obtained from

these routine checks helps the employer put closure on whether or not the clean room is within

specification. There are also set limits which are set by the E.U which help determine serious drifts or

rises of the number of C.F.U in the clean room.

Table 1 : E.U GMP annex 1 action limits for microbial contamination (3)(4)

Recommeneded Limits of microbial contamination Grade Air sample

cfu/m3Settle plates (90mm) cfu/4hrs

Contact plates cfu/plate

Glove print cfu

A <1 <1 <1 <1B 10 5 5 5C 100 50 25 -D 200 100 50 -

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Table 2 : E.U GMP Annex 1 action limits for Particle contamination (3)(4)

Maximum permitted number of particles per m3

At rest In Operation

Grade 0.5µm 5.0 µm 0.5 µm 5.0 µmA 3,520 20 3,520 20

B 3,520 29 352,000 2,900

C 352,000 2,900 3,520,000 29,000D 3,520,000 29,000 Not defined Not defined

The action limits are marked to prevent any damage being done to the product before manufacturing

and production take place. Possible action procedures could include a complete structured cleaning

procedure, stringent gowning and de-gowning procedure with cleaning of personnel. Not meeting

these principle rules can result in possible shutting down of the lab. Financially , this would have an

extremely negative impact on the company.

To ensure the following cleanroom is running up to standard a standardised procedure that consists of

a range of different microbial tests was carried out over a 16 day period with hopes of possibly

identifying at least 6 microbes if present in the room. It will compare results to the E.U GMP Annex 1

( FDA’s Aseptic Processing Guide) for the action levels if a microbial count was to surpass the limit

that is allowed in the room to alert companies that action must be taken

The paper describes a standard procedure for a standardised method that was carried out in the same

fashion from week by week for monitoring and identification of microbiological activity within the

cleanroom. (4)

Chapter 2 .

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Materials

90mm Petrit dishes Nutrient agar ( 0.46g) per plate

5 % blood Base agar

55mm Rodac plate Nutrient Agar (0.345g ) per plate

Oxidase Test Oxidase Reagent

Sterile wooden pick

Filter Paper

(+) control Pseudomonas Aerigonsa

API Strips API 20NE API 20E API 50CH API STAPH

Gram stain kit Microscopic glass slides Staining Tray Reagents ( iodine , decolouriser , safarin , methylene blue , Crystal Violet)

Particle Test Particle Counter

Bio –test kit Bio-test apparatus Nutrient Agar strips

Swab Tests Sterile wooden swabs 0.2µm filtration kit Saline solution ( sodium chloride )

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Each test sample needs innoculm for homogenising the colony and then reagents for adding into each of the wells along each of the strips.

Chapter 3 .

Method:

3.1 Preparation of agar Settle plates and contact plates were used .The settle plates containing up to 25ml and contact plates

up to 15ml. A Powdered Nutrient Agar base supplied by Sigma Aldrich had to be made in different

weights for each of the set of dishes. For the contact plates 6.9 grams of nutrient agar was weighed out

in 300ml( 0.345g per plate) of deionised water which made close to 20 plates. For the settle plates

11.5 grams was weighed out in 500 ml ( 0.46g) of deionised water. Both were made up in duran

bottle and autoclaved at 121oC for 15 minutes at 15 P.S.I. and then allowed to cool for storage at

550C for up to 1 day before they have to be poured. Pouring was carried out in a LAF cabinet wiped

down with 70% IPS to create the perfect sterile background in a bid to prevent contamination of plates

. As plates were poured over the following weeks one settle plate was left opened in the cabinet as the

control and incubated for detection of any contaminants. For swab tests a solution had to be made up

to moisten and store swabs while moving in and out of the cleanroom. A saline solution of 0.54grams

of sodium chloride made up in 60mls was prepared which was autoclaved and had 10ml aliquots

added to different sterile universal tubes. 5% sheep blood from Sigma Aldrich was made up in a blood

agar base by mixing (0.5%) 25ml of pre sterilised sheep blood (370C) into 19.5 grams of blood agar

base. Plates poured when duran bottle was at a point where it could be handled comfortably. 0.2µm

filtration kit was used for

3.2 Settle/contact plate A total of 10 plates , for both the settle and contact plates was carried out each day with 8 plates

carried out in the grade C section and 2 in the grade A . Plates were made as per set up in --- and

placed in their allocated positions along clean room.and allowed to sit in the room for up to 3-4 hours

max. Contact plates were pressed against surfaces using a rolling action before being sealed for

incubation . Area in contact with pate was s wiped down with disinfectant.

3.3 biotestBio- test sample was carried by first by Autoclaving the top half of the instrument that holds the agar

strip . Sample setting placed at 1000 litres and first tested in Grade A section before being brought to

grade C area . This is to stop the contamination and the carrying of microbes from Grade A to C .

3.4 Particle counterThe Particle counter was set for cumulitive count for 15 minute intervals which measured for dust

particles in the air. 3 tests were carried out ,1 in the class A and 2 in the class C. each week in the

same place section

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3,5 Swab Test Swabs have to be moistend in saline solution prior to taking up of sample in 0.9% saline (sodium

chloride + deionised water )The Swab is rubbed over a surface of 25cm3 using a sterile swab

removed from its packaging beside teste area .The Swab is applied to area to the are with firm

pressure and rotated along the syrface suing the shaft . Are being monitored is wabbed horizontaly

and then vertically along the surface to gain the best possible swab of the surface.

3.6 Oxidase test To determine whether an API 20E or 20NE must be used an oxidase test had to be carried out .First a

piece of flter paper was soaked in the oxidase reagent solution. Using a sterile wooden pick some of

the culture of interest was scraped of the plate and onto a marked section of the soaked filter paper.

This was Compared with a known oxidase positive control( Pseudomonas aerigonosa) for and a blue

colour was expected

3.7 API TestGeneral method for all API is to inoculate a large colony of bacterium into it EG 0.85% NaCL

solution and was vortexed and mixied well untill the sample was homogenous. The tube was then

cracked open when the solution was completely homogenised and using a sterile Pasteur pipette

inoculate the bacterial suspension into each of the well of the API Strip. Following instruction for

each of the different API tests each well was filled or not filled and then the strip was incubated for up

to 24 hours. After incubation a set of reagents was used which were added to individual well

according to a specific procedure

3.8 0.2 µm filtration kit for swabs Test was carried out using sterile wooden swab and a filtration kit. Swab sample taken from both

grade A and grade C and vortexed for set amount of seconds before being filtered through. Thin

membrane filter paper is then removed ( should contain bacteria ) and placed onto fresh agar medium

and incubated for set time.

3.9 Incubation / storrage of plates All plates incubated at 37oC for 48 hours before being analysed. Plates were stored after incubation in cold room at 4oC.

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Day Number

Fridge (A)

Mask(A)

Shower

c.room

bench

chiller

Evap

Probe st

chem cab

cab (2)

Day 1 0 0 0 0 0 0 0 0 0 0Day2 0 0 0 0 0 0 0 0 0 0Day 3 0 0 3 1 1 2 1 0 1 2Day 4 0 0 0 1 0 0 1 1 0 1Day 5 1 0 0 5 1 1 1 3 0 3Day 6 1 0 1 4 1 0 2 0 0 2Day 7 0 3 0 16 1 0 0 1 0 1Day 8 0 0 1 1 0 1 0 1 1 0Day 9 4 0 1 9 0 1 0 0 5 4

Day 10 0 0 1 0 0 1 1 0 3 2Day11(R) 2 4 3 3 0 0 4 6 4 9Day 12 0 0 0 0 0 0 0 0 0 0Day 13 Day 14 0 0 0 0 0 0 0 0 0 0Day 15 0 0 0 0 0 0 0 0 0 0Day 16 0 0 0 0 0 0 0 0 0 0

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Table 3: C.F.U( colony forming units) count over a 16 day process for Grade A and grade C cleanroom using both nutrient agar and Red blood agar (R)= red blood agar

Figure 1 : C.F.U formation afer 4 hours of settleing and incubated at 37oC for 48hrs for Grade A and Grade C

Chapter 4

Results

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Day 1

Day2Day 3Day

4Day

5Day

6 Day

7 Day

8 Day

9

Day 10

Day 11Day

12Day

13Day

14Day

15Day

160

10

20

30

40

50

60

c.f.u count /settle plate/4hours Vs time Fridge

Mask

Shower

c.room

bench

chiller

Evap

Probe st

chem cab

cab (2)

limit C

No.

of c

.f.u/

pla

tes/

4 ho

urs

Table 3/ Figure 1. Is a 16 day analysis of c.f.u counts using 90mm nutrient agar plates. The following data shows analysis of 10 different sites , 8 grade C and 2 Grade A sites( Fridge and mask) . Plates were allowed to settle for up to 4 hours with the room being unmanned on each day. Changing room form Grade C area shows the most consistent highest count after the 4 hours with the holding cabinet from Grade C showing the second highest. Mask aligner and fridge (Grade A) contain small counts with the highest reaching 4 c.f.u for blood agar(day 11) mask aligner and 4 c.f.u for nutrient agar at the fridge (day9)

Table 4. C.F.U( colony forming units) count over a 16 day process for Grade A and

Grade A action limit <1

Day Number Fridge Mask

Shower c.room

Probe st.

Acid .B Evap Wafer Hybrid

cab (2)

Day 1 0 0 0 0 0 0 0 0 0 0Day2 0 0 0 0 0 0 0 0 0 0Day 3 0 0 0 0 1 2 0 1 3 2Day 4 0 8 0 2 1 3 0 0 0 0Day 5 1 0 0 1 0 0 0 1 0 1Day 6 1 1 3 0 0 0 17 0 0 0Day 7 2 15 0 5 0 0 2 1 4 25Day 8 1 2 0 0 0 0 1 0 0 1Day 9 1 2 0 1 0 1 1 1 1 1Day 10 1 0 0 0 0 1 2 1 1 1Day 11 0 0 0 4 1 1 1 1 0 1Day 12 0 0 0 0 0 0 0 0 0 0Day 13 Day 14 0 0 0 0 0 0 0 0 0 0Day 15 0 0 0 0 0 0 0 0 0 0Day 16 0 0 0 0 0 0 0 0 0 0

Day 1

Day2 Day 3

Day 4

Day 5

Day 6

Day 7

Day 8

Day 9

Day 10

Day 11

Day 12

Day 13

Day 14

Day 15

Day 16

0

5

10

15

20

25

30

C.f.u count /Contact plate/M3 Vs Time

Fridge

Mask

Shower

c.room

Probe st.

Acid .B

Evap

Wafer

Hybrid

cab (2)

limit C

C.f.u

coun

t /Co

ntac

t pla

te/M

3

Table 3

Day No.

0.3µm 0.5µm 1.0µm 3.0µm 5.0µm 10.0µmday 1 0 0 0 0 0 0day2 0 0 0 0 0 0day 3 0 0 0 0 0 0day 4 0 0 0 0 0 0day 5 9 4 3 2 1 0day 6 8 5 2 1 0 0day 7 9 6 5 3 2 0day 8 9 6 4 2 1 0day 9 6 2 1 0 0 0day 10 24 5 3 1 0 0day 11 124 23 11 5 1 0day 12 0 0 0 0 0 0day 13 0 0 0 0 0 0day 14 14 7 3 2 1 0day 15 0 0 0 0 0 0

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Table4 / Figure 2 represent 16 analyses of contact plates that measure c.f.u counts per m3 for grade C and grade A( fridge/mask) . Holding cabinet shows the most activity in grade C 4 one particular day coming into line with the E.U action limit. Mask aligner for Grade A exceeds E.U action limit <1 on several days and also surpasses somes section within the grade C area for C.F.U. Other section with in the grade C section show consistent but low values staying under the limits for Grade C .

Figure 2. 16 days analysis of C.F.U per m3 in both a Grade A and Grade C clean room *limit C = action limit for C

Table 5. 16 day analyses of particle counts taken in a grade A clean room per m3

day 16 0 0 0 0 0 0

day 1

day2day

3day

4day

5day

6 day

7 day

8 day

9

day 10

day 11

day 12

day 13

day 14

day 15

day 16

0

20

40

60

80

100

120

140

Particle per M3 Grade A Fridge Vs Time (day)

0.3µm

0.5µm

1.0µm

3.0µm

5.0µm

10.0µm

5.0µm action limit

Parti

cle

coun

t pe

r M3

Day No.

0.3µm 0.5µm 1.0µm 3.0µm 5.0µm 10.0µmday 1 0 0 0 0 0 0day2 0 0 0 0 0 0day 3 0 0 0 0 0 0day 4 0 0 0 0 0 0day 5 68 43 30 17 5 1day 6 34 14 7 2 1 0day 7 24 18 13 6 2 0day 8 14 10 7 4 1 0day 9 65 13 5 2 0 0day 10 105 8 2 0 0 0day 11 124 23 11 5 1 0day 12 15 8 6 4 2 0day 13 0 0 0 0 0 0day 14 15 8 6 4 2 0

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Table 5/ figure 3 is a cumulative analyses of particles counts per m3 for class A cleanroom. Main particles of interest are 0.5µm with an E.U limit of 3520 particles per m3 and 5.0µm with an E.U limit of 29 particles per m3. 0.5µm particles stay well within their limit and only go as high as 23 particles per m3 before dropping . Particles of the 5.0µm range only ever go as high as 2 which fall well underneath the E.U limit of 29. Each set of results show relatively low values with each particle size having one day where they peaked slightly.

Figure 3: Cumulitive count of Particles per m3 over 16 day period within a Class A clean room ( action limit of 29 for 5.0µm¿

Table 6. 16 day analyses of particle counts taken in a grade C (chiller) clean room per m3

day 15 0 0 0 0 0 0day 16 0 0 0 0 0 0

day 1

day2

day 3

day 4

day 5

day 6

day 7

day 8

day 9

day 10

day 11

day 12

day 13

day 14

day 15

day 16

0

20

40

60

80

100

120

140

Particle per M3 Grade C Chiller Vs Time (Day)

0.3µm

0.5µm

1.0µm

3.0µm

5.0µm

10.0µm

Parti

cle

per

M3

Day No.

0.3µm 0.5µm 1.0µm 3.0µm 5.0µm10.0µm

day 1 0 0 0 0 0 0day2 0 0 0 0 0 0day 3 0 0 0 0 0 0day 4 0 0 0 0 0 0day 5 17 8 4 2 0 0day 6 5 3 2 1 0 0day 7 11 8 6 3 1 0day 8 13 8 5 3 1 0day 9 6 4 2 1 0 0day 10 12 3 1 0 0 0day 11 8 5 3 2 0 0day 12 0 0 0 0 0 0day 13 0 0 0 0 0 0

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Table 6/ Figure 4 is a cumulative analyses of particles per m3 within a grade C section beside the chiller unit. Action limits for Grade C increase to 352,000 particles per m3for 0.5µm and 2900 particles per m3 for 5.0µm. Both 5.0µm and 0.5 µm fall well within their respective limits with 0.5µm showing the highest individual particle count at 43 m3. 5.0µm only one one occasion fall as far as 5 particles per m3 and keeps low values for the remainder of the days.

Figure 4 Cumulitive count of Particles per m3 over 16 day period within a Class C clean room chiller section

Table 7. 16 day analyses of particle counts taken in a grade C (probe st.) clean room per m3

Action Limits

0.5µm =2900

5.0 = 352,000

day 14 12 9 4 2 1 0day 15 0 0 0 0 0 0day 16 0 0 0 0 0 0

day 1

day 3

day 5

day 7

day 9

day 11

day 13

day 15

0

2

4

6

8

10

12

14

16

18

Particle per M3 Grade C probe st .Vs Time (Day

0.3µm

0.5µm

1.0µm

3.0µm

5.0µm

10.0µm

Parti

cle

per

M3

Day No. Fridge

(A)chem cab (C)

day 1 0 0day2 0 0day 3 1 3day 4 4 3day 5 9 0day 6 4 0day 7 y/m 0 0day 8 y/m 0 0day 9 y/m 0 0

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Table 7 / Figure 5 analyses a cumulative analyses of particles per m3 in a grade C clean room beside the probe station. Action limits for Grade C increase to 352,000 particles per m3for 0.5µm and 2,900 particles per m3 for 5.0µm. Both sets of results for the 0.5µm and 5.0µm fall well within the their limits of the E.U guidelines. 0.5 µmvery rarely has a value of 0 but has low values that fall within the 2900 limit and forms an undulating trend. 5.0µm limit only ever reaches a high of 1 particles per m3 also falling well within it limit of the action limit of 352,000

Figure 5 Cumulitive count of Particles per m3 over 16 day period within a Class C clean room probe st. section

Table 8 : 16 Day analyses of c.f.u/m3 for grade A and grade C using nutrient Agar strips and yeast/mould strips y/m= yeast/mould

Action Limits

0.5µm =2900

5.0 = 352,000

day 10 y/m 0 0day 11 0 0day 12 0 0day 13 0 0day 14 0 0day 15 0 0day 16 0 0

day 1

day 3

day 5

day 7

day 9

day 11

day 13

day 15

0

20

40

60

80

100

120

c.f.u/M3 Biotest Vs Day (time)

Fridge (A)

chem cab (C)

Series3c.f.u

/M3

Bio

test

Action limit (C )

Day No. Tap A Shower Cday 1 0 0day2 0 0day 3 0 2day 4 8 1day 5(fk) 8 0day 6(fk) 4 1day 7 0 0day 8 0 1

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Table 8/Figure 6 . 16 analyses of bio-test samples carried out in Grade A and grade C clean room. E.U limit for Grade C is 100 c.f. per m3 in which Its well under limit with the highest count coming in at 3. Class A exceeds its limit of <1 with particles count of 9 per m3 on one particular day. Due to a shortage on nutrient agar strips Yeast and mould strips were used instead but showed no sign of any growth and were ceased from being used after 2 weeks

Figure 6 : C.F.U /M3 count bio-test for both Grade A and Grade C with nutrient/yeld/mould agar strips

Table 9 :16 day C.F.U count analyses of swabs grade A tap and Grade C shower over a 25cm3 surface. fk= filtration kit (0.2µm)/ (R)= red blood agar

day 9 1 1day 10 0 30day 11 (R) 9 7day 12 0 0day 13 0 0day 14 0 0day 15 0 0day 16 0 0

day 1

day2day

3day

4day

5day

6 day

7 day

8 day

9

day 10day

11day

12day

13day

14day

15

day 16

0

5

10

15

20

25

30

35C.f.u count swab 25cm3Vs day time

Tap A Shower C

C.f.u

coun

t sw

ab 2

5cm

3

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Figure 7 . 16 day investigation of of c.f.u counts over a 25cm3 surface from a Grade A section (Tap) and a Grade C section ( filtration kit test included)

Table 9/Figure 6 shows a 16 study swab samples taken from each grade A and grade C section . Section C shower shows immediately the most prominent c.f.u count with 30 C.F.U but then keep relatively low values on each of the other days. Grade A tap peaks on two separate days using the normal swab method and also using the filtration kit

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Gram positive Coccus (API STAPH)(5)

Identified as Micrococcus spp (99.4%)Gram(+) strictly aerobic cocci . Usually occur in irregular cluster along agar dish. Cell 1-1.8 diameter. Regarded as harmless found mostly along skin and mucosa but can be opportunistic for immunocompromised people . Can cause infection such as bacteremia and infectiions with ventricular shunts. Possible cause of contamination in clean room from personel .

Gram Positive Cocci ( API STAPH)(6)

Identified as Staphylococcus warneir (61.5%)Commonly present in flora of human epithelia and mucosal membranes. Ha been reported as a developing pathogen . Has been suggested as a cause for spontaneous abortion as well as urinary tract infection. Possible cause of contamination in the clean room once again from people coughing etc..

Gram Positive Rod (API 50CH)(7)

Identiified as bacillus megaterium(89.3%)

Motile pertrichous flagella. colonies round to irregular , common saprophyte (bacterium /fungus) mainly found in soil . considered non pathogenic , but it does form endospores which can products . Possible introduction into the room from shoes .

Gram negative rod /cocci ( Oxidase(+) API 20NE)(8)

Identified as Pseudomonas stutzeri (70.3%)Known as a universal microbe as it is present in almost all enviroments such as water and soil. It s know to have both nitrogen fixing and dentrifiers which gives them there divers metabolism . Micorbe can be used in industries such as bioremediation , water and waste treatment , drain cleaners etc

Gram (-) oxidase (-) API 20 E (9) (10)

identified as Pantoea spp Recently seperated from the enterobacter genus (entric bacilli) when gram stained cells all look alike . Found in the GI tract of humans , involved in diseases such as salmonella , shigella . Possible introduction into the room froom cuts on human interaction , movements

Gram (-) oxidase (+) API 20NE (10)

Rod shaped grams negative , Identified as Rhizobium Bacterium Aerobic bacterium , that is associated with human systematic diseases such as urinary tract infection and myositis.

Figure 8 : Systematic diagram of API Test identification wigram stains

Chapter 5

Discussion The available guideline for cleanroom technology in Europe and the USA has helped create safe

environments for workers as well as the products that are created. In the case for the following

experiment the action limits that are set for each of the following tests in each Graded room are taken

from the E.U GMP Annex 1 which states C.F.U limits that a clean room must not pass if it is to be

classified within specification, These action limits and guidelines help form trends that enforce rules

and requirements to help clean rooms meet the right standards/specification.

The analyses of the settle plates figure 1 from both the grade A and Grade C show the room meets the

requirements for the Grade C but fails in the Grade A. This is because it does not meet the action

limits of <1 C.F.U set by FDA Standards .The consistently low C.F.U counts found within the Grade

C section and the fact that the C.F.U count is lower than the E.U action limit indicates that this is

within specification. Only on one occasion does the C.F.U count take a considerable jump which was

in the changing room but this can be expected as exchange of clothing and the introduction of

microbes via doors be opened and closed . On Day 11 a trial run of blood agar base made from 5 %

sheep blood was used to see if a higher/lower C.F.U count could be picked out compared to the

nutrient agar base. The results gathered found a more consistent set of C.F.U counts but nothing in

large quantity to justify that immediate action is needed. The Grade A section was found to break the

limits on several days and reach a high of 4 C.F.U . The fridge section showed the most out of spec

for the grade A breaking the limit on four different test days. This means overall the Grade A section

which is meant to hold the highest degree of cleansiness was more out of specification than the Grade

C section.

Contact plates monitor surfaces with different action limits for both the grade A and Grade C taken

from the E.U. GMP Annex 1. Data gathered from the contact plates indicate that the Grade C section

contains one unsatisfactory result on a particular day. With the E.U limit at ≤25 the holding cabinet

contact plate for day 7 had a count of 25 C.F.U and failed to meet the requirements of the room

therefore putting the room out of specification. Despite the result falling out of specification the result

can be looked at from two point of views. The first point is that because the rest of the day’s results

from the cabinet show little or no C.F.U counts the result could be contaminated. This could have

been through storage of the plate or poor method of applying to the surface. The second point is that

through the whole of day 7 there was a significantly higher set of C.F.U count in each location

during that day which could mean that activity was significantly higher during that week creating

more microbes and particles to be picked up then other weeks. Analysing day 7 on figure 2 there is a

low undulating trend before a significant increase in C.F.U count for the holding cabinet but then it

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then suddenly drops again. In order to address the situation more tests in this particular section should

be carried out to determine whether it is within spec or whether it was a plate error.

Figure 9. Shows damage to HEPA filters along the roof of Grade A cleanroom.

Grade A section from figure 2 shows relatively low and stable C.F.U counts for the fridge section but

still passes over the < 1 action limit. The mask aligner contains 2 sudden jumps that well exceed over

the action limit <1 and therefore put this section out of specification. It must be noted that the highest

count for C.F.U was also on day 7. Once again this high count could have been to poor a poor

operating procedure or down to high activity of personnel within the room. It also could have been

down to the noticeable damage that was found in the HEPA filter along the roof in figure 9 . This

would contribute to the poor overall results in the class A and would be why it is out of specification

This also could have contributed to the poor settle plates results. For the Grade A which read similar

C.F.U counts .

Analysing the particles counts from figure 3, 4 and 5 it can be concluded that both sections of the

clean room are within specification for particle counts with two sets of tests being carried out in the

Grade C and one in the grade A. The main particles of interest were the 0.5µm particles and the 5.0

µm with the Grade A containing an action limit of 3520 for the 0.5 µm and 29 for the 5.0 µm.

Overall the grade A(fig3) had a stable number of particles with only a slight incline occurring on day

11 which read 23 particles but this still falls short of the limit which is at 29. Particles of the 5.0µm

range rarely fall above the baseline further illustrating that the grade A section is within specification.

Both the chiller unit section and probe station section ( fig 4,5 ) contain graphs that are in zig zag

format .i.e The particles counts vary up and down daily. Both sets of data for Grade C clean room fall

well under the E.U limits meaning that particles count within this section are within specification.

Bio-test counts were unable to be classed as out of spec or within spec as a shortage of nutrient agar

strips thwarted this process. Therefore only four weeks of data using nutrient agar strips was able to

be gathered .Yeast and mould strips were used for weeks 4 and 5 but found no results. Figure 6 is the

data collected from the nutrient agar strips for the grade A and grade C section but unfortunately there

was not enough data collected to come up with a viable conclusion. Looking at the results that were

gathered (table 9) results for the Grade C Section were were holding low values that fell in the

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specification. The action limit for Grade C is 100 c.f.u./m3 which the grade C was measuring under

with the few results recorded The Grade A however had already exceeded the <1 action limit on 4

different occasions giving an indication that corrective action would have to be taken in order to

correct this fault.

Swab Tests showed out of spec results for both Grade A + Grade C. Limit of <1 for Grade A was

exceeded on more than 5 different days. The grade C broke its limit on one day counting 30 C.F.U

which was five more over the ≤25 limit. This indicates that surfaces are not being wiped down

properly or cleaned at regular intervals. Grade A was tested on a tap that would be used for machinery

and used at regular intervals .Transfer of dirt onto tap is not only affecting the grade A section but

also other surface that the user may touch. This could be contributing towards the high counts the

mask aligner is receiving as well as some areas in the grade C.

One of the main objectives of this practical was to identify at least 6 different colonies from 6

distinctive plates that could pose a probable treat to products within the room. Figure 10 is a

systematic diagram of 6 colonies that were identified through gram stains first/ oxidase tests and then

API test.

Figure 10 : Process of I.D Plates and ready for gram stain .

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Figure 10 : shows the process that was taken to pick

the 6 best colony formations. Plates were picked

from over the past weeks from different types of

tests that showed the most promising and distinctive

growth. Colonies from each plate were placed onto

glass slide and had a gram stain process carried out

on them. From each slide they were able to be

identified as gram (+) or gram (-) which can be

found in figure 8.This helped to choose the correct

API procedure Plates that were chosen were re

plated and incubate for 24 hrs On fresh medium in

order to be identified for API tests , each labelled

with were they were sampled from and whether

they were a settle/contact swab etc..Plates had to be

replated more than once due to a poor quadrant

streak technique.

Figure 11. Layout of oxidase procedure with known (+) control pseudomonas aeruginosa

Throughout this process six organisms were identified and analysed to see if they posed any treat

within the cleanroom. As a cleanroom rule states all organisms must be treated as harmful unless

proved otherwise. Figure 8 is an evaluation of all the following information just discussed. The types

of organisms that were found were all found to be naturally occurring organisms that are found in

many types of environment such as soil and water and skin of humans. Micrococcus SPP was found

by a settle plate that was sitting in the fridge section of the Class A clean room. Micrococcus SPP is

not relatively harmful to a healthy individual however it can cause nasty skin infections to individuals

who are immune compromised. Finding this type of organism within the class A sections means

medical practising in the class A could be extremely dangerous for contaminating products.(12) This

indicates that staff were not being attentive while putting on cleanroom garments or being very hasty

when working within the environment Eg. coughing without covering mouth , poor use of hair net ,

removal of gloves especially within a class A environment where these procedures are paramount.

There is even further proof of poor procedure from the second I.D 4 organism which came from a

contact plate in the class C environment along the evaporator. Samples were taken along a control

panel where interaction from people was high. An organism called Staphyloccus Warneri was found

which is a common microbe found in mucous membrane and epithelial cells. Possible sources for this

microbe are from workers scratching themselves releasing skin debris into the air , coughing , . Other

possible sources are from entering of raw material which have been handled by personnel wearing no

gloves or gear etc. This is another microbe that can cause serious skin infection if it was to spoil a

product The other types of microbes found in the room (figure 8) come from similar sources , skin

cells , GI tracts and therefore tell us that human negligence for safe practice is the cause . Pantoea spp

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Figure 11 is the process of identifying 3 I.D’s on whether they are oxidase positive oxidase negative and whether An API 20NE or API 20E would be used .Flter paper had to be directly applied to (+) control dish in order to pick up any available colonies for testing and then the oxidase reagent was added to test for colour change . I.D 6 and 8 both had colour change so API 20NE would be suitable for this (ox +) API 20E was used for (ox -) I.D 7

was an organism found from a settle plate in the changing room and this type of organism is

associated with cuts along the skin. Personnel not covering cuts with plaster allows the release of skin

debris which can be brought into the cleanroom with the possibility of exposing medical products to

salmonella and shigella forming the bacteria A combination of action limits being broken on several

tests and the discovery of harmful organisms that could damage products means the IT Tallaght

cleanroom in its current state is not fit for carrying out work in .

When evaluating all of the results together a conclusion can be drawn that both the class A and Class

C do not follow the required specification that meet the E.U standards. Each section passes in most

areas but then fail in another and its these failures that deem the room non fit for work.. The class A

section in the cleanroom may pass when it came to the particles counts but it was alarmingly out of

specification on numerous occasions on the contacts and settle plates. Class A cleanrooms are meant

to be the highest of standards for cleanliness which is why medical companies us them for medical

research but finding an organism which has the potential to spoil and cause harm outside of the room

means the class A should be shut down immediately. The class C does not have as strict limits as the

class A but still has limits broken of its own .

The following monitoring program has proven itself to be suitable for monitoring a class A and class

C cleanroom . It was able to identify area that fell in and out of specification as well as determine that

the room is out of specification

Conclusion

Over the course of this sixteen day monitoring program the main aim was to find out whether the

following cleanroom was within specification .Results gathered have stated that the cleanroom fails in

multiple areas and therefore cannot said to meet the standards of grade A and grade C. The Results

do not keep to the standard published by the FDA E.U GMP Annex 1 however only in certain areas

does the room fail. Extra monitoring and more stringent procedures in practice can bring the room

back up to spec by prioritising in these area. However the cleanroom in its current state must be shut

down as it is not safe enough to carry out practice in , Regular cleaning procedures along the floor

and surface and can help keep the place clean and particle free. An updated monitoring programs with

regular recording of data can help develop trends which can make it easier to determine if the room is

going out of specification. Also from figure 9 fixing damaged HEPA filter and ceiling covers would

also help bring the room back down to spec,. Retraining of staff in how to operate within the

cleanroom and assessment on how to put on cleanroom clothing would also be wise as over half the

organism found in the room are common microbes found on skin.

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Chapter 6

References

1. Lieberman, A. 1992, Contamination control and cleanrooms: problems, engineering

solutions and applications, Van Nostrand Reinhold, New York

2. Whyte, W. 2001, Cleanroom technology: fundamentals of design, testing and operation, J.

Wiley, Chicheste

3. Whyte, W. 1999, Cleanroom design, John Wiley, Chichester

4. E.U GMP Annex 1 2008

5. Characterization of 26 Staphylococcus warneri isolates from orthopedic infections. - PubMed

- NCBI . 2015. Characterization of 26 Staphylococcus warneri isolates from orthopedic

infections. - PubMed – NCBI

6. Characterization of 26 Staphylococcus warneri isolates from orthopedic infections. - PubMed

- NCBI . 2015. Characterization of 26 Staphylococcus warneri isolates from orthopedic

infections. - PubMed – NCBI

7. De Vos, P. et al. Bergey's Manual of Systematic Bacteriology: Volume 3: The Firmicutes.

Springer (2009)

8. Lalucat, J., Bennasar, A., Bosch, R., García-Valdés, E. & Palleroni, N.J. 2006, "Biology of

Pseudomonas stutzeri", Microbiology and Molecular Biology Reviews, vol. 70, no. 2, pp. 510-

547.

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9. Donnenberg, Michael (2009). Mandell, Douglas, and Bennett's Principles and Practice of

Infectious Diseases. Chapter 218: Enterobacteriaceae. p. 2827.

10. Laboratory diagnosis of infectious diseases: essentials of diagnostic microbiology Paul G.

Engelkirk, Janet L. Duben-Engelkirk

11. Clinical and Microbiological Characteristics of Rhizobium radiobacter Infections . 2015.

Oxford journal Volume 38 issue 1 Pp. 149-153

12. Hug DH, Dunkerson DD, Hunter JK The degradation of L-histidine and trans- and cis-urocanic

acid by bacteria from skin and the role of bacterial cis-urocanic acid isomerase. J Photochem

Photobiol B 1999 May;50(1):66-73

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Map of clean room

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Appendix

BSc (Hons) in Bioanalytical science / Pharmaceutical Science

PROJECT WEEKLY PROGRESS REPORT

Student Name: Shane Kearney Supervisor: Sylvia Healey

Date 10/2/15 11/2/15 Week number 2

A. Summary of key results /activities for this week

Bulk of activities carried out this were the making of 90mm and 55mm plates for contact, swabs, and settle plates . The amount of agar in mls was played around with to find a suitable method for pouring plates effectively without error or disruption. It was found that making the agar in 500ml bottles made it easier for pouring but also allowed quicker cool down times when out of the autoclave. Pouring the plates in a Laminar flow cabinet that was cleaned down with 70% IPS made it a lot easier and more efficient to pour large amounts of plates in quick succession and also reduced the time it took for them to set. Settle plates were also poured between the Laminar flow cabinet and the desk under the blue flame of a Bunsen. A saline solution was made for 6 plates for both Tuesday the 10 th and Wednesday the 11th for swab test which would be swobbed onto a plate. Biotest kit was set at a parameter of 1000L (m3) and 3 strips for test in both class A and two in class C. When carrying out tests in the cleanroom settles plates were placed within a 10 minute period and then contacts and swab test were carried out in pre-determined positions. Settle plates were only able to sit for 1 hour and 35 minute on Tuesdat the 10th but got the full 4 hour settle period on Wednesday the 11th. Plates when collected have been placed in the incubator at 37oC for 48 hours and then removed from incubator to the fridge till their ready to be inspected

B. Plans for next weekMy plan for next week is to keep the makings of the agar to the same method as I found it was safe, quick and there was a low risk of contamination. After receiving a particle counter I will hope to carry out a particle count in each of the sections A and C. I am also hoping that there is a 0.2µm filter filtration apparatus available that can be used for a swab test to see is it more affective then the diect swabbing to a agar plate. I will also hope to have better timing of

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Once again the bulk of activities carried out were on the making of agar . Plates made from last week had already been pre made and ready labelled. This week a particle counter was able to be sourced for monitoring of clean room particulates. 1 test was carried out in section A with another 2 carried out in section C. Swab test were carried out using a filter filtration apparatus. Swab from clean room A was carried out using the wrong measure so the expected result from this is to be negative . 1 biotest was carried out on the 11th due to issues with the battery.

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10 th -2-2015

Settle plate Table recording

Ec GMP Grade Sample location At rest (cfu) Operation (cfu) Colony identification

A Fridge 0 ---- ---

A Mask Aligner 0 --------

C(1) Shower corner 3 Circular/raised/entire

C(2) Changing room 1 Circular/raised/entire

C(3) Work bench 1

C(4) Chiller Unit 2

C(5) Evaporator 1

C(6) Probe station 0

C(7) Chemical cabinet 1

C(8) Chemical cabinet (2)

2

Bio-test sample m3

Ec GMP Grade Sample location At rest (cfu/m3) Operation (cfu/m3) (3 people)

A Fridge 1

C(1) Mask Aligner 3

C(2) Shower corner 4

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Contact plates cm2

Ec GMP Grade Sample location At rest (cfu/cm2) Operation (cfu/m2)

A Fridge 0

A Mask Aligner 0


C(2) Changing room 0


C(4) Acid bench 2

C(5) Evaporator 0

C(6) Wafer spinner 1

C(7) Hybrid fume hood 3

C(8) Bench red door 2

Finger DAB cells

DAB Plate Cfu count

Glove A 10

Glove B

swab Plate Cfu count

swab A 2 Circular/dark/yellow

swab c 0

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11 th -2-2015


Ec GMP Grade Sample location At rest (cfu) Operation (cfu) Colony identification

A Fridge 0 ---- ---

A Mask Aligner 0 --------

C(1) Shower corner 0 Circular/raised/entire

C(2) Changing room 1 Circular/raised/entire

C(3) Work bench TNC

C(4) Chiller Unit 0

C(5) Evaporator 1




1

Bio-test sample m3


A Fridge 4

C(1) Mask Aligner 2


Contact plates cm2

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Ec GMP Grade

Sample location At rest (cfu/cm2) Operation (cfu/m2)

A Fridge TNC

A Mask Aligner 8




C(4) Acid bench 3

C(5) Evaporator 0


C(7) Hybrid fume hood

TNC


Finger DAB cells

DAB Plate Cfu count

Glove A 23

Glove B



swab c 8

B. Plans for next weekTo record test for week 3 17/18th-2-2015 and to carry on using the same methods as before with swab test being done again directly onto agar plates. I may try to refine my method a bit more by placing plates out at a quick and efficient rate .

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Once again the bulk of activities carried out were on the making of agar . Plates made from last week had already been pre made and ready labelled. Particle counter reading were taken In both Grade A and grade C. Unfortunetly nutrient agar strips were not available so fungal yeast strips were used instead . Each other test was carried out as per routine.

B. Plans for next week

The plans for week 5 are carry out tests as per stated in my manual. To take plates from weeks 2-4 that have the best growth and re-plate them for up to 48 hours so that that gram stains , API and oxidase test can be carried out for identification.

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Ec GMP Grade Sample location Operation (cfu)

A Fridge 0

A Mask Aligner 3



C(3) Work bench 1

C(4) Chiller Unit 0

C(5) Evaporator 1




1

Bio-test sample m3


A Fridge 0

C(1) Mask Aligner 0


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17/2/15

Contact plates cm2

Ec GMP Grade


A Fridge 2

A Mask Aligner 15

C(1) Shower corner Tnc



C(4) Acid bench 0

C(5) Evaporator 2



4


Finger DAB cells

DAB Plate Cfu count

Glove A TNC

Glove B TNC



swab c 0

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A Fridge 0

A Mask Aligner 0



C(3) Work bench 0

C(4) Chiller Unit 1

C(5) Evaporator 0




0

Bio-test sample m3


A Fridge 0

C(1) Mask Aligner 0


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18/2/15

Contact plates cm2

Ec GMP Grade


A Fridge 1

A Mask Aligner 2




C(4) Acid bench 0

C(5) Evaporator 1



0


Finger DAB cells

DAB Plate Cfu count

Glove A(No glove ) 45

Glove B 11


swab A 0

swab c 1

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Once again the bulk of activities carried out were on the making of agar . Plates made from last week had already been pre made and ready labelled. Particle counter reading were taken In both Grade A and grade C. Unfortunetly nutrient agar strips were not available so fungal yeast strips were used instead . Each other test was carried out as per routine.

Taking week two and week three 4 plates were taken from each week which showed the most distinctive colony formation and re-plating them on a fresh agar medium plates. For week 6 a set of blood base agar plates were made using blood agar base and a sheep blood extract . It was made up by weighing out 19.5kg of agar powder and autoclaving. This was allowed to cool to the point that it was able to be held comfortable. The blood extract was heated to a temperature of 37oC to stop it from coagulating and then added to duran bottle containing agar which was then poured into the agar dishes.

The 8 plates taken from week 2 and 3 that were re-plated were each given I.D numbers and 8 glass slides were then taken and labelled from gram stain analysis. It was carried using the correct protocol and then analysed under the microscope for gram positive /negative identification. Most slides showed gram positive rod/cocci with one slide in particular showing a possible presence actinomyces and this will be further proven with an API identification test

B. Plans for next week

The plans for week 6 are carry out tests as per stated in my manual. More Gram stains are hoping to be carried out on a few more plates with identification of these slides using the API tests .

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A Fridge 4

A Mask Aligner 0



C(3) Work bench 0

C(4) Chiller Unit 1

C(5) Evaporator 0




4

Bio-test sample m3


A Fridge 0

C(1) Mask Aligner 0


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24/2/15

Contact plates cm2

Ec GMP Grade

Sample location Operation (cfu/m2)

A Fridge 1

A Mask Aligner 2




C(4) Acid bench 1

C(5) Evaporator 1



1


Finger DAB cells

DAB Plate Cfu count


Glove B TNC


swab A 1

swab c 1

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A Fridge 0

A Mask Aligner 0



C(3) Work bench 0

C(4) Chiller Unit 1

C(5) Evaporator 1




2

Bio-test sample m3

Ec GMP Grade Sample location Operation (cfu/m3) (3 people)

A Fridge 0

C(1) Mask Aligner 0


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25/2/15

Contact plates cm2

Ec GMP Grade


A Fridge 1

A Mask Aligner 0




C(4) Acid bench 0

C(5) Evaporator 1



1


Finger DAB cells

DAB Plate Cfu count


Glove B TNC


swab A 0

swab c 30

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Results for blood agar plates were taken as well as contact plates and biotest strips .The rest of the API identifications(20NE) were carried out and identified through the computer system

B. Plans for next weekWeek 8 no tests can be carried out as there is not enough time to allow sufficient time for monitoring. So week will consist of gathering all data and collating all data together to be represented in graph format …

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A Fridge 9

A Mask Aligner 4



C(3) Work bench 0

C(4) Chiller Unit 0

C(5) Evaporator 3




2

Bio-test sample m3

Ec GMP Grade Sample location Operation (cfu/m3) (3 people)

A Fridge 0

C(1) Mask Aligner 0


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Blood /nutrient Agar plates 10/3/15

Contact plates cm2

Ec GMP Grade

Sample location Operation (cfu/m2)

A Fridge TNC

A Mask Aligner TNC




C(4) Acid bench 1

C(5) Evaporator 1



0



swab A 9

swab c 7

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4th year thesis microbiological assessment of a cleanroom

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