Some factors affecting the efficiency of settle plates

Journal

Some

of Hospital

Infection

factors

(1984)

affecting

M. P. Russell, Quality

Control

5, 189-199

J. A. Goldsmith

Department, *St

the efficiency

Thomas’

plates

and I. Phillips*

Roche Products Limited, Hertfordshire Hospital Medical School,

Summary:

of settle

An evaluation has been made of some of the affect the efficiency of settle plates. Water loss was found to Although the count was reduced over an 8 h period the statistically significant. No difference in total bacterial detected between four, 4 h exposures and one, 2 h exposure. water and the surface area of the plates had no effect on

Welwyn

Garden

City,

London factors which may be linear with time. reduction was not counts could be The addition of the total count.

Introduction

Settle plates, or sedimentation plates, are widely used, both in the pharmaceutical industry and in hospitals, to measure the number of viable particles falling out of the atmosphere. Although the method is widely used, there is little published information on its efficiency. However, Kingston (1971) mentioned an unpublished study which suggested that bacteria, inoculated on a plate, were killed by subsequent drying. It has also been suggested that exposure of agar plates for more than 30 min reduces their sampling efficiency due to water loss from the agar surface. May (1969) showed that oxyethylene docosonal (OED) increased the count obtained by a slit sampler when the sampling was prolonged. Keuhne and Decker (1957) using aerosols of Serratia marcescens demonstrated that agar concentration and sampling time affected the numbers of bacteria recovered with a slit sampler. Therefore, we investigated rates of water loss; exposure time; effect of a retardant agent; storage; addition of water and surface area. Material

Agar Nutrient Agar (Gibco M35600) was used throughout these experiments, made up according to the manufacturer’s instructions and cooled to approximately 45°C before pouring into 9 cm plates under a horizontal laminar flow hood. The lids were left off until the agar set (approximately 5 min). Approximately 25 ml of agar was added to each plate except in one experiment when 16 ml was used. The plates were stored at 23°C until needed

unless

otherwise

stated.

190

M.

P. Russell

et al.

Oxyethylene docosonal (OED) The compound described by May (1969) is no longer available and therefore one as similar as possible to that described in the literature was prepared (Kulkani et al., 1962). This has a melting point of 69-7O”C, which suggests that it is purer than that described by May (1973). An emulsion of 0.2 g OED 100 ml-’ of distilled water was prepared, which required the addition of 0.5 per cent (v/v) Tween 80 for stabilization. It was sterilized at 121°C for 20 min. The OED plates were made by drying nutrient agar plates for 20 min under horizontal Laminar Air Flow (LAF) before the addition of the OED solution. The plate was then rotated so that the solution covered the entire agar surface and the surplus was poured into the next plate. Thus approximately 0.45 g of OED solution was added to each plate. The plates were then allowed to stand for at least 1 h before use. 0.1 per cent peptone water One gram of bacteriological peptone (Difco 011801) distilled water, and dispensed into 10 ml amounts 121°C for 15 min.

was dissolved in 1 1 of before autoclaving at

Methods Weight loss of agar plates The agar plates used during the routine environmental monitoring of the pharmaceutical production areas were weighed before and after exposure. The exposure period was 2 h and the areas monitored were: LAF workstations; clean rooms and changing rooms. The LAF workstations were operating at a face velocity of 90 ft min-’ and the air change rates in the cabins were 70 h-’ for cabins 1 and 2; changing room 1,4.6; changing room 2, 10.5; filtration room, 30; filtration changing room, 28.2; filtration airlock, 34. The effects of different treatments on weight loss were examined, i.e. (a) freshly poured plates, (b) plates stored at 37°C for 48 h, (c) plates with 16 ml of agar instead of 25 ml (the 16 ml was measured using a dispensing syringe), (d) as in (b) and (c) with added OED, (e) freshly poured, exposed under LAF and (f) freshly poured in laboratory 1. Plates a--d were exposed in laboratory The plates were poured and dried for 5 min under LAF, weighed and then exposed. The plates were weighed, with lids on, at regular intervals. Counts obtained on both four, f h plates and one, 2 h plates Settle plates were exposed for four, + h periods and one, 2 h period alongside each other under the following conditions, (a) under LAF in a pharmaceutical production clean room, (b) in a pharmaceutical production clean room, (c) in a pharmaceutical production clean room changing room, (d) in a pharmaceutical production Class 3 (BS 5295) clean room (washer rooms 1

Evaluation

of settle

plates

191

and 2), (e) in a pharmaceutical production area before commissioning, (f) in the laboratory (i) using freshly poured plates, (ii) using plates stored at 37°C for 24 h before use, (iii) as in (i) and (ii) but with added OED. The numbers of colonies obtained in each situation could then be compared. Comparison of counts obtained on plates exposed for i, 1, 2, 4 and 8 h Settle plates were exposed in the laboratory for 8 h alongside plates which were replaced every ‘+, 1, 2 and 4 h. This was done on 10 occasions and in duplicate. The plates were poured as described earlier with the exception that exactly the same amount of agar was added to each plate with a Jencons Accuramatic dispenser, and they were stored overnight at 30°C before exposure in the laboratory. The plates were covered after the appropriate period and incubated at 30°C for 4 days before counting. Addition of water to exposed plates Plates (17 replicates) were exposed in the laboratory for 2, 4 and 8 h on different days. The plates were then collected and 1 ml of 0.1 per cent peptone water was aseptically added to the surface of half of the plates. The plates were then rotated so as to spread the water over the surface. They were then incubated at 30°C for 4 days and a t-test performed on the counts obtained. Eflect of surface area on count Three sizes of Petri dish were poured using nutrient agar. The sizes were 23 x 23 cm (Nunc Ltd), 14 cm diam. and 9 cm diam. (Sterilin Ltd). These were exposed in the laboratory in duplicate for 2 h and then incubated for 4 days at 30°C before counting. This was done on eight separate occasions. Additional information on the spread of counts obtained on a replicate plate can be gained from Experiment 4. Results

Experiment to investigate weight lossfrom agar plates The results of the seven experiments to investigate rate of loss are presented graphically in Figure 1. The figures are the mean of 10 plates in each case. In this way the effect of changing each parameter can be seen by the change in the slope. The effect of position of exposure, i.e. under LAF, in three clean rooms, (cabins and filtration room in Table I) three associated changing rooms and an airlock (associated with filtration room) on weight loss is shown in Table I. Here the mean losses for each type of area are expressed as a percentage of the initial weight, together with the number of samples and the standard deviation. ‘f-tests were carried out on the data given in Table I. Areas which are similar were compared, e.g. LAF workstations, and also associated areas, e.g.

M. P. Russell

192

et al. .

20 -

. 15.

R

0 8

12 a _o 2 f 3 b e : Y a”

.

_

B

.

IO9-

0

8 7-

6-

30

60

90

120

100

210 240 Time

Figure plates stored 16 ml

300

360

420

470

1. Relationship between percentage weight loss and exposure time in nutrient agar given seven different treatments. , Laboratory 2 freshly poured; 0, laboratory 2 48 h; A. laboratory 2 stored 48 h + OED; 0, laboratory 2,16 ml agar; , laboratory 2, agar +OED; 0, laminar air flow, freshly poured; 0, laboratory 1 freshly poured.

n

Table

I. Percentage

Mean percentage Weight loss No. of samples SD.

Mean percentage Weight loss No. of samples

q

weight loss of 9 cm nutrient agar in d#erent sites in the production A

settle plates area

exposed

for

2h

1 LAF

2 LAF

1 Cabin

2 Cabin

1 C/Rm

2 C/Rm

6.6 90 1.86

8.21 90 2.27

4.95 28 1.15

6.1 24 1.69

4.3 27 2.34

6.35 21 3.26

Filtration vertical LAF

S.D.

270 tmln)

5.56 21 1.84

Filtration room 5.18 21 2.15

Air

lock 3.6 7 1.2

Filtration C/room 8.73 20 8.04

Evaluation

of settle

plates

193

clean rooms and their changing rooms. Those found to be significantly different at the 1 per cent level were: horizontal LAF workstations; clean room cabins; LAF workstation 1 and cabin 1; LAF workstation 2 and cabin 2. DifJeerence in count obtained at four, 3 h vs. one, 2 h exposures Table II shows the mean count for each period and each treatment. 4n analysis of variance for each group of results was performed. A significant difference at the 95 per cent level was found only in one case, that of the ‘old’ clean room. Additionally, F-tests were made on four, i h stored against fresh; one, 2 h stored against fresh; four , 4 h fresh against OED; one, 2 h fresh against OED; four , + h stored against stored + OED; and one, 2 h stored against stored + OED. -4 significant difference was not found in any case at the 5 per cent level. However, in only one case (washer room 1) is the total count for the 2 h exposure higher than for four, 3 h. Experiment to iwestigate the eflect of exposure time on settle plate count Table III shows the count obtained on 10 different days for each exposure period. The count given is the total of the number of replicates shown but normalized to the 8 h period for one plate. This was done by dividing the total count by the number of replicates and multiplying by (8 divided by the exposure period). Thus the counts are directly comparable. Table III also shows the totals for the 10 days for each exposure period. Additionally, this Table

II.

Mean

and

total counts obtained and 2 h in 10 different

&umber exposures

Area ‘Old’ LAF ‘Old’

clean room stations clean room

‘Old’

changing

‘New’

filtration

Washer Washer Laboratory

room room

on settle plates encironments

exposed

for

30 min

Mean

total)

count

after

of Four

(and

f h exposures

One

2 h exposure

52

1.3

0.2

43

y.;, (81) (*4Sj

0.9 (39) 44.8 (1076) 6.3

(12) room

24 22

I

8

II

8

fresh

Laboratory

stored

Laboratory

fresh

Laboratory

stored

+ OED

7.8 (171) 14.5

(116) 25.7

yg)

28

(206) 15.2

y&L

11

(425) 20.4

13

yjy

ww + OED

gy

S

14.0 (70)

(387) 15.9 (17.5) 15.4 :zlqi) (72)

-

134 105 64 81 134

76 8 9 10

Totals Percentage

180

203 183 145 140

1

32 4 5

Total Count

Day

i: 32 30 30

32

:;

No. of Replicates

$h

698 100

is 71

62 53

102 92 ;i

Corrected Count __-___ 90

1:‘:

97 85 73

17.5 220 144 134

341

Total Count

:% 16

:: 14

16 14

20

No. of Replicates

lh

739 106

49 49 42 40 75

17: 83 67

136

Corrected Count

102 80 59 91 269

175 195 128 321

143

-__Total Count

2h

time

tt 8 12 15

8: 8 18

No. of Replicares

Exoosure slates

616 88

51 40 30 30 72

ii98 64 71

Corrected Count

of

381 246 131 224 382

158 423 187 107

103

Total Count

4h

i”2 9 12 13

;: 6 4

3

No. of Replicates

556 80

:7’ 59

48 41

54

63 z:

69

Count

Corrected

371 317 183 268 391

283 238 239 287

136

Total Count

of

8h

s” 7

8i

:3 4

Replicates

No.

533 76

ii 56

46 40

7’; 60 48

68 -r

Count

-___ Corrected

e

8 = 2

a

g

Evaluation

of settle

plates

195

total is expressed in the final row as a percentage of the total obtained for ) h exposures. Thus the count obtained on one‘8 h plate is 76 per cent of that obtained on 16 successive $ h plates. F-tests were performed on each pair of groups and none was found to be significantly different at the 5 per cent level. Experiment to determine the effect of the addition of 0.1 per cent peptone water to exposed settle plates Table IV shows the counts obtained in the laboratory on replicate plates exposed for different periods on different days. Although the mean counts for each treatment are very similar, the standard deviations are very much higher for the plates with added water. Also shown are the results of a t-test performed on the data. No statistical difference is found with the addition of water regardless of exposure period. Table IV additionally gives an indication of the spread of results found on nine plates exposed at the same time and immediately adjacent to each other, with the standard deviations varying from 10 to 17 per cent. Experiment to obserzqethe eflect of surface area on count obtained The results shown in Table V are means of duplicate plates. The mean counts for each size of plate were multiplied so as to give a count per square metre. If the largest count (i.e. that on the largest plate) is taken as 100 per cent then the count on a 9 cm plate is 91 per cent and that on a 14 cm plate 93 per cent. The counts per square metre were tested for significance by a t-test. At the 95 per cent confidence level, there is no significant difference between the plates’ collecting efficiency. Discussion

The graph (Figure 1) shows a linear rate of water loss from the exposed plate which would be unlikely to occur if a skin is formed on the surface of the plate. Such a skin would reduce the access of moisture to ‘caught’ microorganisms preventing their multiplication. Skin formation would be likely to give a non-linear plateau-shaped graph. The slopes of the graphs are all very similar, although not the same. This could be due to the inherent variation in the results but it does indicate that the rate of water loss, at least during short time periods, is not affected by the treatments used. However, the results in Table I demonstrate that the position or environment of exposure is very relevant to weight loss. Since this is very probably due to the differing air patterns above each plate, it indicates that the settling of bacteria from the air on to the surface will also be affected by these air patterns. This demonstrates the importance of exposing settle plates in exactly the same position when comparison of results on a time basis is required. Such a comparison is recommended in the DHSS Guide to Good Manufacturing Practice (1971).

M. P. Russell

196 Table

IV. Settle plate counts with and without

et al.

the addition

of

0.1 per cent peptone water

Time of exposure (h) 2 +H,O

4 -H,O

49 21 17 16 23 15 10 24 Average

21.9 11.9

S.D.

Percentage ‘calculated ‘tabulated Conclusion

20 25 33 27 19 28 26 19 18 22.7 3.7

+H,O 32 55 33 33 33 34 24 40 37 35.6 8.42

0.21 2.13

+H,O

27 37 33

55 68 46 69 38 44 41 36

ifi 29 32 35 33 32 3.04 1O<‘b

16%

S.D.

8 -H,O

1.2

2.13 i%o difference at 9.5”i,

-H,O 62 48 49 54 51 54 48 36 37 48.7 8.19 17”/,

49.6 12.9 0.16 2.13

The graphs also show that the weight loss is influenced only by the environment in which the plate is exposed, and not by storage at 37°C for 24 h or by the volume of agar or by the presence of OED, which was intended to retard moisture loss. The OED was ineffective in reducing moisture loss. This may have been because (1) it was a different compound to that described by May (1969) with different properties, although studies with a slit sampler showed that weight loss with OED was significantly less than without it (t-test at 95 per cent), (2) the incorporation of the 0.5 per cent Tween 80 to stabilize the emulsion prevented the formation of the mono-layer, (3) OED only exerts its effects when very much larger volumes of air are sampled than were in these experiments. The results in Table I (the weight losses in different types of environment) reinforce the arguments for precise positioning of settle plates, since weight loss is dependent upon the environment in which the plate is exposed. Since each area in the Table is made up of a mean of several positions, the standard deviation is a measure of the air turbulence within that area. The very high standard deviation in the filtration changing room is due to the positioning of one plate under an inlet duct. It is of interest to note that weight loss decreases with decreasing air flow, i.e. from the LAF hoods to the changing rooms. The effect of these differences in air flows demonstrates that differences in the efficiency of settle plates may be expected in different environments, i.e. in the different numbers of bacteria settling out in a given time. This is because a particle of given density will be variously affected by different air velocities.

Count/plate as a pekentage 10.9

12

Area

as a percentage

91

63.62

(cm2)

surface

S.D.

=

2142

1572 1100 3144 1886 1100 2358 2672 3301 865

Equivalent m -2

of varying

Count m -2 as a percentage

Area

1: 17 21

2: 12

10

cm

Count/9 plate

13.3

pm am pm pm pm pm pm pm

effect

V. The

Mean

20.08 21.08 21.08 24.08 25.08 26.08 27.08 01.09

Date

Table

area

153.96

32.9

:: 35 35

27 23 54 29

27

29.1

93

on settle

Count/ 14 cm plate

on counts

exposed

2164 S.D.=617

1754 1494 3.507 1883 1819 2208 2323 2323

Equivalent mm2

plates

for

529.0

1:; 127 122 122

123 89 181 107

Count/23 plate

2 h on eight 2 cm

100

100

100

occasions

S.D.

=

2325 1682 3421 2022 1569 2778 2400 2305 2312 596

Equivalent mP2

0

D 5 a 5

198

M. P. Russell

et al.

Only one of the results presented in Table II shows a statistical difference between the total counts obtained on a simultaneous 2-h exposure. This would be expected once in 20 times at the 95 per cent level. However, the means for the 4 h plates are higher than those for the 2 h and because of this it could be argued that four, 3 h exposures are more efficient than one, 2 h exposure. However, the difference was not statistically significant and this is probably due to the random nature of the settling of bacteria. It is certainly far less practical to use four , 3 h plates. Further analysis of the data shows that the mean of the counts obtained on + h plates is significantly less than that from the 2 h plates. Therefore, in an area of low fallout, as in a pharmaceutical clean room, one, 2 h plate is more efficient than one, 3 h plate and more convenient than four, 3 h plates. Additionally a possible reason for the slightly higher totals for the four, 4 h exposures is the additional manipulation associated with changing the plates every 3 h. It has been shown (Table III) that exposure times of up to 8 h can reduce the count by approximately 25 per cent of that obtained by exposure of 16 consecutive 30 min plates. This implies a decline in collection efficiency with exposure time, which is not measureable at 2 h (see Experiment 2). However, at the 95 per cent level, the means are not significantly different on F-tests at 5 per cent. Therefore, this is probably due to the high variation in counts on different occasions. The result obtained at 1 h exposure implies an increase in efficiency, and one possible explanation may be that efficiency is increased by a lowering in the water activity (Chirife, Favetto and Scorza, 1982). The position of exposure is not significant since it was between the + h plate and the 2 h plate. Further investigation is necessary to clarify this result. If water loss by the settle plate is significant in its collection efficiency, then any loss should be at least partly made up by the addition of water. This hypothesis was tested in this experiment and the results are shown in Table IV. It can be seen that the results do not support the hypothesis, there being no statistical difference between the counts on plates with and without water. Thus, this lack of response to the addition of water suggests that microorganisms are dehydrated beyond revival during their passage through air, and that it is not the lack of water in the medium which prevents growth. It would appear from the data presented here that no ‘skin’ forms on the surface of the agar to prevent the rehydration of organisms with water from the agar. The percentage standard deviations on the plates without water, show that the surfaces vary consistently over the exposure period and that the spread of counts over the replicate plates varies consistently. However, the high percentage standard deviation over so many replicates may be taken as further evidence that the position of exposure can be crucial when comparing sequential results. Two opposite reasons can be offered to explain the higher standard deviations obtained for the plates with water added. One is that the action of rotating surface water breaks up clumps of bacteria, increasing the count. The other is that rehydration shock, despite the presence of peptone,

Evaluation

of settle

plates

199

kills some cells. Each factor may or may not operate depending on the history and type of bacteria present. Since the environment plays such an important role in the loss of water from exposed plates (see Experiment 1) the effect of surface areas on count was investigated. The results indicate (Table V) that large plates give very slight increases in count over smaller ones when corrected to an equivalent area. As reported in the results, these differences are not significant. These small differences can probably be attributed to the correction factor and the effect of the rim of the plate on collection efficiency. Thus, the standard 9 cm plate is a good indicator of the number of viable particles falling from the atmosphere. We wish to thank Dr J. M. Osbond of Roche Research Harris, Mr Purdie and Dr A. A. Wagland for comments colleagues at Roche for comments on the manuscript allowing facilities to carry out this work.

for the synthesis of OED and Mr R. on the statistical methods used, other and Roche Products Limited for

References Chirife.

S., Favetto, G. & Scorza, 0. C. (1982). Th e water activity of common liquid bacteriological media. Journal of Applied Bacteriology 53, 219-222. Guide to Good Manufacturing Practice (1971). DHSS. HMSO. Kingston, D. (1971). Selective media in air sampling-A review. Journal of Applied Bacteriology 34, 221.-232. Kuehne, R. W. & Decker, H. H. (1957). Studies on continuous sampling of Seuratia marcescens using a slit sampler. Applied Microbiology 5, 321-323. Kulkoni, S. B., Gharpurey, M. K., Sanjana, N. R., Deo, A. V., Abraham, K. 0. & Rao, B. C. S. (1962). Agents for controlling evaporation of water. Indian Patent 70670. Chemical Abstracts 57, 14939e. May, K. R. (1969). Prolongation of microbiological air sampling by a monolayer on agar gel. Applied Microbiology 18, 5 13-S 14. May, K. R. (1973). Airborne Transmission and Airborne Infection: 4th International Symposium on Aerobiology (J. P. Pl. Hers & K. C. Winkler, Eds), pp. 27-32. Oosthock.