some factors affecting the efficiency of settle plates

Journal of Hospital Infection (1984) 5, 189-199

Some factors affecting the efficiency of settle plates

M. P. Russell, J. A. Goldsmith and I. Phillips* Quality Control Department, Roche Products Limited, Welwyn Garden City,

Hertfordshire *St Thomas’ Hospital Medical School, London

Summary: An evaluation has been made of some of the factors which may affect the efficiency of settle plates. Water loss was found to be linear with time. Although the count was reduced over an 8 h period the reduction was not statistically significant. No difference in total bacterial counts could be detected between four, 4 h exposures and one, 2 h exposure. The addition of water and the surface area of the plates had no effect on 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 However, Kingston (1971) mentioned an unpublished study which suggested that bacteria, inoculated on a plate, were killed by subsequent drying.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) was dissolved in 1 1 of distilled water, and dispensed into 10 ml amounts before autoclaving at 121°C for 15 min.

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 loss from 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.


20 -

15-

12 - a _o _

2 f IO- 3 b 9- e : 8 Y

a” 7-

6-

.

.

R

8

. B

.

0

.

0

30 60 90 120 100 210 240 270 300 360 420 470

Time tmln)

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

Table I. Percentage weight loss of 9 cm nutrient agar settle plates exposed for 2 h in d#erent sites in the production area

A

1 LAF 2 LAF 1 Cabin 2 Cabin 1 C/Rm 2 C/Rm

Mean percentage Weight loss 6.6 8.21 4.95 6.1 4.3 6.35 No. of samples 90 90 28 24 27 21 SD. 1.86 2.27 1.15 1.69 2.34 3.26

Filtration Filtration vertical LAF room Air lock

Filtration C/room

Mean percentage Weight loss No. of samples S.D.

5.56 5.18 3.6 8.73 21 21 7 20

1.84 2.15 1.2 8.04


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 on settle plates exposed for 30 min and 2 h in 10 different encironments

&umber of Mean (and total) count after

-

Area exposures Four f h exposures One 2 h exposure

‘Old’ clean room LAF stations ‘Old’ clean room

‘Old’ changing room

‘New’ filtration

Washer room I

Washer room II

Laboratory fresh

Laboratory stored

Laboratory fresh + OED

Laboratory stored + OED

52 1.3 0.2

(12) 43 y.;, 0.9

(81) (39) 24 (*4Sj 44.8

(1076) 22 7.8 6.3

(171) 8 14.5 gy

(116) 8 25.7 yg)

(206) 28 15.2 y&L

(425) (387) 11 20.4 15.9

13 yjy (17.5) 15.4

ww S 14.0 :zlqi)

(70) (72)

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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 obserzqe the 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’ micro- organisms 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).


Table IV. Settle plate counts with and without the addition of 0.1 per cent peptone water

Time of exposure (h)

2 4 8

+H,O -H,O +H,O -H,O +H,O -H,O

49 20 21 25 17 33 16 27 23 19 15 28 10 26 24 19

18

Average 21.9 22.7

S.D. 11.9 3.7 Percentage S.D. 16% ‘calculated 0.21 ‘tabulated 2.13

32 27 55 37 33 33 33 33 ifi 34 29 24 32 40 35 37 33

35.6 32

8.42 3.04 1 O<‘b

1.2 2.13

55 62 68 48 46 49 69 54 38 51 44 54 41 48 36 36

37

49.6 48.7

12.9 8.19 17”/,

0.16 2.13

Conclusion i%o difference at 9.5”i,

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.

Tabl

e V.

Th

e ef

fect

of

va

rying

su

rface

ar

ea

on

coun

ts

on

settl

e pl

ates

ex

pose

d fo

r 2

h on

ei

ght

occa

sion

s

Date

Co

unt/9

cm

Eq

uiva

lent

Co

unt/

14

cm

m

-2

Equi

vale

nt

Coun

t/23

2 cm

pl

ate

plat

e m

m2

plat

e Eq

uiva

lent

m

P2

20.0

8 pm

21

.08

am

21.0

8 pm

24

.08

pm

25.0

8 pm

26

.08

pm

27.0

8 pm

01

.09

pm

Mea

n

Area

(c

m2)

Coun

t m

-2

as

a

perc

enta

ge

Area

as

a

perc

enta

ge

Coun

t/pla

te

10

2: 12

1:

17

21

13.3

63.6

2 15

3.96

91

93

12

29.1

1572

11

00

3144

18

86

1100

23

58

2672

33

01

2142

S.

D.

= 86

5

27

23

54

29

:: 35

35

32.9

1754

14

94

3.50

7 18

83

1819

22

08

2323

23

23

2164

S.

D.=6

17

123 89

18

1 10

7

1:;

127

122

2325

16

82

3421

20

22

1569

27

78

2400

23

05

122

2312

D

S.D

. =

596

5 a 52

9.0

5 0 10

0

100

as

a pe

kent

age

10.9

27

10

0


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 micro- organisms 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,


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 for the synthesis of OED and Mr R. Harris, Mr Purdie and Dr A. A. Wagland for comments on the statistical methods used, other

colleagues at Roche for comments on the manuscript and Roche Products Limited for allowing facilities to carry out this work.

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 Sym- posium on Aerobiology (J. P. Pl. Hers & K. C. Winkler, Eds), pp. 27-32. Oosthock.

some factors affecting the efficiency of settle plates

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