Vol. 38, No. 3
APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Sept. 1979, p. 466-470 0099-2240/79/09-0466/05$02.00/0
Wipe-Rinse Technique for Quantitating Microbial Contamination on Large Surfaces L. E. KIRSCHNER AND J. R. PULEO* Planetary Protection Laboratory, Jet Propulsion Laboratory, Eastern Test Range, Cape Canaveral, Florida 32920
Received for publication 9 April 1979
The evaluation of an improved wipe-rinse technique for the bioassay of large undertaken due to inherent inadequacies in the cotton swab-rinse technique to which assay of spacecraft is currently restricted. Four types of contamination control cloths were initially tested. A polyester-bonded cloth (PBC) was selected for further evaluation because of its superior efficiency and handling characteristics. Results from comparative tests with PBC and cotton swabs on simulated spacecraft surfaces indicated a significantly higher recovery efficiency for the PBC than for the cotton (90.4 versus 75.2%). Of the sampling area sites studied, PBC was found to be most effective on surface areas not exceeding 0.74 m2 (8.0 feet2). areas was
The need to determine the level of microbial contamination on spacecraft surfaces has necessitated a set of sampling procedures somewhat different from standard bioassay techniques. Many attempts have been made to design and test a simple, accurate, and precise method for quantitating microbial contamination on surfaces; most of these methods have been oriented towards application to clinical or food microbiology. The majority of these methods are not applicable to spacecraft hardware bioassays. This is evident when examining the five basic procedures for recovering microorganisms from surfaces (5). Direct surface agar plate methods (2, 6) and agar contact procedures such as the Rodac method (4, 10) cannot be used because agar culture media is brought in contact with spacecraft hardware surfaces leaving an undesired residue. Rinse techniques (3) are inappropriate for large static surface areas since they call for mechanical agitation of the hardware in a fluid menstruum. A vacuum probe (13) was found to be effective for removing from surfaces airborne microbial fallout, but many spacecraft surfaces do not permit its use. A wipe-rinse technique which uses a rayon cloth (14) for the microbiological assay of skin was not applicable due to the physical properties of the material which prohibits its use on spacecraft surfaces or in clean room environments. Microbial sampling of spacecraft surfaces (18-22) has, therefore, been restricted to the use of the swab-rinse technique. The cotton swab method, although adopted as standard procedure by the American Public Health Association (1), has several disadvantages. The surface area that can be sam-
pled by a swab is relatively small, the method is designed for high levels of microbial contamination, and there is poor correlation among investigators as to the amount of microbial contamination removed and recovered (4). The objective of this study was to develop a surface sampling system that would accommodate a variety of surface textures and configurations, have good removal and recovery efficiency, leave no residue, be commercially available, and be effective with respect to time and cost. The system was specifically intended for use on the large surface areas of the Space Transportation System, but is adaptable to other applications. The cotton swab rinse technique was used as a point of reference to compare to the wipe-rinse technique. MATERIALS AND METHODS CRC. Four commercially available clean room cloths (CRC) (The Texwipe Co., Hillsboro, N.J.) were evaluated. These consisted of two types of cellulose cloth (TX309, TX500), a spun bound polyamid fabric (TX909), and a polyester-bonded cloth (PBC, TX409). Individual CRC (23 by 23 cm) were folded to onequarter of their original size and double wrapped in craft paper. Packages of CRC were sterilized by autoclaving and allowed to dry before use. General sampling technique. To maintain aseptic technique, personnel wore sterile surgical rubber gloves which were rinsed with sterile 70% isopropyl alcohol before opening the inner package containing CRC and between each sample. CRC were moistened with 5 ml of sterile water, before use. A constant, steady pressure was used for a reciprocal motion that covered the entire surface. The CRC were refolded so that the contaminated portion was interior to the new
466
VOL. 38, 1979
WIPE-RINSE TECHNIQUE FOR LARGE SURFACES
sampling surface, and the surface was covered a second and third time with the wiping motion rotated 900 after each complete coverage of the sample area. Each CRC was placed into a dry, sterile glass container with a displacement of 1 liter. After each six samples collected, a handling control was taken to check for possible contamination picked up during the sample procurement. Samples were transported to the laboratory and processed within 1 h. Sterile 400 ml of a buffered rinse solution containing a 0.02% (vol/vol) solution of Tween 80 (polyoxyethylene sorbitan monooleate; Hilltop Research, Inc., Miamiville, Ohio) was added. The containers were placed for 2 min at 25 kHz in an ultrasonic bath (tank, LTH60-3; generator A300; Branson Instruments, Inc., Stamford, Conn.) containing a 0.3% (vol/vol) solution of Tween 80 (16, 17). After ultrasonication, cloths were agitated with a pipette for 5 s, then appropriate duplicate dilutions were plated with Trypticase soy agar (BBL Microbiology Systems, Cockeysville, Md.) Culture plates were incubated at 32°C under aerobic conditions, and colony counts were performed after 72 h. CRC evaluation procedure. Preliminary evaluation of the four CRC utilized 1.3-m2 surface areas to determine recovery efficiencies and handling characteristics. The PBC and TX309 materials were found to have a greater recovery efficiency and were selected for study and compared to the standard cotton swab method (1, 12). Stainless-steel tables (17.8 cm wide, 188 cm long, and 102 cm high), each containing two ribbons (7.5 by 183 cm [0.139 m2]) of Teflon (5 mil, FEP DuPont), used as collection surfaces (15), were exposed for 1 week to the intramural environment of the Vehicle Assembly Building, Kennedy Space Center, Fla. Sterile, double thicknesses of craft paper were used to separate the ribbons from the table tops. Before taking samples, we lifted each test ribbon from the table with sterile forceps and removed the first paper divider, leaving a sterile surface beneath the ribbon. The first assay procedure consisted of three experiments using 10 ribbons per experiment. Four ribbons of each group were designated as No (initial microbial concentration) controls. The remaining six ribbons were divided in half and used as test surfaces for PBC, TX309, and cotton swabs. The general sampling procedure listed above was employed in the PBC and TX309 assays. Half-ribbons used for cotton swab assays were divided into four equal parts of 174.2 cm2 each. Cotton swabs were used with a sampling motion identical to that used for CRC, then dispensed into 10 ml of sterile buffered-distilled water (12). Samples were ultrasonicated for 2 min then plated. Duplicate 1- and 2-ml pour plates were made for each swab. No controls and test ribbons used for cloth and swab assay surfaces were collected in sterile 1-liter glass containers to which 400 ml of sterile buffered rinse solution was added. Ribbons were ultrasonicated for 6 min. The No count was determined by plating the appropriate dilution in duplicate with Trypticase soy agar. Duplicate 50-ml portions from the test ribbon samples were plated with 50 ml of double-strength Trypticase soy agar to determine residual organisms on ribbons after wiping. The PBC and cotton swab methods were further
467
evaluated. Eight groups of six Teflon ribbons were placed in the Vehicle Assembly Building and exposed as previously described. Two ribbons from each group were designated as No controls, with the four remaining ribbons used as test surfaces for the PBC and cotton swab assays. Following standard assay of No samples, ribbons were subjected to two successive rinse assay procedures to determine the effectiveness of 6 min of ultrasonication. The number of microorganisms detected after the first 6 min of ultrasonication was then compared with the total number recovered in the three sequential assays. Application of PBC to spacecraft hardware. The upper quadrants of the exterior surface of a horizontally placed first-stage rocket booster were used as sampling surfaces for the PBC. To determine the microbial contamination level of the hardware surface, eight initial experiments were conducted consisting of six samples each using a surface area of 0.37 m2 (4.0 feet2). The use of 0.37-M2 surface areas as controls was continued when evaluating the 0.74-M2 (8.0 feet2) and 1.49-M2 (16.0 feet2) sample areas. Assay technique for recovery of low levels of microorgansims. Experiments were conducted to determine the accuracy and precision of the wipe-rinse technique for assaying low levels of microbial contamination. Stainless-steel strips (2.5 x 5 cm) were exposed to the intramural laboratory environment for 4 days. The technique used to enumerate the number of microorganisms which accumulate on stainless-steel surfaces have been described previously (6, 8, 9, 11, 12). Because of the small surface area to be tested, the PBC and recovery rinse water was reduced by 75%. Following standard use of PBC, duplicate 50-ml portions of rinse water were plated with doublestrength Trypticase soy agar. All laboratory procedures were performed in a horizontal laminar flow clean bench (7) to eliminate background airborne contamination. Calculations. Removal and recovery efficiencies were calculated by using the following equations:
z, I{NoNo-R,0J
NLn
n
% Removal =
.100
N
Where R, = residual number of organisms for a given observation, No = control for an experiment, n = total number of observations for an experiment, and N = number of experiments; I
NL % Recovery Where I
=
=
n
(I/No)l n N
.100
count after ultrasonication.
RESULTS A cellulose cloth (TX500) and a spun bound polyamid cloth (TX909) were eliminated during preliminary testing because of poor recovery efficiency and handling characteristics.
468
APPL. ENVIRON. MICROBIOL.
KIRSCHNER AND PULEO
The results of more extensive comparisons of PBC, cellulose cloth, and cotton swabs are given in Table 1. These data indicate that removal efficiency for all three devices was essentially the same, but the recovery efficiency for PBC was greater than the others. Coefficients of variation were similar for PBC and cotton swabs, but were much higher for the cellulose cloth. Comparison of PBC and cotton swabs (Table 2) indicated a significantly greater recovery efficiency for the PBC. Data collected to indicate the effectiveness of the 6-min ultrasonication cycle in removing natural airborne fallout (Table 3) from No samples showed that ;90.7% of the microorganisms were removed by the first ultrasonication cycle. The second and third ultrasonication cycles removed a7.4% and ¢1.9%, respectively, of the remaining contamination. After selecting PBC as the desired clean room cloth, application was made to spacecraft hardware. Data presented in Table 4 indicated that good correlation existed between control samples and those from 0.74-m2 areas. Plate counts from 1.49-M2 areas were lower than 0.37-M2 controls. Assays of stainless-steel strips resulted in good removal of fallout (Table 5), but some variation of recovery efficiency was evident. Three of the six recovery figures were found to exceed the overall recovery efficiency for PBC in Table 1. Handling controls were consistently negative throughout all operations.
tested generally had good removal potential, but some were lacking in the ability to release the collected organisms. According to data presented in Table 1, the cellulose cloth removed about the same percentage of organisms as did the PBC, but the percentage recovery was about 19% less, indicating that entrapped organisms TABLE 3. Effectiveness of the 6-min ultrasonication
cycle Expt no.
3.1 0.4 0.1 5.1 0.6 0.1 4.3 0.1 0.1 4 5.1 0.6 0.2 5 6.1 1.0 0.1 6 6.9 0.4 0.1 7 3.5 0.2 0.0 8 5.4 0.2 0.0 a >90.7% removed after first ultrasonication. b removed after second ultrasonication. '31.9% removed after third ultrasonication.
a'7.4%
TABLE 4. Recovery of natural fallout from spacecraft hardware using PBC Spacecraft sample area
sam-
ples PBC Cellulose cloth Cotton swab a For recovery.
12 12 64
Mean recovery'
No. of
(i2)
samples
Test
Controls
0.37 0.74 1.49
48 46 12
3.7 x 104 2.4 x 104 1.8 x 104
2.4 x 104 4.2 x 104
a Colony-forming units.
TABLE 5. Recovery of low numbers of microorganisms using PBC Mean No. off orgaExpt s Expt o.n saples nisms/ per expt cm 2
TABLE 1. Comparison of removal and recovery values for two CRC and cotton swabs Collection device
Secondb ul- Third' ultratrasonicasonication tion
1 2 3
DISCUSSION For a surface assay system to be effective, it must be efficient in terms of both microbial contamination removal and recovery. The CRC
Total no. of
Initiala count (organisms/cm2)
1
2 3 4 5 6
CoeffiRemoval Recovery cienta of (%) (%) variation (%) 97.2 90.4 15.9 97.5 72.0 47.6 89.6 75.2 12.3
5 8 6 5 6 5
Removal Recovery (%()
0.5 1.3 1.0 0.5 1.4 0.9
97.1 100.0 100.0 100.0 100.0 100.0
71.4 118.2 83.3 125.0 107.0 64.3
0.9a
99.5a
95.0a
a Mean.
TABLE 2. Recovery values: PBC versus cotton swabs No. of expt
Collection device
PBC Cotton swab a Organisms per cm2. b Degrees of freedom
=
14.
Total no. of samples
Rcoverya
8
32
4.6
8
128
3.1
t test (t = 0.05)b Theoretical Calculated
2.15
3.35
Significant difference
Yes
VOL. 38, 1979
WIPE-RINSE TECHNIQUE FOR LARGE SURFACES
were not released from the cloth. This ability to release collected organisms, while relatively consistent for each cloth type, is probably a function of the fibrous nature of the cloth and is one of the limiting factors of their effectiveness. The tendency to retain collected organisms can be seen when comparing the PBC and cotton swabs. The coefficient of variation is about 3.6% less for the cotton swab than for the PBC, but its recovery efficiency is also significantly less, indicating that cotton swabs do not release all of the collected organisms after ultrasonication. Previously reported recovery efficiencies for cotton swabs range from 36 to 85% (2, 3), and the value of 75.2% reported here falls within this range. A second advantage of PBC over the cotton swab is the surface area that can be sampled with one unit. According to the National Aeronautics and Space Administration Standard Procedures for the Microbiological Examination of Spacecraft Hardware (12), the area sampled with a cotton swab is 25.4 cm2 (4.0 inches2). When monitoring procedures call for large areas to be assayed, statistically valid sample numbers can become very large, resulting in extended sampling and processing times. The bioassay of a 6,451.6 cm2 (1,000 inches2) area requires 250 cotton swab samples for full area coverage. The same area could be sampled with one PBC. To illustrate further, a prominent structure of a recently launched spacecraft had a surface area of 127 feet2. A total of 72 cotton swabs were required to sample 1.6% of this area. The use of one PBC would have allowed a sample size 6.3% of the total area, or an increase of 74.6% in size of the area sampled. Because of the nature of the spacecraft hardware used in evaluating PBC, it was impossible to determine the exact presample microbial population or the residual population after sampling. Data in Table 3 give an indication, however, of recovery from different size areas. The wide difference in counts between the 0.37-M2 controls and the 1.49-M2 test areas was probably the result of the cloth drying while sampling the larger area, which resulted in its decreased efficiency. In environments where the temperature and relative humidity are controlled, this drying effect might not occur. For the suggested procedure, and with the constraints of these sampling conditions, the maximum effective sampling area was found to be between 0.74 and 1.49 In2. For standardization of technique, the sample size should be reduced to an area of 0.74 M2. To evaluate the appropriateness of PBC for collection of small numbers of organisms, assays were performed on stainless-steel strips having
469
a low organism-to-surface ratio. The mean recovery value of 95% for this series of assays compares well with the recovery figure of ca. 90% given in Table 1 and suggests a linear relationship in collection efficiency from low to high numbers. The recovery values over 100% (Table 5) might be the results of both variation of deposition of natural airborne microbial fallout on stainless-steel strips and disintegration of clumps or particles after ultrasonication. Cost and time factors for materials must also be considered in comparing PBC to cotton swabs. Based on previous cotton swab assays of spacecraft hardware (275 samples), the use of PBC reduces the material for sampling a 6,451.6 cm2 (1,000 inches2) area, by about 99%. Similar time savings are also possible. During normal sampling operations, an experienced bioassay team can collect samples at a rate of approximately one cotton swab per 2-min period. The same team should be able to sample an unobstructed 0.74-M2 area in the same time, resulting in a saving of time and labor cost. The wipe-rinse technique was developed specifically for use on the Space Transportation System (Shuttle) and other spacecraft hardware surfaces, but its overall efficiency makes it applicable to contamination control activities other than spacecraft bioassay. This technique could be used in various areas of environmental microbiology where the ability to quickly, accurately, and economically measure surface contamination is important. ACKNOWLEDGMENT'S This paper presents the results of one phase of research carried out at the Jet Propulsion Laboratory, California Institute of Technology, under contract NAS 7-100 sponsored by the National Aeronautics and Space Administration. We thank P. D. Stabekis, Exotech Research and Analysis, Inc., for the helpful discussion and suggestions, and S. L. Bergstrom, N. D. Fields, and L. A. Maull for their technical assistance. LITERATURE CITED 1. American Public Health Association. 1972. Standard methods for the examination of diary products, 13th ed. American Public Health Association, Inc., New York. 2. Angelotti, R., and M. J. Foter. 1958. A direct surface agar plate laboratory method for quantitatively detecting bacterial contamination on non-porous surfaces. Food Res. 23:170-174. 3. Angelotti, R., M. J. Foter, K. A. Busch, and K. H. Lewis. 1958. A comparative evaluation of methods for determining the bacterial contamination of surfaces. Food Res. 23:175-185. 4. Angelotti, R., J. Wilson, W. Litsky, and W. G. Walter. 1964. Comparative evaluation of the cotton swab and Rodac methods for the recovery of BaciUus subtilis spore contamination from stainless steel surface. Health Lab. Sci. 1:289-296. 5. Baldock, J. D. 1974. Microbiological monitoring of the food plant: methods to assess bacterial contamination on surfaces. J. Milk Food Technol. 37:361-368.
470
APPL. ENVIRON. MICROBIOL.
KIRSCHNER AND PULEO
6. Favero, M. S. 1971. Microbiological assay of space hardware. Environ. Biol. Med. 1:27-36. 7. Favero, M. S., and K. R. Berquist. 1968. Use of laminar air-flow equipment in microbiology. Appl. Microbiol. 16:182-183. 8. Favoero, M. S., J. R. Puleo, J. H. Marshall, and G. S. Oxborrow. 1966. Comparative levels and types of microbial contamination detected in industrial clean rooms. Appl. Microbiol. 14:539-551. 9. Favero, M. S., J. R. Puleo, J. H. Marshall, and G. S. Oxborrow. 1968. Comparison of microbial contamination levels among hospital operating rooms and industrial clean rooms. Appl. Microbiol. 16:480-486. 10. Hall, L. B., and M. J. Hartnett. 1964. Measurement of the bacterial contamination on surfaces in hospitals. Public Health Rep. 79:1021-1024. 11. McDade, J. J., M. S. Favero, and L. B. Hall. 1967. Sterilization requirements for space exploration. J. Milk Food Technol. 30:179-185. 12. National Aeronautics and Space Administration. 1968. National Aeronautics and Space Administration standard procedures for the microbiological examination of space hardware, NHB 5340.1A. National Aeronautics and Space Administration, Washington, D.C. 13. Petersen, N. J., and W. W. Bond. 1969. Microbiological evaluation of the vacuum probe surface sampler. Appl. Microbiol. 18:1002-1006. 14. Petersen, N. J., D. E. Collins, and J. H. Marshall. 1973. A microbiological assay technique for hands. Health Lab. Sci. 10:18-22. 15. Puleo, J. R., M. S. Favero, G. S. Oxborrow, and C. M.
16. 17. 18.
19.
20.
21.
22.
Herring. 1975. Method for collecting naturally occurring airborne bacterial spores for determining their thermal resistance. Appl. Microbiol. 30:786-790. Puleo, J. R., M. S. Favero, and N. J. Petersen. 1967. Use of ultrasonic energy in assessing microbial contamination on surfaces. Appl. Microbiol. 15:1345-1351. Puleo, J. R., M. S. Favero, and G. J. Tritz. 1967. Feasibility of using ultrasonics for removing viable microorganisms from surfaces. Contam. Control 6:58-67. Puleo, J. R., N. D. Fields, S. L. Bergstrom, G. S. Oxborrow, P. D. Stabekis, and R. C. Koukol. 1977. Microbiological profiles of the Viking spacecraft. Appl. Environ. Microbiol. 33:379-384. Puleo, J. R., N. D. Fields, B. Moore, and R. C. Graves. 1970. Microbial contamination associated with the Apollo 6 spacecraft during final assembly and testing. Space Life Sci. 2:48-56. Puelo, J. R., G. S. Oxborrow, N. D. Fields, and H. E. Hall. 1970. Quantitative and qualitative microbiological profiles of the Apollo 10 and 11 spacecraft. Appl. Microbiol. 20:384-389. Puleo, J. R., G. S. Oxborrow, N. D. Fields, C. M. Herring, and L. S. Smith. 1973. Microbiological profiles of four Apollo spacecraft. Appl. Microbiol. 26:838845. Puelo, J. R., G. S. Oxborrow, and R. C. Graves. 1969. Microbial contamination detected on the Apollo 9 spacecraft, p. 80-83. In Proceedings of the 8th Annual Technological Meeting American of the American Association for Contamination Control, New York. American Association for Contamination Control, Boston.
APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Sept. 1979, p. 466-470 0099-2240/79/09-0466/05$02.00/0
Wipe-Rinse Technique for Quantitating Microbial Contamination on Large Surfaces L. E. KIRSCHNER AND J. R. PULEO* Planetary Protection Laboratory, Jet Propulsion Laboratory, Eastern Test Range, Cape Canaveral, Florida 32920
Received for publication 9 April 1979
The evaluation of an improved wipe-rinse technique for the bioassay of large undertaken due to inherent inadequacies in the cotton swab-rinse technique to which assay of spacecraft is currently restricted. Four types of contamination control cloths were initially tested. A polyester-bonded cloth (PBC) was selected for further evaluation because of its superior efficiency and handling characteristics. Results from comparative tests with PBC and cotton swabs on simulated spacecraft surfaces indicated a significantly higher recovery efficiency for the PBC than for the cotton (90.4 versus 75.2%). Of the sampling area sites studied, PBC was found to be most effective on surface areas not exceeding 0.74 m2 (8.0 feet2). areas was
The need to determine the level of microbial contamination on spacecraft surfaces has necessitated a set of sampling procedures somewhat different from standard bioassay techniques. Many attempts have been made to design and test a simple, accurate, and precise method for quantitating microbial contamination on surfaces; most of these methods have been oriented towards application to clinical or food microbiology. The majority of these methods are not applicable to spacecraft hardware bioassays. This is evident when examining the five basic procedures for recovering microorganisms from surfaces (5). Direct surface agar plate methods (2, 6) and agar contact procedures such as the Rodac method (4, 10) cannot be used because agar culture media is brought in contact with spacecraft hardware surfaces leaving an undesired residue. Rinse techniques (3) are inappropriate for large static surface areas since they call for mechanical agitation of the hardware in a fluid menstruum. A vacuum probe (13) was found to be effective for removing from surfaces airborne microbial fallout, but many spacecraft surfaces do not permit its use. A wipe-rinse technique which uses a rayon cloth (14) for the microbiological assay of skin was not applicable due to the physical properties of the material which prohibits its use on spacecraft surfaces or in clean room environments. Microbial sampling of spacecraft surfaces (18-22) has, therefore, been restricted to the use of the swab-rinse technique. The cotton swab method, although adopted as standard procedure by the American Public Health Association (1), has several disadvantages. The surface area that can be sam-
pled by a swab is relatively small, the method is designed for high levels of microbial contamination, and there is poor correlation among investigators as to the amount of microbial contamination removed and recovered (4). The objective of this study was to develop a surface sampling system that would accommodate a variety of surface textures and configurations, have good removal and recovery efficiency, leave no residue, be commercially available, and be effective with respect to time and cost. The system was specifically intended for use on the large surface areas of the Space Transportation System, but is adaptable to other applications. The cotton swab rinse technique was used as a point of reference to compare to the wipe-rinse technique. MATERIALS AND METHODS CRC. Four commercially available clean room cloths (CRC) (The Texwipe Co., Hillsboro, N.J.) were evaluated. These consisted of two types of cellulose cloth (TX309, TX500), a spun bound polyamid fabric (TX909), and a polyester-bonded cloth (PBC, TX409). Individual CRC (23 by 23 cm) were folded to onequarter of their original size and double wrapped in craft paper. Packages of CRC were sterilized by autoclaving and allowed to dry before use. General sampling technique. To maintain aseptic technique, personnel wore sterile surgical rubber gloves which were rinsed with sterile 70% isopropyl alcohol before opening the inner package containing CRC and between each sample. CRC were moistened with 5 ml of sterile water, before use. A constant, steady pressure was used for a reciprocal motion that covered the entire surface. The CRC were refolded so that the contaminated portion was interior to the new
466
VOL. 38, 1979
WIPE-RINSE TECHNIQUE FOR LARGE SURFACES
sampling surface, and the surface was covered a second and third time with the wiping motion rotated 900 after each complete coverage of the sample area. Each CRC was placed into a dry, sterile glass container with a displacement of 1 liter. After each six samples collected, a handling control was taken to check for possible contamination picked up during the sample procurement. Samples were transported to the laboratory and processed within 1 h. Sterile 400 ml of a buffered rinse solution containing a 0.02% (vol/vol) solution of Tween 80 (polyoxyethylene sorbitan monooleate; Hilltop Research, Inc., Miamiville, Ohio) was added. The containers were placed for 2 min at 25 kHz in an ultrasonic bath (tank, LTH60-3; generator A300; Branson Instruments, Inc., Stamford, Conn.) containing a 0.3% (vol/vol) solution of Tween 80 (16, 17). After ultrasonication, cloths were agitated with a pipette for 5 s, then appropriate duplicate dilutions were plated with Trypticase soy agar (BBL Microbiology Systems, Cockeysville, Md.) Culture plates were incubated at 32°C under aerobic conditions, and colony counts were performed after 72 h. CRC evaluation procedure. Preliminary evaluation of the four CRC utilized 1.3-m2 surface areas to determine recovery efficiencies and handling characteristics. The PBC and TX309 materials were found to have a greater recovery efficiency and were selected for study and compared to the standard cotton swab method (1, 12). Stainless-steel tables (17.8 cm wide, 188 cm long, and 102 cm high), each containing two ribbons (7.5 by 183 cm [0.139 m2]) of Teflon (5 mil, FEP DuPont), used as collection surfaces (15), were exposed for 1 week to the intramural environment of the Vehicle Assembly Building, Kennedy Space Center, Fla. Sterile, double thicknesses of craft paper were used to separate the ribbons from the table tops. Before taking samples, we lifted each test ribbon from the table with sterile forceps and removed the first paper divider, leaving a sterile surface beneath the ribbon. The first assay procedure consisted of three experiments using 10 ribbons per experiment. Four ribbons of each group were designated as No (initial microbial concentration) controls. The remaining six ribbons were divided in half and used as test surfaces for PBC, TX309, and cotton swabs. The general sampling procedure listed above was employed in the PBC and TX309 assays. Half-ribbons used for cotton swab assays were divided into four equal parts of 174.2 cm2 each. Cotton swabs were used with a sampling motion identical to that used for CRC, then dispensed into 10 ml of sterile buffered-distilled water (12). Samples were ultrasonicated for 2 min then plated. Duplicate 1- and 2-ml pour plates were made for each swab. No controls and test ribbons used for cloth and swab assay surfaces were collected in sterile 1-liter glass containers to which 400 ml of sterile buffered rinse solution was added. Ribbons were ultrasonicated for 6 min. The No count was determined by plating the appropriate dilution in duplicate with Trypticase soy agar. Duplicate 50-ml portions from the test ribbon samples were plated with 50 ml of double-strength Trypticase soy agar to determine residual organisms on ribbons after wiping. The PBC and cotton swab methods were further
467
evaluated. Eight groups of six Teflon ribbons were placed in the Vehicle Assembly Building and exposed as previously described. Two ribbons from each group were designated as No controls, with the four remaining ribbons used as test surfaces for the PBC and cotton swab assays. Following standard assay of No samples, ribbons were subjected to two successive rinse assay procedures to determine the effectiveness of 6 min of ultrasonication. The number of microorganisms detected after the first 6 min of ultrasonication was then compared with the total number recovered in the three sequential assays. Application of PBC to spacecraft hardware. The upper quadrants of the exterior surface of a horizontally placed first-stage rocket booster were used as sampling surfaces for the PBC. To determine the microbial contamination level of the hardware surface, eight initial experiments were conducted consisting of six samples each using a surface area of 0.37 m2 (4.0 feet2). The use of 0.37-M2 surface areas as controls was continued when evaluating the 0.74-M2 (8.0 feet2) and 1.49-M2 (16.0 feet2) sample areas. Assay technique for recovery of low levels of microorgansims. Experiments were conducted to determine the accuracy and precision of the wipe-rinse technique for assaying low levels of microbial contamination. Stainless-steel strips (2.5 x 5 cm) were exposed to the intramural laboratory environment for 4 days. The technique used to enumerate the number of microorganisms which accumulate on stainless-steel surfaces have been described previously (6, 8, 9, 11, 12). Because of the small surface area to be tested, the PBC and recovery rinse water was reduced by 75%. Following standard use of PBC, duplicate 50-ml portions of rinse water were plated with doublestrength Trypticase soy agar. All laboratory procedures were performed in a horizontal laminar flow clean bench (7) to eliminate background airborne contamination. Calculations. Removal and recovery efficiencies were calculated by using the following equations:
z, I{NoNo-R,0J
NLn
n
% Removal =
.100
N
Where R, = residual number of organisms for a given observation, No = control for an experiment, n = total number of observations for an experiment, and N = number of experiments; I
NL % Recovery Where I
=
=
n
(I/No)l n N
.100
count after ultrasonication.
RESULTS A cellulose cloth (TX500) and a spun bound polyamid cloth (TX909) were eliminated during preliminary testing because of poor recovery efficiency and handling characteristics.
468
APPL. ENVIRON. MICROBIOL.
KIRSCHNER AND PULEO
The results of more extensive comparisons of PBC, cellulose cloth, and cotton swabs are given in Table 1. These data indicate that removal efficiency for all three devices was essentially the same, but the recovery efficiency for PBC was greater than the others. Coefficients of variation were similar for PBC and cotton swabs, but were much higher for the cellulose cloth. Comparison of PBC and cotton swabs (Table 2) indicated a significantly greater recovery efficiency for the PBC. Data collected to indicate the effectiveness of the 6-min ultrasonication cycle in removing natural airborne fallout (Table 3) from No samples showed that ;90.7% of the microorganisms were removed by the first ultrasonication cycle. The second and third ultrasonication cycles removed a7.4% and ¢1.9%, respectively, of the remaining contamination. After selecting PBC as the desired clean room cloth, application was made to spacecraft hardware. Data presented in Table 4 indicated that good correlation existed between control samples and those from 0.74-m2 areas. Plate counts from 1.49-M2 areas were lower than 0.37-M2 controls. Assays of stainless-steel strips resulted in good removal of fallout (Table 5), but some variation of recovery efficiency was evident. Three of the six recovery figures were found to exceed the overall recovery efficiency for PBC in Table 1. Handling controls were consistently negative throughout all operations.
tested generally had good removal potential, but some were lacking in the ability to release the collected organisms. According to data presented in Table 1, the cellulose cloth removed about the same percentage of organisms as did the PBC, but the percentage recovery was about 19% less, indicating that entrapped organisms TABLE 3. Effectiveness of the 6-min ultrasonication
cycle Expt no.
3.1 0.4 0.1 5.1 0.6 0.1 4.3 0.1 0.1 4 5.1 0.6 0.2 5 6.1 1.0 0.1 6 6.9 0.4 0.1 7 3.5 0.2 0.0 8 5.4 0.2 0.0 a >90.7% removed after first ultrasonication. b removed after second ultrasonication. '31.9% removed after third ultrasonication.
a'7.4%
TABLE 4. Recovery of natural fallout from spacecraft hardware using PBC Spacecraft sample area
sam-
ples PBC Cellulose cloth Cotton swab a For recovery.
12 12 64
Mean recovery'
No. of
(i2)
samples
Test
Controls
0.37 0.74 1.49
48 46 12
3.7 x 104 2.4 x 104 1.8 x 104
2.4 x 104 4.2 x 104
a Colony-forming units.
TABLE 5. Recovery of low numbers of microorganisms using PBC Mean No. off orgaExpt s Expt o.n saples nisms/ per expt cm 2
TABLE 1. Comparison of removal and recovery values for two CRC and cotton swabs Collection device
Secondb ul- Third' ultratrasonicasonication tion
1 2 3
DISCUSSION For a surface assay system to be effective, it must be efficient in terms of both microbial contamination removal and recovery. The CRC
Total no. of
Initiala count (organisms/cm2)
1
2 3 4 5 6
CoeffiRemoval Recovery cienta of (%) (%) variation (%) 97.2 90.4 15.9 97.5 72.0 47.6 89.6 75.2 12.3
5 8 6 5 6 5
Removal Recovery (%()
0.5 1.3 1.0 0.5 1.4 0.9
97.1 100.0 100.0 100.0 100.0 100.0
71.4 118.2 83.3 125.0 107.0 64.3
0.9a
99.5a
95.0a
a Mean.
TABLE 2. Recovery values: PBC versus cotton swabs No. of expt
Collection device
PBC Cotton swab a Organisms per cm2. b Degrees of freedom
=
14.
Total no. of samples
Rcoverya
8
32
4.6
8
128
3.1
t test (t = 0.05)b Theoretical Calculated
2.15
3.35
Significant difference
Yes
VOL. 38, 1979
WIPE-RINSE TECHNIQUE FOR LARGE SURFACES
were not released from the cloth. This ability to release collected organisms, while relatively consistent for each cloth type, is probably a function of the fibrous nature of the cloth and is one of the limiting factors of their effectiveness. The tendency to retain collected organisms can be seen when comparing the PBC and cotton swabs. The coefficient of variation is about 3.6% less for the cotton swab than for the PBC, but its recovery efficiency is also significantly less, indicating that cotton swabs do not release all of the collected organisms after ultrasonication. Previously reported recovery efficiencies for cotton swabs range from 36 to 85% (2, 3), and the value of 75.2% reported here falls within this range. A second advantage of PBC over the cotton swab is the surface area that can be sampled with one unit. According to the National Aeronautics and Space Administration Standard Procedures for the Microbiological Examination of Spacecraft Hardware (12), the area sampled with a cotton swab is 25.4 cm2 (4.0 inches2). When monitoring procedures call for large areas to be assayed, statistically valid sample numbers can become very large, resulting in extended sampling and processing times. The bioassay of a 6,451.6 cm2 (1,000 inches2) area requires 250 cotton swab samples for full area coverage. The same area could be sampled with one PBC. To illustrate further, a prominent structure of a recently launched spacecraft had a surface area of 127 feet2. A total of 72 cotton swabs were required to sample 1.6% of this area. The use of one PBC would have allowed a sample size 6.3% of the total area, or an increase of 74.6% in size of the area sampled. Because of the nature of the spacecraft hardware used in evaluating PBC, it was impossible to determine the exact presample microbial population or the residual population after sampling. Data in Table 3 give an indication, however, of recovery from different size areas. The wide difference in counts between the 0.37-M2 controls and the 1.49-M2 test areas was probably the result of the cloth drying while sampling the larger area, which resulted in its decreased efficiency. In environments where the temperature and relative humidity are controlled, this drying effect might not occur. For the suggested procedure, and with the constraints of these sampling conditions, the maximum effective sampling area was found to be between 0.74 and 1.49 In2. For standardization of technique, the sample size should be reduced to an area of 0.74 M2. To evaluate the appropriateness of PBC for collection of small numbers of organisms, assays were performed on stainless-steel strips having
469
a low organism-to-surface ratio. The mean recovery value of 95% for this series of assays compares well with the recovery figure of ca. 90% given in Table 1 and suggests a linear relationship in collection efficiency from low to high numbers. The recovery values over 100% (Table 5) might be the results of both variation of deposition of natural airborne microbial fallout on stainless-steel strips and disintegration of clumps or particles after ultrasonication. Cost and time factors for materials must also be considered in comparing PBC to cotton swabs. Based on previous cotton swab assays of spacecraft hardware (275 samples), the use of PBC reduces the material for sampling a 6,451.6 cm2 (1,000 inches2) area, by about 99%. Similar time savings are also possible. During normal sampling operations, an experienced bioassay team can collect samples at a rate of approximately one cotton swab per 2-min period. The same team should be able to sample an unobstructed 0.74-M2 area in the same time, resulting in a saving of time and labor cost. The wipe-rinse technique was developed specifically for use on the Space Transportation System (Shuttle) and other spacecraft hardware surfaces, but its overall efficiency makes it applicable to contamination control activities other than spacecraft bioassay. This technique could be used in various areas of environmental microbiology where the ability to quickly, accurately, and economically measure surface contamination is important. ACKNOWLEDGMENT'S This paper presents the results of one phase of research carried out at the Jet Propulsion Laboratory, California Institute of Technology, under contract NAS 7-100 sponsored by the National Aeronautics and Space Administration. We thank P. D. Stabekis, Exotech Research and Analysis, Inc., for the helpful discussion and suggestions, and S. L. Bergstrom, N. D. Fields, and L. A. Maull for their technical assistance. LITERATURE CITED 1. American Public Health Association. 1972. Standard methods for the examination of diary products, 13th ed. American Public Health Association, Inc., New York. 2. Angelotti, R., and M. J. Foter. 1958. A direct surface agar plate laboratory method for quantitatively detecting bacterial contamination on non-porous surfaces. Food Res. 23:170-174. 3. Angelotti, R., M. J. Foter, K. A. Busch, and K. H. Lewis. 1958. A comparative evaluation of methods for determining the bacterial contamination of surfaces. Food Res. 23:175-185. 4. Angelotti, R., J. Wilson, W. Litsky, and W. G. Walter. 1964. Comparative evaluation of the cotton swab and Rodac methods for the recovery of BaciUus subtilis spore contamination from stainless steel surface. Health Lab. Sci. 1:289-296. 5. Baldock, J. D. 1974. Microbiological monitoring of the food plant: methods to assess bacterial contamination on surfaces. J. Milk Food Technol. 37:361-368.
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APPL. ENVIRON. MICROBIOL.
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6. Favero, M. S. 1971. Microbiological assay of space hardware. Environ. Biol. Med. 1:27-36. 7. Favero, M. S., and K. R. Berquist. 1968. Use of laminar air-flow equipment in microbiology. Appl. Microbiol. 16:182-183. 8. Favoero, M. S., J. R. Puleo, J. H. Marshall, and G. S. Oxborrow. 1966. Comparative levels and types of microbial contamination detected in industrial clean rooms. Appl. Microbiol. 14:539-551. 9. Favero, M. S., J. R. Puleo, J. H. Marshall, and G. S. Oxborrow. 1968. Comparison of microbial contamination levels among hospital operating rooms and industrial clean rooms. Appl. Microbiol. 16:480-486. 10. Hall, L. B., and M. J. Hartnett. 1964. Measurement of the bacterial contamination on surfaces in hospitals. Public Health Rep. 79:1021-1024. 11. McDade, J. J., M. S. Favero, and L. B. Hall. 1967. Sterilization requirements for space exploration. J. Milk Food Technol. 30:179-185. 12. National Aeronautics and Space Administration. 1968. National Aeronautics and Space Administration standard procedures for the microbiological examination of space hardware, NHB 5340.1A. National Aeronautics and Space Administration, Washington, D.C. 13. Petersen, N. J., and W. W. Bond. 1969. Microbiological evaluation of the vacuum probe surface sampler. Appl. Microbiol. 18:1002-1006. 14. Petersen, N. J., D. E. Collins, and J. H. Marshall. 1973. A microbiological assay technique for hands. Health Lab. Sci. 10:18-22. 15. Puleo, J. R., M. S. Favero, G. S. Oxborrow, and C. M.
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