Determination of Bacterial Numbers on Tableware by Means of Direct Plating RICHARD R. BARTON', HAROLD GORFIEN, AND ROBERT M. CARLO United States Naval Supply Research and Development Facility, Bayonne, New Jersey Received for publication April 6, 1954 It has long been recognized that swabbing methods will not remove all or even a fixed percentage of bacteria from surfaces. Because of this, it is not possible to use these methods for quantitative determinations with any degree of accuracy. Considerable work has been done to develop methods that will permit greater recovery of bacteria from tableware. Higgins (1950) and Cain and Steele (1953) have used swabs made of calcium alginate. After use, these swabs were placed in a diluent containing sodium hexametaphosphate. On shaking, the swabbing material dissolves, thereby eliminating the problem of removing bacteria from the swab. However, there is no informa- tion to indicate that this material is any more effective than cotton in removing bacteria from a surface. Olsen and Hammer (1933) developed an agar disc method for studying bacterial contamination on metal surfaces. While this method will recover greater num- bers of bacteria than swab tests, it will not remove bac- teria from cracks and inaccessible areas. The contact method of Walter and Hucker (1941), which is a modification of Olsen and Hammer's method, and the quantitative method of Guiteras et al. (1954) have relatively the same deficiencies. In an attempt to obviate the deficiencies in the above methods, an investigation was made to modify the agar disc method. The modification investigated was to grow the bacteria in situ by placing liquid agar medium on table ware, allowing it to harden and then incubating the utensil. METHODS AND MATERIALS The tableware used in this study were stainless steel compartmented mess trays, ceramic cups and soup bowls, stainless steel knives, forks, and spoons. These utensils were contaminated by dipping in dilute suspen- sions of Bacillus subtilis morphotype globigii. Before plating, the contaminated utensils were air dried. Of necessity, slightly different techniques were used to apply the agar to the various utensils. The following are the procedures used: Trays. Sufficient liquid agar was poured into one of the compartments to provide a layer of agar about Y8" ' Present address: Takamine Laboratory, Clifton, New Jersey. FIG. 1. A compartmented mess tray partially covered with sterile parchment paper. FIG. 2. A cup covered with the top of a Petri dish showing bacterial colonies after 24 hours of incubation of 37 C. thick (about 25 ml). A sheet of sterilized parchment paper was stretched over the top of the tray and sealed with cellulose tape to prevent contamination (figure 1). 264 on February 18, 2021 by guest http://aem.asm.org/ Downloaded from
Transcript
Determination of Bacterial Numbers on Tablewareby Means of Direct Plating
RICHARD R. BARTON', HAROLD GORFIEN, AND ROBERT M. CARLO
United States Naval Supply Research and Development Facility, Bayonne, New Jersey
Received for publication April 6, 1954
It has long been recognized that swabbing methodswill not remove all or even a fixed percentage of bacteriafrom surfaces. Because of this, it is not possible to usethese methods for quantitative determinations withany degree of accuracy.
Considerable work has been done to develop methodsthat will permit greater recovery of bacteria fromtableware. Higgins (1950) and Cain and Steele (1953)have used swabs made of calcium alginate. After use,these swabs were placed in a diluent containing sodiumhexametaphosphate. On shaking, the swabbing materialdissolves, thereby eliminating the problem of removingbacteria from the swab. However, there is no informa-tion to indicate that this material is any more effectivethan cotton in removing bacteria from a surface.
Olsen and Hammer (1933) developed an agar discmethod for studying bacterial contamination on metalsurfaces. While this method will recover greater num-bers of bacteria than swab tests, it will not remove bac-teria from cracks and inaccessible areas. The contactmethod of Walter and Hucker (1941), which is amodification of Olsen and Hammer's method, and thequantitative method of Guiteras et al. (1954) haverelatively the same deficiencies.
In an attempt to obviate the deficiencies in the abovemethods, an investigation was made to modify theagar disc method. The modification investigated wasto grow the bacteria in situ by placing liquid agarmedium on table ware, allowing it to harden and thenincubating the utensil.
METHODS AND MATERIALS
The tableware used in this study were stainless steelcompartmented mess trays, ceramic cups and soupbowls, stainless steel knives, forks, and spoons. Theseutensils were contaminated by dipping in dilute suspen-sions of Bacillus subtilis morphotype globigii. Beforeplating, the contaminated utensils were air dried. Ofnecessity, slightly different techniques were used toapply the agar to the various utensils. The followingare the procedures used:
Trays. Sufficient liquid agar was poured into one ofthe compartments to provide a layer of agar about Y8"
' Present address: Takamine Laboratory, Clifton, New Jersey.
FIG. 2. A cup covered with the top of a Petri dish showingbacterial colonies after 24 hours of incubation of 37 C.
thick (about 25 ml). A sheet of sterilized parchmentpaper was stretched over the top of the tray and sealedwith cellulose tape to prevent contamination (figure 1).
To determine the effectiveniess of the parchment paperin preventing contamintationi, sterile agar was pouredinto eight sterile trays which were then covered.After 24 hours of incubationO at 37 C, there was onlyone colony on one of the trays.Cups and bowls. Liquid agar at a temperature of 40-
45a C was poured in and the utenisil was gently rotatedthrough onie-fourth of a cir-cle in order to obtain a 2Iuniform layer of agar about 18" thick over the curvedsurface. To prevent contaminiationi, the cups and bowlswere covered with sterile parchment paper or the top ofa sterile Petri dish (figures 2 anid 3).
Fi, 5. The development of bacterial colonies in the agarand howl of a spoon incubated for 24 hours at 37 C.
FIG. 3. A soup bowl covered with the top of a Petri dish -T0}showing the development of cterial colonies after 24 hoursof incubationr at 37 C.
.0~~ ~ ~ ~ ~ ~ ~
FIG. 6. The development of lacterial colonies in the agar~~~J ~~and oni the knife blade incubated for 24 hours at 37 C.
Forks and spoons. Sufficient agar was poured intosterile Petri dishes to give a layer about 18' deep.Before the agar had set, the tines of the forks and thebowls of the spoons were immersed in the agar. Toprevenit contamination, the tops of the dishes wereplaced over the flatware and taped into place. In addi-tion, the space between the top and bottom of the dishcaused by the handle of the cutlery was sealed withtape (figures 4 and 5).
Knives. Melted agar was placed in sterile Kohlflasks and the knife blades were immersed into it be-fore it, hardened. The knives were held in place by the
FIG. 4. The development of colonies in the agar and tines plug of cotton in the neck of the flask (figure 6).of a fork incubated for 24 hours at 37 C. After the agar had set, the utensils were placed in an
incubator at 37 C and incubated for 24 hours. It wasfound that this incubation was sufficient for colonydevelopment of the test organism. If longer periods oftime were used, the colonies on heavily contaminatedsurfaces grew together, making accurate counts impos-sible. When the number of colonies was high, a mag-nifying glass was used to facilitate counting. To de-termine the value of a direct plating procedure, 75comparisons were made against standard swabbingtechniques using cotton swabs. In addition, 125 compar-isons were made between the direct plating procedureand the calcium alginate technique as developed byCain and Steele (1953). Paired coAiparisons were madeon trays, cups, and bowls. In these tests, the bacteriaon 4 square inches were determined. To obtain moreaccurate counts, the test area was divided into seg-ments containing less than 300 colonies by marking theagar with an inoculating needle. Unpaired comparisonswere made on the cutlery. The tines of the forks, bowlsof the spoons, and knife blades were swabbed or directplated. The following medium was used as it was foundto be the optimum for growth of the test organism:
When grown on this medium, the coloinies of the testorganism had a bright orange color. Because of this,any contaminants could readily be identified.
TABLE 1. Comparison between direct plating procedure andswab tests on the detection of bacteria on eating utensils
Utensil
TraySoupbowls
Tray
Cup
Tea-spoon
Fork
Knife
AreaTested
4 sq. in.4 sq. in.
4 sq. in.
4 sq. in.
Bowl ofspoon
Tines offork
Blade
AverageCountDirectPlatingProce-dure
2194
673
465
88
209
87
63
Aver-ageCountSwabTest
536
72
85
3
54
18
3
Ratio ofBacteriaDetectedDirectPlate/Swab
4.09:1
9.35:1
5.47:1
29.33:1
3.87:1
4.83:1
21.00:1
Type ofSwab
CottonCotton
Calciumalgi-nate
Calciumalgi-nate
Calciumalgi-nate
Calciumalgi-nate
Calciumalgi-nate
Type ofComparison
PairedPaired
Paired
Paired
Un-paired
Un-paired
Un-paired
No.of
Com-pari-sons
5025
25
25
25
25
25
RESULTS AND DISCUSSIONFrom table 1, it canl be seen that higher bacterial
counts were obtained using the direct plating methodthan either swabbing method. In all the paired com-parison tests, the direct plating procedure gave ahigher recovery than the swab test of the same item.The increase in numbers varied with the test itemand the number of bacteria initially present on theutensils. For example, when a high number of bacteriawere present on mess trays, ani average of 4.09 bacteriawiras recovered by the direct plating method for everyone recovered by a swabbing procedure. When a smallernumber of bacteria were present, this ratio increasedto 5.47 to 1. Similar results were obtained in the un-paired comparisons made on cutlery. Much highercounts were obtained with the direct plating procedure
FIG. 7. The development of colonies on a heavily contami-nated compartment tray after 24 hours of incubation at 37 C.
than with a swabbing techniique. Againi it can be notedthat as the number of organiisms present on the testitem decreases, the ratio of recovery increases.The direct platinig procedure is of particular value
in the detection of bacteria on rough or scratched sur-faces. Because the liquid agar will flow into scratches,a favorable atmosphere is provided for the developmentand detection of bacteria that would Inot be detectedby a swab test. Figure 7 shows the development ofbacterial colonies oni a mess tray. A number of thesubsurface colonies are elongated in the direction of themachine marks on the surface. Close examination ofthe original tray indicated that these elongated colonieswere located directly over these marks.
SUMMARY
Procedures are described for direct plating proceduresfor in situ detection of bacteria on metal mess trays,soup bowls, cups and cutlery.Two hundred comparisons between the direct
plating procedure and swab tests were made. In these
comparisons, 3.87 to 29.3 times as many bacteria weredetected by the direct plating procedure as by swabtests.
REFERENCES
CAIN, R. M., AND STEELE, H. 1953 The use of calcium al-ginate soluble wool for the examination of cleansed eatingutensils. Can. J. Public Health, 44, (12) 464-467.
GUITERAS, A. F., FLETT, L. H., AND SHAPIRO, R. L. 1954
A quantitative method for determining the bacterialcontamination of dishes. Appl. Microbiol., 2, 100-101.
HIGGINS, M. 1950 A comparison of the recovery rate of or-ganisms from cotton-wool and calcium alginate wool swabs.Public Health Lab. Ser. Bull. (Gr. Brit.), 50-51, Feb. 1950).
OLSEN, H. C., AND HAMMER, B. W. 1933 The agar disc methodfor studying the contamination from metal surfaces. IowaAgr. Exp. Sta., Research Bull., 300, Ames, Iowa.
WALTER, W. G., AND HUCKER, C. J. 1941 The use of the con-tact plate method to determine the microbial contamina-tion on flat surfaces. N. Y. Agr. Exp. Sta. Tech. Bull. 260
A Slit Sampler for Collecting Air-Borne Microorganisms
HERBERT M. DECKER AND MYRL E. WILSON1
Chemical Corps Biological Laboratories, Camp Detrick, Frederick, Maryland
Received for publication April 9, 1954
There is an increasing need by civilian defenseagencies, research laboratories, and hospitals, for aninexpensive, simple, continuous sampling device thatwill recover air-borne microorganisms. Such a deviceshould sample a relatively large quantity of air, shouldpermit direct impingement of the organisms on thegrowth media, and should be easy to use in the fieldor laboratory.
Bourdillion, Lidwell and Schuster (1948) have de-veloped several slit samplers. These have been com-plex mechanical units, difficult to sterilize, delicate,and requiring specially designed agar collection plates.
Luckiesh, Holiday and Taylor (1946) have de-veloped a portable air sampler; however the collection.surface is limited to a standard (100 x 15 mm) Petridish and there is no means of determining the periodin which the maximum biological concentration iscollected. Furthermore, the sampling time is limitedwith the Petri dish as desiccation of the media occursif exposed to prolonged periods of air sampling.
MATERIALS AND METHODS
An inexpensive slit sampler for collecting micro-organisms has been designed which utilizes a 150 x 20mm culture plate possessing 2.2 times the area of thestandard Petri dish. This increased area permits collec-tion of a much greater number of organisms with thistype of sampler and prevents drying out of the media.A simple timing device is incorporated in the designof the sampler which permits rotation at the rate of 1revolution per hour, and renders an accurate timeconcentration relationship.The modified slit sampler and its component parts
1 Second Lieutenant, USAF (MSC).
are shown in figures 1 through 4. The sampler is com-posed of 1) a slit and slit tube, 2) sampling box, 3)agar culture plate (150 x 20 mm), 4) agar plate holder,5) drive shaft, 6) 1-hour interval timer, and 7) intervalP-
