Applicator for uniformly applying a sample to bacterial growth media
United States Patent Wadley et al.  
 APPLICATOR FOR UNIFORMLY APPLYING A SAMPLE TO BACTERIAL GROWTH MEDIA Inventors: Clark Searle Wadley, Lake Forest;  
 Donald Sykes Kenney, Northbrook, both of ill.  
 Abbott Laboratories, North Chicago, 111.  
 Filed: Nov. 15, 1973 Appl. No.: 415,871  
 Related U.S. Application Data Continuation-in-part of Ser. No. 795.805, Feb. 3, 1969. abandoned.  
 Assignee:  
 References Cited UNITED STATES PATENTS 9/1964 Ryan 195/1035 R Primary Examiner-Lionel M. Shapiro Assistant Examiner-Robert .l. Warden Attorney, Agent, or FirmRobert L. Niblack; Gildo E. Fato [57] ABSTRACT An applicator for uniformly applying a sample to bacterial growth media comprising a frame having absorbent material positioned over the tip of the frame and a sleeve of permeable material covering the absorbent material. The applicator is used in test procedures for determining the presence of microorganisms in a sample, the applicator distributing a uniform film of inoculum with even distribution of any organism which may be present in the sample. The applicator thereby permits quantitation of any organisms which may be present in a suspected sample since it applies a speciflc amount of the sample.  
 3 Claims, 5 Drawing Figures PATENTEDAPR HQYS SHEET 1 BF 3 WENTEUAPR 1197s 3,875,015  
 SHEU2UF3 F5 .3 EFFECT OF VARIATIONS m we mp ms TlME 40 Q E 7 r a G300 q a; Q &#34;7/ n 20o 2 3 0 O u loo o\z34-56789\o l5 5 E C. O N O S FIG.4- EFFECT m VARIATIONS m THE EcpmuaRm-wn TIME 30 15 m 1 2 E260 Q v: S loo f- 3 vi 3 HENTEU APR 1 19. 5  
 SHEET 3 OF 3 FIG.5  
 RELATIONSHLP BETWEEN POUR PLATE AND COLONY COUNTS OPERATOR. i OPERATOR 2 STAPH-AUREUS Q O s. can A PROTEUS MIRABILIS s 5 o 5 9 w 5 2 v? UMQ hud m wk qq USQQ loo 2.5 5 I000 COLONY COUNT APPLICATOR FOR UNIFORMLY APPLYING A SAMPLE TO BACTERIAL GROWTH MEDIA This application is a Continuation-in-Part application of L&#39;.S. application &#39;Ser. No. 795,805, filed Feb. 3,  
 1%). now abandoned.  
 BACKGROUND OF THE INVENTION To determine the presence of disease causing bacteria in a sample of urine for example, a sample is spread on bacterial growth medium and the resultant growth of bacteria is observed. Bacteria in urine is diagnostic of urinary tract infection when the level of bacteria per milliliter of urine is in the range of 100,000 or greater. While the conventional pour plate dilution method can be used to determine the number of bacteria per milliliter of sample, more rapid methods are desirable so that treatment of the infection can be rapidly and effectively undertaken. One method to determine the presence of a disease causing baceteria in a sample involves the inoculation onto a blood agar plate or similar media of the suspected urine sample for example. with a calibrated loop. A calibrated loop, however, requires considerable skill and may result in uneven or overlapping growth of colonies if not utilized properly.  
 SL&#39;MMARY OF THE INVENTION The present invention relates to an applicator for uniformly applying a suspected sample to bacterial growth media and comprises a frame having an absorbent material on the tip thereof and preferably a sleeve of permeable material positioned over the absorbent material. When dipped into a sample. the external sleeve permits easy access of the sample to the inner absorbent material. The effect is that a small amount of the sample is quickly taken up by the applicator. After an appropriate dipping time. the time applicator is held in the sample, and the equilibration time, the time after removal of the applicator from the sample and before it is applied to the media. is determined, the applicator can be used to quickly and conveniently apply sample to media. Further. the applicator permits the application of a specific amount of sample to the media so that quantitation of any organisms in the sample can be achieved. Additionally. the applicator can be used to uniformly and successively innoeulate a number of media.  
 DRAWINGS The invention will be better understood upon consideration of the following description with reference to the drawings. in which:  
  FIG. I is a side elcvational view. partly in crosssection of an applicator for applying suspected sample to media.  
  FIG. 2 is a perspective view of a container with media therein and illustrating inoculation by means of the applicator.  
  FIG. 3 is a graph illustrating the effect of variations in the time the applicator is held in the sample.  
  FIG. 4 is a graph illustrating the effect in variations ofthe equilibration time after the applicator is removed from the sample.  
  l-l(i. S is a graph illustrating the relationship between the pour plate dilution method and use of the applicatot&#39;.  
 DETAILED DESCRIPTION Referring to the drawings. FIG. I illustrates a suitable applicator 10 comprising an elongated wire frame I] having a loop 12 at one end for convenience in handling, and a transversely disposed tip I3 at the other end. Fitted over the tip 13 and a portion of the frame I l is an absorbent or wick-like material 14 such as for example, cotton cording. Preferably, a sleeve I5. of a permeable material which may be a woven synthetic fabric such as rayon, is positioned over the absorbent material I4. When dipped into the sample. the absorbent material 14 absorbs a small quantity. The absorbent material 14 permits rapid absorbtion ofthe sample to thereby prevent dripping which would result in improper application of the sample to the media surface with consequent unsatisfactory results. With the applicator 10. using a light downward stroke. a technician can deposit a highly uniform film of inoculum. one that distributes any organisms evenly over the entire top surface of the bacterial growth medium. This greatly facilitates the quantitation of the bacteria since it minimizes the uneven or overlapping growth of colonies that may result from a lack of experience when using a calibrated loop of the prior methods. The width of the tip I3 should approximate the width of the media. For example, with a medium width of one inch. the tip I3 should likewise be one inch. While the use of a sleeve I5 covering the absorbent material I4 is preferred. a single absorbent material is satisfactory. Likewise, while cotton cording is preferred for the absorbent material 14. other materials can be used. With each material or combination of materials. the appropriate conversion factor must be determined. The diameter of the material I4 placed on the tip 13 of the applicator can be selected, together with the width thereof, to provide a suitable dipping and equilibration time.  
  FIGS. 3. 4 and 5 illustrate the determination of a factor for converting colony count on the medium using the applicator It) to bacterial count per milliliter of sample. FIG. 3 illustrates the effect of the dipping time, the time the applicator I0 is held in the urine sample. for example. As illustrated, a straight line function results between the length oftime of dip of the applicator 10 in the sample and the number of bacteria deposited uponthe surface of the medium. From these results, a one second dipping time was selected. FIG. 3 illustrates the results obtained when the equilibration time was varied; If insufficient time is allowed for the urine to be absorbed by the applicator 10, a high count will be obtained. Once the urine sample is absorbed, the amount deposited on the medium is consistent. This absorption occurs within 5 seconds. A 5 second equilibration time was therefore selected since this results in a faster test procedure by reducing the time the applicator 10 must be held between dipping and streaking of the sample on the medium.  
  Using a I second dipping time and 5 second equilibration time, the following method was employed to obtain the necessary factor to convert the number of colonies on the medium to the number of bacteria per milliliters of urine sample. Suspensions of 18 hour broth cultures were prepared in I7! peptone water at 40C. Two fold dilutions were made to cover the range from 10 to 10 bacteria per milliliter. The organisms used were Staphylococcus UZH&#39;L&#39;HS 209P. Escherichia coli Upham Maine. and Proteus mirahilis Finland No. 9.  
 TABLE 1 where X is the pour plate count arbitrarily chosen and Y, is the corresponding colony count obtained on RESULTS OF SAMPLE COUNTS Technician No. 1  
 Pour Plate Count Colony Count (colonies/c up) Organism Sample (Organisms/Ml) 1 2 3 Avg Staph aureus A 1.2 X 10 B 6.2 X 10&#34; C 2.0 X 10&#34; 1100 725 1000 950 D 1.2 X 10&#34; 575 300 230 368 E 6.1 X 10 238 24 291 276 F 4.1 X10 179 118 138 145 G 1.3 X 10 9i) 97 64 84 H 7.6 X 10&#34; 4O 37 46 41 l 3.8 X 10&#34; 13 31 6 13 .l 1.6 X l 8 ll) 4 7 E. coli A 1.5 X 10 B 6.4 X 10&#34; C 2.8 X 10&#34; 445 490 825 587 D 1.6 X 10 380 505 440 442 E 8.5 X 159 210 225 198 F 4.4 X 10 79 108 56 81 G 1.4 X 10 47 53 74 58 H 9.1) X 10&#34; 12 49 5O 37 l 3.6 X 10 8 1 1 l7 l2 .1 1.6 X 10 5 4 2 4 Proteus mirabilis A 3.8 X It) B 1.7 X ll) C 7.7 X 10&#34; 625 925 100i) 850 D 4.2 X 10&#34; 775 1100 800 892 E 1.6 X 10 321) 390 280 330 F 9.6 X 10&#34; 275 282 235 263 G 4.4 X 10 245 125 200 190 H 1.6 X 10&#34; 108 117 74 101) 1 8.6 X 10&#39; 33 30 33 32 J 5.2 X 10&#34; 18 23 22 Technician No. 2 Staph aureus A 1.3 X 10 B 7.: x in C 3.4 x 10* 750 800 675 725 D 1.4 x 10 600 300 530 477 E 5.1 10* 320 300 330 317 F 2.8 X 10 210 167 215 197 G 1.2X 1(1&#34; 85 188 76 116 H 7.5 X 10 36 26 31 l 2.3 X 10 6 29 21 19 .1 1.6X10 7 l3 14 11 E. coli A 1.3 X H)&#34; 13 7.5 X 10 C 3.4 X 10&#34; (1 725 375 517 D 1.6 X 10&#34; 186 700 256 381 E 8.5 X 111 227 601) 238 355 F 4.4 X 10 153 )8 118 123 G 1.6 X 10 52 93 83 76 H 7.6 X 11) 41 44 23 36 l 4.3 X 11)&#34; 6 19 3) 21 J 1.8 X10 11 1t) 25 15 Proteus mirabilis A 5.0 X l() 1225 1300 1050 l 192 B 1.8 X 10 1275 725 850 950 C 1.0 X 1(1 (1 1101) 775 792 D 4.9 X 10 325 425 305 352 E 1.6 X It)&#34; 390 380 385 385 F 1.() X 10&#34; 400 475 425 433 (i 3.8 X It) 157 152 H 1.7 X 10&#39; 57 77 131 88 l 9.0 X 1() 36 81 41 53 .l 4.4 X ll)&#34; 21 I) 2) 23 Ton numerous to count a growth media using the applicator 10. X. and Y. are  
  P10. 5 illustrates the average of the results obtained by the two technicians. Over the range of interest. generally 10 to 10&#34; bacteria per milliliter of sample. the points describe a straight line function. The slope of the straight line function may be expressed as a simple factor using the general formula.  
 similar points arbitrarily chosen at a higher location on the graph.  
  Reading from Table 1. two examples of the calculations are presented in the following table for each orsuch as duplicate samples or special growth media for identification purposes. the applicator 10 can be used TABLE 2 Organism SAMPLE YI Y2 XI X: M  
 Staph aureus H 8: D 7,600 140,000 36 422 342 l &amp; H 3.! 7,600 I6 36 225 E. coli l 8L E 4,000 85,000 lb 276 3l2 l &amp; D 4,000 160.000 16 412 394 Proteus J &amp; F 4.800 98.000 22 348 286 mirabilis t&amp; 6 8.800 41.000 4: WI 250 Average M 302 The average of the slope between the chosen points to successively streak each of the media after only a on the graph of FIG. 5 was found to be about 300 and single dipping ofthe applicator into the sample. Unirepresents the conversion factor. form inoculation ofeach of the media will result. In this The applicator l0 and suitable m dium Can be us d manner. at least four media can be successively inocuto determine the presence ofbacteria in a urine sample. [med for example. as follows. A suspected urine sample is wh i l i d it; first shaken to obtain a uniformily mixed sample. The 1 A applicator f upp|ying a Sample to hc cxum- 20 is lmid m p thc medium ined to bacterial growth media. said applicator com- 2] as illustrated in FIG. 2. The applicator 10 is then prising; PP thc SPLClmCIL .l im&#39;crlng thc mmwcrsc 25 an elongated frame having a tip disposed at one end tip 13 and held in place for 1 second before removal. n-unswmc to said frame; f&#39;&#34; Clulllhrltlml pullscflr 5 scumds&#39; thc medium absorbent material fitted over the tip of said applica- Strcakcd l &#39;j m clmplctcly tor to absorb the sample and including a permeable ering the length ol the medium 21 surface. The external glee, pusimmcd Over the absorbent material, said sleeve l5 otters to the urine sample quick absorption i H n U th &#34;n1 U I w t) th and easy excess to the inner absorbent material 14. The 5 pmuuna- L M l L-Ldh) a} L&#39;s L absorbent material. whereby said applicator. when cltect is that a small amount of the urine sample is y I I quickly taken up by the sleeve l5 and absorbent or plmcd m thL l f dbwlh i f qumm) wick-like material 14 so that the applicator l0 deposits &#34;P&#39; l dlsmbuic th? Sample ummrmly a uniform inoculation on the media with even distributhc Smldcc of the medium tion of any organisms which may be present. After in- Thc Pl of claim I tho absorbent oculation. the tray 20 containing the inoculated media &#34;mtcrlill clmprlscs cm&#39;dlng and the Permcilhlc 21 is incubated. After incubation. quick quantitation of Slog: clmllrlscs synthetic fabricthe bacteria is achieved by counting the colonies of the 3. A method of applying a sample to be examined for 2| and multiplying y lllcim 300 the presence of bacteria to bacterial growth mcdia.said tained as previously described to result in the number method comprising: of bacteria per milliliter of urine sample. After quantiproviding an applicator comprising an elongated fying the bacteria. if the bacteria are present at a level frame having a tip disposed at one end transverse of about l00.000 per milliliter of urine or greater. indito the frame and including absorbent material titcating a urinary tract infection. the particular organism ted over the tip of the applicator to absorb the samresponsible can be identified by conventional methods. ple;  
  As described. the applicator 10 can be employed in dipping the tip of the applicator in the suspected sama simple three step method: l dipping the applicator pie for a preselected time; 10 in the suspected sample for a preselected time; (2) w removing the applicator from the sample; removing the applicator [0 from the sample and allowallowing a preselected equilibration time to permit ing a preselected equilibration time before applying the the absorbent material to absorb the sample; n sample to the medium: and (3) inoculating the medium inoculating the medium by streaking the sample on by streaking the sample on the medium with the applithe medium with the applicator. cator 10. in the event a plurality of media are required.