US 2476093 A
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July 12, 194-9. A. A. HIRSGH 2,476,693
BACTERIOLOGICAL TEST BOTTLE Filed Sept. 25, 1943 INVENTOR.
I TN ESSES:
Patented July 12, 1949 UNITED STATES PATENT OFFICE BACTERIOLOGTCAL TEST BOTTLE Abraham Adler Hirsch, New Orleans, La. Application September 25, 1943, Serial No. 503,786
, 4 Claims. 1
The invention relates to improvements in bac teriologicalsampling and testing containers withspecial application to testing natural and treated waters and other potable materials for the presence of gas-forming bacteria, particularly the coliform group. The objects of my improvement are first, to provide asampling bottle containing fermentation tubes attached to its interior walls; second, to present a series of containers of various volumes to allow estimation of bacterial density from the size or sizes of sample portions show ing gas formation; third, to offer a device and method whereby for water supplies or other liquid products the operations of sampling, inoculation, and culture may be performed simultaneously in a single vessel; and fourth, to provide a means for subsequent confirmatory culture by means of a secondary broth tube which is lowered into the same bottle. These improvements are designed particularly to facilitate performance of tests for the presence of coliform organisms in liquids according to the descriptions and explanations detailed below.
The combination sampling-inoculation-culture bottle is illustrated in the drawing, in which Figure 1 is a cut-away perspective of the assembled bottle and primary fermentation tubes; Figure 2 a section through the bottle along the line .r-sc; and Figure 3 a vertical view of the supplementary culture tube for confirmatory tests.
Referring to Figures 1 and 2: I is a bacterial sample bottle, which may be of conventional shape, covered by a ground glass stopper 2 through which an air hole 3 has been perforated to coincide with a corresponding port 3a in the neck of the sample bottle. A mark 4 is lined on the sample bottle to indicate the proper level for filling.
Permanently fused or otherwise attached as with stainless steel clips or by other suitable means of fastening to the interior of the sample.
secondary fermentation tests; I3 is a hooked end to provide lifting facility for a wire hook; i4 is a bulb to provide wastage volume; I5 is an offset bend to allow nearly true vertical suspension during transfer and also to minimize overall width; I6 is the looped portion for retaining gas bubbles formed insecondary fermentation; I! is a bend to block entry of externally formed gas; I8 is a bulb in which the interface is formed between primary and secondary broths; and I9 is a capillary nozzle to minimize wastage of secondary broth during height of liquid in fermentation tube 6 in order to keep the loop full of secondary broth. To permit insert-ion,- the greatest width across tube [2 must be kept slightly less than the diameter of fermentation tube 6-.
Typical procedure for using this bacteriological test bottle for detection, confirmation and enumeration of coliform bacteria is explained in stepwise fashion below:
1. Clean test bottle I and interior glassware with washing compound, rinse with water and drain, not necessarily dry.
2. Introduce one gram of dehydrated lactose broth powder into the bottle, add about 5 mill-iliters of distilled water to dissolve the media and prevent darkening during sterilization. Stopper the sample bottle with air vent open, and wrap tin foil, or any heat resistant sheeting, loosely around the stopper and mouth of the bottle.
3. Sterilize in an autoclave at 15 pounds per square inch steam pressure for 15 minutes.
4. After cooling off the bottle, twist the stopper slightly to close the air vent.
5. Sterilize sampling fixture and fiush according to accepted practice.
- 6. Remove stopper from sample bottle without contaminating stopper or mouth of bottle, and fill the sample bottle with water or liquid up to mark 4 as shown in Figure 1. To provide sufficient sample for the distribution as described below, the required volume of water or liquid will be L milliliters.- Mark 4 therefore is made at a level to contain milliliters,- since 5 milliliters of distilled water had been added to dissolve the media.
. 7. Stopper the bottle with air vent closed, and re-fit foil wrapper over stopper and mouth of bottle.
8. Swirl bottle, while holding it vertically, to mix the media uniformly throughout the sample. 9-. Invert the sample bottle to displace air from all the internal tubes and fill them with samplemed-ia mixture. Several inversions may be needed corresponding to a like amount of water sample,
is retained in tubes l and II. In this manner a series of 100, 50 and milliliter water inocula are obtained automatically in the sample bottle and furnished with the standard quantity of nutrient enrichment media. Splashing of liquid during later handling from tube 6 into tube I0 is prevented by the deflecting hood 1, and by having the overflow notch 8 open on the side away from tube It. Liquid levels in tubes 6 and ID will drop sufficiently on turning bottle I upright to prevent spill or splash over during ordinary handling.
10. Incubate the assembly at 37 centigrade or place it in a warm spot near this temperature, with air vent open under the loosened foil cover to maintain aerobic conditions in the culture.
11. Observe for gas formation in inverted vials 5, 9 and II at the end of any desired incubation periods, such as 18, 24 and 48 hours. If no gas has formed the test is usually considered negative for members of the coliform group of bacteria, and the test is terminated. If gas has been formed, that portion causing gas is presumed to contain coliform bacteria. This positive presumptive test should be confirmed by the secondary test below under steps 12 to 14, inclusive. Due to the relative size of inocula only rarely will gas form in the 10 milliliter portion in tube [0 without the concurrent appearance of gas in the 50 and/ or 100 milliliter portions.
12. By means of a sterile wire hooked under bend l3 transfer aseptically tube [2, filled with sterile brilliant green lactose bile broth (2%) or other acceptable inhibitory broth, to the bottom of sample bottle I, or lower it carefully into tube 6, should only the latter show gas formation. If only the 10 milliliter inocula is presumptively positive, a relatively rare but not impossible case, tilt the sample bottle to contaminate the 100 milliliter portion with spill-over from tube l0, allow time for gas collection in tube 5, and then introduce the brilliant green lactose bile broth tube I2 into the bottom of bottle l.
13. Incubate the assembly containing the introduced brilliant green bile broth tube I2 at 37 cennoting the sizes of volumes positive or negative for primary gas formation, and noting whether confirmed or not. The most probable number method also may be used. These principles in step 15 are not new but are included to show their applicability to the bacteriological test bottle described herein.
Obvious alternatives to the materials, construction and usages described above are likewise contemplated and embraced in these specifications as 4 follows: first, thermoduric transparent materials other than glass may be employed; second, another type of stopper or closure or vent may be adopted without departing from the principles of this sample-culture bottle; third, the arrangement of interior Durham fermentation tubes and vials may be varied for manufacturing convenience or other purposes without affecting the principles of this apparatus; fourth, the primary gas tubes may be of the Cowles looped type instead of the Durham vial type; fifth, volumes other than those described may be selected; sixth, other numbers of internal fermentation tubes, of
any volumes desired, may be provided; seventh,
that specified in the above procedure may be used;
ninth, other materials than potable water may be tested in this apparatus; tenth, other bacteria than the coliform group may be searched for and cultured; eleventh, other methods for confirming the presence of the coliform group than the device described here may be used; twelfth, other means for trapping entry or exit of gas from the secondary fermentation tube may be provided; and thirteenth, alternative methods for attachment of tubes other than by fusion, as illustrated in the drawings, may be provided by such devices as springs, clips, screws, or dowels of stainless steel or other suitable material.
Prior developments, particularly in the field of water bacteriology, which form the elements for the bacteriological test bottle described in these specifications are not claimed as new; these previous items include the Durham fermentation tube and vial, the Cowles fermentation loop, and the use of primary media introduced into the water sample bottle. These basic devices are modified and combined as described herein to enable procedures, conveniences, economies in time and equipment, speed in execution and simplicity in use not foreseen in the separate use of any of the component elements.
I do claim as new and therefore desire to secure rights under Letters Patent to the following features:
1. In combination with a bacteriological test container, a fermentation tube afiixed therein, said tube having an open upper end provided with an overflow notch, and a hood partially covering said open end, said notch and said hood being diametrically opposite each other with said hood facing said notch whereby the two tend to prevent spillage of the tube contents in a direction opposite to that in which the hood faces.
2. In combination with a bacteriological test container, a closure for said container, fermentation tubes of various volumes attached securely therein on the same side thereof, and inverted vials aflix'ed in said tubes, respectively, for indicating gas formation in individual tubes.
3. In combination with a bacteriological test container, a closure for said container, fermentation tubes of various volumes afiixed in said container on the same side thereof, inverted vials attached securely within said tubes, respectively, for indicating gas formation in individual tubes, and a looped tube having a capillary bottom tip, a hooked top end and a narrow profile to facilitate insertion of said looped tube into said container or said fermentation tubes when closure is removed temporarily, for purpose of making secondary fermentation tests.
4. In a sterile test container having fermentation reservoirs and inverted vials attached therein, a method of performing fermentation tests which consists in introducing sterile broth, placing a closure having an air vent in the container with air vent open, again sterilizing and cooling the broth charged container if so desired, closing the air vent until such time as ready for sampling, introducing the sample to be tested into said container, stoppering the container with air vent closed, swirling the container to mix uniformly the broth and the sample, inverting the container to displace air from the fermentation tubes and inverted vials, initiating a confirmatory test by inserting into fermentation tubes and reservoir inside original test container a looped tube previously filled and sterilized with secondary broth, incubating the assembly in an upright position with the air vent partly open, and at the end of desired incubation periods observing for gas formation appearing in inverted vials or collected within looped tube.
A. ADLER HIRSCH.
REFERENCES CITED The following referenlces are of record in the file of this patent:
UNITED STATES PATENTS Number Name Date 1,262,081 Mojonnier Apr. 9, 1918 1,435,367 Ablahadian Nov. 14, 1922 FOREIGN PATENTS Number Country Date 278,546 Germany Sept. 30, 1914 381,002 Great Britain Sept. 29, 1932 OTHER REFERENCES Eimer and Amend, 85 Anniversary Catalogue, 1936, pp. 363, 827.
Eimer and Amend, op. cit., page 131.
Todd and Sanford, Chemical Diagnosis by Laboratory Methods, 9th edition, Saunders, 1941, pages 711 and 724.
Zinsser and Bayne Jones, Textbook of Bacteriology, 7th ed., Appleton-Century, 1935, page 1085.
Ace General Catalog 40, Ace Glass, Inc. 1940, page 9.