US 2771398 A
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Nov. 20, 1956 T. L SNYDER METHOD AND APPARATUS FOR coUNTING MICROORGANISMS Filed sept. 17, 195s .nc-...1.... I .....n
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UnitedStates Patent O METHOD AND APPARATUS FOR COUNTING MICROORGANISMS Thomas L. Snyder, Frederick, Md., assignor to the United States of America as represented by the Secretary of the Army Application September 17, 1953, Serial No. 380,888
2 Claims. (Cl. 195'-103.5)
(Granted under Title 35, U. S. Code (1952), sec. 266) The invention described herein may be manufactured and used by or for the Government of the United States of America without the payment to me of any royalty thereon.
This invention relates to improvements in method and apparatus for counting microorganisms and in particular is concerned with means for the determination of the number of viable biotics in a known volume of Huid or carrier.
In the past it has been a serious problem in the field lof Bacteriology to ascertain the concentration and nurnlb'er of microorganisms in `a carrier and there have been required complicated and pains-taking efforts in such de- One of the conventional processs comprises incubating a culture on a Petri dish and then counting individually the number of microorganism colonies in the growth. Obviously this procedure is time consuming, laborious and leads to inaccuracies caused b3 the ditliculty in counting. Other means for finding the concentration of microorganisms have been devised which include pipetting small known amounts of the culture into a plurality of nutrient-filled test tubes and after an incubation period counting the number of cloudy tubes and correlating this number to the concentration of viable organisms in the carrier. This method is however quite cumbersomein that a large space is required for the test tubes and in carrying out the incubation stage while the `filling of the individual test tubes to the same level in obtaining equal volumes is another requirement necessitating special manipulations'and additional labor." i
By Ymeans of this invention the-complicated procedures and bulky equipment required have been eliminated. The process and apparatus of this invention include ascertaining the number of microorganisms in a nutrient liquid carrier by immersing a perforated stick in the liquid so that the perforations or openings which are of capillary size are charged with the liquid. The material used in this stick must be of a non-wetting characteristic in order that there be no film of liquid between the perforations.
t That is, the stick must be composed of material which is sulliciently wettable to exhibit capillarity characteristics while also possessing suicient non-wettability so as to prevent the formation of any lm on the hat surface adjacent to these openings. This is of critical significance since if the material is wet with only a lm of liquid of molecular thickness on the surface between the perforations, the microorganisms will be transmitted from one aperture to another using this thinly wetted surface as the conductor or migration medium. This invention contemplates broadly the use of halogenated hydrocarbons such as Teon, made by the E. I. Pont de Nemours Co., Inc., which has been found to possess the desirable nonwetting characteristics as well as being chemically inert to the mediums used. The uniformly perforated capillary openings make possible the inclusion of the same volume of liquid within each opening due to the capillary action which causes these openings automatically to be lled to Plce the same level. Obviously this obviates any special procedures in the illing stage and greatly simplifies the operation. Once the stick has been charged with the microorganism the incubation period is commenced at the end of which the determination is made by counting the number of turbid cells indicating the growth of colonies. The perforations in the stick may be so arranged that a completely automatic counting process can be used by photo-electric means and like scanning processes.
Accordingly, it is an object of this invention to provide a method and apparatus for simply and quickly determining the number of microorganisms in a nutrient liquid carrier.
It is a further object of this invention to provide a method and apparatus for taking 4a multiplicity of samples from a liquid carrier which samples are of the same volume with only one piece of apparatus and in only one operation.
It is still another object of this invention to provide a process and apparatus for determining the concentration of microorganisms in a nutrient liquid carrier by using a perforated relatively non-wettable charge stick and subsequently detecting growth of the progeny in the perforations.
`Yet another object of this invention is to provide an inert perforated liquid charge stick of relatively nonwettable characteristics which can be used in a process for determining the number of microorganisms in a nutrient liquid carrier so that the growth in the perforations can be readily ascertained by automatic means.
`Other objects will be apparent from the description which follows and will be further apparent to those skilled in the art.
ln the drawings:
Fig. l is a plan view of one form of the charge stick.
Fig. 2 is a cross-sectional view of Fig. l.
Fig. 3 is a plan view of a modified form of the charge stick.
Fig 4 is a cross-sectional view of Fig. 3.
Fig. 5 is a graph showing a comparison betweenresults obtained by this invention and the standard plate count.
Referring now to the drawings andFigs. 1 and 2A in vp'ir'ticular, it 'will be. seen that the charge stickh is ,of` a'planiform nature and `has 'a' limited depth with perforations 11 of a capillary size extending therethrough. These perforations, as an example, may be uniformly distributed so that there are 2000 such holes in a 6% by 1% inch area. It has been found that such openings may conveniently have a diameter of 0.025 inch although it is to be understood that there is a variable range depending upon the viscosity of the fluid being handled and upon other factors. Likewise other spacings may be utilized. This charge stick may conveniently be used in automatic counting procedures where the individual holes are scanned by photo-electric cells and the like with the turbidity resulting from growth of the progeny registering as a growth cell whereas the sterile cells remain clear.
The depth (or thickness) of the stick is governed by the amount of turbidity (length of perforation) it is desired to offer the detecting instrument. The stick is always completely immersed in the carrier liquid and the holes completely filled, yagitation being employed if necessary. The stick is withdrawn from the liquid with its plane kept perpendicular to the surface of the liquid, in order to retain a uniform and maximum quantity of liquid in the perforations.
The modification shown in Fig. 3 at 13 is of the same general construction as the charge stick disclosed in Figs. 1 and 2 with the exception that the spacing of the perfor.
ations is broken up so that the perforations are in individual banks 12 with four of these banks across the widthl and ten along the length comprising forty banks in all, with ifty perforations in each bank. This arrangement facilitates visual counting by the naked eye so that each bank may be examined separately.
It is necessary that the material used in the construction of the charge sticks be absolutely inert to the medium and be of a non-wettable nature. The tetra-iluoroorganic resins have been found to be very advantageous in this respect and the material Teflon is used in this construction. This resin is synthetic polymerized tetrauoro ethylene which has the desired non-wettable nature. Other plastics, such as the acrylic resin Lucite, have been tested and found to be wanting in this wetting characteristic since they have formed thereon a thin molecular film on the surface of the charge stick between the perforations which is ruinous for the successful operation of this process. The requirements of this non-wetting characteristic are critical to this invention since as has been pointed out above even a molecular film will serve to facilitate the migration of the microorganisms.
A suitable nutrient suspension or other medium containing microorganisms whose concentration and number is to be determined is furnished and the charge stick is immersed therein. The openings in the charge stick which are of a uniform capillary size will draw therein a constant volume of iiuid which is dependent on the viscosity of the fluid. The charge stick after this charging operation can then be withdrawn and handled in either a vertical or horizontal position since the liquid is retained in the openings by capillary attraction.
The charge stickjcontaining the liquid to be analyzed is then set aside in a saturated aqueous atmosphere for a suitable incubation period after which growth of the progeny in each opening is determined by ascertaining whether or not it has turned cloudy or otherwise changed in form. The number of openings so changing may then be correlated to the concentration and number of microorganisms in the medium in which the charge stick Was was immersed by conventional formulation since the volume of this medium and that of the liquid in the charge stick are ascertainable.
Comparisons have been made between the standard plate counting method and the process of this invention using the charge stick herein disclosed on fourteen bacterial suspensions of Serrata marcescens. The results are illustrated by the graph of Fis- 5 which Shows the method of this invention gave higher values than the standard plate count. It is believed that these higher estimates represent more accurate counts of the viable organisms actually present because: (1) non-linearity of the curve tends to eliminate an explanation of the discrepancy based on error of calibration of the device, (2) preliminary statistical studies do not support the hypothesis of signilicant dispersion of growth from positive holes to adjacent sterile holes, and (3) the standard plate count tends to be depressed at high counts per plate as a result of crowding. Analysis of the individual counts gave a standard deviation of four to ive percent for the stick method of this invention and 11.5 percent for the standard plate count method.
Although this invention has been described with relation to the two embodiments of the charge stick shown in the drawing, it will be understood by those skilled in the art that the shape of the stick and the length, diameter and spacing of the holes may be varied to optimum arrangements suitable for either visual or automatic scanning. Other modications will-readily appear to those skilled in the art and this invention should be limited only by the scope of the appended claims.
l. A device for determining the concentration of microorganisms in a liquid which consists of a slab-like form provided with a plurality of relatively uniform openings therethrough; said openings being of a capillary size for holding liquid samples therein by capillary attraction; said slab-like form being made of polymerized tetra-fluoro ethylene.
2. A method for determining the concentration of microorganisms in -a liquid which consists in immersing a slab-like form of polymerized tetra-fluoro ethylene having a plurality of capillary size apertures therein into the liquid withdrawing said slab with its apertures lled with the liquid and its surface adjacent said apertures void of said liquid, subjecting it to incubating conditions in order to develop any progeny in the liquid filled apertures and determining the number of progeny so developed.
References Cited in the tile of this patent UNITED STATES PATENTS Lovell s- May 4, 1954