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1957 ii analytical microbiology 25 butterfield c t 1932 the selection of a dilution water selected microorganisms in inorganic media j bacteriol for bacteriological examinations j bacteriol 23 355 67 ...

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        1957]                                                                                         II
                                                      ANALYTICAL MICROBIOLOGY.                                                                      25
        BUTTERFIELD, C. T.       1932  The selection of a dilution water               selected microorganisms in inorganic media.      J. Bacteriol.,
             for bacteriological examinations.     J. Bacteriol., 23, 355-             67, 628-634.
             368.                                                                 Quartermaster Food and Container Institute          1955   Military
        COHEN, B. 1922 Disinfection studies.          The effects of tem-              Specification   Meal, Precooked, Frozen, MIL-M-13966
             perature and hydrogen ion concentration upon the via-                     (QMC).
             bility of Bact. coli and Bact. typhosum in water. J. Bac-            STOKES, J. L. AND OSBORNE, W. W.         1955   Aselenite brilliant
             teriol., 7, 183-230.                                                      green medium for the isolation of Salmonella. Appl.
        DE MELLO, G. C., DANIELSON, I. S., AND KISER, J. S.            1951            Microbiol., 3, 217-220.
             The toxic effect of buffered saline solutions on the via-            STOKES, J. L. AND OSBORNE, W. W.           1956   Effect of the egg
             bility of Brucella abortus.  J. Lab. Clin. Med., 37, 579-583.             shell membrane on bacteria.      Food Research, 21, 264-269.
        FITZGERALD, G. A.       1947   Are frozen foods a public health           WINSLOW, C. E. A. AND BROOKE, 0. R. 1927 The viability
             problem? Am. J. Public Health, 37, 695-701.                               of various species of bacteria in aqueous suspensions.       J.
        GUNTER, S. E. 1954 Factors determining the viability of                        Bacteriol., 13, 235-243.
                                   Microbiological Process Report
                                                     Analytical Microbiology
                                                         II. The Diffusion Methods
                                                                      JOHN J. GAVIN
                                            Food Research Laboratories, Inc., Long Island City, New York
                                                        Received for publication August 9, 1956
         I. PRINCIPLE. In these methods the substance to be                          centration of substance corresponding to this dis-
        assayed is allowed to diffuse through solid, inoculated                      tance is read from a standard curve. The standard
        culture medium. If the substance being assayed is a                          curve is prepared from a similar series of columns of
        bacteriostatic or bactericidal agent, a zone of inhibition                   known concentrations of the substance being as-
        results. If the substance is a growth factor, a zone of                      sayed. This method is seldom used. Some Japanese
        growth [zone of exhibition (Bacharach and Cuthbert-                          workers (Torii et al., 1947) use it routinely, and
        son, 1948)] develops. The size of the zone, either of                        several other investigators have developed work-
        inhibition or growth, is a function of the concentra-                        able methods (Florey et al., 1949; Davis and Parke,
        tion or in certain circumstances, the amount (Heatley,                       1950: Davis et al., 1950). Its main disadvantages are:
         1948) of the substance being assayed. This function                         (1) Must necessarily use a facultative anaerobe.
         can be expressed as a linear relationship between the                       (2) Test solutions must be sterile.
         size of the zone and the logarithm of the concentra-                        (3) Difficulty in observing the end point.
        tion of the substance (Bliss, 1944; Davies, 1945-1946;                       (4) There is a relatively narrow spread of readings
        Bacharach and Cuthbertson, 1948). By measuring the                                of the zone lengths.
        distance     the substance diffuses,          as  evidenced by               (5) The method is cumbersome. There is a lengthy
         growth or lack of growth of the test organism, and                               time period involved in cleaning and setting up
         comparing it with that of a known standard prepara-                              the apparatus.
         tion, the potency of the sample may be calculated.                          One major advantage of this method (Davis and
         II.                                                                         Parke, 1950) is the ability to better define and con-
             TYPES OF DIFFUSION. There are two types of dif-                         trol the geometry of the diffusion system. To gain
        fusion,    vertical    (linear    diffusion)    and horizontal               this one advantage, it is necessary to sacrifice others
         (radial diffusion).                                                         such as time, ease of handling, and so forth. From a
           A. Vertical (linear) diffusion. In vertical diffusion                     practical standpoint, the increase in accuracy by
           methods, a given volume of the solution to be                             better control of diffusion is not worth the sacrifice.
           assayed is placed on top of a column of inoculated                        The literature does not indicate the use of this
           agar and allowed to diffuse down the column. The                          method for the assay of substances other than anti-
           length of the column, in which the inhibition or                          biotics.
           exhibition takes place, is measured and the con-                          B. Horizontal (radial) diffusion. There are several
         26                                                    J. J. GAVIN                                                     [VOL. 5
            methods employed using the horizontal type of dif-                    pipetted   into   the  cylinders.   The cylinders
            fusion.  These may be divided into two groups                         should be filled completely with solution to
            (Heatley, 1948), first, those methods in which the                    avoid error. Grenfell et al. (1947) found an 18
            diameter of the zone of inhibition or growth depends                  per cent range in zone size when they varied
            upon the concentration of the substance being                         the volume of solution in the cylinders from
            tested, and second, those methods in which the zone                   0.1 to 0.3 ml.
            is dependent upon the amount of the substance.                          Care should be taken when moving the plates
              1. Zones dependent upon concentration. Two meth-                    to avoid breaking the seal between the cylinder
              ods are included in this group, the cylinder and                    and the agar, or spilling the solution contained
              the cup plate methods. The two are essentially                      in the cylinder. The advantages of the cylinder
              the same. The substance being assayed diffuses                      plate assay are:
              from a central source. The diameter of the zone                     (1) Solution being assayed need not be sterile.
              of exhibition is measured and, as in vertical                       (2) Adaptable to rapid assay.
              diffusion, compared to a series of standard con-                    (3) More sensitive to low dilutions than the
              centrations of the substance being assayed. In                          paper disc assay.
              the cylinder method the solution is contained                       The disadvantages are:
              within a cylinder. In the cup plate method a de-                   i~1) Extreme care must be taken in the handling
              pression to hold the solution is made in the agar.                      of plates to avoid breaking seal or spilling
                 (a) Cylinder ntethod. The cylinders used in this                     the contents.
                 assay may be of pyrex glass, glazed porcelain,                   (2) Handling,    cleaning,   and sterilizing    the
                 aluminum, or stainless steel. They should have                       cylinders.
                 the following approximate dimensions, an out-                    (3) Filling of cups is slow and tedious.
                 side diameter of 8 i 0.1 mm, an inside diameter                  (4) Suspended material interferes with diffusion
                 of 6 4- 0.1 mm, and a length of 10 i: 0.1 mm,                        of liquid from the cylinder.
                with a thickness not exceeding 1 mm. If made                      (b) Cup plate method. The depression, which is
                 of glass, porcelain, or aluminum, they should                    cut in the agar, can be made in either of two
                be beveled internally. The object of the bevel                    ways.
                is to reduce the area of contact with the agar                    (1) A sterile rubber stopper, test tube, marble,
                 and so increase the weight per unit area of con-                     or something similar, is placed in the molten
                 tact, thus giving a more effective seal (Florey                      agar. When the agar solidifies, the object is
                et al., 1949). The beveled end, which is placed                       removed leaving a depression.
                in contact with the agar, should be flat and                          or
                free from chips or scratches. The bevel is un-                    (2) The agar is allowed to harden and a slug is
                necessary when the heavier stainless steel                            removed by means of a sterile cork borer.
                 cylinders are used. Cylinders may be placed on                       The size of the cork borer is optional. It
                the agar cold or they may be warmed before                            has been found that there is no difference
                being applied. Warming the cylinder will give                         in results using cork borers from 91      to 13
                a more effective seal. Control of the depth                           mm(Harris and Ruger, 1953).
                that the cylinders are imbedded in the agar is                    In using this method, there are several factors
                important. If the cylinders penetrate too far,                    which are important if accurate and repro-
                the zone size is reduced (Brownlee et al., 1948).                 ducible results are to be obtained (Vesterdal,
                Differences in depth between cylinders will                       1946).
                lead to high experimental error as there will be                  These factors are:
                a corresponding variation in zone size. In order                  (1) Cups must be cut vertically.
                to control this factor, the heating of the cylinders              (2) Cups must be completely filled with the
                should be uniform. This can be done by using                          solution being assayed.
                a hot plate.                                                      (3) Optimum thickness of the agar is 5 to 6 mm.
                   The cylinders may be placed on the agar by                     The zones obtained using this method may be
                hand or by the use of automatic dispensing                        sources -of error. If the agar on the edge of the
                machines. Devices for the automatic placement                     cup is lifted away from the glass, a false large
                of cylinders have been described by Reeves and                    zone will result from the seepage of the solu-
                Schmidt (1945), Oswald and Randall (1945),                        tion between the agar and the bottom of the
                Beadle et al. (1945), and Chandler and Shaw                       plate. When a single layer of agar is used, a
                 (1946). When a machine is used for the place-                    double zone effect may occur. If the same edge
                ment of cylinders, they are applied cold.                         of each zone is read, the error will be negligible
                   The standard and sample solutions are                          (Brownlee et al., 1948). The curve obtained
      1957]                                ANALYTICAL MICROBIOLOGY. II                                               27
             with this test and the accuracy of the test are            cylinder or cup, is used as the reservoir for the
             the same as for the cylinder plate method.                 substance being assayed by this method. The
             The advantages of the cup plate method are:                solution to be assayed is placed on the disc
             (1) Handling of cylinders is eliminated.                   either by use of a pipette or loop, or the disc
             (2) It is more sensitive to* low dilutions than            may be dipped into the solution. As indicated
                 is the paper disc assay.                               previously, the major difference between the
             (3) Suspended material does not interfere with             cylinder or cup method and the paper disc
                 diffusion of the solution.                             method is that the amount of substance, rather
             (4) Variation in the size of the cylinder is               than the concentration, determines the size of
                 avoided (Brownlee et al., 1948).                       the zone. This factor allows the discs to be
             (5) It can be used as a screening test for sub-            placed on the seeded agar either wet or dry.
                 stances   elaborated   by   microorganisms             Because of this it is also possible to assay mate-
                 (Harris and Ruger, 1953; Raper et al.,                 rials  containing  organic   solvents  (Stansly,
                 1944; Wilkens and Harris, 1944).                       1952). The disc can be wet with the substance
             (6) It is adaptable to rapid assay.                        to be assayed and then the solvent allowed to
             The disadvantages are:                                     evaporate before the disc is placed on the agar.
             (1) Cutting of cups is a laborious operation.              It has been found that 60 per cent methanol,
             (2) Some care must be taken in handling plates             40 per cent ethanol, 40 per cent acetone, 10
                 to avoid spilling the solution.                        per cent dioxane, or 5 per cent pyridine does
           2. Zones dependent upon amount. Two methods                  not interfere with the assay of streptomycin
           are also included in this group, the drop plate              (Loo et al., 1945). This same factor has been
           method and the paper disc method. These two                  responsible for the success of the commercially
           methods are, in a sense, as similar in comparison            available antibiotic sensitivity discs which are
           with each other as the two methods described                 used for diagnostic purposes. A modification of
           previously. In the drop plate method, a drop of              this method has been used for the differential
           the substance to be tested is placed directly on a           assay of the penicillins. Paper chromatography
           plate and diffuses into the agar. A zone of inhibi-          is used to separate various penicillins and the
           tion or exhibition results. In the paper disc method,        strips are then placed on agar (Glister and
           the drops are indirectly placed on the agar by               Grainger, 1950; Goodall and Levi, 1947). Zones
           means of a filter paper disc, with subsequent dif-           of inhibition appear at intervals along the strip
           fusion and the resultant zones.                              depending upon the location of the particular
             (a) Drop plate method. Either of two techniques            type of penicillin.
             may be employed with this method. The first                  The curve obtained is a straight line and the
             involves placing a drop of the solution to be              accuracy and reproducibility of results are the
             assayed on the surface of a seeded agar plate              same as for cylinder plate and cup plate assays.
             (Forgacs and Kuchera, 1946; Thomas et al.,                   The advantages of the paper disc method
             1944). A loop, a pipette, or a syringe may be              over other diffusion methods are:
             used to apply the drop. This method is more                (1) Simplicity. It does not require the tedious
             adaptable for a qualitative than quantitative                  work of filling cylinders or cutting out
             assay. The second technique is that of White                   plugs of agar.                    handling,
             (1945-1946). It is claimed that by this method             (2) Convenience. It does not require
             a difference of 5 per cent in potency can be                   cleaning and sterilizing of cylinders. This
             determined. The dilutions to be tested are                     decreases the over-all time required for
             made up in broth. Each dilution is inoculated                  assay and allows more samples to be
             with a suitable microorganism. One drop of                     handled during any given period of time.
             each dilution is placed on nutrient agar which             (3) Rapidity. The discs can be handled rapidly.
             is incubated. The results are based on the ap-                 As in (2) above, the over-all time for assay
             pearance   of  the  bacterial growth between                   is  decreased and more samples can be
             dilutions.                                                     handled.                               need
               This method has a "restricted application,               (4) Ease of handling plates. There is no
             being most suitable for the accurate assay of a                for the special handling of assay plates as
             few samples of approximately known potency"                    with other diffusion methods.
             (Heatley, 1948). It is obvious that neither of             (5) Accuracy. There are more consistent zones
             these two methods would be the method of                       of inhibition due to improved diffusion as
             choice, except in some special application.                    there is better contact of disc with agar.
             (b) Paper disc method. The disc, rather than the               Variable diffusion due to leaks and chips
         28                                                  J. J. GAVIN                                                  [VOL. 05
                     and   inconsistently  applied   cylinders   is          (Higgins et al., 1953; Kersey and Leghorn, 1953;
                     avoided.                                                Larkin and Stuckey, 1951) have had better re-
                 (6) Ability to assay solutions containing rela-             sults with thinner layers.
                     tively high concentrations of organic sol-                It is not important that an exact numerical
                     vents. High and erratic results are obtained            value is obtained for the depth of an optimum
                     with other methods.                                     layer, but rather there is need for a constant
                 (7) Only small amounts of solution are neces-               thickness for a given series of plates or assays. To
                     sary to saturate the discs. This advantage is           obtain a constant thickness it is necessary to
                     valuable when a limited sample of low con-              control the volume of agar added to each plate
                     centration is assayed. [Direct assay of blood           and select dishes that have flat bottoms and are
                     (Sherwood and de Beer, 1947).]                          of uniform size. Many types of containers have
                   The disadvantage of the paper disc method is              been used: Petri dishes, flat bottom baking dishes
                 that it is less sensitive to low dilutions than             (Epstein et al., 1944), flat pyrex plates with pyrex
                either the cylinder or cup plate method.                     rims (Kersey and Leghorn, 1953) and window
         III. FACTORS THAT INFLUENCE DIFFUSION ASSAYS. In                    glass, with wooden or aluminum rims (Brownlee
         any assay method, there are factors which are peculiar              et al., 1948). It is advantageous to obtain as many
         to that type of assay. Control of these factors will re-            zones as possible from one plate. Environmental
         sult in accurate and reproducible assays. The factors               conditions are more apt to be similar for an assay
         to be discussed are the main conditions that need                   conducted on one plate than for an assay using
          control in the diffusion type assay. There are un-                 several plates. This was statistically proved by
         doubtedly many others, for each substance to be                     Brownlee et al. (1948) using large plates contain-
         assayed, and the form in which it is available, pre-                ing 64 cups.
         sents new and different problems. Some of these                     2. Dryness of agar. Insufficient dryness of the
         factors are applicable only to the diffusion assay,                 agar may cause streakiness and indistinct, ob-
         while others might have broader application.                        scure zones. Some workers feel that drying is
            Although the lack of control on some of these con-              essential while others have dispensed with drying
         ditions will lead to significant errors, variation of a            techniques and have obtained excellent results.
         factor or factors could improve an assay. For example,             Partial drying is desirable if streakiness is to be
         decrease in depth of agar increases the sensitivity. This          avoided (Florey et al., 1949). While drying the
         might be a desirable effect.                                       plates, care should be taken to avoid aerial con-
            A. Agar. The diffusion assay is fundamentally de-               tamination. This may be done by covering the
            pendent upon the diffusion of the substance to be               plates with several thicknesses of cheesecloth
            assayed through the agar. Thus the agar becomes                  (Florey et al., 1949), using porcelain covers, or by
            one of the most important factors in this type of               inverting the plates. Drying may be done by
            assay. In the following discussion, the nutrient value          leaving the plates at room temperature or by plac-
            of the agar, which is considered elsewhere to a                 ing them in an incubator at elevated tempera-
            greater extent, is not evaluated. The concern here              tures. The length of time required is a function of
            is for those factors which materially affect the                temperature and humidity, but the operator's
            physical diffusion through the agar of the substance            experience is the best guide as to the extent the
            to be assayed.                                                  plates must be dried before using. Plates may be
              1. Depth of the agar. Agar has been placed in con-            stored in a refrigerator for several days after
              tainers in a variety of depths ranging from the               seeding, with no adverse effects.
              "microfilm" method of Forgacsand Kuchera (1946)               3. Amount of agar. There are reports in the litera-
              to the 8-mm thickness layer of Hayes (1945). A                ture that the sensitivity of the diffusion method
              thin layer will give larger zones than thicker                can be increased by decreasing the agar content
              layers  thus increasing the sensitivity of the                of the medium. The results of Grady and Wil-
              assay. Most authors recommend a layer of 3 to                 liams (1953) are difficult to interpret in this re-
              5 mm. In a statistical analysis of this problem, de           gard, as they varied the depth of the agar at the
              Beer and Sherwxood (1945) found that the best                 same time that they varied the concentration of
              results were found with layers of 2 to 4 mm. The              agar. It is probable that the amount of agar used
              analysis  of variance indicated that extremely                will affect the assay. This would be dependent,
              thick or thin plates should not be used as the log            however, on the quality and origin of the agar.
              dose-response   curve obtained may not be a                 B. pH of agar and assay solutions. The prime requi-
              straight  line. Hayes (1945) in his statistical             site for the pH of the agar is that the value should
              evaluation, indicated that an 8-mm layer of agar            be in the ranges that are suitable for the optimum
              gave the most reproducible results. Other workers           growth of the test organism. On the other hand, the
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...Ii analytical microbiology butterfield c t the selection of a dilution water selected microorganisms in inorganic media j bacteriol for bacteriological examinations quartermaster food and container institute military cohen b disinfection studies effects tem specification meal precooked frozen mil m perature hydrogen ion concentration upon via qmc bility bact coli typhosum bac stokes l osborne w aselenite brilliant teriol green medium isolation salmonella appl de mello g danielson i s kiser microbiol toxic effect buffered saline solutions on egg brucella abortus lab clin med shell membrane bacteria research fitzgerald are foods public health winslow e brooke r viability problem am various species aqueous suspensions gunter factors determining microbiological process report diffusion methods john gavin laboratories inc long island city new york received publication august principle these substance to be centration corresponding this dis assayed is allowed diffuse through solid inoculated...

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