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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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