To obtain the number of yeasts and spores in the sample, a count is made
in one-half of the ruled squares. Two hundred squares represent a volume
equivalent to 1-20 c mm, which, multiplied by the dilution, would give
the number in 1-60 c mm. It is stated that it is believed that it is
possible for manufacturers to keep the count below 25 per 1-60 c mm.
The same mount is used in estimating the bacteria, but the ×18 ocular
used so as to increase the magnification to approximately 500 diameters.
The “number in several areas, each consisting of five of the small
squares, is counted.” Nothing is said as to the order of the five
squares, whether in a row or other arrangement, nor what number
constitutes “several.” The average number found in five squares
represents the number in 1-800,000 part of a cc, and this multiplied by
3, for the dilution, would make the factor 1-2,400,000 for a cc. It is
stated that it is believed that it is possible for manufacturers to keep
within 12,500,000 bacteria per cc in the pulp and 25,000,000 in ketchup.
The number present is expressed in terms per cc though the yeast and
spores are expressed in 1-60 c mm. Possibly bacteria to the lay mind
mean something dangerous, so by expressing the numbers in millions they
appear appalling. Yeasts and spores are not so generally associated with
dirt and disease so that by giving them a small unit, only 1-60,000 part
of a cc, they may seem much less offensive. If the mind is capable of
conceiving what is meant by millions per cc for bacteria in one case,
there seems to be no good reason why the same unit of volume should not
hold for the other.
To estimate the number of molds present, a drop of the undiluted pulp or
ketchup is placed on an ordinary slide and the ordinary cover-glass
pressed down until a film of 0.1 mm is obtained. The directions state
that after some experience this can be done, but do not state how one’s
efforts may be directed to obtain this result. It is apparent that by
experience in comparing a measured amount with a judged amount that the
tendency would be toward accuracy, but in this case there is no measured
amount for comparison, except the diluted drop in the counting chamber.
Some workers have placed thin cover-glasses under the edges of the mount
so as to have something to help in estimating the thickness of the film,
but as the thinnest ordinary cover-glasses vary from .12 to .17 mm in
thickness, the error varies 20 to 70 per cent from that required. One
manufacturer in advertising No. 1 cover-glasses states that they vary
from 0.13 to 0.17 mm, while another states they vary from 1-200 to 1-150
of an inch (0.127 to 0.169 mm). Careful checks show that it is not
always easy to get exactly .1 mm on the specially prepared counting
chamber; that unless the cover be placed with care and pressed uniformly
on all sides until Newton’s rings appear, a variation of ten per cent or
more in thickness may occur, and without such a guide the error becomes
greater. The micrometer screw adjustment on the microscope can be used
to help in determining the thickness, but none of the workers observed
has used this refinement.
The examination for mold is made with the ×6 ocular and 16 mm objective,
giving a magnification of approximately 90 times. About 50 fields are
supposed to be examined and the result expressed in terms of the per
cent in which mold was found. It is stated that it is believed that
manufacturers can conduct their operations so that mold will not be
present in more than 25 per cent of the fields. There are, therefore,
three units in which to express the results: bacteria in cubic
centimeters, yeasts and spores in one-sixtieth of a cubic millimeter,
and molds in percentage of microscopic fields.
Aside from the errors which may occur in the manipulation of the purely
mechanical part of the technique, there are other considerations which
affect the accuracy of the results. First, the differentiation between
organisms and tissues is not considered possible by most pathologists
and bacteriologists without differential staining. Even in such simple
examinations as those for diphtheria and tuberculosis, a stain is
required. In foods the particles of the plant tissue and the organisms
are not so different that they can be clearly separated without using
similar technique. It is possible to make some separation, but not with
accuracy. Threads of protoplasm may be mistaken for bacilli; the
granular contents of a cell for cocci, yeasts, or spores; bits of cell
wall for hyphae under the magnifications given, and the results obtained
be high or low, depending upon the personal ability of the operator.
Each error magnified by the enormous factor used in calculating the
final result naturally gives figures which may be far above or below the
truth. Those who have had special training in plant structure and
bacteriology are likely to give the higher figures, while those who have
had these subjects as incidentals in a scientific course are apt to give
much lower ones.
Second. The standard is set for what organisms shall be counted and
those which need not be. It is said that micrococci need not be counted
because of the difficulty in distinguishing them from “particles of
clay, etc.,” and not upon their power to produce decomposition. When an
organism is a coccus and when rod shaped is not easily settled, even
with the aid of pure cultures and high power objectives. More than one
organism has found a home first in one group and then in the other, and
differentiation with the low power obtained by an 8 mm objective is
impossible. There are always present some very large rods, but there may
be more very short ones which may not be counted, and there is nearly
always a diplococcus present, which, with the magnification used, is
difficult to differentiate from a rod. There are four forms associated
with rot and tomato diseases which have been carefully studied—all rods,
but very small ones. Ps. fluorescence, 0.68×1.17-1.86; Ps. michiganense,
0.35-0.4×0.8-1.0; B. carotovorus, 0.7-1.0×1.5-5; and B. solanacearum,
0.5×1.5. Bacillus subtilis, .7×2-8 and some lactic acid forming
varieties are always present. It is clearly a matter of judgment on the
part of the examiner as to which organisms he will count and which he
will not attempt to count. A personal equation is thus introduced which
nullifies the possibilities of scientific accuracy.
The yeasts and spores are counted together. They can not be separated
under the microscope, neither can they be differentiated from contracted
protozoa which may be present in large numbers. In counting these, it is
not always possible to distinguish the smaller yeast cells and smaller
spores from the refractive bodies which are formed in some mold hyphae
when these are impoverished, and which are liberated if thorough shaking
of the sample be done. The yeasts found in pulp and ketchup are more
likely to be “wild yeasts” and these are, as a general thing, smaller
than the cultivated, sporulate more readily, and have more highly
refractive spores. Then, some of the so-called molds found form minute
conidia and when these and the yeasts are mixed with the detritus of the
tomato and the mass subjected to heat, with the consequent changes, the
accuracy of the count becomes a somewhat problematical matter. A careful
examination of the kind and condition of the hyphae present might assist
materially in making some distinction.
In counting molds, no distinction is made as to whether a small bit is
in the field or a large mass. In making a mount for molds, the solids
generally tend to stay in the center of the field while the liquid tends
to run to the edge. The fields selected may therefore give a high or low
result determined by their location. One examiner desiring to favor the
manufacturer may select the outer part for most of the fields, while
another, making the examination for the buyer, who may wish to make a
rejection, may reverse the operation. Some persons modify the directions
given by counting only pieces which are one-sixth the diameter of the
field, while others use a smaller fraction. It is easily possible to
have one clump of mold in one field which will be twenty to thirty times
in extent that of another, yet both are given equal value in the final
expression.
Third. No real relation exists between the organisms counted and
decomposition, for mere numbers are not always coincident with
putrefactive activity. A pulp or ketchup may be bad and show less than
30,000,000 bacteria, or it may be good and show 300,000,000. Rotting, or
decomposition, may depend more upon the cocci and the organisms which
are not counted than upon those which are. The only work done in which
microscopical and chemical work were reported on the same samples
appears in Circular No. 78, Bureau of Chemistry. This was not done upon
samples prepared and kept under control, but for the most part upon
commercial pulp and ketchup. The results do not show any close relation
between the number of organisms and the lactic acid content which is
given as the measure of decomposition.
Fourth. Bacteria are expressed in numbers per cc, yeast and spores in
numbers per 1-60 c mm. Since the counting can be done only in the fluid
portion, an error occurs proportional to the number of bacteria in or
attached to the tissue which cannot be counted.
The error of assuming that numbers of organisms alone are a sufficient
index of the wholesomeness of a food product is well illustrated by work
on water analysis. The following statement by an authority on the
subject is illuminative: “The belief is widespread among the general
public that the sanitary character of a water can be estimated pretty
directly by the number of bacteria it contains. Taken by itself,
however, it must be admitted that the number of colonies which develop
when a given sample of water is plated affords no sure basis for judging
its potability. A pure spring water containing at the outset less than
100 bacteria per cubic centimeter may come to contain tens of thousands
per cubic centimeter within twenty-four to forty-eight hours, after
standing in a clean glass flask at a fairly low temperature. There is no
reason for supposing that the wholesomeness of the water has been
impaired in any degree by this multiplication of bacteria.”[3]
-----
Footnote 3:
Jordan, E. O. A text-book of General Bacteriology. 1908.
There are certain steps in the process of manufacture which also
influence the number of organisms which may be counted. A pulp may vary
from an unevaporated tomato juice to a concentration which is
represented by an evaporation of a volume of water up to 60 per cent,
and ketchup may vary from a thin watery consistency to one which is so
heavy that it will scarcely flow from the bottle. It becomes evident
that a method which does not sustain some close relation to the amount
of tomato present would naturally be deficient as a standard for
judging. For example, a tomato juice with an initial count of 10,000,000
if evaporated to one-half its volume will have more than twice the
number of organisms estimated in the original. The pulp is composed of
both liquid and solids and part of the liquid portion only is driven off
by evaporation, leaving in the residue a different proportion to the
solids. As the organisms can be counted only in the liquid portion, it
is obvious that with concentration, the number will be increased at a
much greater ratio than will the reduction of the bulk. A thin pulp with
10,000,000 bacteria may easily be worse than a heavier one with
30,000,000 or 40,000,000, if judged by numbers alone. The same
conclusion is necessarily true for ketchup. It clearly refutes the
argument that a product having twice as many bacteria as another of the
same kind is more than twice as bad. The effect of recommending an
arbitrary low limit for bacterial content, irrespective of the
consistency of the product, is to cause manufacturers to pack thin pulp
and sloppy ketchup, and to discourage the more desirable heavy body. The
examination of a very large number of samples shows that the majority of
the heavy pulps and ketchup upon the market show much higher counts than
the thin ones when the tissues show good stock in both.
It is not possible to concentrate any pulp to the consistency of paste
and have it pass under the present method; that is, considering a
product to be filthy, putrid or decomposed if the bacteria exceed
25,000,000 per cubic centimeter.
There are some soup and ketchup manufacturers who still follow the
draining method for separation and this is generally done to secure a
certain quality in the flavor. This kind of pulp always shows a high
bacterial count, which is usually ascribed to fermentation. As the
draining can be started in about twenty minutes, and is nearly always
completed in forty minutes to one hour, there is little time for
fermentation, and yet such a pulp may show several times the count of
the original whole pulp. The condition is similar to that which takes
place in the separation of cream by gravity. Dr. John F. Anderson, U. S.
Public Health Service,[4] has shown that the bacterial content of
gravity cream is about sixteen times that of bottom milk and that this
discrepancy may be much wider. One test is given in which the cream
showed 386 times as many organisms as the bottom milk. The question
logically arises whether, if a pulp which contains 10,000,000 bacteria
per cubic centimeter and is considered sound, becomes “filthy, putrid or
decomposed” when the same pulp is heavily concentrated and the count
becomes 100,000,000, or a cream is bad when it contains 2,000,000,
though the whole milk from which it was derived contained only 300,000.
There should be a recognized difference in rating a product in which the
number of organisms is influenced by concentration, and one in which
they have developed. Some very erroneous statements have been made upon
increase of bacteria in pulp while standing. Some of these have been
based upon the academic proposition that reproduction in bacteria may
occur every twenty minutes under perfect conditions of food supply,
freedom of movement, and optimum temperature. Such statements are
obviously not based on experiments with pulp. Assuming that such a rate
of reproduction were possible, a pulp with an initial start of only
5,000,000 would increase to 10,000,000 in twenty minutes; 20,000,000 in
forty minutes; 40,000,000 in one hour; 80,000,000 in one hour and twenty
minutes; 160,000,000 in one hour and forty minutes; 320,000,000 in two
hours; and 2,560,000,000 in three hours. No food product like tomato
pulp, cider, or grape juice would be usable in a very short time. To
determine the rate of increase of the organisms in tomato pulp,
experiments were made, using sound tomatoes. In each experiment, the
tomatoes were divided into two lots, one lot used raw, the other
steamed, the steaming varying from two minutes’ time, just sufficient to
slip the skins, and eight minutes, in which the whole tomato is
softened. Samples were taken at hourly intervals for the first six
hours, then at intervals of twelve hours, the samples counted by means
of the plate and direct methods. For the plates tomato gelatin was used
with an acidity of 0.3% and 0.4%, the samples for the direct count were
put in cans, sterilized, and counted later. With the lower acidity there
were liquifiers which prevented the counting of some plates, so that in
the later trials the higher acidity gelatin was used. The count of the
molds was not normal, due to the frequent stirrings, which prevented
spore formation, besides injuring the hyphae.
-----
Footnote 4:
The Journal of Infectious Diseases. 1909. Vol. 6, p. 393.
The results varied, some pulps giving a much higher initial count than
others, but they all agreed in having a comparatively slight increase in
the first three hours, the large numbers which one is led to expect not
being present until the pulp had stood for at least five hours and under
the most favorable conditions; usually it requires a longer time. The
plates and the direct count agreeing in the general trend, though the
numbers obtained by the two methods varied. In the pulp obtained from
the steamed tomatoes, the initial count was much lower in the tomatoes
steamed eight minutes, being only 20 per cc in the plates, but the same
thing was true of these in that the increase was very slow at first. The
figures from all the trials, both raw and steamed pulp, and from the
plates and direct counts, show that the theoretical estimation of the
increase of organisms from the classic twenty minutes required for
reproduction of an organism with the consequent progression,
irrespective of the condition of the organism at the start, or its
environment, will have to be modified. In the plates all colonies, aside
from the molds, were counted as bacteria, but this would not give a very
large error, as yeast does not reproduce at the same rate as do
bacteria.
The state of comminution of the product determines to a considerable
extent the number of organisms which may be counted. The more finely the
comminution, the greater the number. Two pulps made from the same
material, one run through an ordinary cyclone and the other through a
finishing machine, will show from 50 to 100 per cent more in the latter.
Coarse pulp and coarse ketchup may be inferior articles and yet give the
better results by the direct method. The effect on the mold is even more
marked—filaments and clumps will be torn into many small particles. The
total quantity is not increased, but it is distributed more nearly
perfectly and thus occurs in more fields.
In work done on meat to determine the technique which should be employed
in the bacteriological analysis, comparison was made between shaking the
sample and grinding it in a mortar with sand. In the three samples
reported, the shaking gave only 3, 12, and 13 per cent, respectively, of
those obtained from grinding.[5]
-----
Footnote 5:
Weinzirl, John and Newton, E. B. American Journal of Public Health.
Vol. IV, No. 5.
A finely comminuted pulp was vigorously shaken for definite times and
samples taken as quickly as possible after the tenth, fiftieth, one
hundredth, and two hundredth times shaken. The results were as follows:
Mold
Yeast and in Per
No. Times Bacteria Spores Per Cent of
No. Shaken. Per c.c. 1-60 c.c. Fields.
1 10 31,020,000 22 80
2 50 50,040,000 42 76
3 100 84,730,000 106 92
4 200 116,640,000 116 100
In line with this are the results obtained before and after shipping
long distances. When the goods have been handled roughly during shipping
the count is much higher.
The length of time elapsing after manufacture until the counting is done
also has an effect. Pulp put up in the fall will show one count and the
same pulp the following season a different count. This difference is not
due to any multiplication during storage, but to the fact that the
organisms separate from the tissues more readily. The difference made in
the counting from this treatment is not as marked as that produced by
the other factors already treated, but is sufficient to cause a change
in the count.
It is known that the surface of plants is covered by a variety of
bacteria and other fungi that remain dormant under unfavorable
conditions, but that these become active when the food which is
invariably present is rendered available by access of moisture, either
dew or rain, or the rupture of the host, etc. These will vary in numbers
with the season, wet or dry, hot or cold, in different sections of the
country, and, in the case of the tomato, with the variety of the fruit;
whether perfectly smooth or with a slight bloom; whether irregular or
regular in shape; and whether slightly green with a firm skin or fully
ripe. These are all factors that have an influence and should not be
overlooked. Some packers have already learned that by packing tomatoes
which are colored, but not really ripe, that the count will be lower,
and as such a practice extends, it means the use of poorer material
instead of that which is properly developed and with the normal flavor.
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● Transcriber’s Notes:
○ Typographical errors were silently corrected.
○ Inconsistent spelling and hyphenation were made consistent only
when a predominant form was found in this book.
○ Text in bold is enclosed by “equal” signs (=bold=).