Tomato pulp furnishes a medium suitable for the development of many
organisms, as it contains all of the necessary food elements. The raw
pulp has an acidity of from 0.2 to 0.4 per cent usually, though there
may be variation due to fermentation and other causes. On account of its
mild acidity, it is especially suitable for the development of many
yeasts and molds, and some forms of bacteria, consequently there is
present a varied and abundant flora if the pulp be held for an
appreciable time before using, or if it has been made from tomatoes not
properly sorted and washed. Where the black rot occurs on tomatoes, the
tissue is hardened like cork, and if not removed on the sorting belt, is
broken into small pieces by the cyclone, and appears as black specks in
the ketchup, these being readily perceived by the naked eye. The white
rot forms soft spots, which, though not so prominent as the black, carry
much more contamination, as, apart from the bacteria, yeasts, and molds
present, they are often swarming with Protozoa. These are not ordinarily
recognized in the ketchup, as a chemical or physical shock causes them
to contract, assume a spherical shape, and become motionless. In this
condition they resemble the immature conidia of some of the molds.
Rarely only one organism predominates in pulp from rotted fruit, then
the rot consisting of a nearly pure culture. In all cases of soft rot,
there is much more contamination carried, as the organisms are small and
a greater number present in a given area. Whenever the inner tissue of
tomatoes is exposed, organisms develop rapidly, the forms varying with
the locality and the conditions in the pulp. Some of these organisms may
survive the treatment of the pulp when converted into ketchup, or the
original organisms may be destroyed, and a different set gain access and
develop, but in either event all the organisms alive or dead which were
present at the period of manufacture are found in the ketchup. It has
been noted that certain brands of ketchup have predominating organisms
present which are practically constant from year to year.
A method for the microscopic examination of ketchup in order to
determine the number of organisms present is described in Circular No.
68, Bureau of Chemistry. It consists in an adaptation of a method used
in examining blood in physiological and pathological work, and of yeast
in the brewing, wine-making, and distilling industries. The outfit
required consists of two parts, the microscope and the counting chamber,
each with minor accessories. The optical outfit recommended for food
examination consists of a microscope with eye pieces and objectives
which will give approximate magnifications of 90, 180, and 500
diameters. It is advised that these magnifications be obtained by using
16 mm and 8 mm apochromatic objectives, and ×6 and ×18 compensating
oculars (×6 ocular and 16 mm objective equals ×90; ×6 ocular and 8 mm
objective equals ×180; and ×18 ocular and 8 mm objective equals ×500),
higher objectives being impracticable on account of their short working
distances. This equipment is adequate for working upon blood or yeast,
but is wholly inadequate for bacteriological work, except that of the
simplest character and under conditions quite different from those found
in ketchup and other food products.
The counting apparatus or chamber recommended is known as the
Thoma-Zeiss haemacytometer, named from the designer and maker. The
apparatus consists of a heavy glass slip, on which is cemented a glass
0.2 mm thick, having a circular hole in the middle. In the center of the
hole is mounted a smaller disk 0.1 mm thick, leaving an annular space.
In the middle of the small inner disk are etched two sets of twenty-one
parallel lines which cut each other at right angles. The drop of liquid
to be examined is placed on this square, after which it is covered with
a specially heavy cover-glass, which, if perfect and adjusted so closely
that Newton’s rings appear, gives a layer of liquid 0.1 mm in depth. The
drop to be examined must be so small that it remains in the middle of
the chamber, but in contact with the cover-glass and bottom of the cell.
Each side of the ruled square is 0.1 mm, and as there are 20 spaces on a
side, there is a total of 400 small squares, the depth being 0.1 mm,
thus the cubical content of each is 1-4,000 c mm or 1-4,000,000 cc. For
convenience in counting, every fifth space is sub-divided. Other
counting chambers have been devised based on the same principle, but
varying chiefly in their rulings for convenience in counting.
The other apparatus recommended consists of a 50 cc graduated cylinder,
slides, and cover-glasses.
Since the counting chamber has been used extensively in blood
examination and in yeast work, a brief description of the technique as
followed in the latter may serve to give a better understanding of its
limitations. First, in the preparation of the sample, the cylinder and
flasks for mixing, and the pipette must be absolutely clean. The liquid
to be examined is shaken thoroughly and then the measured sample
withdrawn as quickly as possible to prevent the cells from settling and
diluted with weak sulphuric acid (about 10 per cent), which prevents any
further development of cells, and also aids both in the separation of
the cells from one another and in their suspension—the latter factor
being important when only a single drop is taken for examination. When
counting blood cells, a normal or other salt solution is used so as to
have the specific gravity of the diluent approximately that of the blood
serum. The dilution is made as low as possible, since the number
obtained in the count has to be multiplied by the dilution co-efficient,
and any errors made are increased proportionately. A slight error when
multiplied by the factor 4,000,000, the unit for each square, becomes
very large in the total. The sample is shaken very thoroughly after the
diluent is added, a drop of the liquid taken by means of a pipette,
placed in the center of the counting chamber, and the cover-glass put in
place. The withdrawal of the pipette and the transference of the drop to
the chamber are done as quickly as possible to prevent the cells from
sinking. The determination of the number of blood corpuscles, yeasts, or
other cells in one cubic centimeter, the unit of volume generally used,
will depend upon the average found in a number of squares. The number of
squares to be counted is determined by making counts until a constant
average is obtained, for if a true average is not obtained, the
counting, naturally, is of no value. If the mounts do not show
uniformity in the field, they are repeated.
In using the counting chamber for counting yeast cells and blood
corpuscles, for which it was originally devised, the bodies to be
examined are fairly large, well defined, and suspended in a fairly clear
liquid, usually of rather high specific gravity. Even with these
favorable conditions, the work must be done by observing the most
careful technique in order to get relative results, which will be of
value, and they are absolutely useless if any detail has been slighted
or neglected. In attempting to adapt the method to food products, very
different conditions are encountered—conditions which are opposed to
obtaining accurate results. Food products, like ketchup, consist of a
mixture of solids and liquids in which are various forms of organisms,
the latter in varying condition, due to their environment and treatment,
as well as to stages of disorganization.
In estimating the number of yeasts and spores in pulp or ketchup, the
Thoma-Zeiss counting chamber is used and the mount observed under a
magnification of 180 diameters. To prepare the sample, 10 cc of the
material has 20 cc of water added and is “thoroughly mixed.” Before
taking a drop for examination, the sample is allowed to rest for a
“moment” to allow the “coarsest particles” to settle. This step in the
technique is not as clear as could be desired, for what might be
considered as “thoroughly mixed” by one microscopist as a half dozen
shakings of the cylinder, might not be so construed by another even with
sixty shakings. As the material consists of both solids and liquid, this
is a very important detail, as it may easily account for some of the
wide differences in results obtained by different workers on the same
sample. In a bulletin[2] dealing with the examination of solid foods,
the following statement occurs relative to the shaking in order to be
able to obtain the bacterial condition: “The longer the shaking, the
more perfect was the diffusion of particles. It could not, however, be
continued beyond a comparatively short period of time, because of the
multiplication of organisms. With the quantities of tissue above stated,
ten minutes’ shaking was selected as a happy medium between an
undesirable multiplication of the organisms on the one hand and the
retention of the organisms by the tissue and the consequent lowering of
the numbers found, on the other.” The organisms in pulp or ketchup are
dead, or, if alive, do not possess such phenomenal power of
multiplication, therefore, the shaking should be conducted with
sufficient energy and for a sufficient time to insure their separation
from the tissue. Furthermore, “letting stand for a moment” may mean
thirty seconds or two or three minutes to different persons.
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Footnote 2:
No. 115—Bureau of Chemistry, Dept. of Agr.
In all biological work involving the counting of organisms, either by
the plate or direct method, in the case of yeast, the operator works as
rapidly as possible to prevent the organisms from settling, so as to
have them evenly distributed in order that he may obtain an average
sample. A pipette is used for removal of a drop of the liquid and the
drop placed in the chamber as quickly as possible to prevent settling.
No directions are given as to how the drop of the diluted pulp or
ketchup is to be removed to the chamber, so that a stirring rod or other
apparatus is frequently used, as the solid particles interfere with the
use of a fine pipette. If the rod be inserted to the bottom, or nearly
to the bottom of the mixture and withdrawn slowly and another withdrawn
somewhat rapidly, a difference of fifty per cent or even more may result
in the count. It is not possible for different operators to use
pipettes, glass rods, pen knives, toothpicks, and matches for drawing
the samples, and get comparable results. It has been found that in (all
of these have been seen in use) the counting of the organisms in pulp
and ketchup, some persons use distilled water, others tap water, some
clean their measuring flasks and pipettes, while others rinse them, so
that naturally reports are made of such varying numbers that
manufacturers do not look upon the method with confidence. It is only by
using uniform methods and the same care necessary for other biological
work that even an approximation can be made.