A discussion of the microscopic appearance of ketchup in terms which can be readily understood by manufacturers is not an easy task, as it necessarily involves technical knowledge. The subject has become one of importance, owing to the attitude of many food officials in enforcing a microscopic standard for this product, and on the part of many brokers in requiring a guarantee to comply with this standard in making purchases. Many manufacturers have either assumed or found it necessary to have their finished products examined. Some employ “experts” to make the examinations in their own plants, while the majority send their samples to commercial laboratories. The total tax upon the industry for such work amounts to thousands of dollars annually. The result of the work as a whole has been beneficial, as any effort is which attracts attention to details. It has likewise been the means of causing much unpleasantness and not infrequently loss, because of lack of understanding on the part of both manufacturer and examiner as to the cause of certain findings. The manufacturers have proceeded in the usual way without sufficient knowledge of what the resultant product will be unless there is careful supervision of material and methods, while too frequently the examiner is neither experienced in technique of the examination nor in the effects of the different steps in manufacture upon the product. Furthermore, much distrust in microscopic finding is evinced when a half dozen or more samples from the same batch, sent to as many persons, result in as many different reports. It naturally causes a lack of confidence in both paid examiners and in food officials, though those who make these examinations may be absolutely honest in their findings. In order to clarify some of the points, it has become necessary to go into detail, into both the method of examination and into the effect produced by manufacture.

A scientific method of food examination is necessary for food officials in order to determine the condition of a product, but is not necessary for the manufacturer, though it may be advantageous. The latter is in a position to know what enters his factory and what changes take place in the food until it reaches the sealed package. He should have no fear of a method which correlates the findings in the finished product with that of the material used and the changes due to treatment.

Undue importance may seemingly be given to the subject of ketchup, but the principle involved applies as well to other products.

The fundamental basis for the microscopic examination of any food product must depend upon the structure of the material which enters into its composition. Any attempt to determine an abnormal condition, such as decomposition, without a knowledge of the normal, must necessarily be of little value. There is some work which can be done in a mechanical manner by almost anyone capable of looking through a microscope, and if the work is properly supervised, it may have a value, but the lines along which this can be done are very limited. Any attempt to apply such superficial methods to the general examination of food products can not properly protect the public and may be unfair to the producer. It has, therefore, been deemed advisable to incorporate a brief statement concerning the structure of the tomato before discussing the resultant products.

HISTOLOGY OF THE TOMATO AND OF THE RESULTING KETCHUP.

STRUCTURE OF THE TOMATO.

Pericarp. The tomato is a typical berry, the ovary wall, free from the calyx, forming the fleshy pericarp, which encloses chambers filled with a clear matrix, containing the seeds. The pericarp consists of an outer tough membrane, the epidermis, a more or less thick layer of parenchyma tissue, the pulp, and an inner thin, delicate membrane, the lining layer of the loculi or chambers in which are the seeds. The epidermis consists of a single layer of cells which have a very thick continuous cuticle about one-half of the diameter of the whole cell. The cuticle differs in chemical composition from the rest of the cell walls, being impervious to water, and resisting rotting longer than do the cellulose walls. As it is continuous over the whole of the fruit, the skin can be readily separated from the other tissues. Hot water facilitates the removal of the skin, as it causes the cellulose of the walls to swell more than the cuticle, producing an effect as of shrinkage of the outer wall and a consequent curling of the skin. The radial walls of the epidermis are short and irregularly thickened, leaving pits in the walls, and giving them a beaded appearance. The skin constitutes about 1.3 per cent of the tomato.

The layers of parenchyma just beneath the epidermis are closely united and flattened, with their adjoining walls irregularly thickened. On account of their position, they are called hypoderm. In the tomato the hypoderm consists of two or three layers of cells, parts of which usually separate with the epidermis. Below these cells are the thin-walled parenchyma cells, which are approximately globular, vary considerably in size, are very loosely held together, and have many intercellular spaces. These cells constitute the mass of the pulp, and with the juice constitute 96.2 per cent of the tomato.

The layer of cells which lines the chambers has the typical leaf epidermal structure, the wavy outlines, the hollows and protuberances of adjoining cells fitting one another so that they form a continuous layer. They are also flattened laterally. The structure can be understood readily when it is known that the pericarp is really a metamorphosed leaf and that the outer side of the leaf forms the inner wall of the ovary.

The chambers of the tomato are filled with a clear, slimy matrix in which the seeds are embedded. The matrix consists of parenchyma cells of various sizes and with delicate walls, and a small nucleus. The cells are massed loosely, and can be separated readily. In those cells, as well as in the wall cells, are starch grains which vary in size, being round or approximately so, and having the hilum, when visible, a straight line to one side of the center.

Coloring Matters. In the parenchyma cells are two coloring matters, one yellow, which is amorphous in structure, and the other red and of crystalline form. The sap contains a yellow color in solution which differs in its reactions from those in the pulp.

Red Color in Tomatoes. The red coloring matter in tomatoes is in the form of irregularly shaped crystal-like chromoplasts, which occur in masses of various sizes. They are present in largest amounts usually in the protoplasm which lies close to the ectoplasm and in that surrounding the nucleus. They vary from sharp, bright-colored forms to those more or less blunt in outline, and dull in color. They may be situated largely in the periderm, the soft parenchyma beneath the periderm, or through the whole mass of the parenchyma with the exception of the matrix surrounding the seeds in the loculi. In tomatoes having the color in the periderm a considerable amount is lost by adherence to the skin. The chromoplasts are not affected by rotting to the same extent as are the other constituents of the cell; they can be found floating free in the debris from rotted cells, still retaining considerable color. They lose their color gradually, in some varieties much more rapidly than in others. In stored pulp which has fermented, the color may be faded to a dull yellowish brown. In tomatoes intended for ketchup where a bright red color is desirable, care should be used in the selection of a variety having the chromoplasts bright, properly oriented, and in sufficient quantity.

Vascular Bundles. In the pulp of the tomato are found strands of vascular tissue, entering from the stem, and dividing and ramifying through the soft pulp. These consist of long tubes with thin walls, some of which have a strengthening band in spiral form on their interior walls, the associated cells being without any special marking. The strands vary in size from those having a few tubes to those having a large number.

Seeds. The seeds of the tomato are small, flattened, yellow bodies covered by a clear gelatinous membrane. Their peculiar characteristic is the out-growth of hairs of varying lengths. The seeds constitute about 2.5 per cent of the weight of the tomato.

STRUCTURE OF KETCHUP.

Although the tomato pulp is broken into fine particles by the action of the cyclone, and the skin and seeds are removed by the fine sieves, pieces of the various tissues can be readily identified. The skin and seeds have characteristics which would serve to distinguish them from similar parts of other vegetables which might be used for adulteration, but particles of skin and hairs from the seeds are rarely found. The distinctive features which can be relied upon are the red, irregularly-shaped, chromoplastic bodies in the parenchyma cells, and the peculiar wavy-outlined cells of the lining layer of the chambers. As nearly all young vegetable tissues have spiral vessels in their vascular strands, these are not distinctive, except that they might differentiate similar tissues of different size. There is very little starch in mature tomatoes, and moreover, as the cooking causes the starch to swell and lose its structure, the starch could not be used for identification.

Good ketchup made from whole tomatoes, in spite of the minuteness of the particles, has a clean appearance, and can be readily distinguished from poor ketchup. All ketchup will have some micro-organisms present, as it is practically impossible to free the tomatoes from them in the washing, but the number is very small in some of the best, in the manufacture of which careful washing and sorting have been done. The poorer the ketchup, usually, the greater number of organisms—bacteria, yeasts, and molds; sometimes one form predominating, sometimes all three being in great abundance, this latter condition usually prevailing in the poorest ketchup, where more or less rotting has occurred.

As the tomato pulp is a favorable medium for certain organisms, these will develop first, and it has also been determined that while one organism is developing vigorously, others present are checked until the activity of the first ceases. Then again, as the composition of the pulp is being altered by the development of the organisms, the changes induced render it a more suitable medium for other organisms which are present but held in abeyance, so that pulp which has been allowed to stand for some time will usually have present not only a large number, but also different kinds of organisms.

CHANGES PRODUCED IN PULP BY ROTTING.

When tissue is held and allowed to rot spontaneously, the pulp is decomposed into a granular, watery mass. The cells beneath the epidermis are the finest and driest in the sound tomato, considerable pressure of the cover-glass being required to separate them for examination. Even when forced apart, the cells retain their shape. They contain a delicate semi-transparent protoplasm with a rather large nucleus surrounded by protoplasm and having strands from this mass connect with the protoplasm lining the wall. Pieces of the same tissue, on having the skin removed so as to expose the broken tissue to the air, were covered with mold in one day and in three days so badly disorganized that the cells separated with the weight of the cover-glass. The cells were transparent, the walls collapsed into a wrinkled mass, the protoplasm had disappeared, except a skeleton of the nucleus, but the red chromoplastic masses were intact. The middle lamella of the cells is the part which dissolves first, allowing the cells to separate and causing the walls to become thinner. The cell cavity is often filled with bacteria, so that the effect of the rotting can not be seen until the cells have been washed thoroughly. These bacteria have been mistaken for the particles left by the decomposition of the cell contents. The vascular bundles are surrounded usually by small parenchyma cells which do not separate readily from the strand in the healthy tissue, but in the decayed tissue the vessels can be seen clearly, free from other tissue. In advanced stages of rottenness the walls of the vessels may be dissolved, leaving only the spiral thickening, and the parenchyma tissue crumbled into powder-like fragments. The parts of the tomato which resist rotting the longest are the skin, which may be washed clean of adhering particles, the spirals of the vessels, and red particles of the chromoplasts.

The conditions found in the rotted sections and pieces of tomato can be distinguished in the poor ketchup and these factors, together with the large number of organisms present, serve for purposes of differentiation.

ORGANISMS IN KETCHUP.

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.

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.

STRUCTURE OF THE TOMATO.

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]

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.

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]

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:

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.