Part 4

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 The liquid to be examined is shaken thoroughly and then the measured sample withdrawn as quickly as possible to prevent the cells from settling and diluted with weak sulphuric acid (about 10 per cent), which prevents any further development of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.

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.

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]

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.