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 If the rod be inserted to the bottom, or nearly to the bottom of the mixture and withdrawn slowly and another withdrawn somewhat rapidly, a difference of fifty per cent or even more may result in the count.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.