Part 2

It is our experience that skilled analysts can check themselves and each other with reasonable accuracy, and it is our duty to tell the manufacturer whether his product is legal. Should the Bureau of Chemistry adopt some other method as preferable to the Howard method, it would be our duty to use the new method and continue to serve the industry by telling the manufacturer whether samples submitted by him would pass the Government tests.

With a full understanding of our attitude in this matter many manufacturers of tomato products send samples from time to time for examination. It is made plain in every instance that the results obtained by the examination of a particular sample refer only to the batch from which that sample was taken and may give no indication of the character of any other batch.

Some manufacturers of tomato products use the Howard method as a check on their factory control. For this purpose it is not satisfactory to have samples examined in a laboratory located at a distance from the factory. Even if several samples are examined from a day’s run, they probably do not represent all the pulp manufactured on that day. It sometimes happens that one wagonload of tomatoes is almost entirely free of rotting material, whereas the succeeding load contains a considerable amount. Even with inefficient sorting, the pulp made from the first load will show a low microscopic count whereas, unless sorting is exceptionally good, the pulp made from the second load may show a high count. Thus one batch may readily comply with the requirements of the Bureau of Chemistry and the next batch may be outside of those limits. Because of this fact this laboratory recommends that manufacturers of tomato pulp do not rely upon the microscopic results of a single sample. The only way in which the product may be absolutely controlled by means of the microscopic count is to examine a sample from each batch—that is, from each kettleful or tankful that is evaporated. This is manifestly impossible. It would require several analysts for one plant. Moreover, it is entirely unnecessary.

It has been found that much better results can be secured by having an analyst in the plant to examine samples from time to time. Then, whenever the microscopic count becomes excessive, he can locate the trouble and see that it is corrected.

Manufacturers who desire frequent analyses of their products, therefore, should employ an analyst and arrange to have him instructed in a laboratory conversant with the Howard method as used by the Government. The laboratory of the National Canners’ Association makes it a practice to give the necessary instruction in this method to analysts employed by members of the association. These analysts should be carefully selected. Other things being equal, better results should be expected of a college graduate or at least one who has had college training in biology and chemistry. It has been repeatedly demonstrated, however, that a carefully selected man or woman with common school education can learn the method and use it with sufficient accuracy for factory control. The person selected for this work should have good powers of observation and a positive character.

This laboratory has heretofore advised that manufacturers of tomato pulp should not give too much attention to the microscopic count of their product. We have maintained that the expense would be better placed on the sorting belt; that if the sorting and trimming were adequately done, the plant maintained in a sanitary condition and the product manufactured as rapidly as possible, a low microscopic count would be assured. This we still maintain is true. So many cases have come to our attention, however, in which canners have not succeeded in maintaining the degree of sorting necessary with a product of this kind that we have grown to feel that the presence of an analyst working continuously in a plant is an additional safeguard.

The conditions attending the canning of tomatoes are widely different from those attending the manufacture of tomato pulp. The ordinary rot is almost always apparent from the outside of the tomatoes[3] and is removed by the peelers when preparing tomatoes for canning. Practically none of it, therefore, finds its way into the can. With pulp it is quite different. Any rot which is not removed by sorting and trimming goes into the cyclone and passes into the pulp. With trimming stock pulp, the condition is obviously much worse than with whole tomato pulp. One hundred pounds of tomatoes will yield not far from 85 pounds of cyclone juice. If only trimming stock is made into pulp, however, nearly half the tomatoes are used for canning and the remainder (50 or 55 pounds of trimming stock) will only make something like 35 or 40 pounds of cyclone juice. Yet, since the rot is almost entirely on the outside of the tomatoes, this 35 or 40 pounds made from the trimming stock contains the same amount of molds as the 85 pounds manufactured from the whole tomatoes. The mold count of the trimming stock pulp, therefore, is much higher than that of whole tomato pulp made from the same raw product.

[3] Tomatoes are sometimes found with rotten centers of which there is little or no external evidence. This is unusual, however, and the influence of this form of rot under manufacturing conditions is negligible.

The Bureau of Chemistry condemns tomato pulp whose microscopic examination gives results as high as the following figures:

Molds 66 per cent of fields. Bacteria 100 million per cubic centimeter. Yeasts and spores 125 per 1/60 cubic millimeter.

These figures, of course, apply to the Howard method as employed by the Bureau of Chemistry. The method is entirely arbitrary and results agreeing with those obtained by the Bureau of Chemistry can be obtained only by using this method substantially as it is used by the bureau. An examination of the pulp, therefore, by an analyst who is not thoroughly conversant with this method as it is employed by the Bureau of Chemistry not only is useless but may actually afford a manufacturer a false sense of security which will be greatly to his disadvantage.

Microscopic Equipment Required

The apparatus employed by the Bureau of Chemistry includes apochromatic objectives and compensating oculars. In 1914 it became impossible to obtain these accessories[4] because of the European war and equivalent apparatus of American manufacture was found to give the same results. Both of these forms of apparatus are recognized in the official Howard method which is given below.

[4] Apochromatic objectives may now be obtained but are more expensive than the achromatic.

This laboratory made a careful study of the accessories available in order to determine what could best be used. It was found that very satisfactory results could be obtained by employing a 10X Huyghenian ocular and a 4 mm. achromatic objective (working distance 0.6 mm.) and a 16 mm. achromatic objective. These accessories require a careful adjustment of light, but with proper use enable an analyst to secure satisfactory results. It is found that the best results are obtained with a rather dark field.

The apparatus necessary for the Howard method, including the accessories mentioned above, may be obtained of two American manufacturers, the Bausch & Lomb Optical Company, of Rochester, N. Y., and the Spencer Lens Company, of Buffalo, N. Y.

There is given below a full list of the optical apparatus required, including catalog numbers of the two manufacturers, as far as numbers have been assigned by them to the various items. In addition to the apparatus given in this list, the analyst should have a 50 c. c. graduated cylinder for measuring and diluting samples. This may be obtained of any dealer in chemical apparatus and at many drug stores. When ordering the optical apparatus the full description as given below should be included.

Optical Apparatus for the Howard Method

Quantity Item Bausch & desired Lomb Spencer 1 Microscope without oculars, objectives or other a FF 44 1 Abbe condenser with two iris diaphragms (lower and upper) 1740 300 1 Double nosepiece 1844 450 1 16 mm. achromatic objective 1021 108 1 4 mm. achromatic objective with working distance of 0.6 mm. 1029 116 1 8 mm. achromatic objective with working distance of 1.6 mm. 1027 112 1 10X Huyghenian ocular 1104 142 1 Mechanical stage 2116 485 1 Substage lamp with Daylite glass 1774 385-B 1 Blood counting chamber (Haemacytometer with ruling of Thoma, Neubauer, Jappert, Brewer or Turk) 3550 1472 6 Cover glasses for same, 20×21 mm., 0.4 thick 3595 1460 1 Howard’s mold counting chamber (with ¾ inch inner disk) for same 3566 Special 6 Cover glasses for same 33 mm. square, 0.6 mm. thick 3598 Special 2 Cases for counting chambers 3580 1505

All analysts undertaking the Howard method should secure copies of the two bulletins of the United States Department of Agriculture written by Mr. B. J. Howard—Bulletin 569 on Sanitary Control of Tomato Canning Factories and Bulletin 581, Microscopic Studies on Tomato Products. These bulletins may be obtained from the Superintendent of Documents, Government Printing Office, Washington, D. C., on payment of five cents each in coin.

The details of the method as given below are reprinted from the Methods of Analysis of the Official Agricultural Chemists as amended in 1921.

Apparatus

(_a_) _Compound microscope_.—Equipped with apochromatic objectives and compensating oculars, giving magnifications of approximately 90, 180, and 500 diameters. These magnifications can be obtained by the use of 16 and 8 mm. Zeiss apochromatic objectives with X6 and X18 Zeiss compensating oculars, or their equivalents, such as the Spencer 16 and 8 mm. apochromatic objectives[5] with Spencer X10 and X20 compensating oculars, the draw-tube of the microscope being adjusted as directed below.

(_b_) Thoma-Zeiss blood counting cell.[2]

(_c_) Howard mold counting cell.—Constructed like a blood-counting cell but with the inner disk (which need not be ruled) about 19 mm. in diameter.[6]

[5] We are informed that the Bausch and Lomb Optical Co. also furnishes suitable apochromatic objectives and compensating oculars for use in counting molds, yeast and bacteria by the Howard method.

[6] Comment by authors: In using these cells the plane parallel cover glasses furnished with them by maker should be used instead of the ordinary microscope cover-glasses, since the latter are subject to curvatures that introduce errors in the thickness of the mounts.

Molds.—Tentative

Clean the special Howard cell so that Newton’s rings are produced between the slide and the cover-glass. Remove the cover and place, by means of a knife blade or scalpel, a small drop of the sample upon the central disk; spread the drop evenly over the disk and cover with the cover-glass so as to give an even spread to the material. It is of the utmost importance that the drop be mixed thoroughly and spread evenly; otherwise the insoluble matter, and consequently the molds, are most abundant at the center of the drop. Squeezing out of the more liquid portions around the margin must be avoided. In a satisfactory mount Newton’s rings should be apparent when finally mounted and none of the liquid should be drawn across the moat and under the cover-glass.

Place the slide under the microscope and examine with a magnification of about 90 diameters and with such adjustment that each field of view covers 1.5 sq. mm. This area is of vital importance and may be obtained by adjusting the draw-tube in such a way that the diameter of the field becomes 1.382 mm. as determined by measurement with a stage micrometer.[7] A 16 mm. Zeiss apochromatic objective with a Zeiss X6 compensating ocular or a Spencer 16 mm. apochromatic objective with a Spencer X10 compensating ocular, or their equivalents, shall be used to obtain this magnification. Under these conditions the amount of liquid examined is .15 cmm. per field. Observe each field as to the presence or absence of mold filaments and note the result as positive or negative. Examine at least 50 fields, prepared from two or more mounts. No field should be considered positive unless the aggregate length of the filaments present exceeds approximately one-sixth of the diameter of the field. Calculate the proportion of positive fields from the results of the examination of all the observed fields and report as percentage of fields containing mold filaments.

[7] Comment by authors: Obviously after the proper draw-tube length has been secured that adjustment should be noted and always used in making mold counts.

Yeasts and Spores.—Tentative

Fill a graduated cylinder with water to the 20 cc. mark, and then add the sample till the level of the mixture reaches the 30 cc. mark. Close the graduate, or pour the contents into an Erlenmeyer flask, and shake the mixture vigorously for 15 to 20 seconds. To facilitate thorough mixing the mixture should not fill more than three-fourths of the container in which the shaking is performed. For tomato sauce or pastes, or products running very high in the number of organisms, or of heavy consistency, 80 cc. of water should be used with 10 cc. or 10 grams of the sample. In the case of exceptionally thick or dry pastes, it may be necessary to make an even greater dilution.

Pour the mixture into a beaker. Thoroughly clean the Thoma-Zeiss counting cell so as to give good Newton’s rings. Stir thoroughly the contents of the beaker with a scalpel or knife blade, and then, after allowing to stand 3 to 5 seconds, remove a small drop and place upon the central disk of the Thoma-Zeiss counting cell and cover immediately with the cover-glass, observing the same precautions in mounting the sample as given under 28.[8] Allow the slide to stand not less than 10 minutes before beginning to make the count. Make the count with a magnification of about 180 diameters to obtain which the following combination, or their equivalents, should be employed: 8 mm. Zeiss apochromatic objective with X6 Zeiss compensating ocular, or an 8 mm. Spencer apochromatic objective with X10 Spencer compensating ocular with draw-tube not extended.

Count the number of yeasts and spores[9] on one-half of the ruled squares on the disk (this amounts to counting the number in 8 of the blocks, each of which contains 25 of the small ruled squares). The total number thus obtained equals the number of organisms in 1/60,000 cc. if a dilution of 1 part of the sample with 2 parts of water is used. If a dilution of 1 part of the sample with 8 parts of water is used the number must be multiplied by 3. In making the counts, the analyst should avoid counting an organism twice when it rests on a boundary line between two adjacent squares.

[8] This number refers to the section as given in the Methods of Analysis of the Association of Agricultural Chemists.

[9] Comment by authors: The organisms counted as “yeasts and spores” are the yeast cell and yeast and mold spores, not bacteria spores.

Bacteria.—Tentative

Estimate the number of rod-shaped bacteria from the mounted sample used in 29[10] (yeasts and spores), but before examination allow the sample to stand not less than 15 minutes after mounting. Employ a magnification of about 500, which may be obtained by the use of an 8 mm. Zeiss apochromatic objective with X18 Zeiss compensating ocular with draw-tube not extended, or an 8 mm. Spencer apochromatic objective with X20 Spencer compensating ocular and a tube length of 190, or their equivalents.[11]

Count and record the number of bacteria having a length greater than one and one-half times their width in an area consisting of five of the small size squares. Count five such areas, preferably one from near each corner of the ruled portion of the slide and one from near the center. Determine the total number of the rod-shaped bacteria per area in the five areas and multiply by 480,000. This gives the number of this type of bacteria per cc. If a dilution of 1 part of the sample with 8 parts of water instead of 1 part of the sample with 2 parts of water is used in making up the sample, then the total count obtained as above must be multiplied by 1,440,000. Omit the micrococcus type of bacteria in making the count. Thus far it has proved impracticable to count the micrococci present, as they are likely to be confused with other bodies frequently present in such products.

[10] This number refers to the section as given in the Methods of Analysis of the Association of Official Agricultural Chemists.

[11] The 4 mm. achromatic objective and the 10X ocular as given in the list of apparatus may also be used to secure this magnification.

Determination of Total Solids

1. BY THE EXAMINATION OF THE PULP

The total solids in tomato pulp may be determined by drying _in vacuo_ at 70° C.; by drying at atmospheric pressure at the temperature of boiling water; by calculation from the specific gravity of the pulp; or from the per cent of solids, specific gravity or index of refraction of the filtrate. The solids obtained by different methods on 31 samples of pulp are given in Table 4.

(_a_) _By drying._—By drying either _in vacuo_ or at atmospheric pressure, it is our experience that after the sample has reached apparent dryness, four hours’ drying gives complete results. From 2 to 4 grams should be taken for the determination, and enough water added to distribute the sample uniformly over the bottom of a flat-bottomed dish at least 2.5 inches in diameter.

The solids as determined by drying _in vacuo_ at 70° C. are about 108.5 per cent of the result obtained by drying at the temperature of boiling water at atmospheric pressure. This figure is the average of the results obtained by the examination of 20 samples of pulp, in all of which the per cent of solids obtained by drying _in vacuo_ agree quite closely with the per cent obtained by drying at atmospheric pressure multiplied by 1.085. In 15 of the 20 samples examined, the difference did not exceed 0.10 per cent, and in only one case did it exceed 0.20 per cent. The results obtained by the subsequent examination of a considerable number of other samples confirm this relation.

(_b_) _By calculation from the specific gravity of the pulp._—There is a very exact relation between the specific gravity of pulp (determined by the method given above) and the per cent of total solids as determined by drying. The solids corresponding to pulps of various specific gravities are given in Table 5, or may be obtained from the following formula which is derived from the same table:

Per cent Solids = 228 (sp. gr. of pulp - 1.000) + 19.1 (sp. gr. of pulp - 1.015).

2. BY THE EXAMINATION OF THE FILTRATE

If a sample of pulp of considerable size be thrown on a folded filter, a filtrate is obtained whose composition has a definite relation to that of the whole pulp.

(_a_) _By drying._—The per cent of solids in the filtrate may be determined by drying _in vacuo_ at 70° C, or under atmospheric pressure at the temperature of boiling water.

As in the case of the drying of pulp, a constant relation is found to exist between the per cent of solids in the filtered liquor as determined by drying _in vacuo_ at 70° C., and the per cent of solids as determined by drying at atmospheric pressure at the temperature of boiling water. The per cent of solids in the filtrate obtained by drying at atmospheric pressure, multiplied by 1.125, gives the per cent of solids obtained by drying _in vacuo_. This relation is shown in detail in Table 5.

TABLE 2.—_Composition of Whole Tomato Pulps_

======================================= | Composition of pulps | +-------+---------------------+ |Sp. gr.| Total |Insoluble | Sample | at | solids(a)| solids | No. |20° C. | | | | | | | | | | | --------+-------+----------+----------+ | |_Per cent_|_Per cent_| 1290 | 1.0252| 5.94 | 0.66 | 1291 | 1.0273| 6.54 | 0.78 | 1292 | 1.0234| 5.50 | 0.80 | 1293 | 1.0293| 7.02 | 0.74 | 1294 | 1.0272| 6.48 | 0.69 | | | | | 1295 | 1.0361| 8.67 | 0.95 | 1296 | 1.0380| 9.00 | 1.06 | 1297 | 1.0465| 11.20 | 1.19 | 1299 | 1.0417| 10.07 | 1.23 | 1300 | 1.0322| 7.70 | 0.93 | | | | | 1301 | 1.0312| 7.36 | 0.91 | 1302 | 1.0310| 7.45 | 0.91 | 1303 | 1.0340| 8.17 | 0.91 | 1304 | 1.0292| 6.88 | 0.88 | 1305 | 1.0371| 9.03 | 1.19 | | | | | 1306 | 1.0370| 8.95 | 0.98 | 1307(b)| 1.0328| 7.86 | 1.01 | 1481 | 1.0449| 10.82 | | 1482 | 1.0444| 10.83 | | 1483 | 1.0464| 11.21 | | | | | | 1484 | 1.0423| 10.27 | | 1485 | 1.0347| 8.55 | | 1477(c)| 1.0610| 13.86 | | 1479 | 1.0411| 10.00 | 1.21 | 1486 | 1.0169| 4.34 | 0.62 | | | | | 1491 | 1.0198| 4.97 | 0.63 | 1496 | 1.0341| 8.27 | 1.15 | 1515 | 1.0352| 8.56 | 1.15 | 1529 | 1.0209| 5.11 | 0.89 | | | | | 1530 | 1.0252| 6.21 | 0.98 | 1531 | 1.0291| 7.17 | 1.08 | 1224(c)| 1.0486| 11.22 | 0.91 | 1325 | 1.0327| 7.86 | 0.93 | --------+-------+----------+----------+