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I: 1;, JHEbm> This is to certify that the thesis entitled The Effect of Different Storage Temperatures on the Bacterial Flora of Massecuites presented by Glenn Richard McGregor has been accepted towards fulfillment of the requirements for Liaete_r_s__degree in Bacteriology 8: Public liealtf Major professor DateO‘b‘: /71 /fl7é’F “-795 THE EFFECT OF DIFFERENT STORAGE TEMPERATURES ON THE BACTERIAL FLORA OF MASSECUITE By GLENN RIC HARD M‘CGREGOR A THESIS Submitted to the School of Graduate Studies of Michigan State College of Agriculture and Applied Science in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Bacteriology and Public Health 1948 IHEtti \‘u TABLE OF CONTENTS Page Introduction . . . . . ........... . . . ....... . ......... 1 Review of Literature ...... . ..... . . . ............. 2 Procedure and Method . ........................... . 6 Examination for Micro -organisms ............. . . 6 Discussion ............. . . T ........... . ......... 9 Critical Temperature Studies .......... . ........ 13 Summary ....... . ....... . ....... . .............. 46 Review of Literature ............. . ......... . . 47 r) a 73(31 9-.» ~- AC KNOWLEDGMEN T The writer wishes to express his sincere appre- ciation to Dr. F. W. Fabian, Professor of Bacteriology and Public Health, for his interest and helpful guidance throughout this study. The writer is also deeply grateful to Mr. A. H. Jones, Assistant Bacteriologist, Division of Bacteriology and Dairy Research, Department of Agri- culture,0ttawa, Canada,for his many helpful suggestions in this study. Thanks are due Mr. J. M. Brown, Chief Chemist, Revere Sugar Refinery, of Charleston, Massa- chusetts, for furnishing the many samples of massecuite used in this work. INTROD UC TION Over 7,800,000 short tons of sugar are consumed annually in the United States. Of this amount 1,847,738 tons are beet and 5,952,262 tons are cane sugar. The United States produces only 513,260 tons of cane sugar. The balance of this large tonnage is shipped from such countries as Hawaii, Puerto Rico, Virgin Islands, Philippines, and Cuba. In shipping sugar from such far distances there has always been the problem of bacterial deterioration taking place between the raw sugar factory and the refinery in the United States. The problem deal- ing with the deterioration of sucrose in storage has been approached from the standpoint of both beet and cane sugar. The bacteria involved and their control in both instances are similar although there are dis- tinct problems in both the beet and cane sugar refinery. Browne (1) reported losses as high as $1,150,925 a year due to the deterioration of raw cane sugar in shipments from Cuba. Owen (21) confirmed the findings of Browne and estimated the seasonal losses of 1923-24 at $1,450,000. This condition has been improving throughout the years but still remains a problem. Cameron and Williams (7) con- sidered raw sugar coming into the sugar refinery as one of the three major sources of contamination of the refined sugar. If the number of micro-organisms in raw sugar could be kept at a minimum, it would help control one of the initial foci of contamination. Browne (1) did not believe that all these organisms played such a major role in sugar ‘ deterioration since he found that sugar held in storage for a period of two years would be practically sterile at the end of this time. While it is doubtless true that bacteria would die out in raw sugar held for a period of two years, however, most of the raw sugar is not held so long but is refined much sooner. This study was concerned primarily with two things; first, the influence of the storage temperature of raw sugar on the me saphilic and thermOphilic bacterial counts and, second, a comparison of the National Canners Association’s method, hereafter known as the N.C.A. method, of determining thermOphilic bacteria in sugar with the method prOposed by Jones (17). REVIEW 9!: LITERATURE The literature concerning the presence of micro-organisms in sugar and their role in the deterioration of sugar and contamination of foods by sugar which require a sweeting agent is voluminous. One of the most recent literature reviews on the subject is that of Hucker and Pederson (14). No attempt will be made in this paper to cover all the literature but only that immediately applicable to the problem. The work may be divided into two parts, first, the role of the meSOphilic organisms found in sugar factories and, second, the micro— bial deterioration of sugar. The first recorded work in literature per- taining to bacteria in sugar was that of Kircher (18) in 1839 when he found micro-organisms in slimy beet sugar, and discovered that he in IA 4“ w could produce this slimy condition when growing the organisms in a sugar solution. In 1878 Cienkowski (10) working with the organism discovered by Kircher, which was commonly known in the sugar industry as “frog spawn”, named it Ascococcus mesenteroides. Later Van Tieghem (26) in a study of this slime producing organism, changed its name to Leuconostoc mesenteroides by which it is still known. Owen (21) was one of the first workers to recognize the impor- tance of sporogenic group of bacteria as a cause of the deterioration of raw sugar in storage. His conclusion was that the destruction of sucrose was due to the production of levanase by a group of sporogenic bacteria, which resembled the potato bacillus. Van der Bijl (25), in studying the effect of temperature on this group of organisms concluded that they could withstand 100°C. (212°F.) for two hours or more. It was also found that this group of organisms could withstand a 1:50 solution of formalin or a one percent solution of sodium fluoride for 30 minutes. The other group of meSOphiles that were studied from the stand- point of their ability to deteriorate sugar were yeast and molds. Owen (21) (22) and Browne (1) showed the ability of yeasts and molds to invert sucrose, which they considered a fundamental cause in the breakdown of raw sugar. Owen (21) showed the ability of three species of molds to invert sucrose to levulose; Penicillium glaucum, Monilia nigra, and Torula communis . Owen (22) suggested a method of preserving raw sugar by the addition or inoculation of sugar with Torulae. These yeasts were 4 capable of growth in the high tension film of molasses surrounding the crystal of raw sugar, but were incapable of fermenting sucrose. In the growth of this yeast on the sugar film, carbon dioxide was produced from the levulose fermentation. According to Owen the carbon dioxide surrounding the sugar crystal prevented the growth of other bacteria. The second period may be considered one of the most important in some respects as it was during this period that the emphasis was placed on sugar as a source of micro-organisms when added to other food products. The workers during this period were more interested in the number and type of bacteria and their effect on foods that required a sweeting agent, than the effect of the micro-organism on the sugar itself. Weinzirl (27) in working with the spoilage of candy, discovered that 85 percent of 33 samples tested contained anaerobes. This was the first record in literature that sugar was a source of bacterial contami- nation in candy. The work of Barlow (2) in 1912 showed the presence (of thermo- philic bacteria in sugar, but it was not until 1928 when Cameron, Williams, and Thompson (8) traced the spoilage of canned corn to the sugar added to it, that their real significance was recognized. This work (8) led Cameron and Williams (7) and Cameron (3) (4) (5) to make a complete study of the sporogenic, thermOphilic bacteria in sugar. They found three groups of bacteria in sugar of major importance to the canner which they classified as : (a) thermOphilic anaerobes which pro- duce acid but no gas and are commonly known as “flat sours,” (b) thermOphilic anaerobes which form acid and gas (gas is CO2 and H2) and known as “gassy anaerobes" or “hard swells" and (c) thermOphilic anaerobes which produce traces of acid and gas consisting chiefly of hydrogen sulphide and commonly known as “stinkers” or “H23 pro- ducers.” Of the three groups of bacteria group (a), or the flat sour type, is by far the most common and causes the greatest amount of trouble. It is likewise the most insidious of the three groups because it produces no gas - only acid - and therefore gives no warning of its presence in canned foods. The other two groups advertise their presence by the gas they produce and the effect it has on the concave can. Cameron and Yesair (9), and Cameron and Biglow (6) made a thorough investigation of the previous work done and proved the high resistance of the thermophilic organisms found in sugar. In a study on the thermophilic food spoilage organisms in beet sugar, Hall (11) (12) showed that there was a gradual decrease in the thermOphilic flora of refined sugar after eight and 20 months storage. He found that the most rapid reduction occurred shortly after the sugar was manufactured. The sugar was packed in four mediums; paper, burlap, toweling, and glass. There was no noticeable effect in the type of container used and the survival of the micro -organisms present. To eliminate the sporogenic group of bacteria from sugar, Hall and Keane (13) tried the incorporation of ultra violet light, (2537 0A), into the refining process. It was found that the bacterial flora could be reduced by 50 percent by the use of these lights, but even so it is not used by many of the refiners today. It was found that by careful sanitary methods in the refining process that the sporogenic group of bacteria could be kept to a mini- mum. With this knowledge at hand the National Canners Association (19) has set up standards for “Canning Grade Suger.” PROCEDURE AND METHOD In carrying out this study, samples of massecuite were received weekly from the Revere Sugar Refinery, Charlestown, Massachusetts, for a period of one month. Each sample was divided into four batches of five pounds each, and placed in one gallon cardboard Sealright Sani- tary Containers. The raw sugar was stored at four different tempera- tures; 55°C. (131°F.), 21°C. (70°F.), 4.5°C. (40°F.), and -23.3°C. (-10°F.). EXAMINATION FOR NHCRO -ORGANISMS The massecuites held at the various temperatures were analyzed bacteriologically for both the me saphilic and thermOphilic type of bacteria. The method of examination for thermophilic micro-organisms was the one established by the National Canners Association (19) and adopted as the official method by the Association of Official Agricultural Chemists (20). In addition to the National Canners Method of determin- ing flat sour thermophiles, the method preposed by .Jones (17) was also run to see if there was a correlation between the two methods. The media used for determining the different therm0phi1ic groups were Difco dextrose tryptone agar for flat sour bacteria of the Bacillus stearothermoPhilus type according to the National Canners Association method (19) and the same medium, made in the laboratory, without agar according to the Jones method (17). Jones (17) suggested substituting dextrose tryptone broth with brom cresol purple as an indicator, for the detection of flat sour organisms. Using pure cultures he showed that there was a direct correlation between the two methods. Each tube contained the Durham fermentation tube to show the presence of gas if other types of thermophiles were present. The typical flat sour organ- ism is one that produces acid without gas from dextrose. Modified sulfide agar was used for detecting therm0philic hydro- gen sulfide bacteria of the Clostridium nigrificans type according to the National Canners Association method (19) and a modified thioglycollate medium for detecting gassy anaerobes of the Clostridium thermosaccharolyticium type. All plates were incubated at 55°C. for 48 hours. The meSOphiles were determined by plating on Difco tryptone glucose beef extract agar and incubated at 30°C. and counted at the end of 48 to 72 hours. Fig. 1 illustrates diagramatically the schema used for determin- ing the respective groups of bacteria present in the mas secuite. macaw us 20k ‘23ka «auscuoeflcxm agéés m§g§< QSER §8§Q $¥ \f > 8.33 \ m ~t . . 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In this study, the emphasis was placed on this group of organisms, as it is felt by most of the workers that they give the best overall index of contamination. If time and material allowed for only one test to be run, the test for flat sours would be the most important one. Table 1. Showing relative viability of the flat sour bacteria at various storage temperatures over a six month period. (National Canners Association method) Sample 55°C. 21.1°C. 4.40C. p -23.3°C. Percentage reduction of original count. A 40040 -56.6 - 7.6 - 3.3 B -100.0 -76.4 -46.2 -34L8 C - 96.6 -41L9 - 7.7 -10.0 D - 88.4 -10.8 - 841 -30.3 (Jones’ method) Sample 55°C. 21.1°C. 4.4°C. -23.3°C. Percentage reduction of original count A -90.9 - 9.0 - 9.6 + 1.5 B -78.1 -10.4 40.9 - 5.9 c -95.5 ‘ 43.5 .. 4.3 45.9 D -84 5 - 3,0 - 0.9 - 4,8 Qm_a 10 Two methods of analysis were used in determining the effect of different storage temperatures on this group of organisms. These were the N.C.A. methods and the method prOposed by Jones. The greatest reduction in the flat sour bacteria occurred at 55°C., in which'case there was approximately 100 percent reduction in all four samples during 85 days storage period as shown in Table 3, Fig. 2a, b, c, and d, and Fig. 3a, b, c, and d. At 21.1°C. there was an average reduction of 46.4 percent over a six months period, Table 4, Fig. 4a, b, c, and d, and Fig. 5a, b, c, and d. At temperatures of 4.4°C. and -23.3°C. there was an average reduction of 17.4 and 19.6 percent respectively, which indicates at these temperatures the bacteria died very slowly. The Jones’ method for the analysis of flat sour bacteria is analogous to the dilution method used in the analysis of coliforn bacteria in water and milk. However, in the Jones’ method, a positive tube for flat sours indicates the formation of acid without gas, while in the latter it indicates the fermentation of lactose with gas. The method for plotting the viability of the flat sour organisms, as determined by the Jones’ method, was a comparison of the percent positive tubes, to the total number of tubes in all of three dilutions. For example, if there were nine positive tubes out of the 12 total, this would mean that 75 percent of the tubes had viabile bacteria present. This method of cal- culation was used throughout this paper. The trend line was determined by the method of least squares and plotted accordingly. Although it is impossible to calculate the coefficient of correlation between the two 11 methods, the respective graphs showed similar trends with respect to the number of ‘viable bacteria in the massecuite when stored at differ- ent temperatures for the same period of time. Table 3, Fig. 2a, b, c, and d, Jones’ method, and Table 3, Fig. 3a, b, c, and d, N.C.A. method, showed corresponding trends at 55°C. where there was a respective reduction of 87.2 and 100 percent. However, as the temperature de- creased the correlation between the two methods was less marked because the Jones’ method is more sensitive than the N.C.A. method since it picks up more positive flat sour bacteria. For example, at 21.1°C. (Table 4, Fig. 4a, b, c, and d) Jones’ method and Table 4, Fig. 5a, b, c, and d, N.C.A. method there was a reduction of only 9.4 percent by the Jones’ method and 46.4 percent by the N.C.A. method. At the lower temperatures of 4.4°C. Table 5, Fig. 6a, b, c, and d, Jones’ method and Table 5, Fig. 7a, b, c, and d, N .C .A. method, there is a re- duction of 6.4 and 17.4 percent respectively. At -23.3°C. Table 6, Fig. 8a, b, c, and d, Jones’ method, and Table 6, Fig. 9a, b, c, and d, N.C.A. method, the respective reductions were 6.3 and 19.6 percent. This shows that there is a greater correlation at the two lower temperatures (4.40 and -23.3°C.) than at room temperature. A study was also made of the influence of storage temperatures on the total aerobic, thermophilic and meSOphilic bacterial plate counts. These data are given in detail in Table 3 to 6. The data in Table 2 were determined by calculating the trend line in each case by the method of least squares and then computing the increase or decrease of the actual counts in terms of this trend line. The data so obtained show that at . _ 12 55°C. over an 85 day period there was a decrease in both the mesoPhilic and therm0philic counts in all samples. In fact the samples were prac- tically sterile at the end of this time.“ Table 2. Showing relative viability of the therm0phi1ic and meSOphilic ' group of bacteria at various storage temperatures over a six month period. (therm0philic bacteria) Sample 55°C. 21.1°C. 4.4°C. -23.3°c. Percentage increase or reduction of original count. A - 87.1 -11.6 +12.5 +71.1 B ~100.0 -40.2 + 6.3 +40.2 C - 83.1 +26.3 +39.4 +39.4 D - 87.3 +20.3 +76.6 + 5.6 (me sophilic bacteria) Sample 55°C. 21.1°C. 4.4°C. -23.3°C. Percentgg increase or decrease of ogginal count, A 4100.0 +33.5 -53.5 +35.9 B - 76.6 -29.3 -62.4 -76.6 C -100.0 -19.1 -63.3 -32.5 D ~100.0 - 1.5 -76.1 - 1.7 At 21.1°C. over a six month period the therm0philic count showed a decrease in half the samples and an increase in the other half while the meSOphilic count showed a decrease in three out of four sam- ples. At 4.4°C. there was no decrease in thermophiles but a slight increase while in the meSOphiles there was a decrease in all samples. 13 This may have been due to the sporogenic nature of the two groups. At the still lower storage temperature, over the six month period, the thermophiles again showed no decrease but an increase while the me so- philes showed a decrease in all except one sample. Two other groups of therm0philic bacteria were also determined to make the bacteriological picture complete; the anaerobic sulfide spores, “stinkers,” and the anaerobic “hard swell” spores. The “stinker” spores, which were first reported as the cause of spoilage of canned corn, were not predominate in any large numbers in three of the four samples tested. In sample “C” there was a large number of these spores present, which ran as high as 150 spores per 10 grams of raw sugar. Again it was noticed that the greatest reduction took place at 55°C. and then at 21.1°C. At the cooler temperatures of 4.40 and -23.3°C. there was no noticeable reduction over the six month period. The anaerobic “hard swell” spores were predominate in all six tubes over the entire testing period with the exception of the samples stored at 55°C. At this temperature there was an approximate 90 to 100 percent reduction in the 85 day testing period. In massecuite this test is not sensitive enough, due to the large inoculum and the number of spores present. Critical Temperature Studies At the completion of the first study, it was felt that there was a more critical temperature for the reduction of the therm0phi1ic Spore 14 count that would fall between the ranges of 30° to 55°C. An additional 20 pounds of massecuite was obtained from the Revere Sugar Refinery and divided into four batches of five pounds each. These were stored at 30°, 37°, 45°, and 55°C., and examined bacteriologically each week up to a period of 46 days. Table 8. Influence of temperatures between 30° to 55° C. on the thermophilic and meSOphilic counts over a 46 day period. Flat sours Flat sours Temper- N.C.A. Jones’ Total Total atures method method therm0philes me saphiles Percentage increase or decrease in count. 30°C. -3o.o -15.6 - 45.2 +12.3 37°C.. -43.1 --2o.o .. 28.4 +20.6 45°C. - -19.6 -17.3 - 30.8 -73.7 55°C. -96.5 -46.5 -100.0 -89.8 It was found that the greatest reduction in the flat sour Spore count again occurred at 55°C. with a 96.5 percent reduction in the 46 days. On the whole the next most favorable temperature for the reduc- tion of organisms was 45°C. where there was a decrease in most cases. In general as the temperature was lowered there was less decrease in the number of bacteria. This was especially true in the case of the meSOphiles where there was a slight increase at 37° and 30°C. 15 Table 3. ThermOphilic and mes0philic bacterial plate counts of massecuite held at 55°C. for 85 days. SAMPLE A (55°C.) THERMOPHILIC BACTERIA HESOPHILIC N.C.A. METHOD JONES’ BACTERIA Total Sulfide H. S.* Flat METHOD Total count spores percent sour Flat sours count Date per per tubes per D.T. brothak per 1948 10 gm. 10 gm. growth 10 gm. 2.0 1.0 0.1 1.0 gm. + + + + + .- 1-14 155 0.0 100 130 + + + + - 500 + + + + .- 1-23 60 0.0 84 40 + + + + — 150 + + + + + 1-30 75 0.0 100 55 + + - - - 350 + + + - + 2-4 50 0.0 84 20 + + - - - 200 + + + + .- 2-18 45 0.0 50 10 + + .. - - 150 + + - - .- 2-25 40 0.0 67 5 - - - - - 200 4-6 20 0.0 17 0 - - - - - 0 SAMPLE B (55°C.) THERMOPHILIC BACTERIA MESOPHILIC __ ILCA. METHOD JONES’ BACTERIA Total Sulfide H. S.* Flat METHOD Total count spores percent sour Flat sours count Date per per tubes per [)ng broth* per 1948 10 gm. 10 gm. growth 10 gm. 2.0 1.0 O. 1.0 gm. + + + + + 1-14 285 0.0 100 185 + + + + - 350 + + + + - 1-23 120 0.0 84 85 + + + + - 400 + + + + 1-30 125 0.0 84 125 + + + + - 200 + + + + .. 2-4 90 0.0 84 70 + + + + - 100 + + + + + 2-18 60 0.0 67 50 + + + + - 200 + + + + .- 2-25 55 , 0.0 50 25 + + - — - 200 4-6 0 0.0 0 0 — - - - - 100 * H.S. = Hard swells * D.T. = Dextrose tryptone Table 3. (C ont’d.) SAMPLE C (55°C.) 16 * H.S. = Hard swells THERMOPHILIC BACTERIA MESCPHILIC N.C.A. METHOD JONES’ BACTERIA Total Sulfide H. S.* Flat METHOD Total . count spores percent sour Flat sours count Date per per tubes per D.T. broth!!! per 1948 10 gm. 10 gm. growth 10 gm. 2.0 1.0 0. 1.0 gm. + + + ...+ + 1—14 130 0.0 100 95 + + + + + 650 + + + + .. 1-23 65 2.5 100 40 + + + + - 600 + + + + + 1-30 85 0.0 84 70 + + + — .. 100 + + + + 2-4 85 0.0 100 40 + + - - - 200 + + + - - 2-18 85 0.0 50 35 - - - - - 200 ‘ + + - — — 2-25 20 0.0 67 5 + - - - - 200 4,-6 15 0.0 34 5 - - - - - 100 SAMPLE D (55°C.) THERMOPHILIC BACTERIA MESOPHILIC N.C.A. METHOD JONES’ BACTERIA Total Sulfide H. S.* Flat METHOD Total count spores percent sour Flat sours count Date per per tubes per D.T. brothak per 1948 10 gm. 10 gm. growth 10 gm. 2.0 1.0 0. 1.0 gm. + + + + + 1-23 290 0.0 100 145 + + + + _ 400 + + + + - 1-30 160 0.0 84 90 + + + + — 350 T + + + + .. i4 125 0.0 84 70 + + + - — 200 + + + + .- 3—18 50 0.0 50 30 + + - - - 200 + + - - - $25 25 0.0 17 10 + - - - - 100 4-6 0 0.0 0 0 - - - - - 0 * D.T. = Dextrose tryptone 17 Table 4. Thermophilic and mesophilic bacteria plate count Of massecuite held at 21.1°C. for six months. THERMOPHILIC BACTERIA MESOPHILIC N.C.A. METHOD JONES’ BACTERIA Total Sulfide H. S.* Flat METHOD Total count spores percent sour Flat sours count Date per per tubes per D.T. broth* per 1948 10 gm. 10 gm. growth 10 gm. 2.0 1.0 0.1 1.0 gm. ++ ++ ++ 1-4 980 7.5 100 700 + + + + + + 800 I + + + + + + 1-11 830 2.5 100 430 + + + + + — 1000 + + + + + + 1-21 650 7.5 100 545 + + + + + - 950 ++ ++ ++ 1-28 790 0.0 100 560 + + + + + - 1200 ++ ++ ++ 2-6 835 2.5 100 690 + + + + + - 800 - + + + + + + 2-13 850 2.5 100 630 + + + + + - 1150 ++ ++ ++ 2-20 810 5.0 100 525 + + + + + - 1400 + + + + + + 2-28 710 0.0 100 395 + + + + — - 1400 ++ ++ ++ 3-5 865 2.5 100 320 + + + + - - 1300 ++ ++ ++ 3-21 850 ’ 0.0 100 380 + + + + + — 600 W+ + + + + + 3-29 870 0.0 100 405 - + + + + + - 500 . + + + + + + 4-6 815 0.0 . 100 330 ‘+ + + + + - 500 T + + + + + + 4-13 860 0.0 100 380 + + + + - - 600 W + + + + + 37 . 4-20 780 5.0 100 370 + + + + - - 750 T. + +‘ + + + + 5-9 710 0.0 100 400 + + + + + - 800 T + + + + + + 5-20 875 0.0 100 350 + + + + + — 1100 ~ ~ + .+ + + + + 51-22 725 0.0 100 295 + + + + + - 700 , + + + + + + 1.29 800 0.0 100 270 + + + + - - 1300 - + + + + + + l-Q 730 0.0 100 295 + + + + - - 1700 + + + + + + 7-15 675 0.0 100 310 + + + + + - 1700 * H.S. = Hard swells * D.T. = Dextrose tryptone 18 Table 4. (Cont’d.) - SAMPLE B (21.1°C.), THERMOPHILIC BACTERIA A MESOPHILIC N.C.A. METHOD JONES’ BACTERIA Total Sulfide H. S.* Flat METHOD Total count spores percent sour Flat sours count Date per per tubes per D.T. brothak per 1948 10 gm. 10 gm. growth 10 gm. 2.0 1.0 0.1 1.0 gm. ++ ++ ++ 1-4 395 15.0 100 210 + + + + + - 950 ++ ++ ++ 1-11 500 5.0 100 240 + + + + - - 800 + + + + + + 1-21 835 10.0 100 720 + + + + + - 750 ++ ++ ++ 1-28 760 2.5 100 610 + + + + + - 600 ++ ++ ++ 2-6 660 5.0 100 490 + + + + - — 300 ++ ++ ++ 2-13 425 5.0 100 ' 345 + + + + - - 600 ++ ++ ++ 2-20 340 2.5 100 280 + + + + - - 700 T‘ ' ++ ++ ++ 2-28 575 5.0 100 275 + + + + - .. 1000 ++ ++ ++ 3-5 375 5.0 100 200 + + + + - .. 800 ++ ++ +— 3-21 475 10.0 100 170 + + '+ + - .. 600 + + + + + .- 3-29 825 27.5 100 175 + + + + - - 300 T + + + + + - 4-6 490 10.0 100 185 + + + + — - 500 T + + + + + .. 4-13 440 2.5 100 190 + + + + - .. 450 - + + + + + + A 4-20 395 2.5 100 230 + + + + + - 400 T + + + + + + _5-9 405 0.0 100 190 + + + + _ - 400 ++ ++ ++ _5-20 310 0.0 100 210 + + + + - - 400 ++ ++ ++ 3-22 355 0.0 100 150 + + + + + - 500 - + + + + + + J-ZQ 340 7.5 100 125 + + + + _ — 700 + + + + + - _7-9 315 0.0 100 125 + + + + - .. 750 + + + + + — -15 300 2.5 100 * 145 + + + + - - 550 * H.S. = Hard Swells * D.T. = Dextrose tryptone I . .-.',' s 'l - . b" ." . .4...."- 1.!» ~ 5: 19 Table 4. (Cont’d.) SAMPLE C (21.1°C.) THERMOPHILIC BACTERIA MESOPHILIC __ N.CHALMETHOD JONES’ BACTERIA Total Sulfide H. S.* Flat METHOD Total count spores percent sour Flat sours count Date per per tubes per D.T. broth* per 1948 10 gm. 10 gm. growth 10 gm. 2.0 1.0 0.1 1.0 gm. + + + + + + 1-4 345 180.0 100 175 + + + + + — 800 4 ' + + + + + + 1-11 1310 55.0 100 590 + + + + + - 650 ++ ++ ++ 1-21 395 22.5 100 280 + + + + + + 700 ++ ++ ++ 1-28 420 50.0 100 300 + + + + + - 800 + + + + + + 2—6 , 380 47.5 100 325 + + + + - - 450 + + + + + + 2-13 . 540 142.5 100 310 + + + + - - 500 + + + + + + 2—20 650 180.0 100 310 + + + + - - 1100 ++ ++ ++ 2-28 475 60.0 100 200 + + + + + - 800 ' + + + + + + 3-5 580 92.5 100 305 + + + + - - 850 ++ ++ ++ 3-21 620 296.0 100 310 + + + + .- - 600 + + + + + + 3-29 915 82.5 100 330 + + + + - - 600 - + + + + + + 4-6 580 37.5 100 220 + + + + - — 550 ++ ++ ++ 4-13 630 122.5 100 310 + + + + + - 600 + + + + + + 4-20 580 97.5 100 300 + + + + + - 550 . ' + + + + + + 5-9 860 50.0 100 355 + + + + + - 490 + + + + + + .5-20 850 30.0 100 250 + + + + + - 500 + + + + + + 6-22 460 125.0 100 155 + + + + - - 600 + + + + + + 6-29 735 80.0 100 175 + + + + - - 650 ++ ++ ++ 7-9 600 30.0 100 175 + + + + - .. 650 + + + + + + 7-15 690 32.5 100 225 + + + + — - 700 * H.S. = Hard Swells * D.T. = Dextrose tryptone 20 Table 4. (Cont’d.) SAMPLE D (21.1°C.) THERMOPHILIC BACTERIA NEESOPHILIC N._C_.A. METHOD JONES’ BACTERIA Total Sulfide H. S.* Flat METHOD Total count spores percent sour Flat sours count Date per per tubes per D.T. broth* per 1948 10 gm. 10 gm. growth 10 gm. 2.0 1.0 0.1 1.0 gm. ++ ++ ++ 1-21 280 25.0 100 235 + + + + - _ 400 ++ ++ ++ 1-28 375 2.5 100 200 + + + + - - 450 + + + + + + 2-6 405 10.0 100 280 + + + + - — 550 + + + + + + 2-13 415 22.5 100 300 + + + + - - 600 + + + + + + 2-20 455 2.5 100 210 + + + + + - - 600 + + + + + - 2—28 350 0.0 100 180 + + + + - - 700 ++ ++ ++ 3-5 480 15.0 100 250 + + + + - - 600 . + + + + + 3-21 460 7.5 100 180 + + + + ' - - 500 + + + + + .. 3-29 645 7.5 100 175 + + + + - - 300 A + + + + + + 4-6 480 5.0 100 180 + + + + - — 350 + + + + + + 4—13 525 7.5 100 250 + + + + - - 300 ~ ++ ++ ++ 4-20 510 0.0 100 220 + + + + - — 300 L + + + + + + 5-9 430 5.0 100 200 + + + + - - 350 ++ ++ ++ _5-20 480 2.5 100 275 + + + + .. .. 600 ++ ++ ++ _6-22 390 0.0 100 170 + + + + - - 300 . + + + + + + _6-29 450 5.0 100 200 + + + + — - 600 ++ ++ ++ i9 475 5.0 100 220 + + + + - - 600 + + + + + .- i—ls 485 7.5 100 205 + + + + - - 650 * H.S. = Hard Swells * D.T. = Dextrose tryptone 21 Table 5. ThermOphilic and meSOphilic bacteria plate count of massecuite held at 4.4°C. for six months. THERMOPHILIC BACTERIA MESOPHILIC N.C.A. METHOD JONES’ BACTERIA Total Sulfide H. S.* Flat METHOD Total count Spores percent sour Flat sours count Date per per tubes per D.T. brothal: per 1948 10 gm. 10 gm. growth 10 gm. 2.0 1.0 0.1 1.0 gm. + + + + + + 1-17 330 2.5 100 205 + + + + - — 2600 + + 9+ + + + 1-31 745 5.0 100 500 + + + + + - 2450 + + + + + + 2-7 745 0.0 100 605 + + + + + - 1200 + + + + + + 2-14 730 0.0 100 405 + + + + + - 1500 + + + + + + 2-21 1135 2.5 100 , 490 ++ + + + - 1600 + + + + + + 3-1 675 0.0 100 305 + + + + — - 1300 + + + + + + 3-7 705 0.0 100 275 + + + + - - 1300 + + + + + + 3-17 745 7.5 100 400 + + + + + - 1050 + + + + + + 3-30 800 0.0 100 275 ' + + + + - - 900 ++ ++ ++ 4-7 800 2.5 100 275 + + + + - - 1100 + + + + + + 4-14 820 2.5 100 480 + + + + - - 1250 ++ ++ ++ 4-24 780 2.5 100 385 + + + + + — 1200 + + + + + + 5-14 755 0.0 100 400 + + + + + - 850 + + + + + + 5-21 720 0.0 100 350 + + + + + - 750 + + + + + + 6-21 760 0.0 100 355 + + + + - — 1000 + + + + + + 7-1 800 7.5 100 375 + + + + - - 1100 + + + + + + 7-13 770 7.5- 100 385 + + + + - - 1000 + + + + + - 7-20 750 2.5 100 375 + + + + - — 1150 * H.S. = Hard Swells * D.T. = Dextrose tryptone 22 Table 5. (Cont ’d.) SAMPLE B (4.4°C.) THERMOPHILIC BACTERIA MESOPHILIC _ N.C.A. METHOD JONES’ BACTERIA Total Sulfide H. S.* Flat METHOD Total count Spores percent sour Flat sours count Date per per tubes per D.T. brothak per 1948 10 gm. 10 gm. growth 10 gm. 2.0 1.0 0.1 1.0 gm. + + 7+ + + 1-17 500 7.5 100 480 + + + + + - 2100 . ++ ++ ++ 1-31 435 10.0 100 380 + + + + - —- 500 ++ ++ +- 2-7 375 40.0 100 285 + + + + - — 400 ++ ++ +- 2-14 425 7.5 100 325 + + + + - — 950 + + + + + - 2-21 380 5.0 100 225 + + + + - - 550 + + + + + - 3-1 365 7.5 100 200 + + + + - - 1450 + + + + + + 3-7 315 0.0 100 185 + + + + - - 800 « ++ ++ +- 3-17 550 5.0 100 225 + + + + - — 600 + + + + + + 43-30 525 7.5 100 195 + + + + - - 550 + + + + + .- 4-7 475 12.5 100 165 + + + + - - 700 ++ ++ +— 4-14 325 5.0 100 170 + + + + - - . 750 ++ ++ +- _4-24 560 7.5 100 260 + + + + - - 800 + + + + + - _5-14 420 22.5 100 185 + + + + - - 600 ++ ++ ++ _5-21 405 15.0 100 195 + + + + - - 650 + + + + .. .- _6-21 425 12.5 100 200 + + + + — - 500 + + + + + - _7-1 455 20.0 100 185 + + + + - - 550 ++ ++ +- __7-13 475 17.5 100 210 + + + + - - 450 . ++ ++ ++ 1-20 440 12.5 100 230 + + + + - .. 400 * H.S. = Hard Swells * D.T. = Dextrose tryptone 23 Table 5. (C ont’d.) SAMPLE C (4.4°C.) THERMOPHILIC BACTERIA MESTDPHILIC ____+ N.C.A, METHOD JONES’ BACTERIA Total Sulfide H. S.* Flat METHOD Total count spores percent sour Flat sours count Date per per tubes per D.T. brothak per 1948 10 gm. 10 gm. growth 10 gm. 2.0 1.0 0.1 1.0 gm. ++ ++ ++ 1-17 225 125.0 100 195 + + + + + - 1200 + + + + + + 1-31 535 135.0 100 275 + + + + + - 1500 + + + + + + 2-7 520 57.5 100 265 + + + + + - 8050 + + + + + + 2-14 630 152.5 100 385 + + + + + + 11700 . ' ++ ++ ++ 2-21 705 27.5 100 440 + + + + - - 9200 + + + + + + 3-1 650 '_:7.5 100 350 + + + + + - 8750 ++ ++ ++ 3-7 485 80.0 100 150 + + + + + _ 13300 ' ++ ++ ++ 3-17 475 75.0 100 125 + + + + - - 4500 + + + + + + 3-30 485 70.0 100 125 + + + + - - 3700 + + + + + + 4-7 540 67.5 100 150 + + + + - — 2400 + + + + + + 4-14 770 65.0 100 400 + + + + + .. 1600 . + + + + + + _4-24 555 52.5 100 200 + + + + + - 4700 ++ ++ ++ 3-14 590 72.5 100 250 + + + + + - 4300 p + + + + + + _5-21 495 105.0 100 240 + + + + + - 4100 ++ ++ ++ _6-21 690 130.0 100 275 + + + + + - 3800 + + + + + + 1-1 705 135.0 100 260 + + + + + - 3150 ++ ++ ++ _7-13 775 135.0 100 270 + + + + — - 3900 + + + + + + 7-20 790 125.0 100 255 + + + + - - 3850 * H.S. = Hard Swells * D.T. = Dextrose tryptone 24 Table 5. (C ont’d.) SAMPLE D (4.4°C.) THERMOPHILIC BACTERIA MESOPHILIC N.C.A. METHOD JONES’ BACTERIA Total Sulfide H. S.* Flat METHOD Total ‘ count spores percent sour Flat sours count Date per per tubes per D.T. brothak per 1948 10 gm. 10 gm. growth 10 gm. 2.0 1.0 0.1 1.0 gm. + + + + + + 1-17 ‘ 195 10.0 100 130 + + + + - - 900 + + + + + 1-31 370 2.5 100 230 + + + + - - 1050 , + + + + + — 2-7 455 10.0 100 340 + + + + - — 1100 _ + + + + + 2-14 410 7.5 100 325 + + + + - - 1050 + + + + + .— 2-21 380 22.5 100 ' 210 + + + + - - 1100 + + + + + 3-1 415 0.0 100 270 + + + + - - 1000 + + + + + - 3-7 290 7.5 100' 150 + + + + - - 1700 + + + + - - 3-17 255 7.5 100 115 + + + + - - 800 + + + + + - 3-30 405 7.5 100 140 + + + + - - 900 ' .+ + + + + + 4-7 875 10.0 100 205 + + + + + — 700 ++ ++ ++ 4-14 525 7.5 100 255 + + + + - - 600 + + + + + + 4-24 750 7.5 100 235 + + + + - - 550 T + + + + + + 5-14 780 2.5 100 185 + + + + - — 250 T + + + + + + 5-21 495 2.5 100 230 + + + + - - 250 T + + + + - — 6-21 505 2.5 100 230 + + + + - - 350 T . + + + + + + _7.1 525 0.0 100 250 + + + + .. - 350 + + + + + + J43 475 7.5 100 250 + + + + - - 400 ++ ++ ++ 1-20 545 10.0 100 245 + + + + - - 300 * H.S. = Hard Swells * D.T. = Dextrose tryptone 25 Table 6. ThermOphilic and mesophilic bacterial plate counts of massecuite held at -23.3°C. for six months. SAMPLE A (-23.3°C.) THERMOPHILIC BACTERIA MESOPHILIC __ N.C.A. METHOD JONES’ BACTERIA Total Sulfide H. S.* Flat METHOD Total count spores percent sour Flat sours count Date per per tubes per D.T. broth* per 1948 10 gm. 10 gm. growth 10 gm. 2.0 1.0 0.1 1.0 gm. + + + + + + 1-19 335 0.0 100 ‘ 215 + + + + - .. 3700 _ + + + + + + 1-26 280 10.0 100 175 + + + + - - 900 + + + + + + ‘ 2-2 360 0.0 g 100 260 + + + + - - 700 + + + + + 2-9 610 2.5 100 580 + + + + + - 700 + + + + + .- 2—16 595 (0.0 . 100 305 + + + + - - 900 0 + + + + .. - _ 2-23 605 - 2.5 100 305 + + + + - - 1000 p + + + + + .- 2-29 575 0.0 100 305 + + + + - — 1300 + + + + + - 3-18 690 2.5 100 350 + + + + - .- 1300 _ + + + + + -. 4-2 835 2.5 100 335 + + + + - - 2700 + + + + + — 4-10‘ 835 0.0 100 375 + + + + - - 3600 + + + + + + 4-17 920 0.0 100 455 + + + + - - 2000 + + + + + + 5-1 . 810 5.0 100 355 + + + + - - 1950 g + + + + + + 3-15 780 0.0 100 385 + + + + - - 2300 + + + + + + £22 760 0.0 100 345 + + + + — - 1600 . + + + + + + “6-26 670 0.0 100 230 + + + + - - 1400 p + + + + + + 1-7 600 2.5 100 245 + + + + - - 2200 + + + + + + 1414 710 0.0 100 300 + + + + — - 1800 + + + + + - _7-21 760 0.0 100 325 + + + + - - 1900 * H.S. = Hard Swells * D.T. = Dextrose tryptone 26 Table 6. (C ont’d.) SAMPLE B (-23.3°C.) THERMOPHILIC BACTERIA MESOPHILIC NJC.A_.1 METHOD JONES’ BACTERIA Total Sulfide H. S.* Flat METHOD Total count Spores percent sour Flat sours count Date per per tubes per D.T. brothak per 1948 10 gm. 10 gm. growth 10 gm. . 2.0 1.0 0.1 1.0 gm. + + + + + + 1-19 ‘ 535 0.0 100 485 + + + + + + 600 ++ ++ ++ 1-26 230 0.0 100 180 + + + + — - 2400 + + + + + - 2-2 235 5.0 100 165 + + + + - - 600 + + + + + - 2-9 325 7.5 100 260 + + + + - - 600 , + + + +‘ + .- 2-16 260 7.5 100 220 + + + + - - 1200 I + + + + + - 2-23 230 0.0 100 145 + + + + - - 500 + + + + + - 2-29 170 7.5 100 155 + + + + — — 600 . + + + + + - 3-18 175 0.0 100 165 + + + + - - 500 + + + + + - 4-2 500 5.0 100 185 + + + + - - 600 ++ ++ +- 4-10 305 0.0 100 200 + + + + .. - 500 , + + + + + - ‘ 4-17 400 2.5 100 225 + + + + _ - 450 + + + + + - 5-1 350 2.5 100 160 + + + + - - 500 ++ ++ ++ 5-15 300 0.0 100 120 + + + + — - 600 + + + + + + 5-22 340 0.0 100 150 + + + + _ - 450 . ++ ++ ++ 6—26 475 0.0 100 165 + + + + - - 350 + + + + + - 7-7 330 2.5 100 195 + + + + - - 400 + + + + + 7-14 350 0.0 100 175 + + + + - - 300 + + + + — .- 7-21 425 0.0 100 190 + + + + - - 350 * H.S. = Hard Swells * D.T. = Dextrose tryptone 27 Table 6. (C ont’d.) SAMPLE C (-2330). THERMOPHILIC BACTERIA MESCPHILIC ____: N.C.A. METHOD JONES’ BACTERIA Total Sulfide H. S.* Flat ' METHOD Total count Spores percent sour Flat «50an count Date per per tubes per D.T. brothak per 1948 10 gm. 10 gm. growth 10 gm. 2.0 1.0 0.1 1.0 gm. ++ ++ ++ 1-19 265 62.5 100 205 + + + + + + 1600 ++ ++ ++ 1-26 290 67.5 100 225 + + + + + - 1500 ' + + + + + + 2-2 670 60.0 100 445 + + + + + + 2300 + + + + + + 2-9 590 90.0 100 290 + + + + + + 3050 ++ ++ ++ 2-16 640 280.0 100 230 + + '+ + + + 4800 ++ ++ ++ 2-23 620 182.5 100 170 + + + + + + 2400 . ++ ++ ++ 2-29 435 182.5 100 165 + + + + + - 5200 , ,++ ++ ++. 3-18 450 105.0 100 225 + + + + - - 2400 + + + + + + 4-2 595 187.5 100 205 + + + + - - 2400 + + + + + + .4—10 620 102.5 100 300 + + + + - — 1800 T , + + + + + + 4-17 750 65.0 100 295 + + + + + - 1900 ‘ ++ ++ ++ _5-1 780 70.0 100 290 + + + + + - 1700 ++ ++ ++ 5-15 795 50.0 100 220 + + + + + - 2400 ++ ++ ++ 5-22 825 62.5 100 230 + + + + + - 1200 ‘ ++ ++ ++ 6-26 700 42.5 ‘ 100 265 + + + + - — 2400 ++ ++ ++ 7-7 500 120.0 100 200 ‘ + + + + - - 2200 ‘ + + + + + - 7-14 620 125.0 100 230 + + + + - - 2000 + + + + + 7-21 600 127.5 100 250 + + + + — - 1900 * H.S. = Hard Swells * D.T. = Dextrose tryptone 28 Table 6. (Cont’d.) SAMPLE D (23.3°C.) THERMOPHILIC BACTERIA MESOPHILIC _ N.C.A. METHOD JONES’ BACTERIA Total Sulfide H. S.* Flat METHOD Total count spores percent sour Flat sours count Date per per tubes per DTT. brothflr—JH per 1948 10 gm. 10 gm. growth 10 gm. 2.0 1.0 0.1 1.0 gm. + +T + + + 1-26 235 0.0 100 205 + + + + - — 300 + + + + + + 2-2 555 0.0 100 400 + + + + - - 1200 + + + + + + 2-9 425 0.0 100 240 + + + + — _ 1000 + + + + + + 2-16 455 7.5 100 275 + + + + - - 900 + + + + + - 2-23 470 10.0 100 275 + + + + — - 3400 + + + + + - 2-29 390 0.0 100 215 + + + + - - 1300 T + + + + + .- 3-18 375 20.0 100 225 + + + + - - 1500 + + + + + — 4—2 380 2.5 100 230 + + + + - - 1500 + + + + + - -4-10 525 2.5 100 220 + + + + - - 1400 + + + + + + 4-17 520 5.0 100 260 + + + + + - 1200 + + + + + + 5-1 395 5.0 100 215 + + + + - — 1600 + + + + + + 5-15 420 10.0 100 200 + + + + - — 2900 + + + + + - 5-22 445 7.5 100 190 + + + + - - 900 + + + + + + 6-26 555 15.0 100 225 + + + + - - 600 + + + + + .- 7-7 350 10.0 100 205 + + + + - - 1000 + + + + + - 7-14 450 7.5 100 195 + + + + - - 950 + + + + + - 7-21 495 10.0 100 200 + + + + - - 1100 * H.S. = Hard Swells * D.T. = Dextrose tryptone 29 Table 7. ThermOphilic and meSOphilic bacterial plate counts of massecuite held at temperatures of 30, 37, 45 and 55° C. for a period of six weeks. SAMPLE A (30°C.) THERMOPHILIC BACTERIA MESOPHILIC N.C .A. METHOD JONES’ BACTERIA Total Sulfide H. S.* Flat METHOD Total count Spores percent sour Flat sours count Date per per tubes per D.TL broth* per 1948 10 gm. 10 gm. growth 10 gm. 2.0 1.0 0.1 1.0 gm. . + + + + + - 7-16 555 0.0 100 175 + + + + - — 800 , + + + + + .- -7-23 530 2.5 100 180 + + + + - - 750 + + + + + .- 7-30 480 5.0 100 150 + + + + - - 900 ++ ++ ++ 8-5 435 5.0 100 145 + + + + -_ - 700 ++ ++ ++ 8-15 350 2.5 100 155 + + + + - - 900 T + + + + + - 8-22 380 5.0 100 135 + + + + - - 800 + + + + - 8-30 305 2.5 100 120 + + + + - — 900 SAMPLE B (37°C.) THERMOPHILIC BACTERIA MESOPHILIC. N.C.ALMETHOD JONES’ BACTERIA Total Sulfide H. S.* Flat METHOD Total count spores percent sour Flat sours count Date per per tubes per D.T. broth* per 1948 10 gm. 10 gm. growth 10 gm. 2.0 1.0 0.1 1.0 gm. f + + + + - .- 7-16 565 2.5 100 145 + + - - - - 600 + + + + .. - 7-23 610 0.0 100 155 + + + + - - 700 + + + + - — 7-30 575 2.5 100 140 + + + + - - 750 + + + + + .- 8-5 305 5.0 100 125 + + + + — - 700 + + + + + 8-15 650 0.0 100 390 + + + + — — 800 + + + + + - 8-22 615 2.5 100 245 + + + + - - 900 + + + + + — 8-30 255 2.5 100 125 + + + + - - 650 * H.S. = Hard Swells * D.T. = Dextrose tryptone 30 Table 7. (C ont’d.) SAMPLE C (45°C.) THERMOPHILIC BACTERIA MESOPHILIC N.C.A. METHOD JONES’ BACTERIA Total Sulfide H. S.* Flat METHOD Total count spores percent sour Flat sours count Date per per tubes per D.T. broth* per 1948 10 gm. 10 gm. growth 10 gm. 2.0 1.0 0.1 1.0 gm. + + + + + .. 7-16 - 490 5.0 100 135 + + + + - — 1200 + + + + + 7-23 500 5.0 100 185 + + + + - — 900 + + + + — — 7-30 540 2.5 100 140 + + + + + - 850 + + + + — .- 845 385 7.5 100 125 + + + + + + 800 + + + + - - 8-15 570 5.0 92 185 + + + + - - 600 + + + + — - 8-22 450 2.5 100 145 + + + + - - 300 + + + + - - 8-30 250 0.0 92 95 + + + + - - 400 SAMPLE D (55°C.) THERMOPHILIC BACTERIA . MESOPHILIC N.C.A. METHOD JONES' BACTERIA Total Sulfide H. S.* Flat METHOD Total count spores percent sour Flat sours count .Date per per tubes per D.T. broth* per _1948 10 gm. 10 gm. growth 10 gm. 2.0 1.0 0.1 1.0 gm. + + + + + + _7-16 475 2.5 100 150 + + + + - - 400 + + + + + _7—23 495 0.0 100 155 + + + + - - 350 + + + + - - :30 290 0.0 84 170 + + + + — - 200 + + + + — - _8-5 265 0.0 50 180 + + + + - - 200 + + + + — .- _8-15 65 0.0 . 17 25 + + + - - — 100 + + + + - - 35:22 55 0.0 17 15 + + - — - — 150 + + - - - - £30 30 0.0 0 10 + + - - - - 50 * H.S. = Hard Swells * D.T. = Dextrose tryptone 31 «2‘1 V tUQ‘Q 8566 P. r NR ‘ékfihk )..:~AI “ chSss‘fi / Ob ~ .4 on x AA 2 220m 3.2.4 .Oowwtdtmdmsaxw so 6.2 .QNK kzbou :a 431‘ tut ‘3 \E vStka kt 3:32P o / O G 0 Q m. ||BIIIII| 06‘ 2 Qme. k‘flk Comm. 19 AN ul‘vfi. .3u a 6x .QNQ uK>3OU 3v T976 9 .3. .m? . 2km... >623 x3332 3.32.2. VAICI on o o l . §Q\ / bu. \ / o I QN QSOM. kvtqk Gown. thtmqlxév‘m, .33 023 l (me .3233 SC 311‘ I002“ XQvSQQNK Xt‘hz‘x. Jl Q o a 0m. UM QDQW Rusk Comm... :INNQRY .:Q 0. Ox .301 952300 E cofima .mmcoh. 6 38 .o .0. .mm .mfm .523 >653 xevstnuk :53. 3%: xexziuk [J villi! “ / o / o / / / . I o. o / /o o O O 4 as Flarfluuggfllofifia 3.. ii ESE ~22me mon .53 32. ES 030m. quk .90 WWnQIN653 xevstflt xm§z§ _ 35. 33: xtxaeamk \E :3 3. _. } /» 9’ .ON / O O . / I 0‘ _ .llcv / o 0 PT. no / o «.6 / um / an >0~ —¢Q\ Elam ~22ng mmoExmg . $2. is E88 mzokqxfi 38. , . .CQQ So... 5 t. $6.... 59‘ 83.6.3156 as: :3 .9 ~38, Rik mowwééqofixm 9:2 :3 Em E :3 (a) (b) (C) 33 counr: FER. mo cu. SAMPLE-A - 2/. PC. FLAT SOUR ’00 400 300 JANUARY It'll/ARV MARCH W73 PER. CWNTS P55 ”-0 “'- SAMPLE-C- 2i. I'C. FLAT SOUR ‘00 400 J00 £00 [00 JANUARY FEBRUARY MARCH APR! L HAY JUM JULY Fig. 5 a, b, and c, N.C.A. method (d) (a) -) 600 34 WT: PER. ”19‘“ SAMPLE-0" FLAT SOUR 2/. /°C. 500 300 I00 com": PER 10.0 an -A - 4.44 'C. FLAT‘ SOUR 400 200 couurs P50. [0.0 cu. SAMPLE -3- 444°C. FLAT SOUR 000 500 400 300 £00 [00 .' Fig. 5 d, Fig. 6 a and h, N.C.A,’s Irethod (C) (d) (a) COLIN“ PER. [0.0 6”. 5 00 ‘00 300 I00 COUNT PER. 10.0 CH. 500 400 000 200 [00 COUN“ PER. [0.0 6". 000 500 400 800 I00 [00 SAMPLE ‘6' 414°C. FLAT SOUR SAMPLE-0' 4.449C. FLAT SOUR MARCH APRIL MAY JUNE JULY SAMPLE "A - '23-3‘6‘. FLATSOUR Fig. 6 c and d, Fig, 7 5-, N,C,A.’s method 35 (b) (C) 36 COUNTS PER. ’00 5M ’ SAMPLE-'8‘ '23..3°C. FLAT SOUR m 400 m 300 I00 PlR. emu ’19 ‘3' SAMPLE-C- -ZJ.J'C. FLAT SOUR 400 J00 I00 ___-=a-~ -3] 3' 1 a l~ COUNTS PER. L I. ’40 “’- SAMPLE 'D- 'ZJJ’C. FLAT SOUR 500 I00 000 I00 I00 \ Fig. 7 b, c and d, ’\].C,A,’s method PER awr was: W"! “’0 SAMPLE -A - 2L / °C. FLAT SOUR - DEX TROSL' TRVPTONE BRO TH . i (3) I00 9: 90 u PER CENT TUBES mm ACID SAMPL E-B- Z]. I 'C. FLAT SOUR- OEYTROSE T RVPT ONE BROTH (b) u 00 85 O0 75 MARCH APRIL MAY JUL Y PER CENT TURES 'ITII ACID SAMPLE-C'- ZL /° C. FLAT SOUR- DEX T ROSE TRYPTO/VE BROT H ((3)/u 08 00 .5 00 75 Fig. 8 a, b and C, Jones’ method fl“! 38 par cewr runs . "r” “'0 SAHPL E-O-ZL/‘C. FLAT 5009- new mass TRVP TONE 8907” (d) ,5 00 05 O0 73 PER cewr runs mm ACID -A—4.4°C. FLAT (a) 95 a as 00 75 PER awr runs an m ACID -8- 4. 4°C. FL AT SOUR- DEXTROSE TRYP TONE BROTH (b) 90 0 0.5 00 75 70 65 Fig. 8 d, Fig. 9 a and b, Jones” method (C) (d) (a) an car runs _ Wm ACID SAMPLE-C’- 4.4°C. FLAT SOUR- DEXTROSE TRYPTONE mu! . loo 3 - j 03 o o o o o o b '0 — $ \ * .3 o o d o T- o 0 u f i ‘ l 1 1 ”mar FE (IA 7 UARCII 1 APRIL l war 1 JUNE 1 JUL r PER CENT TURES I'ITII ACID 05 75 70 03 PER CENT TUBES VITII ACID SAMPLE 'A "FLAT SOUR “233°C-DEXTROSE T RYPT ONE BROTH to _L . I no I —— — —— a: L 0 O 0 0 IO 0 O 9 O O O 80 —_—==_ 1 7 .5 3 a a :c c . l 1 ‘JANUARV FEJRUARV ”may I APRIL [ m r? JUNE JULY Fig. 9 C and d, Fig, 10 21, Jones’ method 39 (b) ,, ' ' (C) (d) PER CENT TUOES "7" “’0 SAMPLE-B- FLAT 50w? -23.3°c. mar/«>055 TRVPTONE 5mm so * TTTT 05 00 75 70 MARCH APRIL MA Y JUNE JUL V PER CENT T URES ”IT” 4‘70 SAMPLE 'C - FLAT SOUR -2.3. S‘C. - DEX T ROSE TRYPT ONE BROTH { [WK #6; \\ o 0 \p o o o ' 05 "“‘ ‘ o o o o\3 00 7 5 % JANUARY FEJRUA RY MARCH APRIL MAY JUNE JULY PER cewr runes . mm 4cm SAMPL E-D-FLAT SOUR -23. 3°C. -OEX TROSE TRVP TONE BROTH 05 90 I! 00 75 Fig. 10 b, c and d, Jones’ method 40 41 3‘ V t: x stkfihk c Em o .n .m S .mE XtV§§ - QOs ” “ o¢~ gN 6°” k>$8 u‘kbk UshWIQIM360 , av 42 § . “83,7525" SAMPLE-A- 2/./°c. 70m. COUNT cowrs PER. mo cu. SAMPLE -B- 2!. /°C. TOTAL COUNT (b) cm ”N. 10.0 cu - c - TOTAL :1. I 'c. (C) APRIL Fig. 12 a, b and c 43 N. C335“? [-0- ZINC. 70m cowvr (d) m ‘00 S00 400 ”.0 E00 COUNTS PER. ' "-0 “'- SAHPLE-A - 444°C. TOTAL COUNT .« (a) ”00 I000 000 000 700 S00 400 S00 COUNTS PER. ‘ "-0 “’- SAMPLE-B- 414°C. TOTAL COUNT (b) 000 500 4 00 000 I00 Fig. 12 d, Fig, 13 a and b (d) (a) COUNTS PER. I0. 0 CH. SAMPLE'C- 4.44 TOTAL COUNT d? _ _ -______. APRIL mar W JULY cowrs PER 10.0 cu. SAMPLE-D- 4. 44°C. TOTAL COUNT no 0 000 o 0 no can / o (D . 599 . /"“( o _.. no / _ __ _._ no 6 ——~ 0 :00 e __ ________.L “UL—fin ”may WW I: "Tw L my JUNE JULY couur PER. ”-0 6“ SAMPLE-A- TOTAL COUNT -23.3°C. can #9— 0 0 ‘00 . /?’.—_ 0 0 700 #fiflf // I 0 g 3 000 ° ; $./ 500 Z - . _ -- l 3 400 T 0 0 :00 - zoo ‘ _. . i 1 1 JANUARY runway mncn APRIL I my I JUN: I JULY Fig. 13 c and d, Fig. 14 a 44 (b) ((1) CM“ FIR. “-0 CM SAMPLE-'8' TOTAL COUNT '23.3° C. ‘00 500 400 800 100 I00 COUNTS PER- mo 6M SAMPLE -C - TOTAL COUNT - 900 000 700 000 500 400 IN JAM/ARV FEBRUARY COUNTS PER. IO-OGN- SAMPLE '0- TOTAL COUNT '233’C. 600 500 400 .900 [00 Fig. 14 b, c and d 45 46 SUMMARY Under the conditions of the experiment there was the greatest reduction in both the mesophilic and thermophilic bacterial plate count at 55°C. The next greatest reduction was at about room temperature 21.1°C. (70°F.). As the temperature was decreased, there was less reduction in both the meSOphilic and thermophilic counts. A comparison of the N.C.A. with the method prOposed by Jones indicates that the Jones’ method is more sensitive in picking out the flat sour type of bacteria. It is less expensive and easier to read than the N.C .A. method. 47 LITERATURE CITED 1. Browne, C. A., 1918. The deterioration of raw cane sugar. J. Ind. 2. Barlow. Eng. Chem. 10 (3), 178-190. 1912. Univ. of Illinois Thesis., Microbiology of Canned Foods, Tanner, F. W., 115, 979. 3. Cameron, E. J ., 1930. Thermophilic spoilage bacteria in granulated 10 11 12. 13. sugar, Canner 70 (13), 17-20. , 1936a. Report on culture media for non-acid production. J. Assoc. Off. Agr. Chem. 19, 433-438. , 1936b. Report on methods for detecting and estimating numbers of therm0phi1ic bacteria from sugar. Ind. Eng. Chem. 19, 438-440. , and Bigelow, W. D., 1931. Elimination of therm0philic bacteria from sugar. Ind. Eng. Chem. 23, 1330-1333. , and Williams, C. C ., 1928. ThermOphilic flora of sugar in its relation to canning. Zentbl. Bakt. 76, Pt. 2, 28-37. (Abs. in J. Bact. 15, 3132.) , and Thompson, R. J ., 1928. Bacteriological field study in canning. Nat. Canners Assoc. Bul. 25-L. Washington, D. C. , and Yesair, J ., 1931. Sugar contamination; its effect in canned corn. Canner 72 (14), 15-16. . Cienkowski, L., 1878. Die Gallertbildungen des Zuckerrubensaftes. Charkow. . Hall, H. H., 193 9. Survival of therm0phi1ic food-Spoilage organisms in stored white beet sugar. Food research 4, 259-267. , and James, L. H., 1936. Crystal surface contamination and biological quality of sugar. Facts About Sugar, June, 1936. , and Keane, J. G., 1939. Effect of radiant energy on ther- m0philic organisms in sugar. Ind. Eng. Chem. 31, 1168— 1170. 14. Hucker, G. J. and Pederson, C. S., 1942. A review of the micro- biology of commercial sugar and related sweeting agents. Food Research 7 (6), 459-480. 48 15. James, L. H., 1928a. The bacterial content of raw and commercial sugars. Food Indus. 1, 65-69. 16. , 1928b. Distribution of therm0philic spoilage bacteria. J. Bact. 15, 32. 17. Jones, A. H., 1947. A microbiological survey of Canadian canning sugars and other sweeting agents. Canadian Food Indust., Oct. 18. Kircher, J ., 1839. Ueber Mannit and Schleim aus runkelruben. Justus Liebigs Ann. Chem. 31, 337-339. 19. National Canners Association., 1941. Bacteriological standards for sugar (to supercede “Bacterial standards for the year 1935”) Washington, D. C. 20. Official and Tentative Method of Analysis of the Association of Official Agricultural Chemists, 6th ed. 1945. Assoc. of Official and Agr. Chemists, Washington, D. C. 21. Owen, W. L., 1925. Deterioration of raw sugars in storage. Facts About Sugar 20, 178-179; 300-3-1; 442-444; 518-520; 566- 568; 705-707. 22. , 1940. Suggested sugar standards. National Bottlers Gazette, Sept. 23. , and Mobley, R. L., 1932. Thermophilic bacteria in refined cane sugars. Ind. Eng. Chem. 24, 1042-1044. 24. , , 1935. Bacteriological standards for refined cane sugar. Facts About Sugar 30, 451-452. 25. Van der Bijl, P. A., 1929. Preliminary studies on some fungi and bacteria reSponsible for the deterioration of South African ‘ sugars. Union So. Africa Dept. Agr., Sci. Bul. 12. 26. 'Van Tieghem, P., 1878. 8111‘ la gomme de sucreie. Ann. Sci. Nat. Bot., 6 ser. 7, 180-203. 27. Weinzirl, J., 1927, Sugar as a source of the anaerobes causing explosion of chocolate candies. J. Bact. 13, 203 -207. ~ 1 q... 1 r .. . rm... A ‘v . V O 9 p \ MICHIGAN !STATE UNIVERSITY LIBRA