France in the late 1790s was at war and having difficulty feeding its people. Napoleon's fighting forces had a diet of putrid meat and other items of poor quality. The foods available couldn't be stored or transported except in a dry state. Recognizing an important problem prize was announced offering 12,OOO francs and fame to anyone inventing a useful method of food preservation.
 
    Nicolas Appert, a French confectioner. working in a simple kitchen. observed that food heated in. sealed containers was preserved if the container was not reopened or the seal did not leak. He modestly called the process "the art of Appertizing". Appert received the award from Napoleon after spending ten years proving his discovery.
 
 
    It should be appreciated that the cause of spoilage of food was unknown, The great scientists of the day were summoned to evaluate Appert’s process and offer explanations for its apparent success. The conclusion reached was that the process was successful because in some mysterious and magical fashion, air combined with food in a sealed container, preventing putrefaction. This was quite incorrect. Nevertheless, the canning process was discovered and practiced for the next 5O years with some success, but in the darkness of ignorance.
 
    Appert began work on his process in 1795. Peter Durand received patents in England in 1810 for glass and metal containers for packaging foods to be canned. The tin-plated metal containers were called "conisters" from which the term "can" is assumed to be derived. Early metal containers were bulky, crude and difficult to seal. By 1823 a can with a hole in the top was invented, allowing the food to be heated in boiling water baths with the hole covered with a loose lid. The lid was soldered into place after the heat treatment. Hole-in-top cans are in use presently for canned evaporated milk, although the cans are sealed prior to heating.

    By 1824 Appert had developed schedules for proessing some 50 different canned foods. Meats and stews processed by Appert were carried by Sir Edward Perry in 1824 in his search for a northwest passage to India. Several cans of food from this voyage were obtained from the National Maritime Museum in London in 1938 and opened. The food was found nontoxic for animals. Interestingly there were isolated from these canned products bacteria which had been dorman for at least 114 years. Given proper environment and substrate, they grow!
 
    In the 1820s canning plants appeared in the United States in Boston and
New York. By 1830 sweet corn was being processed in Maine. By 1840 canneries
began appearing throughout the United States.

    Temperature vs. Pressure

    In 1851 Chevalier-Appert invented an autoclave which lessened the danger involved in the operation of steam pressure vessels. It was recognized that some foods could be processed for shorter times if higher temperatures were available. It was learned that the temperature of boiling water could be increased by adding salt. Demands for greater production in factories could be met if the cooking times for foods could be reduced. For instance, the boiling water bath cooking of canned meats could be reduced from 6 hr to perhaps 1/2 hr by cooking the cans in a water-calcium chloride solution. Production could be increased thereby from some 2000 to 20,OOO cans per day. Losses due failure of containers were large. No pressure was applied to the cooking vessels. Commercial cans were unable to withstand the internal pressures developed by heating to 115℃.
 
    The temperature at which water will boil is dependent upon the pressure. Using a pressure pressure it was possible to achieve temperatures in the vicinity of 115℃. However, these retorts were still dangerous to operate.
 
    Spoilage of Food Caused by Microorganisms
     
    In 1862 President Lincoln signed the Morrill Act, creating the land grant colleges (Purdue, Michigan, Massachusetts, Illinois, etc. ). The great scientific debate in universities at that time was "spontaneous generation" of life. At this time Louis Pasteur, son of a well-decorated officer in Napoleon's army, became interested in the problems of the great wine and beer industries of France which were threatened with ruin; their products were diseased and souring from "spontaneous generation" of life in bottles and kegs.
 
    To the Academy of Sciences in France in 1864, Pasteur reported that be had found the cause of the disease of wine and beer to be a microscopic vegetation. When given favorable conditions this vegetation grew and spoiled the products. However, boiled wine sealed from contamination in jars with even cotton plugs would not sour. In fact, it was possible to isolate this microscopic vegetation from the cotton plugs! It was this microscopic growth which spoiled foods, and it was neccessary for such organisms to gain entrance to heated foods if they were to spoil! Here was an explanation for the success of Appert more than half a century before. The concept of heat treating foods to inactivate pathogenic organisms is termed appropriately "pasteurization" today.  It is interesting to note that magnifying lenses were used by Bacon in the late 1200s, but had never been focused on a drop of water until the 1600s by Leeuwenhoek. He had noted microscopic growth which he named "animalcules," but they were only a curiosity in water to him. Two more centuries elapsed before this information was organized and synthesized into an explanation for "spontaneous generation" of life.
 
    Appert had established that containers of food must be carefully sealed and heated. Cleanliness was important to his process, although he did not know that microorganisms were the agents of spoilage. Pasteur established several important principles. Most changes in wine depended on the development in it of microorganisms which were themselves the spirits of disease. Germs were brought by air, ingredients. machinery and even by people. Whenever wine contained no living organisms, the material remained undiseased.
 
    Heat Resistance of Microorganisms Important in Canning
 
    There are two important genera of bacteria which form spores. Both genera are rod forms, one (Bacillus) is aerobic and the other (Clostridium) is anaerobic. When a rod is about to sporulate a tiny refractile granule appears in the cell. The granule enlarges, becomes glassy and transparent, and resists the penetration of various chemical substances. All of the protoplasm of the rod seems to condense into the granule, or young spore. in a hard dehydrated, resistant state. The empty cell membrane of the bacterium may separate off, like the hull of a seed, leaving the spore as a free. round or oval body. Actually a spore is an end product of a series of enzymatic processes. There is no unanimity of opinion either of spore function in nature or of the factors concerned in spore formation.
 
    Since no multiplication take place as a result of the vegetative cell-spore-vegetative cell cycle, few bacteriologists accept the concept of the spore as a cell set apart for reproduction. Instead, various explanations of the biological nature and function of bacterial spores have been advanced. These include: the teleological interpretation of the spore as a resistant structure produced to enable the organism to survive an unfavorable environment; the idea that the spore is a normal resting state(a form of hibernation):the notion that spores are stages in
a development cycle of certain organisms, or a provision for the rearrangement of nuclear material. It is interesting to note that the protein of the vegetative cell and the protein of the spore are antigenically different.
 
    Spores appear to be formed by healthy cells facing starvation. Certain chemical agents (glutamic acid) may inhibit the development of spores. No doubt sporulation consists of a sequence of integrated biochemical reactions. The sequence can be interrupted at certain susceptible stages.
 
    The literature on the subject of the heat resistance ofbacteria contains many
contradictions and discrepancies from the records of the earliest works to those of
the present day. This lack of uniformity has been due in part to factors of unknown nature. Until the factors operative in the thermal resistance of bacteria are understood, it will not be possible to control by other than empirical means the processes which require for their success the destruction of bacteria.
 
    Heat may be applied in two ways for the destruction of bacteria. Oven heat may be considered as dry heat, used in the sterilization of glassware. Other materials are heated when moist or in the presence of moisture; this is commonly termed moist heat. Dry cells exhibit no life functions; their enzymes are not active. Cell protein does not coagulate in the absence of moisture.
 
    The gradual increase in the death rate of bacteria exposed to dry heat is
indicative of an oxidation process.
 
    Whereas death by dry heat is reported as an oxidative process. death by moist heat is thought to be due to the coagulation of the protein in the cell. The order of death by moist heat is logarithmic in nature. The explanation of bacteria death as caused by the inactivation of bacterial enzymes cannot be correct. A suspension containing 99% dead cells has 80% of its catalase active. Since the order of death by moist heat is logarithmic in nature, death must be brought about by the destruction of a single molecule. This change is termed a lethal mutation. To a food technologist, death of a bacterium is described by its inability to reproduce. Heat inactivates or coagulates a single mechanism (gene?) preventing reproduction. The decreasing enzyme content of dead bacteria is the consequence of inhibited growth and probably not the cause . Replacement of the enzyme molecules becomes impossible; the enzyme content slowly decreases.
 
    Regardless of the explanation of death of bacterial spores. the logarithmic
order of this death permits the computation of death points, rates or times. independent of any explanation. The death rates or times permit the comparison of the heat resistance of one .species at different temperatures or of different species at the same temperatures. It is also possible to describe in quantitative terms the effect of environmental factors upon the heat resistance of the bacteria.
 
    Originally the standard method of establishing the heat tolerance of different species of bacteria was the thermal death point,i.e. , the lowest temperature at which the organism is killed in 10 min. This method cannot give comparable results unless conditions such as the age of the culture, the concentration of cells, the pH value of the medium, and the incubation temperature are standardized. Food technologists concerned with processing canned foods have adopted the thermal death time, keeping the temperature constant and varying the times of heating. The thermal death time is the shortest time required at a given. temperature to kill the bacteria present. It is necessary to know the time and temperature required to adequately sterilize canned foods. This procedure involves not only the destruction of spores by moist heat, but also the rate of heat penetration and heat conductivity of containers and their contents. The heat resistance of an organism is designated. by the c value(the number of minutes required to destroy the organism at 121℃) and the z value (the numbre of degree centigrade required for the thermal death time curve to traverse one logarithmic cycle). These two valuse establish and describe the thermal death time curve. and are a quantitative measure of the heat resistance of the spores over a range of temperatures. 
 
    It has been recognized that spores of different species, and of strains of the
same species, exhibit marked differences in heat resistance, but little or nothing
is know in explanation. Some workers have believed that there might be a difference in heat resistance among the vegetative cells, which was transmitted to the spores. Comparing the beat resistance of vegetative cells and spores of a number of bacteria, considerable differences in the spore resistances are found among organisms. Differences in vegetative cell heat resistance is in some instances associated with high spore resistance. Other cultures of vegetative cells produce spores of low resistance. There is evidently no significant relationship between the heat resistance of the vegetative cell and that of the spore produced therefrom. As noted previously, even the protein of the vegetative cell and
 
   spore differ for a species.

     Some researchers reason that the spores of a strain are all of the same heat resistance. Others suspect that in a given spore suspension there are a predominant number of spores of relatively low heat resistance, a smaller number with greater heat resistance, and a still smaller number of very heat resistant spores. However, subcultures from heat resistant selections do not yield survivors  of uniformly high heat resistance over the parent strain.

    Factors Influencing The Heat Resistance of Spores
 
    Concentration. The heat resistance of a suspension of bacterial spores is related to the number of organisms present. The greater the number of spores per milliliter, the higher resistance of the suspension.
 
    Environment Factors. The resistance of bacterial spores is not a fixed property,but one which under ordinary conditions may tend to be relatively constant. The extent of change in resistance is determined largely by the physical and chemical forces which operate from outside the spore cell. Aside from purely theoretical interest, a better understanding of the cause of heat resistance of spores is of fundamental importance to the canning industry. There are relatively few types of spore-forming organisms especially endowed with heat resistant properties, but these account for most of the spoilage potential in canning. Spore heredity. the environment in which grow, and a combination of these factors must play some part in the production of highly heat resistant spores .
 
    Different yields of spore crops can be determined in various media. This may be demonstrated by plate count or by direct microscopic count. There is little information indicating a relationship between the physiological factors influencing spore formation and the heat resistance of spores produced. The reaction (pH value) of the medium in which spores are produced has appearently little influence on their heat resistance. 
 
    Continuous drying seems to enhance the resistance of spores, but this is irregular in effect. Freezing tends to weaken spores. The following data for an aerobic spare-forming organism isolated from spoiled canned milk is noteworthy
 
   (Curran 1935):
                                Heat Resistance at 121℃
     Spore Treatment                          Survival in Minutes
     Wetted                                                    5
     Alternately wetted and dried               6
     Dried                                                       7
     Frozen                                                     2
 
    Spores formed and aged in soil are found to be more heat resistant than those formed and aged in broth or agar. Natural environmental conditions are evidently more conducive to the development of heat resistant spores than conditions prevailing in artificial cultures. The prolonged action of metabolic wastes from cells appears to decrease the heat resistance of spores.
 
    Bacteria exposed to sublethal heat are more exacting in their nutrient and temperature requirements than undamaged bacteria. The composition of recovery media which organisms are placed after heating may have considerable effect on the apparent thermal destruction time of the organisms. Depending on the choice of media, heat treated bacteria may be found to be dead in one and alive in another.
 
    Thermophilic bacteria which from spores in artificial media. produce spores
of comparable heat resistance to those formed on equipment and machinery in canning plants.
 
    Spores obtained from soil extractions and remixed with sterile soil are less heat resistant than those heated in the soil directly. The higher natural resistance of spores in soil may be due to some physico-chemical influence of the soil and not to any differences between the soil and cultured spores themselves.
 
    Anthrax spores remain viable and virulent in naturally contaminated water for as many as 18 years. while artificial cultures remain in this condition for perhaps 5 months. Soil organisms on corn may remain viable on naturally contaminated tissue for at least 7 years. while the artificially cultured die in 3 months. Artificial media apparently weakens cultures of organisms If a culture is to be kept alive for a long period it is apparently desirable to have a medium which permits only a limited growth. limiting metabolic byproducts, than media which permit best growth. B. tuberculosis growing on a relatively poor medium may be kept viable for several years while growth on enriched media has viable organisms for only a few weeks. The preserving influence of natural environments may be a similar phenomena.

    18世紀90年代末,法國處于戰(zhàn)爭時期,國民的食物供應發(fā)生了困難.拿破侖部隊吃的是腐敗的肉和其他劣質食物.這些可供利用的食物除干態(tài)的外,都不可能進行儲藏或運輸.認識到這一嚴重問題之后,就宣告了一項獎金,將給予任何發(fā)明食物有效保藏方法的個人以12,000法郎和榮譽.

    一位工作在簡陋廚房中的法國糖食師傅尼古拉·阿培爾發(fā)現(xiàn)：在密封容器中加熱過的食物，如果不重新打開容器或密封不漏，它便被保存下來。他謙虛地把這種處理方法稱為“阿培爾技藝“。阿培爾在花了10年確認他的發(fā)明之后，才從拿破侖那里拿到這項獎賞。

    要知道，那時并不明白食品變質的道理。于是召集了當時的大科學家，對阿培爾的處理方法進行了評價，并對這種方法的明顯成功作出解釋。得到的結論是：這種處理方法之所以成功，原因是在密封的容器內，空氣以某種神秘難測的方式于食物相結合，防止了食物的腐敗。這當然不正確。盡管這樣，這種罐藏工藝終于被發(fā)現(xiàn)了，并經過了那時以后50年的實踐，取得了一定程度的成功，但還是處在無知的黑暗之中。

    阿培爾于1795年在他所提出的工藝方面開始工作。1810年彼特·杜蘭德在英國獲得用于包裝罐頭食物的玻璃容器和金屬容器專利。人們過去稱鍍錫鋼板的容器為“canister”(金屬罐）現(xiàn)在“can”這一用語被認為是從canister派生出來的詞。早期的金屬容器笨重、粗陋且難以封口。到1823年，發(fā)明了一種頂上帶小孔的金屬罐，用不緊密的蓋子封住小孔，同時讓食物在沸水浴中加熱。熱處理之后，將蓋子就原位焊牢。這種孔蓋式金屬罐目前仍用于淡煉奶罐頭，只不過這種鐵罐是在加熱之前密封的。

    到1824年，阿培爾已經制定了加工約50多種不同罐頭食品的作業(yè)計劃。經阿培爾處理的肉類和燉菜由埃德華·彼里爵士于1824年他探索通往印度西北航道是帶去。1838年，人們從倫敦的國立海事博物館得到了幾罐來自這次航行的罐頭食品，并將這些罐頭打開。發(fā)現(xiàn)這些食物對動物無毒。有趣的是，從這些罐食品中分離出一些細菌，它們已至少休眠了114年。給以適當?shù)沫h(huán)境和基質后，它們又生長了!

    19世紀20年代末在美國的波士頓和紐約出現(xiàn)了罐頭制造廠。1830年，緬因州開始加工甜玉米。到1840年，罐頭食品廠開始在美國到處出現(xiàn)。

    溫度和壓力

    1851年，查弗利爾—阿培爾創(chuàng)制了一種高壓鍋，它可以減少汽壓容器操作中所涉及的危險。人們早就知道，如果更高的溫度能辦到，有些食品的熱處理時間就可以縮短。人們也知道：用加鹽的方法可提高沸水的溫度。如果能減少食品的熱處理時間，那么工廠提高產量的要求就能夠得到滿足。例如，可以把肉類罐頭的沸水浴熱處理時間從6小時縮短到用氯化鈣水溶液熱處理時的0.5小時左右，從而可使產量大體從每天2000罐增加到20000罐。容器損壞所造成的損失是大的。因為對熱處理的容器不加壓，所以商業(yè)金屬罐也就不能耐受因加熱到115℃而產生的內壓。

    水沸騰時的溫度取決于壓力。使用壓力容器就可以達到115℃左右的溫度。雖如此，這類高壓殺菌鍋的操作仍有危險。

    微生物引起的食品腐敗

    1862年，林肯總統(tǒng)簽署了莫里爾法令，創(chuàng)辦了幾所政府贈地的高等學校（如普多、密歇根、馬薩諸塞、伊利諾斯等）。那時大學里科學辯論的重大課題是生命的“自發(fā)生說”。此時，拿破侖軍隊里一位功勛顯赫的軍官的兒子路易·巴斯德開始對法國巨大的葡萄酒和啤酒工業(yè)面臨毀滅危險這一問題產生了興趣，這些工業(yè)的產品由于酒瓶和酒桶里的生命“自發(fā)生”問題而得了毛病，正在變酸。

    1864年，巴斯德向法國科學院提出報告說，他已發(fā)現(xiàn)了導致葡萄酒和啤酒變壞的原因是某種微小營養(yǎng)體造成的。只要給予有利的條件，這種營養(yǎng)體便生長，使產品腐敗。但煮沸后的瓶裝葡萄酒，即使采用哪怕是棉花塞密封以隔絕污染，也不會變酸。的確，用棉花塞隔離這種微小營養(yǎng)體是可能的！正是這些微小的生長物造成了食品的腐壞！這些生物體若要腐壞加熱過的食品，它就得進入食品！以上就是50多年前阿培爾的成功的一種解釋。今天人們把這種對食品進行熱處理使致病生物體失活的概念恰當?shù)胤Q為“巴氏滅菌”。

    有趣的是我們注意到，雖然培根在13世紀末期就使用放大鏡了，但直到17世紀才由列文虎克將放大鏡對準一滴水。列文虎克發(fā)現(xiàn)了稱之為“微小物”的微小生長物,不過對他來說，這些微動物只不過是水中稀奇的東西罷了。經過兩個多世紀之后，這一知識被條理化了，被綜合成為對生命“自發(fā)生說”的一種解釋。

    阿培爾肯定：食品容器必須嚴格密封和加熱。清潔對他的工藝方法很重要，不過他并不知道微生物是腐敗的媒介。巴斯德確立了一些重要的原則。葡萄酒的許多變化取決于微生物在其中的生長，而微生物本身則是葡萄酒出問題的實質。生命的胚芽是由空氣、配料、機器甚至人帶來的。只要酒中不含活的生物體，此物一定可保持不變質。

    罐頭制造中重要微生物的耐熱性 

    形成芽孢的細菌主要有兩屬。這兩屬都是桿狀菌。一屬（芽孢桿菌屬）是需氧的，另一屬（梭狀芽孢桿菌屬）是厭氧的。當一個桿菌即將形成芽孢時，細胞內便出現(xiàn)一粒帶折射性的微小顆粒。這顆粒逐漸擴大，變成玻璃狀透明，并能抵抗多種化學物質的侵入。桿菌中的所有原生質似乎都凝聚到此顆粒（即幼芽孢）中，使之處于一種干硬而有抵抗力的狀態(tài)。此細菌的空細胞膜象種子殼一樣可被分離掉，留下呈圓形或蛋形散粒物的芽孢。實際上一個芽孢是一系列酶促過程的最終產物。不論關于芽孢在自然界中的作用，或有關芽孢形成的原因，都沒有一致的看法。

    因為“營養(yǎng)細胞—芽孢—營養(yǎng)細胞”循環(huán)的結果不產生增殖作用，所以幾乎沒有細菌學家認為芽孢是一種分開來供繁殖用的細胞。相反，對細菌芽孢的生物學本質和功能卻提出了多種解釋。這些解釋包括：有關芽孢是一種正常休眠狀態(tài)（一種蟄伏狀態(tài)）的看法;有關芽孢是某些生物體生長循環(huán)中的一個階段，即為重新調整細胞核物質作準備的意見。有趣的是我們注意到，營養(yǎng)細胞蛋白質與芽孢蛋白質在抗原上是不同的。

    健康細胞面臨饑餓時就要形成芽孢。某些化學物質（谷氨酸）可以抑制芽孢的發(fā)育。無疑芽孢的形成包括一系列完整的生物化學反應。這一系列反應在某些易受外界影響的階段可能被中斷。

    有關細菌的耐熱性問題的文獻中載有從早期工作者資料到目前資料中的許多矛盾和分歧的地方。這種不一致的部分原因是本質未明的因素造成的。要等到這些對細菌耐熱性起作用的因素被人們認識之后，人們才能用經驗方法以外的方法來成功地控制殺菌過程。

    為了殺滅細菌，加熱方法可以有兩種。烘爐的熱可認為是干熱，用于玻璃器皿的滅菌。其他物料是在潮濕（即有水份存在）的時候加熱的，就是通常說的濕熱。干的細胞不顯現(xiàn)生命功能，其酶系統(tǒng)無活性。細胞蛋白質在沒有水份存在的情況下不凝固。

    暴露在干熱狀態(tài)下的細菌，其死亡率的逐漸上升是某種氧化過程的表現(xiàn)。

    盡管據(jù)報道干熱致死是一種氧化過程，但一般認為濕熱致死則是由細胞中蛋白質凝固造成的。濕熱致死的量級本質上是對數(shù)性的。把細菌死亡解釋為由細菌酶鈍化引起是不正確的。某種含99％死細胞的懸浮液，其中過氧化氫酶有80％是有活性的。由于濕熱至死的量級是對數(shù)的，所以死亡的發(fā)生必定按單個微粒一一死亡的方式。這種變化稱為致死突變。對食品工藝學家來說，細菌的死亡被說成是無力繁殖。熱使某種簡單的機制（基因？）不起作用或凝住，從而不使細菌再生繁殖。死亡細菌酶含量不斷減少是生長受抑制的結果，可能不是生長受抑制的原因。酶分子的補充成為不可能，故酶含量慢慢地減少。

    不論對細菌芽孢死亡的解釋如何，芽孢死亡的對數(shù)量級使致死溫度、致死率和致死時間的計算成為可能，而與任何解釋無關。致死率或致死時間使同一菌種不同溫度下或不同菌種在同一溫度下的耐熱性有可能進行比較。同時，還有可能以定量方式描述環(huán)境因素對細菌耐熱性的影響。

    最初，確定不同菌種的耐熱性的標準方法是熱死溫度法，即這種生物體在10分鐘內被殺死的最低溫度。此法不能得出可比的結果，除非象菌種齡期、細胞濃度、培養(yǎng)基pH值和培養(yǎng)溫度都是統(tǒng)一規(guī)定的。關心罐頭食品加工的食品工業(yè)學家卻采用了熱死時間法，即保持溫度不變、改變加熱時間的方法。熱死時間使在給定溫度下殺滅現(xiàn)存細菌所需的最短時間。

    有必要知道罐頭食品充分滅菌所需要的時間和溫度。這一步驟不僅涉及用濕熱法殺滅芽孢，也涉及到傳熱速率和容器及其內容物熱傳導率。生物體的耐熱性由C值（在120℃下殺滅此生物體所需的分鐘數(shù)）和E值（沿熱死時間曲線移動一個對數(shù)周期所需的攝氏度數(shù)）表示。這兩個值確定并描繪了熱致死時間曲線，是芽孢在一定溫度范圍內耐熱性的定量的量度。

    人們已經認識到：不同菌種芽孢和同菌種不同菌株的芽孢，都表現(xiàn)出明顯不同的耐熱性，但幾乎不知道如何解釋。有些工作者認為，營養(yǎng)細胞之間也會在耐熱性下有一定差異，此差異傳給了芽孢。比較許多細菌的營養(yǎng)細胞和芽孢的耐熱性后發(fā)現(xiàn)生物體中間在芽孢耐熱性上有顯著差異。有的情況營養(yǎng)細胞耐熱性差異與芽孢耐熱性強有聯(lián)系。也有一些營養(yǎng)細胞的培養(yǎng)物產生耐熱性弱的芽孢。顯然，營養(yǎng)細胞耐熱性和由營養(yǎng)細胞產生的芽孢的耐熱性之間沒有明顯的聯(lián)系。如以前所指出的，即使同一菌種的營養(yǎng)細胞和芽孢中的蛋白質也不同。

    一些研究者推論：同菌株芽孢的耐熱性都是相同的。但另外一些人認為：在給定的芽孢懸浮液中，耐熱性弱的芽孢的數(shù)目占有優(yōu)勢;耐熱性愈強，數(shù)目愈少;耐熱性最強的，數(shù)就更少。然而，從耐熱性選育得到的次代培養(yǎng)物不產生比親株均勻耐熱性更強的存活者。

    影響芽孢耐熱性的因素 

    濃度——細胞懸浮液的耐熱性于現(xiàn)存生物體的數(shù)量有關。每毫升中芽孢數(shù)越多，懸浮液耐熱性越強。

    環(huán)境因素——細胞芽孢的耐熱性不是一種固定不變的性質，而是一種在一般條件下趡于相對恒定的選擇。耐熱性變化的幅度主要取決于受芽孢細胞外部影響的物理力和化學力。對于罐頭制造工業(yè)來說，除了純理論的興趣外，更深入了解芽孢耐熱性的起因使十分重要的事情。只有比較少數(shù)的幾種產芽孢微生物特別賦有耐熱特性，而這種微生物則是造成罐頭制造上大多數(shù)潛在腐敗的主要原因。芽孢的遺傳性、它生長所在的環(huán)境、以及這些因素的綜合都必然在強耐熱性芽孢的產生方面有一定作用。

    我們能夠測定各種培養(yǎng)基中芽孢培養(yǎng)物的不同收得量。這可由平板計數(shù)或直接顯微鏡計數(shù)來顯示。幾乎沒有什么測定數(shù)據(jù)能說明影響芽孢形成的生理因素與產生的芽孢的耐熱性之間的關系。產芽孢的培養(yǎng)基的作用（pH值）很明顯不影響芽孢的耐熱性。

    持續(xù)的干燥似乎增強了芽孢的耐性，但這實際上沒有什么規(guī)律。冷凍趡于使芽孢耐熱性減弱。下面是從腐敗牛奶罐頭中分離出來的需氧產芽孢菌的數(shù)量，值得我們注意（柯倫，1935年）：

    121℃時的耐熱性
    芽孢處理法                           存活時間（min）
    潮濕                                        5
    干、濕交替處理                    6
    干燥                                        7         
    冷凍                                        2
 
    我們發(fā)現(xiàn)在土壤中形成和成熟的芽孢比在肉湯或瓊脂中形成和成熟的芽孢有更強的耐熱性。顯然，自然環(huán)境條件比常見人工培養(yǎng)條件更有助于耐熱芽孢的發(fā)育。看來來自細胞新陳代謝廢物的長時間作用會使芽孢的耐熱性減弱。

    受亞致死熱量作用的芽孢比未受損害的芽孢有更加嚴格的營養(yǎng)要求和溫度要求。對于經加熱之后接入微生物的回收培養(yǎng)基，它的組成對該微生物的表現(xiàn)熱致死時間會有明顯的影響。根據(jù)選用培養(yǎng)基的不同，可以發(fā)現(xiàn)熱處理后的細菌在一種培養(yǎng)基里死亡，而在另一種培養(yǎng)基里存活。

    在人工培養(yǎng)基中形成芽孢的嗜熱細菌，它產生的芽孢于在罐頭工廠機器設備上形成的芽孢，在耐熱性上差不多。

    從土壤分離出來再混以無菌泥土的芽孢，其耐熱性比直接在土壤中加熱的芽孢弱。土壤中芽孢的天然耐熱性較強可能是由于土壤的某些物理化學影響造成的。而不是土壤芽孢與人工培養(yǎng)芽孢本身之間的什么差異造成的。

    炭菹菌芽孢在天然污染的水中保持活性和毒性長達18年，而人工培養(yǎng)物則保持此狀態(tài)約5個月。玉米上的土壤微生物在天然污染的生物組織上至少可以存活7年，而人工培養(yǎng)的則在3個月內就死亡。顯然人工培養(yǎng)的微生物的活性減弱。

    如果要長期保持培養(yǎng)物的活力，顯然就要有一種只許有限生長的培養(yǎng)基，來限制新陳代謝的副產物，而不要那些允許旺盛生長的培養(yǎng)基。生長在相對劣質培養(yǎng)基中的結核桿菌可以存活好幾年，而在營養(yǎng)豐富培養(yǎng)基中生長時，僅存活幾周。天然環(huán)境的防腐作用大概也是一種類似的現(xiàn)象。