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世纪90年代末,法国处于战争时期,国民的食物供应发生了困难.拿破仑部队吃的是腐败的肉和其他劣质食物.这些可供利用的食物除干态的外,都不可能进行储藏或运输.认识到这一严重问题之后,就宣告了一项奖金,将给予任何发明食物有效保藏方法的个人以12,000法郎和荣誉.

    一位工作在简陋厨房中的法国糖食师傅尼古拉·阿培尔发现：在密封容器中加热过的食物，如果不重新打开容器或密封不漏，它便被保存下来。他谦虚地把这种处理方法称为“阿培尔技艺“。阿培尔在花了10年确认他的发明之后，才从拿破仑那里拿到这项奖赏。

    要知道，那时并不明白食品变质的道理。于是召集了当时的大科学家，对阿培尔的处理方法进行了评价，并对这种方法的明显成功作出解释。得到的结论是：这种处理方法之所以成功，原因是在密封的容器内，空气以某种神秘难测的方式于食物相结合，防止了食物的腐败。这当然不正确。尽管这样，这种罐藏工艺终于被发现了，并经过了那时以后50年的实践，取得了一定程度的成功，但还是处在无知的黑暗之中。

    阿培尔于1795年在他所提出的工艺方面开始工作。1810年彼特·杜兰德在英国获得用于包装罐头食物的玻璃容器和金属容器专利。人们过去称镀锡钢板的容器为“canister”(金属罐）现在“can”这一用语被认为是从canister派生出来的词。早期的金属容器笨重、粗陋且难以封口。到1823年，发明了一种顶上带小孔的金属罐，用不紧密的盖子封住小孔，同时让食物在沸水浴中加热。热处理之后，将盖子就原位焊牢。这种孔盖式金属罐目前仍用于淡炼奶罐头，只不过这种铁罐是在加热之前密封的。

    到1824年，阿培尔已经制定了加工约50多种不同罐头食品的作业计划。经阿培尔处理的肉类和炖菜由埃德华·彼里爵士于1824年他探索通往印度西北航道是带去。1838年，人们从伦敦的国立海事博物馆得到了几罐来自这次航行的罐头食品，并将这些罐头打开。发现这些食物对动物无毒。有趣的是，从这些罐食品中分离出一些细菌，它们已至少休眠了114年。给以适当的环境和基质后，它们又生长了!

    19世纪20年代末在美国的波士顿和纽约出现了罐头制造厂。1830年，缅因州开始加工甜玉米。到1840年，罐头食品厂开始在美国到处出现。

    温度和压力

    1851年，查弗利尔—阿培尔创制了一种高压锅，它可以减少汽压容器操作中所涉及的危险。人们早就知道，如果更高的温度能办到，有些食品的热处理时间就可以缩短。人们也知道：用加盐的方法可提高沸水的温度。如果能减少食品的热处理时间，那么工厂提高产量的要求就能够得到满足。例如，可以把肉类罐头的沸水浴热处理时间从6小时缩短到用氯化钙水溶液热处理时的0.5小时左右，从而可使产量大体从每天2000罐增加到20000罐。容器损坏所造成的损失是大的。因为对热处理的容器不加压，所以商业金属罐也就不能耐受因加热到115℃而产生的内压。

    水沸腾时的温度取决于压力。使用压力容器就可以达到115℃左右的温度。虽如此，这类高压杀菌锅的操作仍有危险。

    微生物引起的食品腐败

    1862年，林肯总统签署了莫里尔法令，创办了几所政府赠地的高等学校（如普多、密歇根、马萨诸塞、伊利诺斯等）。那时大学里科学辩论的重大课题是生命的“自发生说”。此时，拿破仑军队里一位功勋显赫的军官的儿子路易·巴斯德开始对法国巨大的葡萄酒和啤酒工业面临毁灭危险这一问题产生了兴趣，这些工业的产品由于酒瓶和酒桶里的生命“自发生”问题而得了毛病，正在变酸。

    1864年，巴斯德向法国科学院提出报告说，他已发现了导致葡萄酒和啤酒变坏的原因是某种微小营养体造成的。只要给予有利的条件，这种营养体便生长，使产品腐败。但煮沸后的瓶装葡萄酒，即使采用哪怕是棉花塞密封以隔绝污染，也不会变酸。的确，用棉花塞隔离这种微小营养体是可能的！正是这些微小的生长物造成了食品的腐坏！这些生物体若要腐坏加热过的食品，它就得进入食品！以上就是50多年前阿培尔的成功的一种解释。今天人们把这种对食品进行热处理使致病生物体失活的概念恰当地称为“巴氏灭菌”。

    有趣的是我们注意到，虽然培根在13世纪末期就使用放大镜了，但直到17世纪才由列文虎克将放大镜对准一滴水。列文虎克发现了称之为“微小物”的微小生长物,不过对他来说，这些微动物只不过是水中稀奇的东西罢了。经过两个多世纪之后，这一知识被条理化了，被综合成为对生命“自发生说”的一种解释。

    阿培尔肯定：食品容器必须严格密封和加热。清洁对他的工艺方法很重要，不过他并不知道微生物是腐败的媒介。巴斯德确立了一些重要的原则。葡萄酒的许多变化取决于微生物在其中的生长，而微生物本身则是葡萄酒出问题的实质。生命的胚芽是由空气、配料、机器甚至人带来的。只要酒中不含活的生物体，此物一定可保持不变质。

    罐头制造中重要微生物的耐热性 

    形成芽孢的细菌主要有两属。这两属都是杆状菌。一属（芽孢杆菌属）是需氧的，另一属（梭状芽孢杆菌属）是厌氧的。当一个杆菌即将形成芽孢时，细胞内便出现一粒带折射性的微小颗粒。这颗粒逐渐扩大，变成玻璃状透明，并能抵抗多种化学物质的侵入。杆菌中的所有原生质似乎都凝聚到此颗粒（即幼芽孢）中，使之处于一种干硬而有抵抗力的状态。此细菌的空细胞膜象种子壳一样可被分离掉，留下呈圆形或蛋形散粒物的芽孢。实际上一个芽孢是一系列酶促过程的最终产物。不论关于芽孢在自然界中的作用，或有关芽孢形成的原因，都没有一致的看法。

    因为“营养细胞—芽孢—营养细胞”循环的结果不产生增殖作用，所以几乎没有细菌学家认为芽孢是一种分开来供繁殖用的细胞。相反，对细菌芽孢的生物学本质和功能却提出了多种解释。这些解释包括：有关芽孢是一种正常休眠状态（一种蛰伏状态）的看法;有关芽孢是某些生物体生长循环中的一个阶段，即为重新调整细胞核物质作准备的意见。有趣的是我们注意到，营养细胞蛋白质与芽孢蛋白质在抗原上是不同的。

    健康细胞面临饥饿时就要形成芽孢。某些化学物质（谷氨酸）可以抑制芽孢的发育。无疑芽孢的形成包括一系列完整的生物化学反应。这一系列反应在某些易受外界影响的阶段可能被中断。

    有关细菌的耐热性问题的文献中载有从早期工作者资料到目前资料中的许多矛盾和分歧的地方。这种不一致的部分原因是本质未明的因素造成的。要等到这些对细菌耐热性起作用的因素被人们认识之后，人们才能用经验方法以外的方法来成功地控制杀菌过程。

    为了杀灭细菌，加热方法可以有两种。烘炉的热可认为是干热，用于玻璃器皿的灭菌。其他物料是在潮湿（即有水份存在）的时候加热的，就是通常说的湿热。干的细胞不显现生命功能，其酶系统无活性。细胞蛋白质在没有水份存在的情况下不凝固。

    暴露在干热状态下的细菌，其死亡率的逐渐上升是某种氧化过程的表现。

    尽管据报道干热致死是一种氧化过程，但一般认为湿热致死则是由细胞中蛋白质凝固造成的。湿热致死的量级本质上是对数性的。把细菌死亡解释为由细菌酶钝化引起是不正确的。某种含99％死细胞的悬浮液，其中过氧化氢酶有80％是有活性的。由于湿热至死的量级是对数的，所以死亡的发生必定按单个微粒一一死亡的方式。这种变化称为致死突变。对食品工艺学家来说，细菌的死亡被说成是无力繁殖。热使某种简单的机制（基因？）不起作用或凝住，从而不使细菌再生繁殖。死亡细菌酶含量不断减少是生长受抑制的结果，可能不是生长受抑制的原因。酶分子的补充成为不可能，故酶含量慢慢地减少。

    不论对细菌芽孢死亡的解释如何，芽孢死亡的对数量级使致死温度、致死率和致死时间的计算成为可能，而与任何解释无关。致死率或致死时间使同一菌种不同温度下或不同菌种在同一温度下的耐热性有可能进行比较。同时，还有可能以定量方式描述环境因素对细菌耐热性的影响。

    最初，确定不同菌种的耐热性的标准方法是热死温度法，即这种生物体在10分钟内被杀死的最低温度。此法不能得出可比的结果，除非象菌种龄期、细胞浓度、培养基pH值和培养温度都是统一规定的。关心罐头食品加工的食品工业学家却采用了热死时间法，即保持温度不变、改变加热时间的方法。热死时间使在给定温度下杀灭现存细菌所需的最短时间。

    有必要知道罐头食品充分灭菌所需要的时间和温度。这一步骤不仅涉及用湿热法杀灭芽孢，也涉及到传热速率和容器及其内容物热传导率。生物体的耐热性由C值（在120℃下杀灭此生物体所需的分钟数）和E值（沿热死时间曲线移动一个对数周期所需的摄氏度数）表示。这两个值确定并描绘了热致死时间曲线，是芽孢在一定温度范围内耐热性的定量的量度。

    人们已经认识到：不同菌种芽孢和同菌种不同菌株的芽孢，都表现出明显不同的耐热性，但几乎不知道如何解释。有些工作者认为，营养细胞之间也会在耐热性下有一定差异，此差异传给了芽孢。比较许多细菌的营养细胞和芽孢的耐热性后发现生物体中间在芽孢耐热性上有显著差异。有的情况营养细胞耐热性差异与芽孢耐热性强有联系。也有一些营养细胞的培养物产生耐热性弱的芽孢。显然，营养细胞耐热性和由营养细胞产生的芽孢的耐热性之间没有明显的联系。如以前所指出的，即使同一菌种的营养细胞和芽孢中的蛋白质也不同。

    一些研究者推论：同菌株芽孢的耐热性都是相同的。但另外一些人认为：在给定的芽孢悬浮液中，耐热性弱的芽孢的数目占有优势;耐热性愈强，数目愈少;耐热性最强的，数就更少。然而，从耐热性选育得到的次代培养物不产生比亲株均匀耐热性更强的存活者。

    影响芽孢耐热性的因素 

    浓度——细胞悬浮液的耐热性于现存生物体的数量有关。每毫升中芽孢数越多，悬浮液耐热性越强。

    环境因素——细胞芽孢的耐热性不是一种固定不变的性质，而是一种在一般条件下趡于相对恒定的选择。耐热性变化的幅度主要取决于受芽孢细胞外部影响的物理力和化学力。对于罐头制造工业来说，除了纯理论的兴趣外，更深入了解芽孢耐热性的起因使十分重要的事情。只有比较少数的几种产芽孢微生物特别赋有耐热特性，而这种微生物则是造成罐头制造上大多数潜在腐败的主要原因。芽孢的遗传性、它生长所在的环境、以及这些因素的综合都必然在强耐热性芽孢的产生方面有一定作用。

    我们能够测定各种培养基中芽孢培养物的不同收得量。这可由平板计数或直接显微镜计数来显示。几乎没有什么测定数据能说明影响芽孢形成的生理因素与产生的芽孢的耐热性之间的关系。产芽孢的培养基的作用（pH值）很明显不影响芽孢的耐热性。

    持续的干燥似乎增强了芽孢的耐性，但这实际上没有什么规律。冷冻趡于使芽孢耐热性减弱。下面是从腐败牛奶罐头中分离出来的需氧产芽孢菌的数量，值得我们注意（柯伦，1935年）：

    121℃时的耐热性
    芽孢处理法                           存活时间（min）
    潮湿                                        5
    干、湿交替处理                    6
    干燥                                        7         
    冷冻                                        2
 
    我们发现在土壤中形成和成熟的芽孢比在肉汤或琼脂中形成和成熟的芽孢有更强的耐热性。显然，自然环境条件比常见人工培养条件更有助于耐热芽孢的发育。看来来自细胞新陈代谢废物的长时间作用会使芽孢的耐热性减弱。

    受亚致死热量作用的芽孢比未受损害的芽孢有更加严格的营养要求和温度要求。对于经加热之后接入微生物的回收培养基，它的组成对该微生物的表现热致死时间会有明显的影响。根据选用培养基的不同，可以发现热处理后的细菌在一种培养基里死亡，而在另一种培养基里存活。

    在人工培养基中形成芽孢的嗜热细菌，它产生的芽孢于在罐头工厂机器设备上形成的芽孢，在耐热性上差不多。

    从土壤分离出来再混以无菌泥土的芽孢，其耐热性比直接在土壤中加热的芽孢弱。土壤中芽孢的天然耐热性较强可能是由于土壤的某些物理化学影响造成的。而不是土壤芽孢与人工培养芽孢本身之间的什么差异造成的。

    炭菹菌芽孢在天然污染的水中保持活性和毒性长达18年，而人工培养物则保持此状态约5个月。玉米上的土壤微生物在天然污染的生物组织上至少可以存活7年，而人工培养的则在3个月内就死亡。显然人工培养的微生物的活性减弱。

    如果要长期保持培养物的活力，显然就要有一种只许有限生长的培养基，来限制新陈代谢的副产物，而不要那些允许旺盛生长的培养基。生长在相对劣质培养基中的结核杆菌可以存活好几年，而在营养丰富培养基中生长时，仅存活几周。天然环境的防腐作用大概也是一种类似的现象。