Resistance of Bacillus subtilis var. niger Spores Occluded in Water-insoluble Crystals to Three Sterilization Agents
封闭于不溶于水的结晶体中枯草杆菌黑色变种芽孢对三种灭菌剂的耐受性
JOHN E. DOYLE AND ROBERT R. ERNST
Research Laboratory, Castle Company, Rochester, New York 14623
Received for publication 23 December 1966
The resistance to destruction of spores of Bacillus subtilis var. niger occluded in crystals of calcium carbonate and exposed to ethylene oxide and moist and dry heat was determined and compared with the destruction of unoccluded spores. Occluded spores could not be inactivated with ethylene oxide. Resistance to inactivation was approximately 900 and 9 times higher for occluded than for unoccluded spores subjected to moist and dry heat, respectively, at 121 C. The protective effect may be due either to the unavailability of oxygen for destruction by oxidation or to inhibition of the loss of essential cell constituents by vaporization. Evidence also implicates the crystal structure as a thermal conductivity barrier. Occluded spores retained viability over a 3-year period compared with unoccluded spores which decreased over 90% during this period. Occluded spores in insoluble materials are seldom encountered in the technology of sterilization, but could be the most critical factor in the sterilization of interplanetary vehicles. Entrapped spores in insoluble materials are usually difficult to detect, and are very stable as well as extremely resistant to destruction by heat and ethylene oxide.
检测了封闭在碳酸钙晶体中的枯草杆菌黑色变种芽孢暴露于环氧乙烷、湿热和干热中的耐受性，并与未封闭孢子的破坏相比。封闭的孢子不能被环氧乙烷灭活。封闭孢子对121ºC的湿热和干热灭活的耐受性大约是未封闭孢子的900和9倍。其保护作用可能是因为缺乏氧化杀灭的氧气，或抑制了蒸汽造成的基本细胞成分的丧失。证据还表明晶体结构作为导热性障碍。闭塞孢子保持生存能力3年以上，与之相比未封闭的孢子在此期间减少了90%以上。不溶材料中的闭塞孢子在灭菌技术中很少见，但在星际运输的灭菌中可能是最关键的因素。不溶材料中的孢子通常难以检出，而且非常稳定，还有极强的抗热和环氧乙烷破坏的抵抗性。
Many papers have been written on factors which influence the resistance of microorganisms to a sterilizing agent. Sobernheim and Mundel (13) found that spores in soil were six to eight times more resistant to sterilization by moist heat than the same number of spores in a laboratory culture. We have found soils having a much higher resistance to dry heat, moist heat, and ethylene oxide than spore isolates cultured from the same soil.
关于微生物对灭菌剂耐受性的影响因素已有很多论文。Sobernheim和Mundel (13)发现污物中的孢子与实验室中培养的相同数目孢子相比，对湿热灭菌的耐受性高6至8倍。我们发现，污物与从同一污物中分离培养的孢子相比，对干热、湿热和环氧乙烷有更高的耐受性。
The occlusion and survival of microorganisms in crystals have been reported previously. Abbot, Cockton, and Jones (1), using various water-soluble crystals, observed an increased resistance to destruction in formaldehyde and ethylene oxide. They demonstrated the inclusion of organisms in crystals by use of the electron microscope. Royce and Bowler (10) also noted that protection from gaseous ethylene oxide was provided by water-soluble crystals.
在晶体中封闭并生存的微生物先前已有报道。Abbot, Cockton和Jones(1)利用各种水溶性晶体观察到其对甲醛和环氧乙烷的耐受性增强。他们用电子显微镜证明在晶体中包含的有机体。Royce和Bowle(10)也指出，水溶性晶体提供了对气态环氧乙烷破坏的保护。
Bacteria were seen in crystals of calcite more than 100 million years old by Bradley (4). Ehlers, Stiles, and Birle (7) and Schopf et al. (11) have reported iron bacteria in pyrite. Barghoorn and Schopf (2) and Barghoorn and Tyler (3) have also reported on microorganisms present in geological formations. No attempt was made by the preceding investigators to see whether the microorganisms were viable.
Bradley在100多万年前的方解石晶体中发现了细菌(4)。Ehlers, Stiles, 和Birle (7)以及Schopf等(11)报道了黄铁矿中的铁细菌。Barghoorn和Schopf(2) 及Barghoorn和Tyler(3)也报告了微生物存在于地质层。以前没有研究者尝试研究微生物是否存活。
Apparently no one has investigated the viability or resistance of microorganisms in water-insoluble crystals. If a crystal is grown very rapidly so that impurities, such as microorganisms, do not have time to be desorbed, they may be entrapped within the growing crystal. Recovery would be impossible with ordinary culturing techniques, since they would not be released from the crystalline matrix. For this study, calcium carbonate was chosen because it is relatively insoluble in water and easily dissolved in dilute acid or ammonium chloride. Other insoluble crystalline materials were also studied.
显然，没有人研究不溶于水的晶体中微生物的活性和抵抗性。如果晶体生成非常迅速,使得诸如微生物之类的杂质没有时间得到充分解吸附，微生物可能在生长的晶体中被捕获。普通培养技术不可能使之还原，因为它们不会从晶状基质中释放出来。在本研究中选用了碳酸钙，因为它相对不溶于水，且易溶解于稀酸或氯化铵。还研究了其他不溶于水的晶体材料。
MATERIALS AND METHODS
材料及方法
Organism. Bacillus subtilis var niger 356 S.C. no. 4 N. R. Smith strain was used (12). This strain has a relatively high dry-heat and ethylene oxide resistance, but relatively low moist-heat resistance.
有机体：使用了枯草杆菌黑色变种356 S.C. no. 4 N. R. Smith菌株(12)。这一菌株对干热和环氧乙烷耐受性相对较高，但对湿热耐受性相对较低。
Culture medium. Spores of the test organism were heat-shocked at 65 C for 30 min and were used as inocula for starter cultures in casein acid digest medium of the following composition: casein acid hydrolysate powder (General Biochemicals, Inc., Chagrin Falls, Ohio), 10.0 g; yeast extract (Difco), 5.0 g; glucose, 5.0 g; KH2PO4, 5.0 g; CaCl2-2H2O, 0.66 g; MnSO4-H20, 0.03 g; tap water, 1,000 ml. The medium was filtered, adjusted to pH 7.0, and sterilized in steam at 121 C for 20 min.
培养基：将测试有机体的芽孢在65 C下加热30分钟，作为在组分如下的酸水解酪蛋白培养基中进行起子培养的接种物：酸水解酪蛋白粉(General Biochemicals, Inc., Chagrin Falls, Ohio)，10.0克；酵母提取物(Difco)，5.0克；葡萄糖5.0 g；KH2PO4, 5.0 g；CaCl2-2H2O, 0.66 g; MnSO4-H20, 0.03 g; 自来水1,000 ml。过滤培养基，调整到pH值为7.0，在121 C蒸汽中灭菌20分钟。
FIG. 1. Diagrammatic representation of the autoclave used for determination of moist-heat parameters.
图1：用于测定湿热参数的高压灭菌器示意图
After shaking for 24 hr at 32 C, the starter culture was used to inoculate large flasks of the same medium. These were incubated with shaking for 4 days at 32 C, and the sporulated culture was kept at 45 C overnight to allow autolysis of vegetative cells. The spores were then harvested by centrifugation, washed eight times in distilled water, and checked by phase microscopy to ascertain absence of debris.
在32 C下摇动24小时后，将起子培养物移至装有同样培养基的大烧瓶中。这些培养物在32C下摇动着培养了了4天，孢子培养是保持在45C下一整夜，以便植物细胞自溶。然后用离心法采集孢子，用蒸馏水洗涤8次，并用相差显微镜检查以确定没有碎片。
Preparation of occluded spores. A 10-ml solution of 1.1 % CaCl2 was prepared containing 108 spores/ml. To this solution, 10 ml of 1.06% Na2CO3 was added very rapidly and the mixture was vigorously shaken. Crystals of Ca2CO3 were immediately formed, occluding large quantities of spores per crystal. The crystals were then centrifuged at 20,500 × g, washed three times with distilled water, and made up to original volume. Methocel (0.2 g, 25 centipoises/sec; Dow Chemical Co., Midland, Mich.) was added to the crystal suspension to make the solution viscous enough to suspend the crystals evenly, and to bind the crystals to the paper on which they were inoculated. Samples of 0.01 ml were used to inoculate the paper strips. The strips were first dried at room temperature, then at 90 C for 16 hr to remove any occluded water. When the strips were not air-dried at 90 C, the resistance of the spores to moist or dry heat did not increase, though it did for ethylene oxide.
封闭孢子的制备：制备1.1 %的CaCl2溶液10-ml，每ml含108孢子。在此溶液中极为迅速地加入10 ml1.06% Na2CO3并剧烈摇动。立即形成了Ca2CO3晶体，每个晶体中都封闭了大量孢子。然后将晶体按0,500 × g进行离心，用蒸馏水洗涤三次，并恢复为原有体积。在晶体悬浮液中加入Methocel (0.2 g, 25厘泊/sec; Dow Chemical Co., Midland, Mich.)，使溶液足够粘稠，以均匀地悬浮着晶体，将晶体包在纸上以进行移植。将0.01毫升的样品移植在纸带上。首先将纸带在室温下晾干，然后在90摄氏度下烘干16小时以消除所有封存的水分。若纸带不在90C下晾干，虽然孢子对环氧乙烷的耐受性会增加，但它对湿热或干热的耐受性不会增加。
To verify that the spores were occluded and not adsorbed to the crystals, the crystallization procedure was followed by use of sterile solutions. Then, after crystallization, spores were added and the procedure was followed as before. Strips made with this procedure had no increased moist-heat, dry-heat, or ethylene oxide resistance. Microscopic examination showed free spores among the crystals.
为了验证孢子被封入晶体中而没有被晶体吸附，结晶过程使用无菌溶液。然后在结晶后添加孢子，操作如前。按此操作制作的纸带不会增加对湿热、干热和环氧乙烷的耐受性。显微镜检查表明晶体中无孢子。
Recovery of occluded spores for assaying and sterility end-point determination. After exposure to the sterilizing agent, the spore strips were placed in 25 ml of sterile 3% NH4C1 for 3 days at 0 C to dissolve the CaCO3. The solution and strip were then placed in a Waring Blendor at high speed for 3 min. The solution was then sonic-treated at 21 kc for 5 min to eliminate clumping. Appropriate dilutions were made on tryptone-glucose-yeast extract-agar (Difco) and incubated at 37 C for 48 hr before counting. This technique did not affect the counts on the unoccluded spore strip controls. For sterility end-point determinations, the above procedure was followed except that the spore strips and NH4Cl solution were added to 25 ml of double-strength Trypticase Soy Broth (BBL) after the crystals were dissolved.
回收封闭孢子以进行含量测定及灭菌终点检测：暴露于灭菌剂后，将孢子纸带放于0C的25毫升3%无菌NH4C1中3天，以溶解CaCO3。然后将溶液和纸带放于韦林氏混匀器中高速搅拌3分钟。然后用21 kc的声波处理5分钟以消除凝块。用胰胨-葡萄糖-酵母浸膏琼脂(Difco)作适当稀释，计数前在37C下培养48小时。这一方法并不影响对在对照纸带上未封闭孢子的计数。灭菌终点检测如上述操作，除了孢子纸带及其加入的25毫升NH4Cl溶液为双倍浓度。晶体溶解后用胰蛋白胨大豆肉汤(BBL)培养。
Determination of resistance to ethylene oxide. Apparatus and procedures similar to those reported by Ernst and Shull (7) were used.
对环氧乙烷耐受性的测定：器械及操作与Ernst和Shull (7)所用的相同。
Determination of resistance to moist heat. An autoclave, 9 inches inside diameter and 16 inches deep (Fig. 1), was used for determining moist-heat resistance. A similar device was described by Cook and Brown (6). Initially, the large chamber was evacuated, after which steam was added to the desired pressure and temperature. The test spore strips were placed in the plunger chamber which was then evacuated. The plunger was pushed into the steam chamber, thus immediately exposing the spore strips to the saturated steam at the desired temperature. After a predetermined time interval, the plunger was pulled out of the steam chamber and the pressure and temperature were reduced instantaneously.
对湿热耐受性的测定：使用内径9英寸、深16厘米的高压灭菌器(图1 )来测定湿热耐受性。Cook和Brown (6)描述了类似装置。最初将大腔抽成真空，然后加入蒸汽直至理想的压力和温度。将测试孢子纸带放于活塞腔随即抽真空。活塞被推入蒸汽室，从而立即将孢子纸带在理想的温度下暴露于饱和蒸汽。经过预定的时间间隔，将活塞拉出蒸汽室，同时减少压力和温度。
Determination of resistance to dry heat. A cylindrical aluminum block, 4 inches in diameter and 63/8 inches in height (Fig. 2), was used for determining dry-heat resistance, and was similar to that used by Bruch, Koesterer, and Bruch (5). It was equipped with a central heating element and a temperature regulator. The temperature was uniform throughout the entire tube and in the six replicate tubes and could be monitored routinely with thermocouples or thermometers (or both). When the tubes were placed in the unit, the temperature came up in less than 1 min. After the predetermined exposure time, the tubes were removed from the unit and submerged in ice water.
对干热耐受性的测定：用直径4英寸、高度为63/8英寸的圆柱形铝砧(图2 )来测定干热耐受性，与Bruch, Koesterer, 和Bruch (5)所用的相同。它装有中央加热元件及温度调节器。整个管和六个复制管温度均匀，可对热电偶或温度计(或两者兼而有之)进行常规监测。当试管放于装置中，不到1分钟内温度上升。经过预设的暴露时间后，从装置中取出试管浸于冰水之中。
FIG. 2. Diagrammatic representation of the aluminum block unit used for determination of dry-heat parameters.
图2：用于测定干热参数的铝砧装置示意图
FIG. 3. Photomicrographs of crystals and various stages of dissolution. × 970. (a) Crystals prior to addition of 0.1 N HCl. (B) Crystals in the process of dissolving, approximately I min after addition of0.1 N HCI. (C) Clumped spores that are left after the crystals are dissolved, approximately 5 min after addition of 0.1 N HCI.
图3 晶体及不同溶解阶段显微照片。× 970。(a)加入0.1 N HCl前的晶体。(B)加入0.1 N HCI约一分钟后溶解过程中的晶体。(C) 加入0.1 N HCI约5分钟后，晶体溶解，剩下的成群芽孢。
RESULTS
结果
Microscopic examination. A loopfull of a water suspension of crystals containing spores was placed on a slide and air-dried. A thin film of 1.5% agar was placed over the crystals and allowed to harden, and a cover slip was placed over the agar. A drop of 0.1 N HCI was added to the edge of the agar. Shortly thereafter, the acid diffused into the crystals, causing them to dissolve slowly. A large number of spores was occluded in a single crystal (Fig. 3).
显微镜检查：将含孢子的水悬浮晶体全环放载玻片上并风干。用1.5 %琼脂薄膜盖住晶体使其变硬，用复盖纸盖住琼脂。在琼脂边缘加一滴0.1 N HCI。此后不久，酸扩散到晶体中使其慢慢溶解。一个单晶中封闭了大量孢子(图3)。
Ethylene oxide resistance. Figure 4 compares the ethylene oxide resistance of unoccluded versus occluded spores of B. subtilis var. niger. At 54 C, 40% relative humidity (RH), and with 1,200 mg of ethylene oxide per liter, 8×103 spores were killed in 30 sec when they were not occluded. The initial drop in count on the strips containing occluded spores was probably due to the killing of unoccluded spores present. Continued exposure to ethylene oxide for 2 weeks did not reduce the count beyond that of the first 5 sec. Of a population of 8×103 spores, 25%, or 2×103 spores, were not killed and, hence, may have been occluded. Since this method probably killed the unoccluded spores, the strips for the moist- and dry-heat experiments were first treated, at 54 C, 40% RH, with 1,200 mg of ethylene oxide for 1 hr.
环氧乙烷耐受性：图4比较了封闭和未封闭的枯草杆菌黑色变种芽胞对环氧乙烷的耐受性。在54C、40 %的相对湿度下(RH)，每升1,200 mg的环氧乙烷中，在30秒内有8×103未封闭的孢子被杀死。最初在含封闭孢子的纸带上滴下可能是因为要杀死存在的未封闭的孢子。继续暴露于环氧乙烷2周，孢子计数并没有比最初5秒少。对于总数为8×103的孢子，有25 %即2×103个孢子并没有被杀灭，因此有可能是被封闭起来的。由于这种方法可杀死未封闭的孢子，因此进行湿热和干热试验的纸带均在54C、40% RH下用1,200 mg环氧乙烷首先处理了1小时。
Moist-heat resistance. Figure 5 compares the resistance of unoccluded and occluded spores of B. subtilis var. niger to saturated steam at 121 C. The unoccluded spores were killed so quickly in saturated steam that the curve was drawn based on the starting number and sterility end point. The occluded spores, on the other hand, were quite resistant to moist heat. After 1 hr, an average of less than 10 spores per paper strip was evident. This low number of spores required an additional 1.5 hr of exposure before inactivation was attained.
湿热耐受性：图5比较了封闭和未封闭的枯草杆菌黑色变种芽胞对121C饱和蒸汽的耐受性。封闭孢子在饱和蒸汽中被杀灭得非常快，以至于曲线从起始数直拉到灭菌终点。另一方面，封闭的孢子颇耐湿热。1小时后每张纸带显然平均不到10个孢子。这些少量孢子需要再暴露1.5小时才能实现灭活。
ETHYLENE OXIDE 1200MG/LITER TEMPERATURE 54C.
环氧乙烷1200毫克/升，温度为54C
RELATIVE HUMIDITY40%
相对湿度4%
A. UWOCCLUDED SPORES A.未封闭的孢子
B. OCCLUDED SPORES B.封闭的孢子
NO. SURVIVING SPORES PER PAPER STRIP
每张纸带上存活的孢子数
EXPOSURE TIME, SECONDS
暴露时间（秒）
FIG. 4. Survivor curves for unoccluded and occluded spores of Bacillus subtilis var. niger exposed to ethylene oxide.
图4 暴露于环氧乙烷的封闭及未封闭枯草杆菌黑色变种芽胞存活曲线
It was observed that occluded spores are about 900 times more resistant than unoccluded spores to moist heat at 121 C.
据观察，封闭孢子对121C湿热的耐受性约为未封闭孢子的900倍。
Dry-heat resistance. Figure 6 compares the resistance of unoccluded and occluded spores of B. subtilis var. niger to dry heat at 121 C. This temperature was chosen so that steam resistance could be compared with dry-heat resistance. The unoccluded spores were killed in a logarithmic manner in 5.5 hr. When the mortality curve for the occluded spores was extrapolated to zero-time, a lower apparent starting number, 8.9×102 spores, was obtained. This we believe represents spores completely protected from dry heat. The remaining 1.1×103 spores are partially protected. The latter were perhaps not tightly surrounded with the crystalline matrix. However, these were incapable of being inactivated with ethylene oxide as stated previously.
对干热的耐受性：图5比较了封闭和未封闭的枯草杆菌黑色变种芽胞对121C干热的耐受性。选择这一温度以便比较蒸汽耐受性和干热耐受性。未封闭孢子在5.5小时内以对数级被杀灭。当封闭孢子死亡率曲线延伸到零时，获得了明显较低的起数，8.9×102个孢子。我们相信这代表孢子完全不受干热破坏。其余1.1×103个孢子受到部分保护。后者也许没被晶状基质紧紧包住。不过如前所述，这些孢子也无法被环氧乙烷灭活。
Occluded spores in crystals were at least nine times more resistant than unoccluded spores to dry heat at 121 C. Moist heat was more effective than dry heat on occluded spores, but occlusion of spores retarded inactivation by steam much more severely than inactivation by dry heat. However, saturated steam was a much faster sterilizing agent than dry heat whether the spores were occluded or not.
晶体中的封闭孢子对121C干热的耐受性至少比未封闭的孢子强九倍。湿热比干热对封闭孢子更有效，但孢子的封闭性对蒸汽灭活的阻碍比对干热严格得多。然而，不管孢子是否被封闭，饱和蒸汽都是比干热见效快得多的灭菌剂。
Viability of spores in crystals. An interesting sidelight of these experiments is the fact that the occluded spores showed no loss of viability or drop in count after 3 years of storage at room temperature. Washed spores of this strain of B. subtilis on paper tended to decrease in number with time. After 3 years, one can expect at least 2 logs decrease in count. It is not unusual for spores to survive long periods in soil. However, we believe that maintaining viability of all the cells of a particular population is unusual.
晶体中孢子的生存能力：这些实验的一个有趣侧面是，封闭孢子在室温贮藏3年后均未失去生存能力或计数下降。纸上洗过的枯草杆菌孢子菌株随着时间流逝，其数量呈减少趋势。三年后人们可以期望其计数至少减少2logs。对于污物中的孢子长期存活并不罕见。不过，我们确信某一特定群体所有细胞保持活力是罕见的。
A. UWOCCLUDED SPORES A.未封闭的孢子
B. OCCLUDED SPORES B.封闭的孢子
NO. SURVIVING SPORES PER PAPER STRIP
每张纸带上存活的孢子数
EXPOSURE TIME, MIUNUTES
暴露时间（分钟）
FIG. 5. Survivor curves for unoccluded and occluded spores of Bacillus subtilis var. niger exposed to saturated steam at 121 C.
图5 暴露于121C饱和蒸汽的封闭及未封闭枯草杆菌黑色变种芽胞存活曲线
A. UWOCCLUDED SPORES A.未封闭的孢子
B. OCCLUDED SPORES B.封闭的孢子
NO. SURVIVING SPORES PER PAPER STRIP
每张纸带上存活的孢子数
EXPOSURE TIME. HOURS
暴露时间（小时）
FIG. 6. Survivor curves for unoccluded and occluded spores of Bacillus subtilis var. niger exposed to dry heat at 121 C.
图6 暴露于121C干热的封闭及未封闭枯草杆菌黑色变种芽胞存活曲线
DISCUSSION
讨论
It has been shown that ethylene oxide cannot inactivate spores entrapped in water-insoluble crystals. Spores occluded in water-soluble crystals are also difficult to inactivate with ethylene oxide. However, they may be killed when the crystals are dissolved in water or when the partial pressure of the relative humidity around the crystals exceeds the vapor pressure of a saturated solution of the crystals. This is because the crystals would be continually dissolving and reforming in such a system. Ethylene oxide treatment of a population may provide a good tool for the determination of the percentage of a spore population which is occluded.
已经表明环氧乙烷不能灭活封闭在不溶于水的晶体中的孢子。封闭在水溶性晶体中的孢子很难被环氧乙烷灭活。然而，在晶体溶解于水时或相对湿度的局部压力超过晶体饱和溶液蒸汽压时，可能将其杀灭。这是因为在这样的系统中晶体会不断溶解和重组。对群体进行环氧乙烷处理可为测定封闭孢子的百分比提供一个很好的工具。
There are two possibilities why moist heat is still more effective than dry heat on occluded spores. (i) Because there is more energy per molecule of saturated steam than there is per molecule of dry air at any given temperature, steam provides a greater heat transfer through the crystals. (ii) The crystalline matrix impedes the diffusion of steam to the spores, and only when steam saturates the spore do we get killing with moist heat. Prior to saturation, killing would be primarily due to dry heat or a combination of moist and dry heat.
湿热比干热对封闭孢子更为有效有两种可能性：(一)因为在任何特定的温度下，饱和蒸汽的每个分子比干燥空气的每个分子有更多能量，蒸汽通过晶体提供了更多的热传递。(二)晶状基质妨碍了蒸汽扩散到孢子，只有当蒸汽渗透到孢子我们才能用湿热杀菌。渗透前，杀菌主要是由干热或湿热和干热结合完成的。
There are three possibilities why dry heat takes so much longer to destroy occluded spores compared with unoccluded spores. First, if the nature of dry-heat sterilization is due to oxidation, the crystalline material may impede the diffusion of oxygen to the cell, thus extending the time to sterilize. The difficulty with this idea is that inert gases have been reported to be slightly more effective than air or oxygen (9), and nitrogen to be equal to air (J. A. Rowe and M. G. Koesterer, Bacteriol. Proc., p. 8, 1965) on the rate of destruction of spores by dry heat.
干热消灭封闭孢子为何需要比消灭封闭孢子长得多的时间有三个可能性。第一，如果干热灭菌的本质是由于氧化，晶体材料可能阻碍氧扩散到细胞，从而延长了灭菌时间。这一办法的争论是已有报道说惰性气体比空气或氧气更有效(9)，而且氮的干热杀孢子效力等于空气(J. A. Rowe and M. G. Koesterer, Bacteriol. Proc., p. 8, 1965)。
A second possibility is that dry-heat sterilization is due in part to vaporization of essential cell components. The crystalline matrix could impede this diffusion, thus extending the time to sterilize. An analogous situation exists in the diffusion of gases through plastic films. For example, nylon has a high permeability to water vapor. If polyethylene is laminated to the nylon, its water vapor transmission rate is decreased because polyethylene has a low water vapor permeability.
第二个可能性是干热灭菌的部分原因是汽化基本细胞成分。晶状基质可阻碍这种扩散，从而延长灭菌时间。气体透过塑料薄膜扩散存也在类似情况。例如，尼龙具有很高的水蒸汽渗透性。如果用聚乙烯在尼龙上层压，其水汽传输速率下降，因为聚乙烯的水蒸汽渗透性低。
However, the most likely explanation is that heat is transferred poorly within the crystal. This possibility is supported by the observation in our laboratory that flowing air at 121 C sterilizes in about 50% of the time required by static air. The phenomenon of protection to a sterilizing agent and viability of spores occluded in water-insoluble crystals perhaps has not been observed before because ordinary culturing techniques would not dissolve such crystals, thereby preventing outgrowth of the organisms. We have observed similar protection and viability with other water-insoluble compounds such as calcium alginate, dimethylglyoxime, zinc carbonate, barium carbonate, and 1-aminoanthraquinone. This phenomenon is expected to be prevalent in nature, but is difficult to prove since the procedures required to recover such organisms may well enhance their destruction as well.
不过，最有可能的解释是晶体内热传递低。我们在实验室里的观察支持这种可能性，静止空气在121C灭菌时大约50 %的时间需要流动空气。以前也许没有观察到封闭在不溶于水晶体中孢子的抗灭菌剂现象及生存能力，是因为通常的培养技术不会溶解这种晶体，从而防止了有机体的自然发展。我们已观察到其他非水溶性化合物（如海藻酸钙、丁二酮肟、碳酸锌、碳酸钡及1-氨基蒽醌）也有类似的保护和生存能力。这一现象可望是一种普遍性质，但很难证明，因为回收这些有机体所需的操作同时也可能啬对其的破坏。
We (unpublished data) have attempted to recover known inocula of spores from materials such as wax, vaseline, and mineral oil by solvent extraction, followed by membrane filtration to retain the organisms and subsequent culturing or assaying of the filter. Some solvents, such as carbon tetrachloride, benzene, and petroleum ether, were deleterious to the spores and, consequently, were inadequate for recovering them. However, other solvents, such as acetone and trichlorotrifluoroethane, were not and the procedure was adequate for recovering a known inoculum of spores.
我们(未公布的数据)企图用溶剂萃取来从诸如石蜡、凡士林、矿物油等材料中收回已知孢子接种物，然后用膜滤法来保留有机体并随后对过滤器进行培养或测定。有些溶剂（如四氯化碳、苯、石油醚）对孢子有害，因此不适用于回收孢子。不过其他溶剂（如丙酮和三氯三氟乙烷）就不会，操作适用于回收孢子接种物。
The fact that spores can exist within crystals and maintain their viability for a long period of time may not present a problem to the field of sterilization generally, because the spores would not ordinarily be released to a nutrient. There are, however, some implications in the field of spacecraft sterilization. Low numbers of spores entrapped within relatively impermeable material may also exhibit abnormally high resistance to dry heat. It may be difficult to assure sterility of a spacecraft because the viability of occluded spores is so difficult to determine.
孢子可以存在于晶体中并保持其生命力的事实在相当长一段时间内可能不会对一般灭菌领域造成任何问题，因为孢子通常释放到养分中。然而，这对航天器灭菌还是有一些影响。相对不渗透的材料中捕获的少量孢子也可表现出对干热异常高的耐受性。可能难以确保航天器的无菌，因为封闭孢子的活性非常难以测定。
ACKNOWLEDGMENT
致谢
We express appreciation to Gary Granata and William Mehrhof for technical assistance.
我们对Gary Granata和William Mehrhof的技术协助表示感谢。
LITERATURE CITED
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