Sterility

20th June 2001 Author W A Hyde, F.I.B.M.S, F.I.F.S.T

Definition
The old definition of sterility (1) was given as, "the destruction of all microorganisms spores and viruses". At the time this definition served its purpose, but now we are more aware of the effect of bioburden on the possible survival of micro- organisms, and the incredible ability of prions to survive most conventional inactivation treatments, this definition has been reviewed. Sterility Assurance Level. The calculated probability that an organism will survive a sterilization process, rendering a proportion of the material sterilized unsterile. We no longer regard sterility as an absolute concept, but use the term Sterility Assurance Level ( SAL) when assessing sterility, as Max Sussman stated, " The sterility of a given item in a large batch cannot be regarded as certain " (2). We now know that the total destruction of bacteria in an object during irradiation is constant in a given time interval, and is a proportion of those bacteria which were present before the onset of sterilization (3)(4). The spores used to determine the times used to irradiate, are themselves very resistant to sterilization, these give a worst case scenario. The initial spore count (the bioburden) is estimated and the object is sterilized for various times, the number of spores surviving are estimated. Inactivation Factor The initial bioburden divided by the count after sterilization. If the bioburden is high, say at 108, and 100 bacteria survive, the inactivation factor is 10-6, and each organism has a 1/1000,000 chance of survival. It is then very obvious that the lower the initial bioburden, the lower number there is of possible survivors. These factors are also influenced by the design of the plant and the material to be sterilized and it is obvious that sterility is not an absolute state and that degrees of sterility exist (2).

The accepted minimum SAL of medical devices is 10-6 these are regarded as sterile by medical professionals, indicating that there is 1 in 1000,000 chance of a surviving organism in the load. A SAL of 10- is applied to all canned non-acid foods to ensure destruction of Cl. botulinum. This is in contrast to the 10-4 required for the expected small number of space vehicles landing on Mars! (3). The production methods used to produce and manufacture medical devices with very low bioburden in clean rooms are such that the product may reach a SAL of 10-3 and lower. The estimation of this level is very difficult in normal laboratories, whose facilities are often nowhere near the standard in which the product has been manufactured and it is likely that the cost of this estimation cannot be justified because of the very large numbers needed to be tested to achieve a statistically significant result.

Heat Sterilization
Moist Heat Moist heat sterilization has conundrums, the temperature and time for sterilization used in clinical laboratories to sterilize culture media is typically 121C for 15 minutes, the basis for this combination seems to be historical. Most culture media manufacturer's manuals give details referring to a volume of 1 litre, most autoclave manufacturers measure this temperature in the drain or the chamber of the machine, taking no account of the construction of the autoclave, its controls, or its method of heating. Work carried out at The Food Research Institute at Chipping Campden (5) showed that: 1). Culture media prepared in 1 litre volumes and sterilized at a temperature of 121C (with the probe in either drain or chamber) for 15 minutes will be correctly processed. 2). Volumes greater or less than 1 litre need to be controlled with simulated in load probe. 3). Mixed load volumes should be avoided.

4). Process times started when chamber or drain temperature reach 121C differ from those times using in load probe. 5). The problems of varying heating patterns found in autoclaves using direct steam or internal heating with steam or externally heated, can only be overcome by using Fo values. This value is the total heat input into the load.

6). Most culture media resist a degree of overheating without affecting performance.

D Values & Z Values

D Value - The time needed to achieve 90% mortality of a standard spore indicator. This a measure of the killing time Z-Value - The number of degrees centigrade through which the sterilizing temperature must be raised or lowered to effect the D value by a factor of 10. The time to kill micro- organisms decreases as the temperature is raised. Z values for bacterial spores Z value Range Average Moist Heat 7 - 24C 10C Dry Heat 10 - 60C 21C

Fo Values
This is a method introduced to ensure the sterility of canned foods and is primarily targeted at Cl. botulinum spores, taking into account the total heat input into the food during the heating up, sterilization and the cooling periods. The heat treatment of canned foods is described by the symbol F. F is the time at specified temperature which is necessary to achieve the required level of safety and keeping quality. The symbol Fo is a process in which 121C is used as the reference temperature and 10C is the Z value for bacterial spores . For non acid foods the F value is 4 minutes at 121C. The holding time of 4 minutes may be shortened if the sterilizing temperature is raised, or the time and temperatures of the heating and cooling cycles are taken into consideration using the lethality tables set out below. The process is calculated from the results obtained by placing a thermocouple probe in the centre of a can at the slowest heating point of the sterilizer load and recording the temperature continuously throughout the cycle.

The F value is calculated using the spores of the spores of Cl. botulinum having a D value of 0.21 minutes at 121C. If each can contained a single spore of Cl. botulinum, 2.52 minutes (12 x 0.21) will be required. In practice this is extended to 4 minutes to ensure safety and keeping quality. Stumbo (5) in 1973 introduced, "lethal rate tables" these are derived from the temperature recorded from a thermocouple placed inside the medium, the area under the temperature curve between 100C and on through the sterilization phase at 121C and then down to the 100C cooling phase this is the heating profile of that product in that machine which is divided into 1 minute intervals, using the lethal rate table or the mathematical equation which gives the equivalent time for that amount of heat inputted at 121C. L = Log -1 ( T-121) Z L = equivalent time at 121C. T = Actual temperature for 1 minute. Z = Temperature coefficient. (10C for spores). Log - 1 = antilog of the number. The Lethal Rate table which can be produced from this equation is shown below. Temperature 100 101 101 103 104 105 106 107 108 109 110 Time 0.008 0.010 0.012 0.015 0.019 0.025 0.031 0.039 0.049 0.062 0.078 Temperature 111 112 113 114 115 116 117 118 119 120 121 Time 0.098 0.123 0.155 0.195 0.245 0.309 0.389 0.490 0.617 0.776 1.000

Temperature = temperature for 1 minute intervals . Time = the equivalent time at 121C.

Using this method of calculating, the total heat input into the material to be sterilized can be measured taking into account the variations in times to heat up and cool down the material. Autoclaves can be fitted with internal calculators which can print out the cycle and the Fo value. The method can only be used when material of the same viscosity and volumes are sterilized together. It is inevitable that if materials of varying consistency, such as agar solutions or aqueous solutions, or varying volumes are sterilized together then some will be either under or over heated. This should be avoided whenever possible and in particular 5 ml volumes in bijoux autoclaved together with 2 litre volumes! Examples of the adjustment found necessary using a small electrically heated autoclave are seen in the following table: Volume 1 - 19ml 20ml 100ml 200ml 500ml 1000ml Time Additions To Sterilization At 121C 0 min 1 min 4 mins 5 mins 8 mins 10 mins

The introduction of computer controlled production lines with sterilization in a microbiologically controlled environment now means that batch runs of 25,000 plates with no contamination can be achieved, solving most of the sterility problems of mass production of culture plates.

References
1.) Gardener, J.F. Peel, M.M. (1986) Introduction to Sterilization and Disinfection. Churchill Livingstone. 2.) 3.) Sussman, M (1994) Microbio. Europe. Sterilization of Health Care Products ISO Standard 11137:1995(E).

4.) Industrial Pharmacists Group (1980) Sterilization : Need for new approach , Pharmaceutical Journal, Jan.12 1980 . 5.) Autoclaves and Media Processing, Notes on Joint meeting of QA/QC and Media and Methods.

6.) Stumbo, C.R. (1973) Thermobacteriology in Food Processing. Academic Press New York. 7.) Working Parties at Food and Drinks RA, At Chipping Campden. 21st Jan 1991. For More Information Contact:

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