English is currently available.
Biological & Biosystems Engineering DISCUSSION
Our lab protocol says to autoclave culture medium at 121 °C for 15 to 20 minutes, but a 10 L vessel of medium treated that way was contaminated, while small bottles are always fine. Reading about it, I found the terms D-value, z-value and F0 and do not understand how they fit together.
How is a sterilisation time calculated from these, and why would a large volume fail with a cycle that works for small ones?
Thermal death of microorganisms is close to first order: at a fixed temperature the surviving population falls by the same factor in each equal time interval. The D-value is the time at a given temperature for a tenfold (90 percent, one log) reduction. Log reductions = t / D.
Bacterial spores are the target because they are the most heat resistant. As an illustration, take a resistant spore with D = 1.5 minutes at 121 °C: a 12-log reduction needs 12 × 1.5 = 18 minutes at that temperature. The required number of logs depends on the starting population and the acceptable probability of a survivor, since first-order death never reaches exactly zero.
The z-value describes how D changes with temperature: it is the temperature rise that makes D ten times shorter, about 10 °C for bacterial spores in moist heat. So the lethal rate relative to 121.1 °C is L = 10^((T - 121.1) / z).
At 115 °C, L = 10^(-0.61) = 0.245: each minute counts as only a quarter of a minute at 121.1 °C, and an equivalent of 15 minutes takes about 61 minutes. At 111 °C each minute counts as about a tenth. A few degrees short of the setpoint is therefore a large loss of lethality, not a small one.
F0 ties these together. It is the total lethality of a cycle expressed as equivalent minutes at 121.1 °C with z = 10 °C, found by adding up L for each minute of the measured temperature record, including heat-up and cool-down. A cycle with F0 = 15 has delivered the same kill as 15 minutes held exactly at 121.1 °C.
The important detail is that the temperature that counts is the temperature of the coldest point of the load, not the chamber temperature shown on the display.
That is the explanation for your 10 L vessel. The autoclave timer starts when the chamber reaches 121 °C, but a large liquid volume heats by conduction and slow natural convection, and its centre can lag the chamber by tens of minutes. A small bottle reaches temperature within a few minutes, so most of a 20 minute hold is effective; the centre of 10 L may spend most of the hold still climbing and collect only a small fraction of the intended F0.
The remedies are a load probe placed in the liquid so that the hold is timed from the load temperature, a longer validated cycle for large volumes, or splitting the medium into smaller containers.
Saturated steam is the other condition. Moist heat kills far faster than dry heat at the same temperature, and the 121 °C figure assumes steam at about 2 bar absolute in contact with the load. Trapped air lowers the steam temperature at a given pressure and insulates the load, so tightly sealed containers, deep pockets and blocked drains all lead to cold spots. Loosen caps and use a liquid cycle with slow exhaust, because rapid depressurisation makes superheated liquid boil over violently.
Follow the autoclave manufacturer's operating and safety instructions, let the load cool before handling, and confirm cycles periodically with biological or chemical indicators.
Back to Biological & Biosystems Engineering community
