Sterility and Death Kinetics PDF

# Sterility and Death Kinetics ## Dr Laura Urbano * F154 Hillside House * Email: [email protected] * Tel: 01707 281372 ## Learning Objectives 1. Define sterility 2. Describe principles of sterilization and death kinetics of microorganisms 3. Describe commonly employed sterilization techniques...

# Sterility and Death Kinetics ## Dr Laura Urbano * F154 Hillside House * Email: [email protected] * Tel: 01707 281372 ## Learning Objectives 1. Define sterility 2. Describe principles of sterilization and death kinetics of microorganisms 3. Describe commonly employed sterilization techniques and their applications 4. Discuss factors affecting efficiency of sterilization processes 5. Calculate and interpret key kinetic values associated with microbial cell death ## What is Sterility? * **Sterile:** Complete absence of ALL microorganisms * **Contaminated:** Presence of impurities (including microorganisms) "Sterility" is the complete destruction or elimination of all viable microorganisms. **But does "sterilization" completely destroy all infectious matter?** ### Sterility Assurance Level (SAL) Sterility assurance level (SAL) is the probability that a single unit that has been subjected to sterilization nevertheless remains nonsterile. * A SAL of 10⁻⁶ means that for every 1,000,000 items sterilized there may be one that contains bacteria. * SAL is a statistical probability that is used because it is impossible to prove that all bacteria have been killed during the sterilization process. ## Reduction in Numbers * **High level** (sterilization activity) * **Intermediate level** (inactivation of Mycobacterium tuberculosis and the most resistant types of viruses, such as the ones without protein membranes in their structure) * **Low level** (reduction of bioburden) **N.B.:** Disinfection at intermediate and low levels is not effective against spores. * **Sanitization:** Elimination of pathogenic microorganisms from public eating utensils/objects * A 10⁻⁶ SAL or greater (1 in 1,000,000) is used for: * Products in contact with breached skin or compromised tissue * Invasive products that enter normally sterile tissue * Products with claims of sterile fluid pathways * Surgically implanted devices * A 10⁻³ SAL or greater assurance of sterility is used for: * Products not intended to come into contact with breached skin or compromised tissue * Topical products that contact intact skin or mucous membranes ## Bacterial Death Kinetics * Death is not instantaneous but occurs over time, therefore the death rate is very important. * Death is (typically) a first-order reaction; cells die at a rate K. ### Rate of Death Depending On: * Microorganisms do not die instantly! * **Linear decline** * **Slope of the line = rate of cell death** * An infinite time is required to achieve sterile conditions * Probability of achieving sterility ### Bacterial Death Kinetics - Factors Influencing Death Rates * **Temperature** * **pH** * **Concentration of disinfectant** * **Type of microbe** * **etc.** ## Bacterial Death Kinetics - Visual Representation * **Log # of bacteria** (y-axis) vs. **Time** (x-axis) * The graph shows a linear decline in the log number of bacteria over time, indicating a first-order reaction. ### First Order Reaction Formula Ka **Viable cells** ➞ **Dead cells** * The rate of cell death is proportional to the number of viable cells. * **-dN/dt = kɑN** where: * **kɑ = specific death rate** * **N = number of viable cells** ### First Order Reaction Formula - Integration * **-Kt = log₁₀(n₁/nƒ)** * **n₁ = initial number of cells** * **nƒ = final number of cells** * **t = time** ## Bacterial Death Kinetics - Alternative Cell Survival Plots * **Log % Cells Survived** (y-axis) vs. **Time (minutes)** (x-axis) * The graph depicts various scenarios for cell death, including first-order kinetics, heat inactivation of spores, cell aggregation, two populations, and mutants or release of cryoprotective agents from lysing cells. ## What is Sterility? - Revisited * **log₁₀(n₁/nƒ)** * **-Kt = log₁₀(n₁/nƒ)** * **t = - log₁₀(n₁/nƒ) / K** **What's the specific death rate?** ## Decimal Reduction Time (D Value) * **Time (in minutes) required to effect a tenfold (90%) reduction in the number of viable cells.** * A graph is used to represent a linear decline in the Surviving fraction over time. * **D = t / log₁₀(n₁/nƒ)** * **t = time (in minutes)** * **n₁ = initial number of cells** * **nƒ = final number of cells** ### Decimal Reduction Time (D Value) - Simplified Explanation * Imagine you start with 1 million cells. * After one D value, you have 100,000 cells remaining (90% reduction). * After another D value, you have 10,000 cells remaining (another 90% reduction). * After a third D value, you have 1,000 cells remaining (again, another 90% reduction). ## Decimal Reduction Time (D Value) - Fixing the Concept * **Log₁₀ (number of survivors)** (y-axis) vs. **Time (minutes)** (x-axis) * The graph illustrates a logarithmic decline in the number of survivors over time. ### Questions to Consider 1. What is D? 2. How many cycles are required to reduce organisms from 1 million to 1000? 3. How long would this take? 4. How does this vary with temperature? ## Decimal Reduction Time (D Value) - Practice Exercise * **C. botulinum** has a D of 0.204 minutes at 121°C. * You have 10¹² spores in a tube. * **How long will it take at 121°C to get down to 1 spore?** 1. **How many cycles?** * 10¹² spores → 1 spore = 12 cycles! 2. **So how long?** * Number of cycles x D value * 12 x 0.204 minutes = 2.45 minutes ## D Value Varies With Temperature * **Survival fraction (log scale)** (y-axis) vs. **Time (minutes)** (x-axis) * The graph shows the decimal reduction time (D value) at different temperatures. * **D at each temperature:** * 2 minutes at 70°C * 11 minutes at 60°C * 40 minutes at 50°C ## Z Value * **Amount of temperature increase necessary to decrease the thermal death time by ten-fold (10 fold change in D).** * A graph is used to represent the D-value (minutes-logarithmic scale) over time, showing a logarithmic decline. ### Z Value - Explanation * **What does it mean if the Z value is 10°C?** * **Determine D at three different temperatures and plot logD versus temperature.** ### Z Value - Mathematical Explanation * **The Z value is the negative reciprocal of the slope of the line.** * **Plot logD v temperature** * **Slope = - D/temp** * **Invert this equation: Temp/1 D** * **This is Z value** ## Z Value - Practice Exercise * **C. botulinum** has a D of 0.204 minutes at 121°C. * **How long will it take at 111°C if the Z value is 10°C?** * **With a DECREASE of 1Z, it takes 10 times longer!** * **121 – 111 °C = 10 °C = 1 Z** * **D₁₁₁°C = 10 x 0.204 minutes = 2.04 minutes** * **Ans = 12 x 2.04 minutes = 24.5 minutes** ## Thermal Death Time (TDT) of F Value * **F value for a process is the number of minutes required to kill a known population of microorganisms under specified conditions.** ### Thermal Death Time (TDT) of F Value - Key Considerations * **Estimate the lethality delivered by an actual thermal process called Thermal death time or F value.** * **Used as a basis for comparing and optimizing heat sterilization procedure.** * **Time needed to reduce microbial numbers by a multiple of the D value. (Typically set to 12D)** * **The F value may also be thought of as time needed to reduce microbial numbers by a multiple of.** ### Example of F Value * A process operation at 115°C based on a microorganism with a Z value of 10°C: **F₁₀¹¹⁵** ## Thermal Death Time (TDT) of F Value - Time and Temperature Values * **Bacterium** | **Time (min)** | **Temp (°C)** --- | --- | --- Neisseria gonorrhoeae | 2-3 | 50 Salmonella typhi| 4.3| 60 Staphylococcus aureus| 18.8| 60 Escherichia coli| 20-30| 57.3 Streptococcus thermophilus| 15| 70-75 Lactobacillus bulgaricus| 30| 71 ## Thermal Death Time (TDT) of F Value - Spore Time and Temperature Values * **Spores of** | **Time to kill at 100°C (min)** --- | --- Bacillus anthracis | 1.7 Bacillus subtilis | 15-20 Clostridium botulinum | 100-330 Clostridium calidotolerans | 520 Flat sour bacteria | Over 1,030 ## Essential Knowledge * The lethal temperature varies in microorganisms. * The time required to kill depends on: * Number of organisms * Species * Nature of the product being heated * pH * Temperature * Know your values and terms: * Thermal death time * Decimal reduction * Z value * F value ## Now You Can 1. Define sterility 2. Describe principles of sterilization and death kinetics of microorganisms 3. Describe commonly employed sterilization techniques and their applications 4. Discuss factors affecting efficiency of sterilization processes 5. Calculate and interpret key kinetic values associated with microbial cell death 6. Understand the concept of D value and Z value 7. Calculate F value ## References 1. Pharmaceutics: the design and manufacture of medicines / edited by Michael E. Aulton., 3rd edi, Churchill Livingstone, 2007. 2. Hugo and Russell's pharmaceutical microbiology edited by Stephen P. Denyer, Norman A. Hodges, Sean P. Gorman, 7th Ed., Blackwell Science, 2004.