Control of Microbial Growth

2. Basic techniques in Microbiology 104 Control of Microbial Growth S M Bakhtiar Ul Islam Microbiology ( BSc. & MSc.) University of Dhaka BD Molecular Biology (MSc.) Umea University SE Email: smbakhtiar@gmail.com April, 2012

3. Lecture 27 , April , 2012 The term control refers to the reduction in number or of activity of the total microbial flora. Purpose: Purpose of controlling microbes are: • To prevent contamination or growth of undesirable microbes. • To prevent transmission of disease and infection • To prevent spoilage of materials by microbes.

4. The Rate of Microbial Death A microbial population is not killed instantly when exposed to a lethal agent. Population death is generally exponential or logarithmic that is the population will be reduced by the same time fraction at a constant intervals. When a bacterial population is heated or treated with antimicrobial chemicals, they usually die at a constant rate. For example, suppose a population of 1 million microbes has been treated for 1 minute, and 90% of the population has died. We are now left with 100,000 microbes. If the population is treated for another minute, 90% of those microbes die, and we are left with 10,000 survivors. In other words, for each minute the treatment is applied, 90% of the remaining population is killed.

5. Microbial Death Curve The concept of a killing curve for microbial populations, including the elements of time and the size of the initial population, is especially useful in food preservation and in the sterilization of media or medical supplies.

6. Actions of Microbial Control Agents • Alteration of Membrane Permeability A microorganism's plasma membrane is the target of many microbial control agents. This membrane actively regulates the passage of nutrients into the cell and the elimination of wastes from the cell. Damage to the lipids or proteins of the plasma membrane by antimicrobial agents causes cellular contents to leak into the surrounding medium and interferes with the growth of the cell. • Damage to Proteins and Nucleic Acids Bacteria are several kind of enzymes. These enzymes, which are primarily protein, are vital to all cellular activities. The three dimensional shape give rise to functional proteins. This shape is maintained by chemical bonds that link adjoining portions of the amino acid chain as it folds back and forth upon itself. Some of those bonds are hydrogen bonds, which are susceptible to breakage by heat or certain chemicals; breakage results in denaturation of the protein. Covalent bonds are stronger but are also subject to attack. For example, disulfide bridges, which play an important role in protein structure by joining amino acids with exposed sulfhydryl (-SH) groups, can be broken by certain chemicals or sufficient heat. The nucleic acids DNA and RNA are the carriers of the cell's genetic information. Damage to these nucleic acids by heat, radiation, or chemicals is frequently lethal to the cell. Therefore, the cell can no longer replicate, nor can it carry out normal metabolic functions such as the synthesis of enzymes.

7. Factors influence the effectiveness of antimicrobial treatments • Population size or the number of microbes: Large number microbes require longer time to eliminate the entire population . • Microbial characteristics.: The microbial characteristics (gram +ve, gram –ve , acid fast) also affect the choice of chemical and physical control methods. Many disinfectants have a greater effect on gram +ve bacteria than a gram –ve bacteria. Bacterial endospores are much more resistant to most antimicrobial agents than are vegetative forms, and younger cells are usually more readily destroyed than mature organisms. • Time of exposure: Chemical antimicrobials often require extended exposure to affect more resistant microbes or endospores. • Concentration of antimicrobial agents: Agents effectiveness is not directly related to concentration of that agent. Sometimes, more concentrated antimicrobial agents destroy microbes more quickly. • Temperature: An increase in the temperature at which a chemical acts often enhances its activity. Frequently a lower concentration of disinfectant or sterilizing agent can be used at a higher temperature. • Environmental influence: The presence of organic matter often inhibits the action of chemical antimicrobials. In hospitals, the presence of organic matter in blood, vomit, or feces influences the selection of disinfectants. Microbes in surface biofilms are difficult for biocides to reach effectively. Because their activity is due to temperature dependent chemical reactions, disinfectants work somewhat better under warm conditions. The nature of the suspending medium is also a factor in heat treatment. Fats and proteins are especially protective, and a medium rich in these substances protects microbes, which will then have a higher survival rate. Heat is also measurably more effective under acidic conditions.

8. Lecture 27 , April , 2012 Physical Methods of Microbial Control Several physical methods are applied to kill microorganisms from an object . These methods can be categories as follows: • Heat • Moist Heat Sterilization • Pasteurization • Dry Heat Sterilization • Filtration • Low Temperatures • High Pressure • Desiccation • Osmotic Pressure • Radiation

9. Heat The use of heat is one of the most effective and widely used means of killing microorganisms. Heat is also usually used to sterilize laboratory media and glassware and hospital instruments. Heat appears to kill microorganisms by denaturing their enzymes which changes the 3-D shapes of the proteins and inactivate them. Heat resistance varies among different microbes which can be expressed through the concept of thermal death point. Thermal death point (TDP) is the lowest temperature at which all the microorganisms in a particular liquid suspension will be killed in 10 minutes. Another factor to be considered in sterilization is the length of time required. This is expressed as thermal death time (TDT), the minimal length of time for all bacteria in a particular liquid culture to be killed at a given temperature. Both TDP and TDT are useful guidelines that indicate the severity of treatment required to kill a given population of bacteria. Decimal reduction time (DRT, or D value) is a third concept related to bacterial heat resistance. DRT is the time, in minutes, in which 90% of a population of bacteria at a given temperature will be killed. D values are used to estimate the relative resistance of a microorganism to different temperatures through calculation of the z value. The z value is the increase in temperature required to reduce D to 1/10 its value or to reduce it by one log cycle when log D is plotted against temperature. Another way to describe heating effectiveness is with the F value. The F value is the time in minutes at a specific temperature (usually 250°F or 121.1°C) needed to kill a population of cells or spores.

10. Moist Heat Sterilization Moist heat has been recognized as an effective biocidal agent. It destroys microbes by : • By coagulating proteins (denaturation), which is caused by breakage of the hydrogen bonds that hold the proteins in their three-dimensional structure. • Denaturaing enzymes and proteins of the cell. • Degrading nucleic acid • Disrupting the cell membrane . Moist heat sterilization can be performed by three process: • Boiling • Steam under pressure (Autoclaving) • Pasteurization. • Boiling: Boiling kills vegetative forms of bacterial pathogens, almost all viruses, and fungi and their spores . Exposure to boiling water for 10 minutes is sufficient to destroy vegetative cells and eucaryotic spores. Mode of action: Protein denaturation. Use: Use in disinfection of instruments. Advantage: Effective and common process. Economic and easily controlled. Disadvantage: It can not kill endospores or some viruses i.e. it is not sporicidal or virucidal. Boiling is therefore not always a reliable sterilization procedure.

11. Autoclaving • Autoclaving is the preferred method of sterilization, unless the material to be sterilized can be damaged by heat or moisture. • Moist heat sterilization must be carried out at temperatures above 100°C in order to destroy bacterial endospores, and this requires the use of saturated steam under pressure. Steam sterilization • is carried out with an autoclave . The development of the autoclave by Chamberland in 1884 tremendously stimulated the growth of microbiology. • Water is boiled to produce steam, which is released through the jacket and into the autoclave’s chamber. The air initially present in the chamber is forced out until the chamber is filled with saturated steam and the outlets are closed. Hot, saturated steam continues to enter until the chamber reaches the desired temperature and pressure, usually 121°C and 15 pounds of pressure. • At this temperature saturated steam destroys all vegetative cells and endospores in a small volume of liquid within 10 to 12 minutes. Treatment is continued for about 15 minutes to provide a margin of safety. Larger containers of liquid such as flasks and carboys will require much longer treatment times. • Indicator organism:Bacillus stearothermophilus . • Advantage: • It is a broad spectrum sterilization procedure. • Can kill vegetative as well as endospores of bacteria. • It may provide environment for some heat required chemical reactions. • Limitation: • Cann’t applied to heat sensible chemicals or materials. • Moisture sensitive materials may be damaged after autoclaving.

14. Use: Autoclaving is used to sterilize culture media, instruments, dressings, intravenous equipment, applicators, solutions, syringes, transfusion equipment, and numerous other items that can with - • stand high temperatures and pressures. • Autoclaving must be carried out properly or the processed materials will not be sterile. The precautions are: • In order to sterilize a solid, steam must be actually in contact with it. The dry material should not be wraped with aluminium foil which is impervious to steam. To avoid this paper should be used and for culture media, cotton should be used for conical flask and test tubes. • If all air has not been flushed out of the chamber, it will not reach 121°C even though it may reach a pressure of 15 pounds. The chamber should not be packed too tightly because the steam needs to circulate freely and contact everything in the autoclave. • Testing a autoclave that it is running well: • Several commercially available methods can indicate whether heat treatment has achieved sterilization or the autoclave is running well. • There are pellet contained in a glass vial melt as the temperature reaches in appropriate position. • A widely used test consists of preparations of specified species of bacterial endospores impregnated into paper strips. After autoclaving, these can be aseptically inoculated into culture media. If growth occur, it indicate that the autoclave is not running well. • After sterilization any culture media, if growth occurs after 24 hours incubation in an incubator, it indicate that the autoclave is not running well.

15. Pasteurization Pasteurization is a process of heating liquid food and beverage to a controlled temperature to keep enhanced quality of stored food and destroy harmful organisms. This method was found by Louis Pasteur for preventing spoilage of beer and wine in the early days of microbiology. The intent of pasteurization of milk was to eliminate pathogenic microorganisms. Depending on the product to pasteurize this method is categorize as follows: • In the older method the milk is held at 63°C for 30 minutes. • Flash pasteurization or high-temperature short-term (HTST) pasteurization: In this method Large quantities of milk are usually subjected to quick heating to about 72°C for 15 seconds, then rapid cooling. In addition to killing pathogens, HTST pasteurization lowers total bacterial counts, so the milk keeps well under refrigeration. • Ultrahigh-temperature (UHT) sterilization: The dairy industry also sometimes uses ultrahigh-temperature (UHT) sterilization. Milk and milk products are heated at 140 to 150°C for 1 to 3 seconds. UHT-processed milk does not require refrigeration and can be stored at room temperature for about 2 months without flavor changes.

16. Dry heat

17. Lecture 28 , April , 2012 Chemical Methods of Microbial Control