Fundamentals of Microbiology

Lesson 19. CONTROL OF MICROBIAL GROWTH BY PHYSICAL METHODS

Module 5. Microbial growth and nutrition

Lesson 19

CONTROL OF MICROBIAL GROWTH BY PHYSICAL METHODS

19.1 Introduction

The control of microbial growth is necessary in many practical situations and significant advances in agriculture, medicine, and food science have been made through study of this area of microbiology. ‘Control of microbial growth’, as used here, means to inhibit or prevent growth of microorganisms. This control is affected in two basic ways: (1) by killing microorganisms or (2) by inhibiting the growth of microorganisms. Control of growth usually involves the use of physical or chemical agents which either kill or prevent the growth of microorganisms. Agents which kill cells are called cidal agents; agents which inhibit the growth of cells (without killing them) are referred to as static agents. Thus, the term bactericidal refers to killing bacteria, and bacteriostatic refers to inhibiting the growth of bacterial cells. A bactericide kills bacteria; a fungicide kills fungi, and so on.

19.2 Mode of Actions of Microbial Control Agents

Two possible antimicrobial effects include:

19.2.1 Alteration of membrane permeability

• The susceptibility of the plasma membrane is due to its lipid and protein components.
• Certain chemical control agents damage the plasma membrane by altering its permeability.

19.2.2 Damage to proteins and nucleic acids

• Some microbial control agents damage cellular proteins by breaking hydrogen bonds and covalent bonds.
• Other agents interfere with DNA and RNA replication and protein synthesis.

Several factors influence the effectiveness of antimicrobial treatment (Fig. 19.1).

• Number of Microbes: The more microbes present, the more time it takes to eliminate population.
• Type of Microbes: Endospores are very difficult to destroy. Vegetative pathogens vary widely in susceptibility to different methods of microbial control.
• Environmental influences: Presence of organic material (blood, feces, saliva) tends to inhibit antimicrobials, pH etc.
• Time of Exposure: Chemical antimicrobials and radiation treatments are more effective at longer times. In heat treatments, longer exposure compensates for lower temperatures.

 

Fig. 19.1 Factors affecting effectiveness of antimicrobial agent

19.3 Physical Methods of Control of Microbial growth

The control methods can be broadly divided into two categories physical and chemical methods(Fig.19.2). In this chapter, former methods are discussed while later being dealt in next chapter.

Physical method can be listed as follows(Table19.1):

• Heat: Moist and Dry Heat
• Filtration
• Low Temperature: Refrigeration, Deep freezing, Lyophilization
• Desiccation
• Osmotic pressure
• Radiation: Ionizing and Non-Ionizing

Fig. 19.2 Overview of methods of control of microbial growth

19.4 Physical Methods

19.4.1 Heat

Heat is frequently used to eliminate microorganisms. Moist heat kills microbes by denaturing proteins (enzymes). Dry heat kills organisms by oxidation. For sterilization one must consider the type of heat, and most importantly, the time of application and temperature to ensure destruction of all microorganisms. Endospores of bacteria are considered the most thermoduric of all cells so their destruction guarantees sterility.

Thermal Death Point (TDP) is the lowest temperature at which all the microbes in a liquid culture will be killed in 10 minutes. Thermal Death Time (TDT) is the length of time required to kill all bacterial in a liquid culture at a given temperature. Decimal Reduction Time (DRT) is the length of time required to kill 90% of a bacterial population at a given temperature; D value. Z value is an increase in temperature required to reduce D by 1/10. F value is time in minutes at a specific temperature needed to kill a population of cells or spores.

19.4.1.1 Moist heat

Moist heat is thought to kill microorganisms by causing denaturation of essential proteins. Death rate is directly proportional to the concentration of microorganisms at any given time. Increasing the temperature decreases TDT, and lowering the temperature increases TDT. Processes conducted at high temperatures for short periods of time are preferred over lower temperatures for longer times. Approximate effective moist heat conditions are given in (Table 19.1)

Table 19.1 Effective moist heat conditions

Environmental conditions also influence TDT. Increased heat causes increased toxicity of metabolic products and toxins. TDT decreases with pronounced acidic or basic pH. However, fats and oils slow heat penetration and increase TDT. It must be remembered that thermal death times are not precise values; they measure the effectiveness and rapidity of a sterilization process. Autoclaving 121°C/15 psi for 15 minutes exceeds the thermal death time for most organisms except some extraordinary spore formers.

Common Examples of methods based on moist heat are:

a) Boiling

It involves heating at 100^oC for 30 minutes. This method kills everything except some endospores. To kill endospores, and therefore sterilize a solution, very long (> 6 hours) boiling or intermittent boiling is required.

b) Autoclaving

Autoclaving is the most effective and most efficient means of sterilization. All autoclaves operate on a time/temperature relationship. These two variables are extremely important. Higher temperatures ensure more rapid killing. The usual standard temperature/pressure employed is 121ºC/15 psi for 15 minutes (Table 19.3). Longer times are needed for larger loads, large volumes of liquid, and more dense materials. Autoclaving is ideal for sterilizing biohazardous waste, surgical dressings, glassware, many type of microbiologic media, liquids, and many other things. However, certain items, such as plastics and certain medical instruments (e.g. fiber-optic endoscopes), cannot withstand autoclaving and should be sterilized with chemical or gas sterilants. When proper conditions and time are employed, no living organisms will survive a trip through an autoclave. The autoclave is a large pressure cooker; it operates by using steam under pressure as the sterilizing agent (Fig. 19.3). High pressures enable steam to reach high temperatures, thus increasing its heat content and killing power. Most of the heating power of steam comes from its latent heat of vaporization. This is the amount of heat required to convert boiling water to steam. This amount of heat is large compared to that required to make water hot. For example, it takes 80 calories to make 1 liter of water boil, but 540 calories to convert that boiling water to steam. Therefore, steam at 100ºC has almost seven times more heat than boiling water.

Table 19.2 Relationship between temperature and pressure of steam at sea level

Fig. 19.3 Construction and operation of autoclave

c) Pasteurization

This heat treatment developed by famous microbiologist Louis Pasteur is used to destroy mostly pathogenic bacteria present in liquid medium e.g. milk and wine. Pasteurization is the use of mild heat to reduce the number of microorganisms in a product or food. In the case of pasteurization of milk (Fig. 19.4), the time and temperature depend on killing potential pathogens that are transmitted in milk, i.e. staphylococci, streptococci, Brucella abortus and Mycobacterium tuberculosis. But pasteurization kills many spoilage organisms, as well, and therefore increases the shelf life of milk especially at refrigeration temperatures (2°C).

Milk is usually pasteurized by heating, typically at 63°C for 30 min (batch method) or at 71°C for 15 s (flash method), to kill bacteria and extend the milk's usable life. The process kills pathogens but leaves relatively benign microorganisms that can sour improperly stored milk. Various time-temperature combinations used for pasteurization are given in Table (19.3).

Table 19.3 Time temperature combinations for pasteurization

Fig. 19.4 Pasteurization of milk

19.4.1.2 Dry heat

a) Hot air oven

Basically in the cooking oven (Fig. 19.5a), the rules of relating time and temperature apply, but dry heat is not as effective as moist heat (i.e. higher temperatures are needed for longer periods of time). For example 160°/2h or 170°/1h is necessary for sterilization. The dry heat oven is used for glassware, metal, and objects that won't melt; used on substances that would be damaged by moist heat sterilization e.g. gauzes, dressings or powders.

Fig. 19.5 (a) Hot air oven Fig. 19.5 (b) Bunsen burner

b) Incineration

Burns organisms and physically destroys them (Fig. 19.5b). Used for needles, inoculating wires, glassware, etc. and objects not destroyed in the incineration process.

19.4.2 Filtration

Filtration is the passage of a liquid or gas through a screen like material with pores small enough to retain microorganisms. A vacuum that is created in the receiving flask aids gravity in pulling the liquid through the filter. Some operating theaters occupied by burn patients receive filtered air. High efficiency particulate air (HEPA) filter remove almost all microorganisms larger than 0.3 µm in diameters. Filtration is especially important for sterilization of solutions which would be denatured by heat (e.g. antibiotics, injectable drugs, amino acids, vitamins, etc.). Portable units can be used in the field for water purification and industrial units can be used to ‘pasteurize’ beverages. Essentially, solutions or gases are passed through a filter of sufficient pore diameter (generally 0.22 µm) to remove the smallest known bacterial cells (Table 19.4, Fig.19.6). Filtration is the primary method of eliminating pathogens from the air supply:

• Operating Rooms
• Burn Units
• Fume Hoods
• Isolation Rooms
• Bio-cabinets
• Pharmaceutical Manufacturing Facilities

Table 19.4 Effective size of membrane filter for exclusion of microorganism

Fig. 19.6 Microfiltration assembly unit

19.4.3 Low temperature

Low temperature (refrigeration and freezing): Most organisms grow very little or not at all at 0^oC. Perishable foods are stored at low temperatures to slow rate of growth and consequent spoilage (e.g. milk). Low temperatures are not bactericidal. Psychrotrophs, rather than true psychrophiles, are the usual cause of food spoilage in refrigerated foods. Although a few microbes will grow in supercooled solutions as low as minus 20°C, most foods are preserved against microbial growth in the household freezer. The effectiveness of low temperatures depends on the particular microorganism and the intensity of the application. Most microorganisms do not reproduce at ordinary refrigerator temperatures (0-7°C); bacteriostatic. Many microbes survive (but do not grow) at the subzero temperatures used to store foods.

19.4.3.1 Freeze drying

Microbes are placed in a suspending medium and frozen quickly at temperatures between -52 and 95°C. Water is removed by vacuum (sublimation) lyophilization. Powder-like product can be reconstituted to bring culture back to viable conditions.

19.4.4 Desiccation

Drying (removal of H2O): Most microorganisms cannot grow at reduced water activity (aw < 0.90). Drying is often used to preserve foods (e.g. fruits, grains, etc.). Methods involve removal of water from product by heat, evaporation, freeze-drying, and addition of salt or sugar.

19.4.5 Osmotic pressure

The use of high concentrations of salts and sugars in foods is used to increase the osmotic pressure and create a hypertonic environment.

Plasmolysis: As water leaves the cell, plasma membrane shrinks away from cell wall. Cell may not die, but usually stops growing. Yeasts and molds: More resistant to high osmotic pressures. Staphylococci spp. that live on skin are fairly resistant to high osmotic pressure.

19.4.6 Irradiation

Irradiation (UV, X-ray, Gamma radiation): The effects of radiation (Fig. 19.7) depend on its wavelength, intensity, and duration. Ionizing radiation (Gamma rays, X-rays, and high-energy electron beams) has a high degree of penetration and exerts its effect primarily by ionizing water and forming highly reactive hydroxyl radicals. Ultraviolet (UV) radiation, a form of non-ionizing radiation, has a low degree of penetration and causes cell damage by making thymine dimers in DNA that interfere with DNA replication (Fig. 19.8). The most effective germicidal wavelength is 260 nm.

Fig. 19.7 Radiations spectrum

Fig. 19.8 Damage of microbial DNA by UV radiations

In some parts of Europe, fruits and vegetables are irradiated to increase their shelf life up to 500 percent. The practice has not been accepted in the U.S. UV light can be used to pasteurize fruit juices by flowing the juice over a high intensity ultraviolet light source. UV systems for water treatment are available for personal, residential and commercial applications and may be used to control bacteria, viruses and protozoan cysts.

The food and drug administration (FDA) has approved irradiation of poultry and pork to control pathogens, as well as foods such as fruits, vegetables, and grains to control insects, and spices, seasonings, and dry enzymes used in food processing to control microorganisms. Food products are treated by subjecting them to radiation from radioactive sources, which kills significant numbers of insects, pathogenic bacteria and parasites.

According to the FDA, irradiation does not make food radioactive, nor does it noticeably change taste, texture, or appearance. Irradiation of food products to control food-borne disease in humans has been generally endorsed by the United Nation's World Health Organization and the American Medical Association. Two important Disease-causing bacteria that can be controlled by irradiation include Escherichia coli 0157:H7 and Salmonella species.

Microwave cooking ovens were originally researched and developed by German scientists to support mobile operations during the invasion of the Soviet Union. Microwaves can kill microbes indirectly as materials get hot.

19.4.7 Microwave radiation

Wavelength ranges from 1 mm to 1 m. Heat is absorbed by water molecules. It may kill vegetative cells in moist foods. Bacterial endospores, which do not contain water, are not damaged by microwave radiation. Solid foods are unevenly penetrated by microwaves.

19.4.8 Gamma radiation and electron beam radiation

These radiations are formed of ionizing radiation used primarily in the health care industry. Gamma rays, emitted from cobalt-60, are similar in many ways to microwaves and x-rays. Gamma rays delivered during sterilization break chemical bonds by interacting with the electrons of atomic constituents. Gamma rays are highly effective in killing microorganisms and do not leave residues or have sufficient energy to impart radioactivity.

Electron beam (e-beam) radiation, a form of ionizing energy, is generally characterized by low penetration and high-dose rates. E-beam irradiation is similar to gamma radiation in that it alters various chemical and molecular bonds on contact. Beams produced for e-beam sterilization are concentrated, highly-charged streams of electrons generated by the acceleration and conversion of electricity. E-beam and Gamma radiation are for sterilization of items ranging from syringes to cardiothoracic devices.