Chapter 8

Factors That Affect Microbial Growth Availability of nutrients All living organisms require nutrients to sustain life. Nutrients are energy sources. Organisms obtain energy by breaking chemical bonds. Moisture Water is essential for life. It is needed to carry out normal metabolic processes. Certain microbial stages (e.g., bacterial endospores and protozoal cysts) can survive a drying process (desiccation).

Factors That Affect Microbial Growth (cont.) Temperature Every organism has an optimum growth temperature. Thermophiles are microorganisms that grow best at high temperatures. Mesophiles are microbes that grow best at moderate temperatures (e.g., 37C, human body temperature). Psychrophiles prefer cold temperatures (like deep ocean water). Psychrotrophs, a particular group of psychrophiles, prefer refrigerator temperature (4C). Psychroduric organisms prefer warm temperatures but can endure very cold or even freezing temperatures.

Factors That Affect Microbial Growth (cont.) pH pH refers to the acidity or alkalinity of a solution. Most microorganisms prefer a neutral or slightly alkaline growth medium (pH 7.0–7.4). Acidophiles prefer a pH of 2 to 5 Alkaliphiles prefer a pH of >8.5

Factors That Affect Microbial Growth (cont.) Osmotic pressure and salinity Osmotic pressure is the pressure that is exerted on a cell membrane by solutions both inside and outside the cell. Osmosis is the movement of a solvent, through a permeable membrane, from a lower concentration of solutes (dissolved substances) to a higher concentration of solutes. When the concentration of solutes in the external environment of a cell is greater than that of solutes inside the cell, the solution in which the cell is suspended is said to be hypertonic. Plasmolysis is a condition in which the cell membrane and cytoplasm of a cell shrink away from the cell wall; it occurs when bacteria with rigid cell walls are placed in a hypertonic solution.

Factors That Affect Microbial Growth (cont.) Osmotic pressure and salinity (cont.) When the concentration of solutes outside a cell is less than that of solutes inside a cell, the solution in which the cell is suspended is said to be hypotonic. If a bacterial cell is placed into a hypotonic solution, it may not burst (because of the rigid cell wall). If it does burst, the cytoplasm escapes; this process is known as plasmoptysis. A solution is said to be isotonic when the concentration of solutes outside a cell equals the concentration of solutes inside the cell. Organisms that prefer to live in salty environments are called halophilic organisms. Those that do not prefer to live in salty environments but are capable of surviving there (e.g., Staphylococcus aureus) are called haloduric organisms.

Encouraging the Growth of Microbes In Vitro

Culturing Bacteria in the Laboratory Bacterial Growth Think of bacterial growth as an increase in the number of organisms rather than an increase in their size. Bacteria divide by binary fission (one cell divides to become two cells) when they reach their optimum size. Binary fission continues through many generations until a colony is produced on solid culture medium. Binary fission continues for as long as there is a sufficient supply of nutrients, water, and space. The time it takes for one cell to become two cells is called the generation time (e.g., E. coli = 20 minutes).

Culturing Bacteria in the Laboratory Culture Media Media (sing., medium) are used in microbiology laboratories to culture (i.e., grow) bacteria; media prepared in the laboratories are referred to as artificial media or synthetic media. A chemically defined medium is one in which all ingredients are known. Culture media can be liquid or solid. An enriched medium is a broth or solid containing a rich supply of special nutrients that promote the growth of fastidious organisms, for example, chocolate agar. A selective medium has added inhibitors that discourage growth of certain organisms while allowing the growth of a desired organism, for example, PEA agar. A differential medium permits the differentiation of organisms that grow on the medium, for example, MacConkey agar. The various categories of media are not mutually exclusive; for example, blood agar is enriched and differential.

Examples of Solid and Liquid Culture Media

Culturing Bacteria in the Laboratory Inoculation of Culture Media Culture media are inoculated with clinical specimens (i.e., specimens collected from patients with a suspected infectious disease). Inoculation involves adding a portion of a specimen to the medium. Inoculation is accomplished using a sterile inoculating loop.

Culturing Bacteria in the Laboratory Importance of Using “Aseptic Technique” Aseptic technique is practiced when it is necessary to exclude microbes from a particular area (e.g., when inoculating culture media). Unwanted organisms are referred to as contaminants; the growth medium or plate is said to be contaminated. The sterility of the media must be maintained before inoculation. Avoid touching the surface of the agar! Inoculating media within a biologic safety cabinet minimizes contamination and protects the laboratorian.

Culturing Bacteria in the Laboratory Incubation After media are inoculated, they must be placed into an incubator which will maintain the appropriate atmosphere, temperature, and moisture level; the process is known as incubation. Three types of incubators are used in clinical microbiology laboratories: A CO2 incubator (contains 5%–10% CO2) A non-CO2 incubator (contains room air) An anaerobic incubator (the atmosphere is devoid of oxygen)

Culturing Bacteria in the Laboratory Bacterial Population Counts Microbiologists sometimes need to know how many bacteria are present in a particular liquid at a given time (e.g., to determine bacterial contamination of drinking water). One can determine either the total number of bacterial cells or the number of viable (living) cells. A spectrophotometer can be used to determine growth by measuring the turbidity of the medium. A viable plate count is used to determine the number of viable bacteria in a liquid sample by making serial dilutions of the liquid and inoculating onto nutrient agar; after overnight incubation, the number of colonies is counted.

Culturing Bacteria in the Laboratory Bacterial Population Growth Curve A population growth curve for any particular species of bacterium may be determined by growing a pure culture of the organism in a liquid medium at a constant temperature. Samples of the culture are collected at fixed intervals to determine the number of viable organisms. A graph is prepared by plotting the logarithmic number of viable organisms (on the vertical or y- axis) against the incubation time (on the horizontal or x-axis).

A Population Growth Curve of Living Organisms

Culturing Obligate Intracellular Pathogens in the Laboratory Obligate intracellular pathogens are microbes that can survive and multiply only within living cells (called host cells). Obligate intracellular pathogens include viruses and two groups of Gram-negative bacteriarickettsias and chlamydias. Culturing these organisms in the laboratory is a challenge; they must be grown in embryonated chicken eggs, laboratory animals, or cell cultures.

Culturing Fungi in the Laboratory Fungi (including yeasts, moulds, and dimorphic fungi) grow on and in a variety of solid and liquid culture media. There is no single medium that is best for all medically important fungi. Examples of culture media for fungi include brain heart infusion (BHI) agar, BHI with blood, and Sabouraud dextrose agar (SDA); SDA is selective for fungi because of its low pH. Caution must be exercised when culturing fungisome are highly infectious!

Inhibiting the Growth of Microbes In Vitro

Definition of Terms Sterilization is the complete destruction of all microbes, including cells, spores, and viruses. Accomplished by dry heat, autoclaving (steam under pressure), gas, various chemicals, and certain types of radiation. Disinfection is the destruction or removal of pathogens from nonliving objects by physical or chemical methods; pasteurization is an example of a disinfection technique. Disinfectants are chemical substances that eliminate pathogens on inanimate objects. Antiseptics are solutions used to disinfect skin and other living tissues.

Definition of Terms (cont.) A microbistatic agent is a drug or chemical that inhibits growth and reproduction of microbes. A bacteriostatic agent is one that specifically inhibits the metabolism and reproduction of bacteria. Lyophilization is a process that combines dehydration (drying) and freezing. This process is widely used in industry to preserve foods, antibiotics, microorganisms, and other biologic materials. Sepsis refers to the presence of pathogens in blood or tissues, whereas asepsis means the absence of pathogens. Antisepsis is the prevention of infection.

Using Physical Methods to Inhibit Microbial Growth Heat Two factorstemperature and timedetermine the effectiveness of heat for sterilization. The thermal death point (TDP) of any species is the lowest temperature that will kill all of the organisms in a standardized pure culture within a specified time. Types of heat Dry heat (e.g., oven, electrical incinerator, or flame) Moist heat (boiling or use of an autoclave)

Using Physical Methods to Inhibit Microbial Growth, cont. The autoclave A large metal pressure cooker that uses steam under pressure to completely destroy all microbial life. Increased pressure raises the temperature above the temperature of boiling water (above 100C) and forces steam into materials being sterilized. Autoclaving at a pressure of 15 psi at 121.5C for 20 minutes destroys vegetative microorganisms, bacterial endospores, and viruses. Can use pressure-sensitive tape or spore strips or solutions as a quality control measure to ensure proper autoclaving.

A Large, Built-in Autoclave

Autoclave Tape: Before and After Autoclaving

Using Physical Methods to Inhibit Microbial Growth, cont. Coldmost microorganisms are not killed, but their metabolic activities are slowed. Desiccationmany dried microorganisms remain viable, but they cannot reproduce. Radiationan ultra-violet (UV) lamp is useful for reducing the number of microbes in the air. Ultrasonic wavesused in hospitals and medical and dental clinics to clean equipment. Filtersused to separate cells/microbes from liquids or gases. Gaseous atmospherecan be altered to inhibit growth.

Using Chemical Agents to Inhibit Microbial Growth Chemical disinfection refers to the use of chemical agents to inhibit the growth of pathogens, either temporarily or permanently. Disinfectants are affected by Prior cleaning of the object or surface The organic load (e.g., feces, blood, pus) The bioburden (types and numbers of microbes) Concentration of the disinfectant Contact time Physical nature of the object being disinfected Temperature and pH

Using Chemical Agents to Inhibit Microbial Growth (cont.) Antiseptics May be used safely on human tissues. Reduce the number of organisms on the surface of the skin; do not penetrate pores and hair follicles. Antiseptic soaps and scrubbing are used by healthcare personnel to remove organisms lodged in pores or folds of the skin.