Burton's Microbiology for the Health Sciences Section IV Burton's Microbiology for the Health Sciences Section IV. Controlling the Growth of Microbes

Burton's Microbiology for the Health Sciences Chapter 8 Burton's Microbiology for the Health Sciences Chapter 8. Controlling Microbial Growth in Vitro

Chapter 8 Outline Introduction Factors that Affect Microbial Growth Encouraging the Growth of Microbes in Vitro Inhibiting the Growth of Microbes in Vitro

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 (dessication).

Factors That Affect Microbial Growth, cont. Temperature Every organism has an optimum growth temperature. The temperature (and pH) ranges over which an organism grows best are largely determined by its enzymes. Thermophiles are microorganisms that grow best at high temperatures. Mesophiles are microbes that grow best at moderate temperatures (e.g., 37o C).

Factors That Affect Microbial Growth, cont. Temperature, cont. Psychrophiles prefer cold temperatures (like deep ocean water). Psychrotrophs, a particular group of psychrophiles, prefer refrigerator temperature (4oC). 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 > 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.

Factors That Affect Microbial Growth, cont. Osmotic Pressure and Salinity, cont. Plasmolysis is a condition in which the cell membrane and cytoplasm of a cell shrink away from the cell wall; occurs when bacteria with rigid cell walls are placed into a hypertonic solution. 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.

Factors That Affect Microbial Growth, cont. Osmotic Pressure and Salinity, cont. 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 which are capable of surviving there (e.g., Staphylococcus aureus) are called haloduric organisms. Barometric Pressure Microbes that can survive in high atmospheric pressure (> 14.7 psi) are know as piezophiles.

Changes in Osmotic Pressure

Factors That Affect Microbial Growth cont. Gaseous Atmosphere Microorganisms vary with respect to the type of gaseous atmosphere that they require. Obligate aerobes prefer the same atmosphere that humans do (~20-21% O2 and 78-79% N2, other gases < 1%). Microaerophiles require reduced concentrations of oxygen (~5% O2). Obligate anaerobes are killed by the presence of oxygen. Capnophiles require increased concentrations of CO2 (5-10% CO2).

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).

Binary fission of staphylococci.

Culturing Bacteria in the Laboratory Culture Media Media (sing., medium) are used in microbiology labs to culture (i.e., grow) bacteria; media prepared in the lab 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; example = chocolate agar. A selective medium has added inhibitors that discourage growth of certain organisms while allowing the growth of a desired organism; example = PEA agar.

Culturing Bacteria in the Laboratory Culture Media, cont. A differential medium permits the differentiation of organisms that grow on the medium; example = MacConkey agar. The various categories of media are not mutually exclusive; e.g., blood agar is enriched and differential. Thioglycollate broth (THIO) is a popular liquid medium in bacteriology labs; it supports the growth of all categories of bacteria from obligate aerobes to obligate anaerobes. How is that possible? There is a concentration gradient of dissolved oxygen in the tube; organisms grow only in that part of the broth where the oxygen concentration meets their needs.

A Thioglycollate (THIO) Broth Tube

Bacterial colonies on MacConkey agar (a selective & differential medium) S. aureus on mannitol-salt agar (a selective & differential medium)

Colonies of a β-hemolytic Streptococcus species on a blood agar plate (in this case, the blood agar is both enriched and differential) Copyright © 2011 Wolters Kluwer Health | Lippincott Williams & Wilkins

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. 3 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). 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. Lag phase Logarithmic growth phase Stationary phase Death phase Copyright © 2011 Wolters Kluwer Health | Lippincott Williams & Wilkins

A Chemostat is used for continuous cultures.

Culturing Obligate Intracellular Pathogens in the Laboratory Obligate intracellular pathogens are microbes that can only survive and multiply within living cells (called host cells). Obligate intracellular pathogens include viruses and 2 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, lab 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); due to its low pH, SDA is selective for fungi. Caution must be exercised when culturing fungi – some are highly infectious!

Culturing Protozoa in the Laboratory Most microbiology laboratories do not culture protozoa; some research and reference labs do, however. Examples of protozoa that can be cultured in vitro are amebae, Giardia lamblia, Leishmania spp., Toxoplasma gondii, Trichomonas vaginalis and Trypanosoma cruzi. Due to the severity of diseases that they cause, it is of greatest importance to culture amebae: Acanthamoeba spp., Balamuthia spp. and Naegleria fowleri.

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. The suffix –cide or –cidal refers to “killing.” Germicidal agents, biocidal agents, and microbicidal agents are chemicals that kill microbes. Bactericidal agents are chemicals that specifically kill bacteria, but not necessarily bacterial endospores. Sporicidal agents kill bacterial endospores. Fungicidal agents kill fungi, including fungal spores. Algicidal agents kill algae. Viricidal agents destroy viruses.

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 2 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

Dry Heat Sterilization Using a Bunsen burner flame Using an electrical heating device

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 100oC) and forces steam into materials being sterilized. Autoclaving at a pressure of 15 psi at 121.5oC 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.

Pressure-sensitive autoclave tape showing dark stripes after sterilization. Copyright © 2011 Wolters Kluwer Health | Lippincott Williams & Wilkins

Biological Indicators for Monitoring the Effectiveness of Steam Sterilization

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. Characteristics of an ideal chemical antimicrobial agent: Should have a broad antimicrobial spectrum Fast acting Not affected by the presence of organic matter Nontoxic to human tissues and noncorrosive Should leave a residual antimicrobial film on surface Soluble in water and easy to apply Inexpensive and easy to prepare Stable as both a concentrate and a working solution Odorless

Using Chemical Agents to Inhibit Microbial Growth (cont.) Antiseptics May safely be used 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.

Inhibiting the Growth of Pathogens in Our Kitchens (from the CD-ROM) Many foods brought into our kitchens are contaminated with pathogens; examples = E. coli O157:H7, Salmonella and Campylobacter spp. on poultry and ground beef. Problems arise when handling foods before cooking. Remain aware of pathogens when preparing foods. Wash hands frequently. Thoroughly clean plates and counter tops that have had poultry or meat on them with hot soapy water The use of antibacterial kitchen sprays is controversial.

Controversies Relating to the Use of Antimicrobial Agents in Animal Feed and Household Products 40% of the antibiotics manufactured in the U.S. are used in animal feed; microorganisms resistant to these antibiotics survive! Drug resistant organisms are transmitted in animal feces and in food products. Efforts are underway to eliminate or reduce the practice of adding antibiotics to animal feed. Use of antimicrobial agents is widespread in toys, cutting boards, in hand soaps, and many other household products; resistant microorganisms survive! Controversy: Should children be exposed to all sorts of microorganisms for their immune systems to develop properly?