Controlling the Growth of Microorganisms Hani Masaadeh, MD, Ph.D

LEARNING OBJECTIVES • List several factors that affect the growth of microorganisms • Describe the following types of microorganisms: psychrophilic, mesophilic, thermophilic, halophilic, haloduric, alkaliphilic, acidophilic, and piezophilic • Explain the importance of using “aseptic technique” in the microbiology laboratory • Draw a bacterial growth curve and label its four phases • Differentiate among sterilization, disinfection, and sanitization

• Explain the processes of pasteurization and lyophilization • List several physical methods used to inhibit the growth of microorganisms • Differentiate between bactericidal and bacteriostatic agents • Cite three ways in which disinfectants kill Explain briefly why the use of antibiotics in animal feed and household products is controversial

General Microbiology for Pharmacy, Isra Private University Microbial Growth Microbial growth is the increase in number of cells, not cell size. Growing microbes accumulating into colonies (groups of cells large enough to be seen without a microscope) of hundreds of thousands of cells, or populations of billions of cells. Prepared by Dr. Ahmad Saleh

Factors influencing microbial growth Tempreture. Moisture. Osmotic pressure. PH. Barometric pressure. Gases. Radiation. Chemicals. The presence of neighboring microbes.

Physical requirements: TEMPERATURE General Microbiology for Pharmacy, Isra Private University Physical requirements: TEMPERATURE The requirements for microbial growth can be divided into two main catagories: Physical and Chemical. Physical requirments include temperature, pH, and osmotic pressure. Chemical requirments include source of carbon, nitrogen, sulfur, The minimum growth temperature is the lowest at which the species will grow. The optimum growth temperature is the temperature at which the species grows best. The maximum growth temperature is the highest temperature at which growth is possible. Prepared by Dr. Ahmad Saleh

Physical requirements: Temperature General Microbiology for Pharmacy, Isra Private University Physical requirements: Temperature Figure 6.1 Prepared by Dr. Ahmad Saleh

Physical requirements: pH General Microbiology for Pharmacy, Isra Private University Physical requirements: pH pH Refers to the acidity or alkalinity of a solution. Most bacteria grow between pH 6.5 and 7.5 (near neutrality) Acidophiles grow in acidic environments Molds and yeasts grow over a greater pH range than bacteria, but the optimum pH is between pH 5 and 6 Alkalinility also inhibits microbial growth but is rarely used to preserve foods. Prepared by Dr. Ahmad Saleh

Physical requirements: Osmotic Pressure Microorganisms obtain almost all their nutrients in solution from the surrounding water. They require water for growth and are made up of 80 to 90% water. Extreme or obligate halophiles are organisms that have adapted so well to high salt concentrations that they require for growth (in Dead Sea). Facultative halophiles, do not require high salt concentrations but are able to grow at salt concentrations of 2 to 15%.

Chemical Requirements: General Microbiology for Pharmacy, Isra Private University Chemical Requirements: Carbon Nitrogen Sulfur Phosphorus Potassium, magnesium, and calcium Oxygen Prepared by Dr. Ahmad Saleh

Chemical Requirements: Oxygen General Microbiology for Pharmacy, Isra Private University Chemical Requirements: Oxygen Oxygen (O2) Table 6.1 Prepared by Dr. Ahmad Saleh

Chemical Requirements: Organic growth factors General Microbiology for Pharmacy, Isra Private University Chemical Requirements: Organic growth factors Organic growth factors Essential organic compounds an organism is unable to synthesize, they must be obtained from the environment Vitamins, amino acids, purines, and pyrimidines Prepared by Dr. Ahmad Saleh

General Microbiology for Pharmacy, Isra Private University Culture Media Tables 6.2, 6.4 Prepared by Dr. Ahmad Saleh

Anaerobic Culture Methods General Microbiology for Pharmacy, Isra Private University Anaerobic Culture Methods Because anaerobic bacteria might be killed by exposure to oxygen, special media called reducing media must be used. Reducing media Contain chemicals (sodium thioglycolate) that combine O2 and deplete the oxygen in the culture medium. Special anaerobic jars are used to grow anaerobes on Pteri plates to observe individual colonies. Culture plates are placed in the jar, and oxygen is removed by addition of a packet of chemicals which react with oxygen to form water. Use of transparent anaerobic champers Prepared by Dr. Ahmad Saleh

Capnophiles Require High CO2 General Microbiology for Pharmacy, Isra Private University Capnophiles Require High CO2 Microbes that grow better at high CO2 concentrations are called capnophiles. Candle jar: Cultures are placed in a sealed jar containing a lighted candle, which consumes O2 and produces CO2. CO2-packet The packet consists of a bag containing a Petri plate and CO2 generator. Figure 6.7 Prepared by Dr. Ahmad Saleh

General Microbiology for Pharmacy, Isra Private University Selective Media Selective and differential media are used to detect the presence of specific microorganisms associated with disease or poor sanitation. Selective media are designed to suppress the growth of unwanted microbes and encourage growth of the desired microbes. Example: bismuth sulfite agar is used to isolate the typhoid bacterium (Salmonella typhi) from feces. Example: Sabourraud’s dextrose agar (pH 5.6), is used to isolate fungi that outgrow most bacteria at this pH. Figure 6.9b–c Prepared by Dr. Ahmad Saleh

General Microbiology for Pharmacy, Isra Private University Differential Media Make it easy to distinguish colonies of the desired organism from other colonies growing on the same plate. Sometimes, selective and differential characteristics are combined in a single medium. Example: Mannitol salt agar contains 7.5% sodium chloride, which will discourage the growth of competing organisms and thus select for Saphylococcus aureus. Enrichment Media The enrichment media is usually liquid and provides nutrients and environmental conditions that favor the growth of a particular microbes but not others. Prepared by Dr. Ahmad Saleh

Obtaining Pure Cultures General Microbiology for Pharmacy, Isra Private University Obtaining Pure Cultures A pure culture contains only one species or strain. A colony is a population of cells arising from a single cell or spore or from a group of attached cells. Microbial colonies often have a distinctive appearance that distinguishes one microbe from another. A colony is often called a colony-forming unit (CFU). The isolation method most commonly used to get pure cultures is the streak plate method. Prepared by Dr. Ahmad Saleh

General Microbiology for Pharmacy, Isra Private University Streak Plate Figure 6.10a–b Prepared by Dr. Ahmad Saleh

Direct Measurements of Microbial Growth General Microbiology for Pharmacy, Isra Private University Direct Measurements of Microbial Growth Plate counts: Perform serial dilutions of a sample Advantage: it measures the number of viable cells Disadvantage: it takes some time, about 24 hours. Plate counts assume that each living bacterium grows and divides to produce a single colony. Figure 6.15, step 1 Prepared by Dr. Ahmad Saleh

General Microbiology for Pharmacy, Isra Private University Phases of Growth Figure 6.14 Prepared by Dr. Ahmad Saleh

The first phase of the growth curve is the lag phase, during which the bacteria absorb nutrients, synthesize enzymes, and prepare for cell division. The bacteria do not increase in number during the lag phase. 2. The second phase of the growth curve is the logarithmic growth phase. In the log phase, the bacteria multiply so rapidly that the number of organisms doubles with each generation time

3. As the nutrients in the liquid medium are used up and the concentration of toxic waste products from the metabolizing bacteria build up, the rate of division slows, such that the number of bacteria that are dividing equals the number that are dying. 4. As overcrowding occurs, the concentration of toxic waste products continues to increase and the nutrient supply decreases. The microorganisms then die at a rapid rate.

General Microbiology for Pharmacy, Isra Private University The Control of Microbial Growth Sepsis refers to microbial contamination. Asepsis is the absence of significant contamination. Aseptic surgery techniques prevent microbial contamination of surgical wounds. Prepared by Dr. Ahmad Saleh

General Microbiology for Pharmacy, Isra Private University Terminology Sterilization: Removal or destruction of all forms of microbial life Methods of sterilization: heating, filtration of liquids and gases. Disinfection: Removal or destruction of vegetative (non spore forming) pathogens Disinfection might use of chemicals, ultraviolet radiation, boiling water or steam. Antisepsis: Removal of pathogens from living tissue, and the chemical is antiseptic. Sanitization: Lower microbial counts on eating utensils Biocide/Germicide: Kills microbes Bacteriostasis: Inhibiting, not killing, microbes Prepared by Dr. Ahmad Saleh

Terminology

Physical Methods of Microbial Control General Microbiology for Pharmacy, Isra Private University Physical Methods of Microbial Control Heat Heat appears to kill microorganisms by denaturing their enzymes, changing their three dimensional shapes leading to functional inactivation. Used for sterilization of canned foods, glass-ware, hospital instruments. Thermal death point (TDP): Lowest temperature at which all cells in a liquid culture suspension are killed in 10 min. Thermal death time (TDT): the minimal length of time to kill all bacterial cells in a particular liquid culture at a given temperature. Prepared by Dr. Ahmad Saleh

General Microbiology for Pharmacy, Isra Private University Moist Heat Moist heat kills microorganisms by coagulation of proteins (denaturation). Types of moist heat sterilization: Boiling: kills vegetative bacteria, viruses, fungi and their spores within 10 minutes. Autoclave: Steam under pressure, the higher the pressure the higher the temperature. pressure= 15 pounds, temperature 121 C. Under these conditions all organisms and their spores are killed in about 15 minutes. Autoclaving is used sterilize culture media, instruments, dressings, intravenous equipment, solutions, syringes, other items that can withstand high temperature and pressures. Prepared by Dr. Ahmad Saleh

General Microbiology for Pharmacy, Isra Private University Pasteurization Pasteurization reduces spoilage organisms and pathogens Equivalent treatments Classical pasteurization: 63°C for 30 min High-temperature short-time pasteurization: 72°C for 15 sec Ultra high temperature treatments: 140°C for <1 sec Thermoduric organisms survive pasteurization, but these are unlikely to cause disease or cause refrigerated milk to spoil. Prepared by Dr. Ahmad Saleh

Physical Methods of Microbial Control General Microbiology for Pharmacy, Isra Private University Physical Methods of Microbial Control Dry heat sterilization kills by oxidation effects. Flaming Incineration Hot-air sterilization: in an oven at temperature 170 C for 2 hours Hot-air Autoclave Equivalent treatments 170˚C, 2 hr 121˚C, 15 min Prepared by Dr. Ahmad Saleh

Physical Methods of Microbial Control General Microbiology for Pharmacy, Isra Private University Physical Methods of Microbial Control Filtration removes microbes The passage of a liquid or gas through a screenlike material with pores small enough to retain microorganisms. Filtration is used to sterilize heat sensitive materials, such as some culture media, enzymes, vaccines, and antibiotic solutions. Membrane filters, composed of substances such as cellulose esters or plastic polymers, with pore sizes of 0.22 µm and 0.45 µm are used to retain bacteria. Low temperature inhibits microbial growth Refrigeration Deep freezing Lyophilization Prepared by Dr. Ahmad Saleh

Physical Methods of Microbial Control General Microbiology for Pharmacy, Isra Private University Physical Methods of Microbial Control Desiccation (absence of water) prevents metabolism microorganisms can not grow or reproduce without water but can remain viable for years. Osmotic pressure: the use of high concentrations of salts and sugars to preserve food. Osmotic pressure causes plasmolysis of microbial cells. Concentrated salt solutions are used to preserve meats, and thick sugar solutions are used to preserve fruits. Yeasts and molds are capable of growing under high osmotic pressure than bacteria. Prepared by Dr. Ahmad Saleh

Physical Methods of Microbial Control General Microbiology for Pharmacy, Isra Private University Physical Methods of Microbial Control Radiation causes DNA damage. Ionizing radiation (X rays, gamma rays, electron beams) Has a wavelength shorter than that of nonionizing radiation, and carries much more energy. The principal effect of ionizing radiation is the ionization of water, which forms highly reactive hydroxyl radicals. Used for the sterilization of pharmaceuticals and disposable dental and medical supplies. Prepared by Dr. Ahmad Saleh

Physical Methods of Microbial Control General Microbiology for Pharmacy, Isra Private University Physical Methods of Microbial Control Nonionizing radiation (UV), has a wavelength longer than that of ionizing radiation. UV light damages the DNA of exposed cells by causing thymine dimers It is used to kill microbes in the air (hospital rooms, nurseries, operating rooms, cafeterias) and to disinfect vaccines and other medical products. (Microwaves kill by moisture heating; not especially antimicrobial) Figure 7.5 Prepared by Dr. Ahmad Saleh

Physical Methods of Microbial Control

Physical Methods of Microbial Control

Chemical Methods of Microbial Control General Microbiology for Pharmacy, Isra Private University Chemical Methods of Microbial Control Chemical agents are used to control the growth of microbes on both living and nonliving objects. Most of chemical agents are disinfectants that reduce microbial populations to safe levels. Principles of effective disinfection Concentration of disinfectant Organic matter pH Time Prepared by Dr. Ahmad Saleh

Chemical Methods of Microbial Control General Microbiology for Pharmacy, Isra Private University Chemical Methods of Microbial Control Evaluating a disinfectant Disk-diffusion method Figure 7.6 Prepared by Dr. Ahmad Saleh

Preserving Bacteria Cultures General Microbiology for Pharmacy, Isra Private University Preserving Bacteria Cultures Refrigerators can be used for the short-term storage of bacterial cultures. Two common methods for preserving microbial cultures for long periods are: Deep-freezing: pure culture of microbes is placed in a suspending liquid and quick-frozen at –50°to –95°C. Lyophilization (freeze-drying): A suspension of microbes is quickly frozen at (–54° to –72°C) and the water is removed by a high vacuum (Sublimation). Prepared by Dr. Ahmad Saleh

The Growth of Bacterial Culture General Microbiology for Pharmacy, Isra Private University The Growth of Bacterial Culture Reproduction in Prokaryotes Binary fission Budding Conidiospores (actinomycetes) Fragmentation of filaments Generation time: the time required for a cell to divide (and its population to double). Prepared by Dr. Ahmad Saleh

General Microbiology for Pharmacy, Isra Private University Binary Fission Figure 6.11 Prepared by Dr. Ahmad Saleh

Effectiveness of Antimicrobial Treatment General Microbiology for Pharmacy, Isra Private University Effectiveness of Antimicrobial Treatment Depends on: Number of microbes: the more microbes, the longer it takes to eliminate the population. Environmental influence: (organic matter, temperature, biofilms) inhibit the action of chemical antimicrobials. Time of exposure: extended exposure for more resistant microbes or endospores are required. Microbial characteristics Figure 7.1b Prepared by Dr. Ahmad Saleh

Chemical Methods of Microbial Control

Chemical Methods of Microbial Control

Chemical Methods of Microbial Control