Microbiology Course: Controlling Microbial Growth In Vitro Lecture Notes PDF

M ICROBIOLOGY Course Controlling Microbial Growth In Vitro CPC C L O W N I N P L U S C R O W N  Factors that affect microbial growth 1- Availability of nutrients : Nutrients serve as sources of energy as well as chemical elements. The amount of elements ne...

M ICROBIOLOGY Course Controlling Microbial Growth In Vitro CPC C L O W N I N P L U S C R O W N  Factors that affect microbial growth 1- Availability of nutrients : Nutrients serve as sources of energy as well as chemical elements. The amount of elements needed differs according to the type of element : Macronutrients (required in large amounts) : C ,O ,H ,N ,P & S Micronutrients (required in smaller amounts) : Na ,Cl ,K ,Ca & Mg Trace elements : iron, iodine & zinc 2- Moisture : Water is essential for life as cells are 70 to 95% water. Bacterial spores & protozoan cysts are able to survive even through desiccation (complete dehydration) 3- Osmotic pressure and salinity: Osmotic pressure is exerted on the cell membrane by the solutions surrounding it. Osmosis is the movement of the solvent through a semipermeable membrane from a hypotonic solution to a hypertonic solution. Halophilic organisms : prefer salty environments for growth Haloduric organisms : don't prefer salty environments, but are able to grow there. In Isotonic solutions (Pressure inside = Pressure outside) : NO effect on cells In hypertonic solutions (Pressure inside < Pressure outside) : Cells without cell wall (RBC & Mycoplasma spp.) : crenation (cell shrinkage) Cells with cell wall (bacteria) : plasmolysis (cell membrane and cytoplasm shrink away from the Cell wall) In hypotonic solutions like distilled water (Pressure inside > Pressure outside) : Cells without cell wall (RBC & Mycoplasma spp.) : hemolysis (cell will burst) Cells with cell wall (bacteria) : plasmoptysis (escape of cytoplasm from the cell) 4- Temperature: Optimum growth temperature : temperature at which the organism grows best Minimum growth temperature : below it the MOs will ceases to grow Maximum growth temperature : above it the MOs will die Microbes are classified according to their preferable temperature of growth into: Thermophiles: prefer high temperatures. Hyperthermophiles (Extreme thermophiles): prefer temperatures above 100°C (live in hot springs) Mesophiles: prefer moderate temperatures (normal body temperature 37°C : pathogenic MOs) Psychrophiles: prefer cold temperatures. Psychrotrophs: prefer a temperature around 4°C for growth. Psychroduric organisms: tolerate cold temperatures, but prefer warmer temperatures for growth. 1 5- pH : Microbes are classified according to their preferable pH of growth into : Alkaliphiles: prefer a pH more than 8.5 (Vibrio cholerae). Most microbes prefer a neutral or slightly alkaline medium : — pH between 7 and 7.4 — Most medically important MOs (normal body pH) Acidophiles: prefer a pH between 2 and 5. Notes : Vibrio cholerae is the only human pathogen that grows well above pH 8. Fungi prefer acidic environments 6- Gaseous atmosphere : Microbes differ according to their preference of gaseous atmosphere. The gases mostly linked to microbial growth are : A- Oxygen (Microbes may be classified according to their Oxygen preference into) : — Obligate aerobes : require O2 in atmospheric concentrations (21%). — Microaerophiles: require O2 in subatmospheric concentrations (about 5 %). — Facultative anaerobes: grow both in the presence or absence of O2, but growth is better in its presence. — Aerotolerant anaerobes: grow better in the absence of O2, but can tolerate its presence. — Obligate anaerobes : only grow in the absence of O2. B- Carbon dioxide: — Capnophiles : microbes which require the presence of CO2 in high concentrations (5-10 %). 7- Atmospheric pressure : Piezophiles : microbes which are able to grow under high atmospheric pressures (in bottoms of the oceans). Encouraging the growth of microbes in vitro Bacterial growth : increase in the number of organisms rather than an increase in their size (proliferation or multiplication of bacteria) When bacterial cell reaches its optimum size, it divides by binary fission into two daughter cells has exactly the same genetic makeup as the parent cell Generation time : the time taken by a particular bacterial species to undergo binary fission Bacteria with short generation times are referred to as rapid growers Bacteria with long generation times are referred to as slow growers Fastidious microbes : MOs that have complex nutritional requirements & difficult to grow in the laboratory Broth : Liquid media or tubed media If a sample contains more than one type of bacteria, they will be mixed together and cannot be separated. 2 Agar : Complex polysaccharide that is obtained from a red marine alga Used as a solidifying agent, much like gelatin Solid media are prepared by adding agar to liquid media and then pouring the media into tubes or Petri dishes In agar the microbes will form separated clusters or colonies Assists in identifying microbes within mixed cultures (different colonies) Chemically defined medium : All the ingredients are known Prepared in the laboratory by adding a certain number of grams of each component (COH , AA, salts) Complex medium : The exact contents are not known Contain ground-up or digested extracts from animal organs (hearts, livers, brains), fish, yeasts, & plants Enriched medium : Broth or solid medium containing a rich supply of special nutrients Promotes the growth of fastidious organisms Prepared by adding extra nutrients to a medium called nutrient agar Examples of enriched media : Blood agar : nutrient agar plus 5% sheep’s red blood cells (contain hemoglobin) Chocolate agar : — Nutrient agar plus powdered hemoglobin — Considered to be more enriched than blood agar because the hemoglobin is more readily accessible — Used to culture fastidious bacterial pathogens which will not grow on blood agar : Neisseria gonorrhoeae Haemophilus influenzae Selective medium : Adding inhibitors that discourage the growth of certain organisms (the target organisms will grow) MacConkey agar : inhibits growth of Gram-positive (selective for Gram-negative) Phenylethyl alcohol (PEA) agar & colistin–nalidixic acid (CNA) agar : inhibit growth of Gram-negative (selective for Gram-positive) Thayer–Martin agar and Martin-Lewis agar : selective for N. gonorrhoeae Mannitol salt agar (MSA) : only salt-tolerant (haloduric) bacteria can grow on Differential medium : Permits the differentiation of organisms that grow on the medium MacConkey agar differentiates between : Lactose-fermenting : produce pink colonies Nonlactose-fermenting : produce colorless colonies MSA is used to screen for S. aureus : turns the originally pink medium to yellow (ferment mannitol) 3  Blood agar (used to determine the type of hemolysis) : Beta (β-hemolytic) → clear zone due to complete RBCs’ lysis Alpha (α-hemolytic) → partial hemolysis Gamma (γ-hemolytic) → No hemolysis Notes : Blood agar is enriched & differential media MacConkey agar and MSA are selective & differential media PEA and CNA are enriched & selective Thayer–Martin and Martin-Lewis agars are highly enriched & highly selective Thioglycollate broth (THIO) : Supports the growth of all categories of bacteria from obligate aerobes to obligate anaerobes The concentration of oxygen decreases with depth Incubation Sterile technique to ensure that the MOs in the sample are the only ones that will grow Types of incubators : 1- Ordinary (non-CO2) incubators : containing room air (20% to 21% oxygen) 2- CO2 incubator : used to isolate capnophiles (5% to 10% CO2) 3- Anaerobic incubator : containing an atmosphere devoid of oxygen (No O2) Notes : Pure culture: Contains only one type of microorganism. Mixed culture: Contains two or more different types of microorganisms. Bacterial Population Growth Curve Shows changes in number and character with time The bacterial population growth curve is plotted by plotting time on the X-axis The logarithm of concentration of viable microbes on the Y-axis It consists of the following phases : 1- Lag phase: bacteria get prepared for cell division without increasing in number. 2- Log phase: Bacteria divide rapidly and their number increases exponentially Growth rate is the greatest during the log phase 3- Stationary phase : The number of viable bacteria becomes constant during this phase Because the number of bacteria forming equals the number of bacteria dying. The culture is at its greatest population density in stationary phase 4- Death (Decline) phase : The number of viable bacteria declines rapidly as they die in large numbers. Witnesses morphological changes (formation of L-forms and spores). Chemostat : Continuously cultured in a controlled environment Regulates the supply of nutrients and the removal of wastes 4  Microbes other than bacteria 1- Obligate intracellular pathogens : Need to be cultured in cell cultures (embryonated chicken eggs or lab animals) Cell cultures come in specific lines: Monkey kidney cells Human lung cells Cancer cells Positive growth is indicated by specific morphological microscopic changes (cytopathic effects) Examples: Viruses, Chlamydia spp., Rickettsia spp. 2- Fungi : Selection for fungal growth against bacteria is provided by : Adding antibacterial agents Reducing the pH Cultured on such media as : Brain-heart infusion (BHI) agar Sabouraud dextrose agar (SDA) Care must be taken in the lab against the spread of potentially infective spores. 3- Protozoa : Protozoal cultures are usually reserved for research and reference labs Of special interest to clinical microbiology is the attempt to culture amebas and ameba-like organisms responsible for causing potentially fatal CNS infections: Acanthamoeba spp. Naegleria spp. This may be done by using: Axenic culture : dose not contain foreign MOs other than protozoa Xenic cultures : — Contain foreign MOs other than protozoa — Using cell cultures or bacteria as a food source for the protozoa Inhibiting the Growth of Microbes In Vitro Sterilization : the complete eradication of all living organisms. Disinfection : The elimination of pathogens on nonliving objects. Disinfectant: chemical substance used to eliminate pathogens on nonliving objects. Antiseptic technique : The use of antiseptics to fight pathogens on living surfaces. Antiseptic : chemical substance used to eliminate pathogens on living surfaces Living surfaces: skin and mucous membranes Aseptic technique: the use of a method (physical or chemical) to fight microbes while carrying out a procedure in the healthcare setting. Sanitization : reduction of microbial populations to levels considered safe by public health standards, such as (restaurants & water) 5 Pasteurization : Physical method of disinfection Moist heat at 63°C for 30 minutes or 72°C for 15 seconds Immediate cooling to below 10 degree. Microbicidal agents : The suffix -cidal- refers to “killing” (killing MOs) Disinfectants or antiseptics that kill microbes Germicidal agents ,biocidal agents & microbicidal agents Bactericidal agents : kill bacteria, but not necessarily bacterial endospores (spore coats are thick) Sporicidal agents : required to kill bacterial endospores (sterilization) Fungicidal agents : kill fungi, including fungal spores Algicidal agents : used to kill algae in swimming pools and hot tubs Viricidal agents : destroy viruses Pseudomonicidal agents : kill Pseudomonas spp. (Pseudomonas aeruginosa) Tuberculocidal agents : kill the causative agent of TB (Myctobacterium tuberculosis → lipid rich cell wall) Microbistatic agents : Inhibits reproduction and metabolism of MOs, but does not necessarily kill them Examples: Lyophilization (Freeze-drying) Rapid freezing (liquid nitrogen) Physical Methods to Inhibit Microbial Growth 1- Heat : The combined importance of temperature and time is assessed by : The thermal death point : the lowest temperature that will kill all the organisms in a standardized pure culture within a specified period The thermal death time : the length of time necessary to sterilize a pure culture at a specified temperature. The concomitant absence or use of moisture divides heating techniques into: A- Dry heat : — Used for objects which can withstand high temperatures — Usually performed at 160-165°C for 2 hours or 170- 180°C for 1 hour — Incineration : burn and completely destroy contaminated, disposable objects — Flaming : another dry heat method used to achieve sterility 6  B- Moist heat: — Achieves results at lower temperatures than dry heat. — Boiling: kills most vegetative microbes after 30 minutes. — Autoclaving : Method for achieving sterility in healthcare settings Pressure is applied in addition to heat and moisture to enhance their killing effect Usually performed at 121.5°C and 15 psi for 20 minutes Care must be taken to allow steam to adequately penetrate the material Indicators are used to ensure the necessary conditions have been met 2- Cold : Variety of techniques use cold to inhibit microbial growth. Refrigeration slows down bacterial growth Slow freezing kills bacteria Rapid freezing keeps bacteria in suspended animation (useful in preserving bacterial cultures) 3- Desiccation : Removes the moisture necessary for bacterial growth. Lyophilization : combining the effects of cold and drying to preserve a wide variety of objects 4- Radiation : UV light (non-ionizing radiation) : Damages microbial DNA, leading to their death UV lamps may be used in a wide variety of settings Must take care as they may cause skin and eye damage Ionizing radiation : Radiations with shorter wavelengths (easier penetration) Such as X-rays & gamma rays Must be made with still more caution. 5- Ultrasonic waves : used to transmit energy to substances stuck on object surfaces (facilitating their removal) 6- Filtration: used in settings where the use of heat is inappropriate. 7- Gaseous atmosphere: Modifying gas concentrations in the environment may make it less appropriate for the growth of certain MOs Depending on their specific oxygen and carbon dioxide requirements. Chemical Methods to Inhibit Microbial Growth Factors considered when evaluating a disinfectant: 1- Concentration of the disinfectant. 2- Physical nature of the object to be disinfected. 3- Prior cleaning of the object to be disinfected from organic matter. 4- Bioburden: the type and quantity of the microbe to be eradicated. 5- Contact time. 6- Temperature. 7- pH. 7 Properties of the ideal disinfectant: 1- Easy to prepare and apply. 2- Soluble in water. 3- Broad antimicrobial spectrum. 4- Fast-acting. 5- Not affected by organic matter. 6- Nontoxic to human tissue. 7- Nondestructive to inanimate objects. 8- Leave a residual antimicrobial film. 9- Stable. 10- Odorless. 11- Inexpensive. Note : There is no ideal disinfectant The power of a certain disinfectant is assessed by comparing it to that of phenol (phenol coefficient test) Mechanisms of action of disinfectants: 1- Destruction of cell membranes : Surface-active soaps Detergents Alcohols & Phenols 2- Destruction of structural proteins and enzymes : Hydrogen peroxide Alcohols & Phenols Formaldehyde Halogens (Fluorine , Iodine , Chlorine) Heavy metal salts (Mercury , Arsenic ,Silver ,Copper & Zinc) 3- Attacking nucleic acids (Ethylene oxide) 8