Microbial Growth and The Control of Microbial Growth

1. Microbial Growth and The Control of Microbial Growth Microbiology

2. Microbial Growth • Microbial requirements for growth: • 1. Physical • A. Temperature • Optimal growth temperature • Permissible range • human pathogens optimal = 37°C

3. Microbial Growth • 1. Physical continued: • B. pH • most organisms optimal pH 6.5-7.5 (near neutral) • C. Osmotic pressure- draws the water from the cells • Most require isotonic solutions • human blood = 0.9% NaCl, isotonic • human skin = ~3-6% NaCl, hypertonic

4. Microbial Growth • 2. Chemical • A. Carbon • B. Nitrogen • C. Sulfur • D. Phosphorus • (all above: to build organic molecules) • E. Trace elements: K+, Mg2+, Ca2+, Fe2+... • cofactors for enzymes • F. Oxygen • aerobe, anaerobe, facultative • G. Organic factors: vitamins, amino acids... • (essential organic compound an organism is unable to synthesize)

5. Microbial Growth • Microbial Growth • -for unicellular organisms, growth = increase in cell number (population) not individual cell size • -generation time = time required for a cell to divide • -both growth and death of the population is exponential, graphed on a log scale

6. Bacterial Growth Curve • 1. Lag Phase: little to no growth, bacteria acclimate to new environment • 2. Log Phase: period of exponential growth with constant generation time • 3. Stationary Phase: cell growth is equal to cell death • 4. Death Phase: cell death exceeds cell growth

7. Terminology for Microbial Control • Sterilization- removal or destruction of all forms of microbial life • Commercial sterilization- subjects canned food to only enough heat to destroy the endospores of Clostridium botulinum • Disinfection- is the destruction of vegetative pathogens on a surface, usually with chemicals • Spores and viruses are not necessarily destroyed • Antisepsis- is the chemical disinfection of living tissue, such as skin or mucous membranes

8. Terminology for Microbial Control • Asepsis- is the absence of pathogens on an object or area, as in antiseptic surgery • Degerming (degermation)- is the removal of transient microbes from the skin by mechanical cleansing or by antiseptic • Sanitation- is the reduction of microbial populations on objects to safe public health levels • A biocide or germicide- kills microorganisms • Fungicides kill fungi, virucides kill viruses • Suffix – cidemeans the killer of a specified microorganism • Suffix- statused in this way indicates only that the substance inhibits – for example bacteriostasis

9. Rate of Microbial Death • Bacterial populations killed by heat or chemicals tend to die at constant rates—for example, 90% every 10 minutes. Plotted logarithmically, these figures form straight descending lines.

10. Factors that influence effectiveness of anantimicrobial treatment: • 1. Number of microbes • -more cells, more time needed to kill all • 2. Environmental influences • -organics often inhibit chemical agents • -temperature • -pH • 3. Time of exposure • -same agent may need longer on resistant organisms or spores • -with heat, lower temps require longer to kill • 4. Microbial characteristics • -resistance genes, protective structures • (e.g.capsules) etc. caninhibit action • -bioflims prevent penetration

11. Type and Age of Microbe • Bacteria- susceptible to protein denaturing BUT mycobacteria is not because of its hydrophobic coat • Growth cycles- physiologically young bacteria(early in growth cycle) susceptible to heat • Endospores more resistant to heat the older they get

12. Properties of Materials: • Plastic and rubber- not heat tolerant • Cutting edges of surgical instruments- no moisture or corrosive chemicals • Edges can become pitted due to rust and corrosion, microbes can hide there • Fabrics- no chemical disinfection; some too fragile

13. Actions of Microbial Control Agents • 1. Alteration of membrane permeability • The plasma membrane controls the passage of nutrients and wastes into and out of the cell. Damage to the plasma membrane causes leakage of cellular contents and interferes with cell growth. • = Leak lysis, death

14. Actions of Microbial Control Agents • 2. Damage to proteins and Nucleic Acids • denature enzymes (no reactions) • prevent replication, transcription, or translation

15. Physical Methods of Microbial Control • -to disinfect objects, food, and solutions common methods: • -temperature: kill or inhibit growth • -filtration: physical removal • -desiccation: inhibit growth • -radiation: kill

16. For methods that involve heat: • Thermal Death Point (TDP) = lowest temp at which all microbes in liquid suspension will be killed in 10 min • Thermal Death Time (TDT)= minimal length of time for all microbes in liquid suspension to be killed at given temp • *Both are different for different species due to microbial variation in heat tolerance • Concept of equivalent treatments: • With any heat treatment, the higher the temperature used the shorter the exposure time needed to achieve the same effect • Moist heat will always kill faster than dry heat at the same temperature

17. For methods that involve heat: • Moist Heat – coagulation of cell proteins • Boiling (100°C) kills vegetative forms of bacterial pathogens, many viruses, and fungi within 10 minutes. • Endospores and some viruses survive boiling for longer times. • Steam under pressure • allows temperatures above boiling to be reached. • Autoclaves, retorts, and pressure cookers are vessels in which high steam pressures can be contained. A typical operating condition for sterilization is 15 psi (pounds per square inch) at 121°C for 15 minutes. Moisture must touch all surfaces in order to bring about sterilization. Air must be completely exhausted from the container. An autoclave is shown in Figure 7.1.

18. Autoclave

19. Low Temperature • Lower temp inhibits growth, rapid freezing limits moisture • Refrigerator temperatures (0° to 7°C) slow the metabolic rate of microbes; however, psychrotrophicspecies still grow slowly. • Some organisms grow at temperatures slightly below freezing, but microbes at the usual temperatures of freezer compartments are completely dormant.

20. Filtration • Liquids sensitive to heat can be passed through a thin membrane filter that has carefully controlled pore sizes to retain microorganisms. • Operating theaters and special clean rooms receive air passed through high-efficiency particulate air filters (HEPA). • Used with solutions for: renal dialysis, heart bypass machines, I.V.’s • Catch particles autoclaves miss

21. Desiccation • Microbes require water for growth, and adequately dried (desiccated) foods will not support their growth, therefore inhibiting growth • Removing all moisture • Lyophilization- rapid freeze drying • Used for blood products , serum products, enzymes, cultures • Avoids ice crystal formation; cells burst when water expands • Liquid nitrogen- (-196) degrees C

22. Osmotic Pressure • High salt or sugar concentrations cause water to leave the cell; this is an example of osmosis. Generally, molds and yeasts resist osmotic pressures better than bacteria.

23. Radiation • U.V. light : rearranges proteins and DNA, bases altered to make dimer bonds; (T-T or T-C- lethal mutations) BUT limited- does not penetrate surfaces, and damages human DNA • X-rays, gamma rays, cosmic radiation- microbicidal • Visible light- can destroy some in presence of oxygen, but only if they have pigments- most pathogens lack pigments • Ionizing radiation- breaks bonds in DNA, creates oxidizing and reducing agents by reacting with water • Microwave- not effective, limited to use in beer and wine • Can be used to disinfect cutting boards, sponges and dish cloths • Ionizing radiation such as X rays, gamma rays, and high-energy electron beams carry high energy and break DNA strands. • Ionizing radiation forms reactive hydroxyl radicals. Such radiation is used to sterilize pharmaceuticals. • Nonionizing radiation such as ultraviolet (UV) light has a longer wavelength and less energy. • UV light causes bonds to form between adjacent thymines in DNA chains. Penetration is low. • Sunlight has some biocidal activity, mainly due to formation of singlet oxygen in the cytoplasm.

24. Chemical Agents • Used for disinfection, not sterilization • Protein denaturing- proteins are unfolded and refolded improperly; irreversible • Organic solvents- alcohol, ether, acetone denature all cell membranes • Limited against spores and viruses • Alcohols- skin, thermometers, anesthesia equipment • Isopropyl- 70 % in water • Ethers, benzenes- highly germicidal, BUT irritating and carcinogenic

25. Chemical Agents • Heavy metals- Hg- toxic, irritating, used to be used in ointment for newborn eyes to prevent gonorrhea, now use penicillin and erythromycin • Ag- irritating, corrosive, but used in band-aids as an antimicrobial • 1% solution of silver nitrate for eye ointment • Cu- fungicidal, algicidal • Zn- superficial fungal and bacterial infections, diaper rash • As- “606” some protozoal infections

26. Chemical Agents • Oxidizing agents- halogens, hydrogen peroxides, potassium permanganate, peracetic acid • Oxidize SH groups and NH2 groups of proteins • Cl- and I- used on vegetative(growing) cells, spores, fungi, viruses • Br- is too toxic- but is used to disinfect spas and hot tubs • Iodine solutions used on skin and mucous membranes, suture materials, thermometers, surgical instruments, eating utensils • Chlorine bleach- 5.25% NaClO- floors, bathrooms, linens, dishes, cutting boards • Dakin’s solution- 0.5% Cl-wound cleanser • Cyst and oocysts(parasites) survive chlorine- Giardia • H2O2- 3% solution – disinfects spacecraft and contact lenses • O2 released quickly in presence of catalase enzyme in human tissue, causes bubbling- clears wound of dirt, reduces deep tissue anaerobes • Lethal to bacteria, viruses, fungi, and reduces # of spores

27. Chemical Agents • Dyes- found in coal tar, have + charge and affinity for nucleic acids • Alkylating agents- formaldehyde- replace H atoms on NH2, OH, COOH, SH groups • Formalin- disinfect instruments, used in gas sterilization • Gluteraldehyde- 2% solution- used for respiratory therapy equipment, unstable at room temp • Ethylene oxide- gas sterilization, carcinogenic, explosive in air, • Usually mixed with inert gas, CO2 or N- artificial heart valves and bedding

28. Chemical Agents • Membrane altering agents- • Surface active- reduce surface tension(detergents) • Increase penetration of liquid or air surface of molecules • Anionic (-) or cationic(+)- cationic example is quaternary salts, or “quats”-alter charges of phospholipids, they are stable, non-irritating, odorless and cheap- only effective on some G-, all G+, no opportunistic bacteria. • Phenolic derivatives- carbolic acid- toxic, irritating, not effective on spores, • 5% aqueous phenol solutions used on sputum, urine, feces, contaminated glass slides • Hexachlorophene is the main ingredient in pHisoHex and is used in nurseries to control gram-positive skin bacteria such as staphylococci and streptococci, surgical scrubs, but excessive use has been linked to neurological damage • Chlorhexidine- It is frequently used for surgical skin preparation and surgical hand scrubs. Betasept, Hibiclens, effective on G- and G+, unaffected by presence of blood or other organic matter