MICROBIAL GROWTH Requirements for microbial growth • Physical • Chemical Physical • Temperature • pH • Osmotic pressure Temperature • -10 to C, Psychrophiles o 20 Temperature • 0 to C, • Psychrotrophs o 30 Temperature • 10 to 50o C, • Mesophiles –Most pathogens Temperature • 40 to C, thermophiles o • 65 to 110 C, Extreme thermophiles o 70 Extreme thermophiles Thermophiles Mesophiles Psychrotrophs Psychrophiles -10 0 10 20 30 40 50 60 70 80 90 100 110 Degrees Centigrade pH • Most bacteria grow at pH 6.5 to 7.5 • Acidophiles (pH 1.0 - 5.5) Lactobacillus acidophilus pH • Alkalophiles (pH 8.5 - 11.5) Bacillus alcalophilus Osmotic Pressure (OP) • Bacteria are better adapted to low OP (i.e. tap water) • Plasmolysis occurs at high OP Osmotic Pressure (OP) (cont.) • Solutes (i.e. sugar and salt) limit water availability Osmotolerant Organisms (halophiles) • Less tolerant –Most Gram-negatives • More tolerant –Staphylococcus aureus –Fungi 1. SEA ICE cold-loving microbes (Psychrophiles) Polaromonas vacuolata 2. DEEP-SEA VENT heat-loving microbes (Thermophiles and Hyperthermophiles) Methanopyrus kandleri 3. SULFURIC SPRING acid-loving microbes (Acidophiles) Sulfolobus acidocaldarius 4. SALT LAKE salt-loving microbes (Halophiles) Haloferax volcanii 5. SODA LAKE alkali-loving microbes (Alkaliphiles) Natronobacteri um gregoryi Chemical Requirements • Carbon (all organic compounds) • Nitrogen (proteins) Chemical Requirements (cont.) • Sulfur (proteins and vitamins) • Phosphorous (DNA, RNA, ATP) Trace Elements • Needed in minute amounts -Iron -Copper -Molybdenum -Zinc Trace Elements (cont.) • Present in high enough amounts in nature • Enzymatic activity –Cofactors Organic growth factors • Essential for growing –Vitamins –amino acids –Purines and pyrimidines Oxygen (O2) • Electron acceptor during aerobic respiration • More efficient than other acceptors (NO3, SO4, and CO3) Oxygen (O2) • Highly reactive oxygen radicals –Singlet oxygen –Superoxide free radicals Neutralization of oxygen radicals • Superoxide Dismutase (SOD) • Catalase Culture Medium Forms an oxygen gradient More Oxygen on top O2 Obligate Aerobes • Aerobic only (O2) O2 Mycobacterium tuberculosis Facultative Anaerobes • Primarily aerobic O2 • Alternatively anaerobic Escherichia coli Obligate Anaerobes • Killed by oxygen O2 Clostridium spp. Aerotolerant Anaerobes • Growth not affected by Oxygen O2 Enterococcus faecalis Microaerophiles • Aerobic • Killed by O2 high (20%) O2 concentration Campylobacter spp. The Requirements for Growth: Chemical Requirements • Oxygen (O2) Facultative anaerobe Obligate aerobe Aerotolerant aerobe Obligate anaerobe Microaerophilic Bacterial Growth Binary fission • Asexual reproduction in which a cell separates into two cells Resting cell Cell elongation and DNA replication Cell wall and cell membrane grow inward Cell wall and Cell membrane meet Individual cells are formed Generation or Doubling Time • Time required for a cell to double in number • Highly variable, 20 minutes to 24 hours Generation Time (cont.) • Escherichia coli –20 minutes • Mycobacterium tuberculosis –12 hours Escherichia coli’s generation time 8 16 32 4 2 1 0 20 40 60 80 100 120 140 Minutes Logarithmic growth Number of bacteria Bacterial Growth Curve Lag phase Time Number of bacteria Bacterial Growth Curve Log Phase Time Number of bacteria Bacterial Growth Curve Stationary Phase Time Number of bacteria Bacterial Growth Curve Death Phase Time Measurement of Microbial Growth • Plate counts • Pour plates Direct Measurements of Microbial Growth • Plate Counts: Perform serial dilutions of a sample Figure 6.15, top portion Serial Dilutions • Transfer of part of a sample through a series of specific volumes of diluent Serial Dilutions (cont.) • Reduce high numbers of microorganisms gradually (i.e. form 100,000,000 to 10) Membrane filtration Sterile cup Vacuum Membrane 0.45 mm Membrane filtration Culture medium Direct Measurements of Microbial Growth • Filtration Figure 6.17a, b Most Probable Number (MPN) • Statistical estimation of the number of organisms present in a sample Most Probable Number (MPN) (cont.) Water samples Positives 0.1 ml 1/5 1 ml 2/5 10 ml 4/5 CULTURE MEDIA Nutritional types of Microorganisms (energy) • Phototrophs (light) • Chemotrophs (organic or inorganic compounds) Nutritional types of Microorganisms (Carbon source) (Cont.) • Autotrophs (CO2) • Heterotrophs (organic carbon) Culture Medium • Material prepared in the laboratory for the growth of microorganisms Culture • Microorganisms that grow in or on a culture medium Streak Plate for obtaining a pure culture Figure 6.10a, b Sterile • Absence of life Chemically Defined Media • Exact chemical composition is known • Mostly autotrophic organisms Chemically Defined Media (cont.) • Ammonium sulfate 0.5 g • Potassium phosphate 0.2 • Calcium chloride 0.04 • Magnesium sulfate 0.04 • Water 1 liter Complex Media • Exact composition is unknown • Slight differences from batch to batch • Extracts from meats, milk, vegetables and yeast Complex Media Composition • Soy peptone 5.0 g • Beef extract 3.0 • Sodium chloride 8.0 • Agar 15.0 • Water 1 liter Agar • Complex polysaccharide • Solidifying agent • Extracted from red algae Deserts using agar Agar-agar – jello made out of seaweed, much firmer than the kind you get in hospitals. Agar used in the lab Robert Koch and Agar (History and Microscopy lecture) • He invented new methods – • of cultivating pure cultures of bacteria on solid media such as potato, and on agar kept in the special kind of flat dish invented by his colleague Petri, which is still in common use • (He also developed new methods of staining bacteria which made them more easily visible and helped to identify them) Anaerobic Growth Media (broth) • Sodium thioglycolate scavenges O2 by combining with it Growth Media Sodium thioglycolate O2 O2 O 2 O2 O2 O2 Clostridium spp. Aerobic Anaerobic Capnophiles require high • Candle jar CO2 • CO2-packet Figure 6.7 Anaerobic Growth Media (solid) • Anaerobic atmospheres (without O2) are obtained using special jars Anaerobic Growth Media (solid) CO2 H2 Pouch with CO2 , H2 and Na borohydride Anaerobic jar Anaerobic Culture Methods • Anaerobic chamber Figure 6.6 Fastidious Organisms • Do not grow on known media • Difficult to grow Fastidious Organisms (cont.) • Mycobacterium leprae –Animals • Treponema pallidum • Rickettsias and Chlamydias Selective Media • Suppresses the growth of unwanted bacteria, without affecting the target organism Selective Media (cont.) • Bismuth sulfite agar –Salmonella typhi • Mannitol salt agar –Staphylococcus aureus Selective Media • Suppress unwanted microbes and encourage desired microbes. Figure 6.9b, c Differential Media • The target organism forms distinctive colonies, usually of different color or appearance Differential Media (cont.) Blood agar Streptococcus pyogenes Differential Media • Make it easy to distinguish colonies of different microbes. Figure 6.9a Differential-Selective Media Mannitol-salt agar Staphylococcus aureus Staphylococcus epidermidis Chromogenic medium Enrichment Media (broth) • Selective medium that favors the growth of the target organism when present in low numbers