Microbial Growth Lecture Notes PDF

Dynamics of microbial growth  Principles of microbial growth Bacteria multiply by binary fission and increase in cell number is exponential. Microbial growth: An increase in number of cells in a population, not by size of cells. Doubling/generation time: Time taken for a popu...

Dynamics of microbial growth  Principles of microbial growth Bacteria multiply by binary fission and increase in cell number is exponential. Microbial growth: An increase in number of cells in a population, not by size of cells. Doubling/generation time: Time taken for a population to double in numbers. Generation time varies depending on the species of organism & growth conditions. – E. coli generation time is ~20 minutes – Mycobacterium tuberculosis requires 12 hrs to double. The exponential multiplication of bacteria has important health consequences. Fig 4.1.Binary Fission  Principles of prokaryotic growth Equation for exponential multiplication of bacteria (Number of cells in a population at a given time) Nt = N0 X 2n Keep your potato salad in cooler when you go for picnic!  Microbial growth in nature Natural conditions differ greatly from the lab conditions with respect to microbial growth & behavior. Bacterial growth is more dynamic in natural environment as – nutrients are continuously supplied in diluted form. – waste products are continuously replaced. Under natural conditions, bacteria remain in log phase for a longer time but multiply more slowly. When growing in running water, bacteria may produce slime layers or other structures that allow them to attach. In contrast, they may not produce adherent structures when growing in the lab.  Biofilms Bacteria living suspended in the aqueous environment are called planktonic or free floating. Bacteria may attach to surfaces & live in a polymer- encased communities called biofilms e.g. Slipperiness of rocks in water stream, Slimy ‘gunk’ in kitchen drains, Scum in toilet bowls and Dental plaque.  Biofilms Adhered bacteria release polysaccharides, DNA & other hydrophilic polymers, referred as extracellular polymeric substances (EPS), to which other cells may attach. EPS gives biofilm a slimy appearance.  Biofilms Biofilms are very important in human health e.g. -Dental plaque may lead to tooth decay & gum disease -Persistent ear infection -Cystic fibrosis -Medical implants and urinary catheters Majority of human bacterial infections involve biofilms. Treatment of infections involving biofilms is difficult as bacteria encased in biofilms are more resistant to disinfectants, antibiotics and body defenses. Biofilms may be beneficial in many bioremediation efforts especially where microbes can degrade harmful chemicals. Water treatment facilities are looking for ways to foster biofilms development.  Interactions of mixed microbial communities In nature, bacteria often live-in close association with other microbes. Sometimes these interactions are cooperative. Some bacteria which otherwise can not survive can live in certain environments in the presence of other bacteria e.g. – In the mouth, aerobes create conditions suitable for the growth of anaerobes. Metabolic waste products of one species can be used as a nutrient by another bacterial species. Mouth contains aerobes, facultative anaerobes & anaerobes. Similarly, gastrointestinal tract has many kinds of microorganisms living together.  Microbial growth in laboratory conditions Bacteria are isolated and grown in ‘pure culture’ to study the characteristics and functions of a particular species. A ‘pure culture’ is defined as a population of organisms (colony) descended from a single bacterial cell. Basic requirements to obtain pure cultures are to use – aseptic technique, – cultivating bacteria on solid media (agar base) or in culture medium and – a method to separate individual cells. Only ~1% of all prokaryotes can be cultured successfully. Fortunately, most medically important bacteria can be grown in pure cultures.  Culture Techniques Agar, a polysaccharide extracted from marine algae, is used to solidify the nutrient solution. Advantages of using Agar include – Bacteria can not degrade agar. – Not destroyed at high temp & can be sterilized by heating – Remains liquid until 45°C so nutrients can be added. – Once solidified, it remains solid until 95°C. – Agar is translucent so colonies can be visualized easily.  Culture Techniques The culture medium is contained in Petri dish to exclude airborne microbial contaminations. The ‘streak-plate method’ is the simplest and most used technique for isolating bacteria. Bacterial stock cultures can be stored – In refrigerator as growth on the surface of an agar slant. – at -70oC in a glycerol-containing solution for long-term storage that prevents ice crystal forming & damaging cells. – Freeze-dried.  Prokaryotic growth in lab Bacteria in the lab are generally grown in broth contained in a tube/flask, or on an agar plate. These are considered closed systems or batch cultures as neither nutrients are renewed nor are wastes removed. Pattern of bacterial growth followed in the closed system is called ‘growth curve’ (five distinct stages). Log or exponential phase is medically important as bacteria are most susceptible to antibiotics & other chemicals during this time. Generation time is measured during the log phase.  Bacterial growth in lab Primary metabolites: produced during active multiplication. Secondary metabolite : mainly produced when surrounding environment changes significantly in late log & stationary phase. Medically important secondary metabolites are antibiotics & they are produced by Streptomyces in the late log phase. To maintain the cells in a state of continuous growth, nutrients must be continuously added & waste products removed. It is called an open system/continuous culture.  Environmental factors that influence microbial growth Major environmental factors are – Temperature – Atmosphere/Amount of oxygen – pH – Water availability Bacteria can be described based on their optimal growth at specific temperatures, gas requirements, pH requirements, and susceptibility to water availability. Each species of bacteria has a range of temp (~25°C range) within which they grow & outside of which growth stops. Within this range lies the optimal growth temperature at which microorganisms grow most rapidly.  Temperature requirements Prokaryotes are mainly divided into 5 groups based on their optimal growth temperature (merely a guideline). – Psychrophiles: temp -5°C to 15°C. Grow in the cold Arctic and Antarctic regions and in lakes fed by glaciers. – Psychrotrophs: optimal temp 15°C to 30°C but grow well at lower/refrigeration temp. Important causes for food spoilage. – Mesophiles: 25°C to 45°C (most disease-causing bacteria exist here as normal body temp for humans is 37°C). Also include some bacteria that inhabit soil. – Thermophiles: 45°C to 70°C. Occur in hot springs & compost heaps. – Hyperthermophiles: 70°C-110°C (members of Archaea).  Temp, food preservation and disease Foods are stored at refrigeration temp (~4°C) to limit the growth of mesophiles. Psychrophiles & psychrotrophs can still multiply so spoilage still occurs but more slowly. Therefore, food is frozen for long-term storage.  Temp, food preservation and disease Wide variation exists in the temperature of various parts of the human/animal body. Infection tends to occur at temperatures optimal for the growth of pathogen e.g. – Mycobacterium leprae (leprosy) - lower temperature promotes growth, so it tends to infect the extremities. – The same situation applies in case of syphilis (Treponema pallidum) where lesions appear in genitalia, lips and tongue. – Major treatment of syphilis was to deliberately induce fever. Fever is a natural body response that can limit bacterial growth by raising body temperature.  Oxygen (O2) requirements Based on O2 requirement, prokaryotes are divided into five groups. 1. Obligate Aerobes: have an absolute requirement for O2 to generate their energy (aerobic respiration) – e.g. Pseudomonas & Micrococcus luteus 2. Obligate Anaerobes: cannot multiply if O2 is present & are often killed because of toxic oxygen derivatives. – e. g. Bacteroides (present in large intestine) and Clostridium botulinum (causes botulism). 3. Facultative anaerobes: grow better if O2 is present but can survive in the absence of O2 too. – Facultative anaerobes use aerobic respiration if O2 is present but use fermentation or anaerobic respiration in absence of O2 e. g. E. coli & Saccharomyces (yeast).  Oxygen (O2) requirements 4. Microaerophiles: require small amounts of O2 (2-10%) for aerobic respiration; higher concentrations are inhibitory. – e. g. Helicobacter pylori. 5. Aerotolerant anaerobes: can exist in O2, but don’t use it for energy (indifferent to O2). As they do not use aerobic & anaerobic respiration, they are also called obligate fermenters e. g. Streptococcus pyogenes (Strep throat). Oxygen (O2) requirements  Reactive Oxygen Species When organisms use O2 in aerobic respiration, several harmful derivatives called ‘reactive oxygen species (ROS)’ are formed as by-products e.g. – Superoxide (O2-) & – Hydrogen peroxide (H2O2). ROS can damage cell components so cells that grow aerobically must have enzymes to degrade ROS into non- toxic forms.  Reactive Oxygen Species In obligates aerobes & facultative anaerobes: Superoxide dismutase enzyme degrades superoxide to O2 & H2O2 Catalase enzyme breaks down H2O2 to water and oxygen. An important exception is ‘aerotolerant anaerobes’ as they do not produce catalase. A test for catalase enzyme is used to distinguish 2 groups of medically important Gram +ve cocci: – Staphyococcus sp. (catalase +ve) – Streptococci sp. (catalase -ve).  pH Our normal internal pH is 7.0 (or a little higher-7.0-7.4) Bacterial cells which grow within the range of pH 5.0-8.0 & have a pH optimum near neutral (pH 7) are called neutrophiles. This is exploited by phagocytes to kill ingested bacteria in acidic endosomes. Similarly, preservation methods acidify foods to inhibit growth of these organisms. Some bacteria adapt to grow at low pH (e. g. Helicobacter pylori grows in the stomach, & causes ulcers) Acidophiles grow optimally at a pH below 5.5. Alkalophiles grow optimally at a pH above 8.5.  Water availability All cells require water for growth. The local concentration of dissolved ions such as salt & sugars can affect the osmotic balance of the cell. If the concentration of solutes is low outside the cell, water tends to flow inside the cell. If the concentration of solutes is high outside the cell, water will tend to flow out of the cell due to osmosis. This causes cytoplasm to dehydrate & shrink from the cell wall, a phenomenon called plasmolysis. Bacteria that can tolerate high salt conc (~10% NaCl), are called halotolerant e. g. Staphylococcus species present on the skin. High conc. of salt and sugars are used in food preservation.  Nutritional factors that influence microbial growth A variety of nutrients such as fatty acids, sugars, amino acids, nucleotides and carbon and nitrogen elements are required for the growth of microbes. Major elements: elements that make up cell constituents (Carbon, oxygen, hydrogen, nitrogen, sulfur, phosphorus, potassium, magnesium, calcium & iron). Limiting nutrients: e. g. Phosphorus & Iron as they are present at the lowest concentration relative to need. Trace elements: required in very minute amounts by all cells (Cobalt, zinc, copper, molybdenum & manganese). These elements are required for enzyme functions.  Carbon and energy sources Carbon Sources: – Prokaryotes that use organic carbon are called heterotrophs. Medically important bacteria use organic carbon e.g., glucose. – Prokaryotes that use inorganic carbon in the form of C02 are called autotrophs. Important in carbon fixation. Energy sources: – Organisms that harvest energy of sunlight are called phototrophs e.g., plants, algae & photosynthetic bacteria. – Organisms that obtain energy by metabolizing chemical compounds (sugar, amino acids & fatty acids) are called chemotrophs e.g., mammalian cells, fungi & many bacteria.  Growth factors Some bacteria are unable to synthesize certain organic molecules and thus, some compounds, called growth factors, must be provided from outside. Microbes display a wide spectrum of growth factors requirements. Fewer enzymes an organism carries for the biosynthesis of small molecules, more growth factors it requires. E. coli is quite versatile & does not need any growth factor if only provided with glucose & six inorganic salts. In contrast, species of Neisseria require many ingredients for growth. Such bacteria that need many growth factors for their growth are called fastidious.  Cultivating microorganisms in the lab Types of media 1. Complex media: composed of variety of ingredients as Meat juices (beef extract) & Digested proteins (peptone) or A mixture of peptone & beef extract (nutrient broth, nutrient agar). 2. Chemically defined media: Composed of precise amounts of pure chemicals. They are not used routinely in lab work. But they are invaluable when studying nutritional requirements of bacteria e.g., Glucose salts.  Cultivating microorganisms in the lab Special type of culture media 1. Selective media : inhibits the growth of organisms other than the one being sought e.g., Thayer-Martin agar for Neisseria gonorrhoeae. 2. Differential media: contain a substance that certain bacteria change in a recognizable way.  Differential media Blood agar: is a complex and differential media used to detect bacteria that produce a hemolysin. Not selective. The type of hemolysis is used as an identifying feature e.g. – Streptococcus pyogenes (causes strep throat) produces clear zone of hemolysis (β-hemolysis). – Streptococcus species that reside in throat harmlessly causes alpha hemolysis characterized by zone of greenish clearing. - Some streptococci have no effect on red blood cells.  Selective and Differential media MacConkey Agar is complex media that is used to isolate Gram-ve rods reside in the intestine. This media is both selective & differential. – Selective as it contains crystal violet dye & bile salts which inhibit Gram +ve & non-intestinal bacteria, respectively. – Differential as it also contains lactose & a pH indicator. Bacteria that ferment sugar produce acid which turns the pH indicator pink e.g. – Lactose-fermenting E. coli form pink colony but lactose negative bacteria form tan or colorless colonies. Syphilis organism Treponema pallidum can not be grown on culture media. MacConkey Agar  Next lecture Read chapter 10-selected topics Read chapter 12-selected topics