Bacterial growth Assist. Prof. Emrah Ruh NEU Faculty of Medicine Department of Medical Microbiology

Bacterial growth Microbial growth: Growth rate: Increase in the number of microbial cells in a population Growth rate: The change in cell number or cell mass per unit time During the cell division cycle, all the structural components of the cell double

Bacterial growth Generation: Generation time (doubling time): The interval for the formation of two cells from one cell Generation time (doubling time): The time required for the cell population to double Vary widely among organisms (many bacteria: 1-3 hr; few bacteria: 10 min or several days!)

Bacterial growth The requirements: Physical Chemical Temperature, pH, salt Chemical Carbon, nitrogen, oxygen

Bacterial growth Physical requirements – Temperature Psychrophiles: can grow at 0°C but optimum is about 15°C Psychotrophs: can grow at 0°C also but optimum is 20 - 30°C Mesophiles: grow best at moderate around 37°C (many pathogenic microorganisms) Thermophiles: have a growth optimum at around 60°C Hyperthermophiles: have growth optimum of 80°C or higher (archaea)

Bacterial growth Physical requirements – Temperature

Bacterial growth Physical requirements – Temperature

Bacterial growth Physical requirements – Temperature Effect of temperature on growth rate and the molecular consequences for the cell

Bacterial growth Physical requirements – pH Most bacteria grow best at pH value between 6.5 and 7.5 Acidophiles: organisms that grow best at low pH (as low as pH 1) Alkaliphiles: organisms that grow best at high pH (as high as pH 10) (archaebacteria)

Bacterial growth Physical requirements – Salt In a hypertonic solution, most microbes undergo plasmolysis Isotonic Hypertonic

Bacterial growth Physical requirements – Salt Halophiles: specific requirement for the sodium ion (microorganisms found in the sea) Mild halophile  1-6% NaCl Moderate halophile  6-15% NaCl Extreme halophile  15-30% NaCl (archaebacteria) Halotolerant: grow best in the absence of NaCl, but can tolerate some NaCl

Bacterial growth Physical requirements Osmophiles: are able to live in environments high in sugar Xerophiles: are able to grow in very dry environments

Bacterial growth Chemical requirements Macronutrients Micronutrients (trace elements) Growth factors

Bacterial growth Chemical requirements Macronutrients Required in large amounts Micronutrients (trace elements) Required in tiny (trace) amounts Metals; enzyme structure

Bacterial growth Chemical requirements Growth factors (organic compounds) Vitamins, amino acids, purines and pyrimidines Required in tiny amounts; only by some bacteria Most microorganisms are able to synthesize all of these compounds; some microorganisms require one or more of them performed from the environment Vitamins are the most commonly needed growth factors

Bacterial growth Chemical requirements Macronutrients Micronutrients Vitamins Carbon (C) Chromium (Cr) p-Aminobenzoic acid Hydrogen (H) Cobalt (Co) Folic acid Oxygen (O) Copper (Cu) Biotin Nitrogen (N) Manganese (Mn) Cobalamin Phosphorus (P) Molybdenum (Mn) Lipoic acid Sulfur (S) Nickel (Ni) Nicotinic acid (niacin) Potassium (P) Selenium (Se) Pantothenic acid Magnesium (Mg) Tungsten (Tu) Riboflavin Sodium (Na) Vanadium (V) Thiamine Calcium (Ca) Zinc (Zn) Vitamins (B6) Iron (Fe) Vitamin K group; quinones Hydroxamates

Bacterial growth Chemical requirements – Carbon The major element in all classes of macromolecules A typical cell is about 50% carbon All organisms require a carbon source; chemoheterotrophs use an organic molecule and autotrophs typically use carbon dioxide

Bacterial growth Chemical requirements – Nitrogen A typical bacterial cell is about 12% nitrogen Important element in proteins, nucleic acids,... Bacteria obtain nitrogen from ammonia (NH3) or nitrate (NO3-); a few bacteria (nitrogen-fixing bacteria) obtain from nitrogen gas (N2)

Bacterial growth Chemical requirements – Oxygen Oxygen requirement: Obligate aerobes Microaerophiles Facultative anaerobes Aerotolerant anaerobes Obligate anaerobes Requirement for oxygen

Bacterial growth Chemical requirements – Oxygen

Bacterial growth Chemical requirements – Oxygen Aerobic bacteria: grow on the top of the tube in order to absorb maximal amount of O2 for survival. Microaerophilic bacteria: grow just below surface as they require O2 at low concentration. They are sensitive to O2 -derived free radicals. Facultative anaerobic bacteria: grow throughout the tube. These bacteria can use O2. But in the absence of O2, they have anaerobic methods of energy production, so they can live all along the test tube.

Bacterial growth Chemical requirements – Oxygen Aerotolerant anaerobic bacteria: cannot use O2 for growth but also are not affected by it and able to survive in the presence of some O2. So, they can spread a little bit upward from the bottom of the test tube. Anaerobic bacteria: cannot use O2 for growth and are even harmed by it. So, they grow at the bottom in order to avoid O2.

Bacterial growth Chemical requirements – Oxygen Aerobes, facultative anaerobes, and aerotolerant anaerobes must have the following enzymes to avoid toxic O2 radicals: Superoxide dismutase (2O2- + 2H+ O2 + H2O2) Catalase (2H2O2 2H2O + O2) or peroxidase (H2O2 + 2H+ 2H2O)

Bacterial growth Media Medium: is any material prepared for the growth of bacteria in a laboratory Culture: Microbes that grow and multiply in a medium

Bacterial growth Media Media used in microbiology labs are divided into four groups according to their agar content Agar is a biological substance extracted from sea algae Agar solidifies the media > 90oC: liquid phase < 40oC: solid phase

Bacterial growth Media Liquid media; do not include agar Semi-liquid media; include 0,05-0,2 % agar Semi-solid media; include 0,3-0,5 % agar Solid media; include 1,5-2 % agar

Bacterial growth Media Liquid medium Semi-liquid medium Semi-solid medium Solid medium

Bacterial growth Media Bacterial growth in solid culture is observed as colony formation Colony

Bacterial growth Media Bacterial growth in liquid culture is observed as turbidity Turbidity Medium (no bacteria) Culture (bacteria)

Bacterial growth Measurement of bacterial growth Counts are reported as the number of colony- forming units (CFU) instead of the number of cells If the concentration of bacteria is too great, the colonies will grow into each other and the plate will be uncountable To insure a countable culture plate, a series of dilutions should be prepared

Bacterial growth Measurement of bacterial growth Plate no. 4  32 colonies X 10,000 = 320,000 cfu/ml

Bacterial growth Media - Ingredients Simple growth requirements: Nutrient broth ( + agar: Nutrient agar) Constituent Peptone (provides vitamins and minerals) Beef extract (supplies energy, C, N and S) Sodium chloride Agar Water

Bacterial growth Media – General purpose media Simple growth requirements Nutrient broth Nutrient agar

Bacterial growth Media – General purpose media Simple growth requirements + blood: Blood agar The most common media in microbiology!

Bacterial growth Media – Special purpose media Selective media: supress unwanted microbes; encourage desired microbes Differential media: differentiate the colonies of desired microbes from others Enrichment media: increase the numbers of desired microbes to detectable levels

Bacterial growth Media – Special purpose media Endo agar, EMB agar and MacConkey agar: Selective and differentiative media Selective for Gram-negative bacteria (suppress Gram-positive bacteria) Differentiative for lactose-fermenting bacteria (Lactose (+) bacteria produce colourful colonies)

Bacterial growth Media – Special purpose media Lactose fermentation test Lactose (+) bacteria produce colourful colonies

Bacterial growth Bacterial division Binary fission: The normal reproductive method of bacteria A single cell divides into two identical cells

Bacterial growth Bacterial division

Bacterial growth Logarithmic representation Logarithmic representation of bacterial populations: Bacterial division occurs according to a logarithmic progression (two cells, four cells, eight cells,...)

Bacterial growth Logarithmic representation Logarithmic representation of bacterial populations:

Bacterial growth Phases of growth Lag phase Log (exponential) phase Stationary phase Death phase

Bacterial growth Phases of growth

Bacterial growth Phases of growth Lag phase: No change in the number of cells; metabolic activity is high DNA and enzyme synthesis occurs

Bacterial growth Phases of growth Log (exponential) phase: Bacteria multiply at the fastest rate (under the optimal conditions) Logarithmic or exponential increase in population

Bacterial growth Phases of growth Stationary phase: Equilibrium between cell division and death Nutrients are exhausted and waste products build up; pH increases

Bacterial growth Phases of growth Death phase: The number of deaths exceeds the number of new cells formed Population decreases at a logarithmic rate