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Microbiology

Bacterial Growth and Nutrition

Faculty of Medicine Hashemite University Dr Mohammad Al-Tamimi, MD, PhD

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Objectives
Growth definition and classification Physical parameters that affect growth Chemical parameters that affect growth Population growth - growth curve Population growth Methods Bacterial growth measurement

Introduction
Growth: Orderly increase in the sum of all the components of an organism, which reflects increase in number of cells Importance of understanding bacterial growth: Bacterial survival and transmission In vitro diagnostic (laboratory culture) Cessation of bacterial growth for treatment

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Microbial growth/Binary Fission

Rapid Growth of Bacteria

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Generation time under optimal conditions

Generation time is the time it takes for a single cell to grow and divide
Organism Generation Time Escherichia coli 12.5 min Staphylococcus aureus 27-30 min Mycobacterium tuberculosis (agent of Tuberculosis) 18 24 hrs Treponema pallidum (agent of Syphilis) 30 hrs

The Growth Curve

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During lag phase, cells are recovering from a period of no growth and are making macromolecules in preparation for growth During log phase cultures are growing maximally Stationary phase occurs when nutrients are depleted and wastes accumulate (Growth rate = death rate) During death phase death rate is greater than growth rate

Factors Affecting Bacterial Growth

Temperature pH Osmotic pressure Oxygen Nutrition

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Temperature
Hydrogen bonds will break at high temperatures leads to protein denaturation

Lipids will be more liquid

Outside membrane cannot preserve the integrity of the cell and it will disintegrate

Minimum Temperature: Temperature below which growth ceases, or lowest temperature at which microbes will grow Optimum Temperature: Temperature at which its growth rate is the fastest Maximum Temperature: Temperature above which growth ceases, or highest temperature at which microbes will grow

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Classification of Microorganisms by Temperature Requirements

Temperature Classes of Organisms

Psychrophiles ( 0-20C) Cold temperature optima Most extreme representatives inhabit permanently cold environments Mesophiles ( 20 45C) Midrange temperature optima Found in warm-blooded animals and in aquatic environments in temperate and tropical latitudes Thermophiles ( 50- 80C) Growth temperature optima between 45C and 80C Hyperthermophiles Optima greater than 80C These organisms inhabit hot environments including boiling hot springs

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Psychrotrophs

pH and Microbial Growth

Each organism has a pH range and a pH optimum
Acidophiles: Grow optimally between ~pH 0 and 5.5 Neutrophiles: Growoptimally between pH 5.5 and 8 Alkalophiles: Grow optimally between pH 8 11.5

Most bacteria grow between pH 6.5 and 7.5 Molds and yeasts grow between pH 5 and 6 Human blood and tissues has pH 7.2+0.2

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Osmotic Effects on Microbial Growth

Osmotic pressure depends on the surrounding solute concentration and water

availability
Hypertonic environments, increase salt or sugar, cause plasmolysis

Classification
Osmophiles: organisms which thrive in high solute Osmotolerant: organisms which tolerate high solute Halophiles: organisms which thrive in high salt Halotolerant: organisms which tolerate high salt Barophiles: organisms which thrive in high pressure Barotolerant: organisms which tolerate high pressure

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Oxygen and Microbial Growth

Using oxygen (O2) in metabolism creates toxic waste Microbes that are able to use aerobic respiration produce enzymes to detoxify oxygen: Catalase: H2O2 --- H20 and 02 Superoxide dismutase (SOD): oxygen radical --- H20 and O2 Microbes that dont make these enzymes cannot exist in the presence of oxygen.

Classification of Organisms Based on O2 Utilization

Aerobes :
Obligate: require oxygen to grow Facultative: can live with or without oxygen but grow better with oxygen Microaerphiles: require reduced level of oxygen

Anaerobes :
Aerotolerant anaerobes: can tolerate oxygen but grow better without oxygen. Obligate: do not require oxygen. Obligate anaerobes are killed by oxygen

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Microbial Nutrition
Organisms use a variety of nutrients for:
their energy needs to build organic molecules & cellular structures

Energy Source
Phototroph: Uses light as an energy source Chemotroph: Uses energy from the oxidation of reduced chemical compounds Required nutrients: Macronutrients Micronutrients Special requirements

Macronutrients
Elements required in fairly large amounts:
Carbon: Structural organic molecules as energy source

Carbon source
Autotroph: Can use CO2 as a sole carbon source (Carbon fixation) Heterotroph: Requires an organic carbon source; cannot use CO 2 as a carbon source

Nitrogen
In amino acids, proteins Most bacteria decompose proteins Some bacteria use NH4+ or NO3 A few bacteria use N2 in nitrogen fixation

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Sulfur
In amino acids, thiamine, biotin Most bacteria decompose proteins Some bacteria use SO42 or H2S

Phosphorus
In DNA, RNA, ATP, and membranes PO43 is a source of phosphorus

Micronutrients
Metals and organic compounds needed in very small amounts, usually as enzyme and cofactors: Calcium, Copper, Iron, Magnesium, Manganese, and Iron

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Special requirements

Amino acids Nucleotide bases Enzymatic cofactors or vitamins

Culture Media

Culture Medium: Nutrients prepared for microbial growth (C,

N,

Phosphorus, trace elements, etc)

Sterile: No living microbes Inoculum: Introduction of microbes into medium Culture: Microbes growing in/on culture medium

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Agar
Complex polysaccharide Used as solidifying agent for culture media in Petri plates, slants, and deeps Generally not metabolized by microbes Liquefies at 100C Solidifies ~40C

Growth of Staphylococcus aureus on Mannitol Salt Agar results in a color change in the media from pink to yellow

Bacterial colonies on solid media

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Types of Media
Media can be classified on three primary levels 1. Physical State 2. Chemical Composition 3. Functional Type

Physical State of Media

Liquid Media Semisolid Solid (Can be converted into a liquid) Solid (Cannot be converted into a liquid)

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Liquid Media
Water-based solutions Do not solidify at temperatures above freezing / tend to be free flowing Includes broths, milks, and infusions Measure turbidity Example: Nutrient Broth

Semi-sold Meida
Exhibits a clot-like consistency at ordinary room temperature Determines motility Used to localize a reaction at a specific site Example: Sulfide Indole Motility (SIM) for hydrogen sulfide production and indole reaction and motility test

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Sold Media
Firm surface for discrete colony growth Advantageous for isolating and culturing Two Types 1. Liquefiable (Reversible) 2. Non-liquefiable Examples: Gelatin and Agar (Liquefiable) Cooked Meat Media Potato Slices (Non-liquefiable)

Chemical Composition of Culture Media

Synthetic Media
Chemically defined Contain pure organic and inorganic compounds Exact formula (little variation) Contains at least one ingredient that is not chemically definable (extracts from plants and animals) No exact formula / tend to be general and grow a wide variety of organisms

Complex or Non-synthetic Media

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Selective Media
Contains one or more agents that inhibit the growth of a certain microbe and thereby encourages, or selects, a specific microbe Example: Mannitol Salt Agar [MSA] encourages the growth of S. aureus. MSA contain 7.5% NaCl which inhibit the growth of other Gram +ve bacteria

Differential Media
Differential shows up as visible changes or variations in colony size or color, in media color changes, or in the formation of gas bubbles and precipitates Example: Mannitol Salt Agar

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Enrichment Media
Is used to encourage the growth of a particular microorganism in a mixed culture. Example: Manitol Salt Agar for S. aureus, blood agar , chocolate agar

Growth in Continuous Culture

A continuous culture is an open system in which fresh media is continuously added to the culture at a constant rate, and old broth is removed at the same rate This method is accomplished in a device called a chemostat Typically, the concentration of cells will reach an equilibrium level that remains constant as long as the nutrient feed is maintained

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Basic Chemostate System

Anaerobic Culture Methods

Anaerobic jar

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Cultures with High CO2

Candle jar

CO2-packet

Methods Used to Measure Microbial Growth

Count colonies on plate or filter (counts live cells) Microscopic counts Mass determination Turbitity Measurement of enzymatic activity or other cell components Flow cytometry (FACS)

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Viable Count

1. Perform serial dilution of original sample (1:10 dilution)

2. Incubate plates with samples from each serial dilution

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3. Count plates that have 25-250 colonies and correct The dilution factor

Microscopic counts

Need a microscope, special slides, high power objective lens Typically only counting total microbe numbers, but differential counts can also be done

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Turbidity
Cells act like large particles that scatter visible light A spectrophotometer sends a beam of visible light through a culture and measures how much light is scattered Scales read in either absorbance or % transmission Measures both live and dead cells

Mass Determination
Cells are removed from a broth culture by centrifugation and weighed to determine the wet mass. The cells can be dried out and weighed to determine the dry mass

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Thank you

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