Lesson 2: Microbial Growth January 20, 2015

Microbial Growth Microbial growth refers to the increase in the number of cells and not the cell size As microbial cells increase, they form colonies. Colonies are groups of cells large enough to be seen without a microscope Population of microbial cells can grow incredibly large in a very short period of time Understanding the conditions necessary for growth we can determine how to control the growth of these organisms that eventually cause disease and food spoilage This lecture will examine the requirements for microbial growth and the methods of measuring this growth

The Requirements for Growth Physical requirements Temperature pH Osmotic pressure Chemical requirements Carbon Nitrogen, sulfur, and phosphorous Trace elements Oxygen Organic growth factor

Physical Requirements Temperature Microorganisms can grow at a variety of different temperatures Most microbes grow at room temperature (20-25 C) Each bacterial species grows better or worse at particular temperatures Minimum growth temperature Optimum growth temperature Maximum growth temperature

Microorganisms are classified into three “primary” groups based on their preferred range of temperature Psychrophiles (cryophiles) prefer cold temperatures (-15 to 10 C). Arthrobacter and psychrobacter Mesophiles are moderate-temperature microbes (20-45 C). Optimum growth at 37 C. Most bacterial species. Human pathogens are mesophiles. Thermophiles are heat-loving microbes (45-112 C). Thermus aquaticus

What do you think is the cause for the immediate growth Typical growth rates of different types of microorganisms in response to temperature. Thermophiles Hyperthermophiles Mesophiles Psychrotrophs Psychrophiles What do you think is the cause for the immediate growth drop-off after the optimum growth temperature?

Food Preservation Temperatures Refrigeration is the most common method of preserving food Psychrotrophs are microbes that are responsible for refrigerator food spoilage Certain psychrophiles have an optimum temperature slightly above a refrigerator’s temperature Growth is not fully inhibited by the temps of the refrigerator. Growth is slow Results in mold and slime on food surfaces, off-tastes, off-colors, and odors (gases being produced by the microbe)

Temperatures in this range destroy most microbes, although lower temperatures take more time. Very slow bacterial growth. Rapid growth of bacteria; some may produce toxins. Danger zone Many bacteria survive; some may grow. Refrigerator temperatures; may allow slow growth of spoilage bacteria, very few pathogens. No significant growth below freezing.

*Safer to store smaller amounts of food* The effect of the amount of food on its cooling rate in a refrigerator and its chance of spoilage. 15 cm (6′′) deep 5 cm (2′′) deep Approximate temperature range at which Bacillus cereus multiplies in rice *Fried Rice Syndrome* Refrigerator air *Safer to store smaller amounts of food*

pH Most bacteria prefer neutral pH. Growth b/w pH 6.5 and 7.5 Acidic foods (sauerkraut and cheese) prohibits growth of certain microbes Most molds and yeasts are less susceptible to growth inhibition than bacteria. Wider range of pH growth. Grow between pH 5 and 8 Acidophiles grow in acidic environments Stomach (pH 1-5)is a conducive environment for several pathogens (Helicobacter pylori)

Osmotic Pressure—pressure that is applied to a system to stop osmosis Hypertonic environments, or an increase in salt or sugar, causes plasmolysis in the cell Plasmolysis—shrinkage of cells cytoplasm

Plasmolysis inhibits the growth of the cell Addition of salts (preservation) results in the increase in osmotic pressure thus preventing growth of organisms Removes all of the water from the cell Extreme or obligate halophiles REQUIRE high osmotic pressure Found in salt water (Dead Sea or the Great Salt Lake) Facultative halophiles TOLERATE high osmotic pressure

Chemical Requirements Carbon Serves as a structural backbone for organic molecules that make up a living cell. Chemoheterotrophs acquires their carbon from the breakdown of organic compounds (proteins, carbohydrates, lipids). Incapable of carbon fixation Chemoautotrophs acquire their carbon from CO2. Capable of carbon fixation

Chemical Requirements Nitrogen Required to synthesize amino acids and DNA Most bacteria decompose proteins (recycling) as their source of nitrogen A few bacteria, such as the photosynthetic cyanobacteria, use atmospheric N2 in nitrogen fixation Nitrogen fixation is the conversion of N2 to ammonia (NH3). Ammonia is more chemically reactive than N2

Chemical Requirements Sulfur Used in the production of amino acids, thiamine, and biotin Most bacteria decompose proteins to generate sulfur Some bacteria use SO42– or H2S as sulfur sources Phosphorus In DNA, RNA, ATP, and the phospholipids of membranes PO43– (phosphates) are a source of phosphorus

Chemical Requirements Oxygen Final electron acceptor in Electron Transport Chain Microbes that use molecular oxygen (aerobes) extract more energy from nutrients than microbes that do not use oxygen (anaerobes) Obligate aerobes require oxygen to live Facultative aerobes can grow in the presence or absence of oxygen Obligate anaerobe unable to use oxygen for energy-yielding reactions. Will not grow in presence of Oxygen

Toxic Oxygen Molecular oxygen (O2) can be viewed as a poisonous gas due to its ability to cause damage in cells Hydrogen atoms in cells are added to O2 to neutralize its deleterious effects in the cell Various types of oxygen radicals (unpaired electrons) are collectively called reactive oxygen species (ROS) Disrupt cell membranes; destroys lipids, proteins, and DNA Obligate aerobes, facultative anaerobes, aerotolerant aerobes, and microaerophiles contain enzymes to breakdown these ROS Obligate anaerobes lack these enzymes

Toxic Oxygen Superoxide free radicals: O2 Peroxide anion: O22– Hydroxyl radical (OH•) Superoxide dismutase (SOD) O2 + O2 + 2 H+ H2O2 + O2 Catalase 2 H2O2 2 H2O + O2 Peroxidase H2O2 + 2 H+ 2 H2O

Table 6.1 The Effect of Oxygen on the Growth of Various Types of Bacteria

Organic Growth Factors Organic compounds obtained from the environment Cannot be synthesized by the organism Vitamins, amino acids, purines, and pyrimidines Functions as coenzymes and building blocks for other macromolecules

Culture Media To identify and study bacterial growth patterns, bacteria must be isolated Isolated bacteria need an artificial growth environment to survive. Cultured Media. Not all bacteria can be “cultured” Some bacteria require special nutrients in order to grow

Culture Media Culture medium contains the nutrients needed for microbial growth Different bacteria need their own set of nutrients Culture medium must be sterile: contains no living microbes Inoculum—microbes that are introduced into a medium for growth Culture—microbes growing in/on culture medium

Culture Media

Culture Media There are several different varieties of culture media Chemically defined media—exact chemical composition is known Chemically undefined media—exact chemical composition is not known Complex media contain extracts and digests of yeasts, meat, or plants Nutrient broth—liquid form of media Nutrient agar—solid form of media

Table 6.2 A Chemically Defined Medium for Growing a Typical Chemoheterotroph, Such as Escherichia coli

Table 6.4 Composition of Nutrient Agar, a Complex Medium for the Growth of Heterotrophic Bacteria

Anaerobic Culture Methods Cultivation of anaerobic bacteria poses a problem to scientists Must absorb all the oxygen from medium/environment in order to grow microbes Reducing media Contain chemicals (thioglycolate or oxyrase) that combine O2 and removes all available oxygen Usually contained in screw cap test tubes or jars Media is heated before use to drive off O2

Envelope containing sodium bicarbonate and sodium borohydride Figure 6.6 A jar for cultivating anaerobic bacteria on Petri plates. Clamp with clamp screw Lid with O-ring gasket Envelope containing sodium bicarbonate and sodium borohydride CO2 H2 Palladium catalyst pellets Anaerobic indicator (methylene blue) Petri plates

Figure 6.7 An anaerobic chamber. Air lock Arm ports

Capnophiles Microbes that require high CO2 conditions CO2 packet Camplyobacter spp. CO2 packet Chemical packets are used to generate carbon dioxide within containers Candle jar Contains a lit candle that depletes the oxygen in an environment and generates carbon dioxide Low-oxygen, high-CO2 conditions resemble the conditions of the intestinal and respiratory tract