1. Lesson 2: Microbial Growth
January 20, 2015

2. 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

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

4. 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

5. 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 ( C). Thermus aquaticus

6. 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?

7. 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)

8. 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.

9. *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*

10. 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)

11. 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

12. 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

13. 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

14. 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

15. 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

16. 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

17. 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

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

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

20. 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

21. 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

22. 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

23. Culture Media

24. 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

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

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

27. 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

28. 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

29. Figure 6.7 An anaerobic chamber.
Air lock
Arm ports

30. 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