CH. 6 | Microbial Growth

6 - 1 Classify microbes into five groups on the basis of preferred temperature range.

Microorganisms are classified by their preferred temperature range. The five primary groups are:

• Psychrophiles – cold-loving; can grow at 0°C, optimum ~15°C. Found in ocean depths and polar regions. Rarely cause food spoilage problems.

• Psychrotrophs – also grow at 0°C but have a higher optimum (20–30°C); cannot grow above ~40°C. More common and are the main culprits in low-temperature food spoilage (refrigerator temps).

• Mesophiles – optimum 25–40°C; most common type of microbe. Most pathogens fall here (optimum ~37°C, body temperature).

• Thermophiles – heat-loving; optimum 50–60°C. Found in hot springs, compost piles. Endospores can survive normal canning heat treatment.

• Hyperthermophiles (Extreme thermophiles) – optimum 80°C or higher; mostly Archaea, found near volcanic hot springs. Record growth temp ~121°C at deep-sea hydrothermal vents.

6 - 2 Identify how and why the pH of culture media is controlled.

• Most bacteria grow best at a near-neutral pH (6.5–7.5); very few grow below pH 4.

• Acidophiles are exceptions — tolerant of high acidity (some survive at pH 1).

• Molds and yeasts tolerate a wider pH range, with an optimum around pH 5–6.

• In lab culture, bacteria produce acids that can inhibit their own growth, so chemical buffers (especially phosphate salts) are added to maintain proper pH.

• Phosphate salts are ideal buffers because they are nontoxic and also supply phosphorus, an essential nutrient.

• Acidic foods (sauerkraut, pickles, cheese) resist spoilage because low pH inhibits most bacterial growth.

6 - 3 Explain the importance of osmotic pressure to microbial growth.

• Microbes get nutrients from surrounding water; they are 80–90% water and require it for growth.

• High osmotic pressure (hypertonic environment) pulls water out of the cell → causes plasmolysis (shrinkage of cytoplasm) → inhibits growth.

• This principle is used to preserve foods: salted fish, honey, sweetened condensed milk all use high salt/sugar to draw water out of any microbial cells.

• Extreme (obligate) halophiles actually require high salt (~30%) — found in places like the Dead Sea.

• Facultative halophiles don't require high salt but can tolerate up to 2% (some up to 15%).

• Low osmotic pressure (hypotonic) causes water to rush into the cell — can lyse cells with weak walls.

6 - 4 Name a use for each of the four elements (carbon, nitrogen, sulfur, and phosphorus) needed in large amounts for microbial growth.

6 - 5 Explain how microbes are classified on the basis of oxygen requirements.

There are five groups based on how microbes respond to oxygen (O₂):

• Obligate Aerobes – require oxygen; grow only where O₂ is present.

• Facultative Anaerobes – prefer oxygen but can survive without it (switch to fermentation or anaerobic respiration). Example: E. coli, most yeasts.

• Obligate Anaerobes – cannot use oxygen; it is actually toxic to them. Example: Clostridium (causes tetanus and botulism).

• Aerotolerant Anaerobes – cannot use oxygen but can tolerate it. Example: lactobacilli (used in acidic fermented foods like pickles and cheese).

• Microaerophiles – require oxygen but only at low concentrations; damaged by normal atmospheric O₂ levels.

6 - 6 Identify ways in which aerobes avoid damage by toxic forms of oxygen.

Oxygen produces toxic byproducts that cells must neutralize:

• Singlet oxygen (¹O₂) — highly reactive, boosted-energy form of O₂.

• Superoxide radicals (O₂⁻) — formed during normal aerobic respiration; extremely unstable and destructive.

• Peroxide anion (O₂²⁻) / Hydrogen peroxide (H₂O₂) — toxic; used as an antimicrobial agent.

6 - 7 Describe the formation of biofilms and their potential for causing infection.

Biofilm

• A thin, slimy layer encasing bacteria that adheres to a surface

• Can consist of a single species or multiple species of microorganisms, and can form on many surfaces

  • Rocks in ponds, teeth (dental plaque), medical catheters, heart valves, etc

• Aggregation of microbes
  
  

Benefits Biofilms Provide to Bacteria

Within a biofilm, bacteria gain significant advantages:

• Shared nutrients among community members

• Protection from desiccation, antibiotics, and the immune system

• Genetic exchange — close proximity facilitates conjugation (DNA transfer between cells)
  
  

Potential for Causing Infection

Biofilms are a major concern in human health:

• Microbes in biofilms are approximately 1,000 times more resistant to microbicides (disinfectants/antibiotics) than free-living bacteria

• The CDC estimates ~70% of human bacterial infections involve biofilms

• Most healthcare-associated infections are linked to biofilms on medical devices, including catheters, mechanical heart valves, and other indwelling devices

6 - 8 Distinguish chemically defined and complex media.

Chemically defined media

• The exact chemical composition is known
  
  

Complex media

• The chemical composition varies from batch to batch

  • Nutrient broth

  • Nutrient agar

6 - 10 Justify the use of each of the following: anaerobic techniques, living host cells, and candle jars.

Anaerobic Techniques

• Used to grow obligate anaerobes, which are killed by oxygen.

• Oxygen must be chemically removed from the growth environment using reducing media (sodium thioglycolate), sealed jars with oxygen-removing packets, or systems like OxyPlate™ (uses the enzyme oxyrase to convert oxygen into water).

  • Without these techniques, obligate anaerobes cannot survive long enough to be cultured, studied, or identified — making them essential for diagnosing infections
    
    

Living Host Cells

• Used for obligate intracellular pathogens (viruses, rickettsias, chlamydias) that cannot grow on artificial media — they can only reproduce inside a living host cell.

• Methods include embryonated eggs, tissue cultures, and lab animals.

  • Since these organisms have no independent metabolism and cannot synthesize their own ATP or proteins outside a host cell, no artificial medium can substitute for a living host
    
    

Candle Jars

• Used for microaerophiles and capnophiles that need low oxygen and elevated CO₂.

• A lit candle in a sealed jar consumes O₂ and produces CO₂.

• Modern chemical gas packets are now more commonly used as they provide more precise gas concentrations.

  • Candle jars and their modern equivalents are necessary because microaerophiles and capnophiles cannot grow under normal atmospheric conditions — they need a carefully controlled gas environment to survive and be cultivated.

6 - 11 Differentiate biosafety levels 1, 2, 3, and 4

BSL-1 — Lowest risk. Used for non-hazardous microorganisms that pose little to no threat (e.g., basic teaching labs). Standard lab practices, no special containment needed.

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BSL-2 — Moderate risk. Used for organisms that present a moderate infection risk. Work is done on open benchtops with gloves, lab coats, and eye/face protection when needed.

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BSL-3 — High risk. Used for highly infectious airborne pathogens (e.g., tuberculosis). Requires biological safety cabinets, negative air pressure in the lab, and air filters to prevent pathogen release.

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BSL-4 — Extreme risk. Used for the most dangerous pathogens with no known cure (e.g., Ebolavirus). Full "space suits" with air supply, sealed negative-pressure environment, HEPA-filtered intake and exhaust air (exhaust filtered twice), and all waste rendered noninfectious before leaving. Only 4 such labs exist in the U.S.

6 - 12 Define Pure Culture

A culture containing only one type of microorganism

• To study a single organism without interference from others

6 - 13 Describe how pure cultures can be isolated by using the streak plate method

• A sterile inoculating loop is dipped into a mixed culture (containing multiple types of microbes)

• The loop is streaked in a pattern across the surface of a nutrient medium plate

• With each streak, fewer and fewer bacteria are deposited on the loop

• By the end of the pattern, cells are spread far enough apart to grow into isolated individual colonies

• A single isolated colony is then picked up and transferred to a nutrient medium, producing a pure culture of one bacterium type

6 - 14 Explain how microorganisms are preserved by deep-freezing and lyophilization (freeze-drying)

Deep-Freezing

• A pure culture is placed in a suspending liquid and quickly frozen at −50°C to −95°C.

• The culture can be thawed and grown again even years later. Best for long-term storage that exceeds the capabilities of refrigeration.
  
  

Lyophilization (Freeze-Drying)

• The microbe suspension is quickly frozen at −54°C to −72°C

• A high vacuum is applied, causing the ice to convert directly to vapor (sublimation) — removing all water

• The container is sealed under vacuum, leaving a dry, powdery residue of surviving microbes
  
  

Main Takeaway

• Refrigeration only works short-term. Deep-freezing and lyophilization preserve microbes long-term by either keeping them frozen or removing all water — both methods halt metabolic activity without killing the organisms.

6 - 15 Describe bacterial growth, including binary fission

Bacterial Growth

• An increase in bacterial numbers, not in the size of individual cells
  
  

Binary Fission (most common method):

1. Cell elongates and DNA is replicated

2. Plasma membrane constricts inward and a new wall begins to form

3. A cross-wall forms, completely separating the two DNA copies

4. The two cells separate into identical daughter cells

6 - 16 Compare the phases of microbial growth, and describe their relation to generation time

Lag Phase

• Little to no cell division occurs

• Cells are metabolically active — synthesizing enzymes and molecules to prepare for growth

• Duration varies: can last 1 hour to several days
  
  

Log Phase

• Cells divide rapidly and at a constant rate

• Generation time is at its minimum and most consistent

• Population doubles at regular intervals → plotted as a straight line on a logarithmic graph

• Cells are most metabolically active — important for industrial microbiology
  
  

Stationary Phase

• Growth slows as the population reaches the environment's carrying capacity

• Rate of new cell growth = rate of cell death → population stabilizes

• Caused by: nutrient depletion, waste accumulation, and lack of space
  
  

Death Phase

• Deaths exceed new cell formation

• Population steadily declines, sometimes until nearly or completely extinct

• Speed of decline varies by species

6 - 17 Explain four direct methods of measuring cell growth

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6 - 18 Differentiate direct and indirect methods of measuring cell growth

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6 - 19 Explain three indirect methods of measuring cell growth

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