Chapter 6: Microbial Growth

Chapter 6:Microbial Growth

Microbial Growth

• Microbial growth = growth in population

─ Increase in number of cells, not cell size

• Two main categories of requirements for microbial growth:

─ Physical requirements (environmental conditions)

◦ Temperature, pH, osmotic pressure

─ Chemical requirements

• Temperature

─ Minimum growth temperature

─ Optimum growth temperature

─ Maximum growth temperature

• Three main classifications

─ Psychrophiles (optimum ~120C)

─ Psychrotrophs (optimum ~230C)

─ Mesophiles (optimum ~370C)

─ Thermophiles (optimum above 500C)

Physical Requirements for Growth:

Temperature

Figure 6.1

Cause majority of food spoilage

Physical Requirements for Growth:

Temperature

Refrigeration

Hansen’s Disease(Leprosy)

• Mycobacterium leprae

• Optimal growth temperature: 30°C

─ Grows in peripheral nerves, nasal mucosa and skin cells

Figure 22.8

• pH

─ Most bacteria grow between pH 6.5 and 7.5

─ Molds and yeasts grow optimally between pH 5 and 6

─ Acidophiles grow in acidic environments (pH<5.5)

─ Alkaliphiles grow in basic environments (pH>8.5)

• Acidic foods (pickles, sauerkraut) preserved by acids from bacterial fermentation

• Growth media used in the laboratory contain buffers

Physical Requirements for Growth: pH

Physical Requirements for Growth: Osmotic Pressure

• Osmotic Pressure─ Hypertonic environments (=high osmotic pressure),

increased salt or sugar, cause plasmolysis◦ Obligate halophiles require high osmotic pressure

◦ Facultative halophiles tolerate high osmotic pressure (>2% salt)

─ Nutrient agar has a high percentage of water to maintain low osmotic pressure (bacterial cells are 80-90% water)

Low osmotic pressure High osmotic pressure

Low solute concentration/High water concentration

High solute concentration/Low water concentration

Water flow

Physical Requirements for Growth: Osmotic Pressure

• Plasmolysis: cell growth is inhibited when the plasma membrane pulls away from the cell wall

─ Added salt or sugar is another method of preserving food

Figure 6.4

Isotonic solution Hypertonic solution(high osmotic pressure)

• Carbon

─ Structural organic molecules, energy source

◦ Heterotrophs use organic carbon sources

◦ Autotrophs use CO2

• Nitrogen, Sulfur, Phosphorus

─ For synthesis of amino acids, nucleotides, vitamins, phospholipids

─ Most bacteria decompose proteins to obtain N

─ Inorganic ions are sources for these elements (NH4

+, NO3-, PO4

3-, SO42-)

Chemical Requirements for Growth

• Trace Elements (Iron, Copper, Zinc)─ Inorganic elements required in small amounts,

usually as enzyme cofactors─ Often present in tap water

• Organic Growth Factors─ Organic compounds obtained from the

environment (i.e. the organism cannot synthesize them)

─ Vitamins, amino acids

Chemical Requirements for Growth

• Oxygen (O2)

Chemical Requirements for Growth: Oxygen

Obligate aerobes

Facultative

anaerobes

Obligate anaerobes

Aerotolerant anaerobes Microaerophiles

• Aerotolerance of individual organisms depends on their ability to handle oxygen toxicity

─ Oxygen radical species: O2-, O2

2-, OH

─ Presence/lack of enzymes that neutralize toxic oxygen species

◦ SOD (Superoxide dismutase)

◦ Catalase/peroxidase

Chemical Requirements for Growth: Oxygen

.

• Oxygen (O2)

Chemical Requirements for Growth: Oxygen

Obligate aerobes

Facultative anaerobes

Obligate anaerobes

Aerotolerant anaerobes Microaerophiles

Don’t express SOD/catalase

Tolerate oxygen (express

SOD/catalase) but incapable of using

it for growth

Require oxygen, but at lower levels

than in the air

Express SOD and catalase

• Culture Medium: Nutrients prepared for microbial growth

─ Source of energy, carbon, nitrogen, sulfur, phosphorus, trace elements and organic growth factors

• Sterile: No living microbes

• Inoculum: Introduction of microbes into medium to initiate growth

• Culture: Microbes growing in/on culture medium

Culture Media

• Complex polysaccharide

• Used as solidifying agent for culture media in Petri plates, slants, and deeps

• Generally not metabolized by microbes

─ Agar is not a nutrient

• Liquefies above 100°C

─ Can incubate at a wide range of temperatures

Culture Media:Agar

Culture Media

• Reducing broth media

─ Contain chemicals (thioglycollate) that combine with dissolved O2 to deplete it from the media

Anaerobic Culture Media:Broth cultures

• Anaerobic jar

─ Oxygen and H2 combine to form water

Anaerobic Culture Methods:Agar Cultures

Figure 6.5

• Selective media: suppress growth of unwanted microbes and encourage growth of desired microbes

• Differential media: make it easy to distinguish colonies of different microbes

Culture Media:Selective and Differential Media

Figure 6.9b, c

E. coli on EMB

Enterobacter aerogenes on EMB

• A pure culture contains only one species or strain

• A colony is a population of cells arising from a single cell or spore or from a group of attached (identical) cells

─ One colony arises from one colony-forming unit (CFU)

• Specimens (pus, sputum, food) typically contain many different microorganisms

─ Common way to isolate a single species from a mixture of microorganisms: Streak plate method

Obtaining Pure Cultures

Streak Plate Method for Isolation of a Pure Species

• Use loop to pick colony

• Inoculate broth

• Pure culture

Figure 6.10a, b

Figure 6.5

Microbial Growth in Hosts:

Biofilms

• Microbial communities

─ 3-dimensional “slime”

─ i.e. dental plaque, soap scum

• Share nutrients

• Sheltered from harmful factors

• Cell-to-cell communication: quorum sensing

Bacterial biofilm growing on a micro-fibrous material

• Quorum sensing allows a form of bacterial communication

─ Individual cells can sense the accumulation of signaling molecules (autoinducers)

◦ Informs individual cells about surrounding cell density

◦ May change the behavior (gene expression) of individual cells

−Results in a coordinated response by the whole population

Microbial Growth in Hosts:

Biofilms & Quorum Sensing

http://biofilmbook.hypertextbookshop.com/public_version/

Prokaryotic Reproduction:Binary Fission

Figure 6.11

Generation time: the time required for one population doubling

─ Varies with species and environmental conditions

Reproduction in Prokaryotes:Generation Time

Reproduction in Prokaryotes:Generation Number

• Generation number: the number of times a cell population has doubled in a given time under given conditions

Figure 6.12b

Figure 6.13

Reproduction in Prokaryotes:Growth Plot

Arithmetic

Logarithmic

Bacterial Growth Curve

• Lag: little/no cell division

─ Adapting to new medium

─ *Metabolically active*

• Log: exponential growth

─ Most metabolically active

─ Gen. time at constant minimum

• Stationary: equilibrium phase

─ Growth rate = death rate

─ Nutrients exhausted, waste accumulation, pH changes

• Death: logarithmic decline

Figure 6.14

Measuring Microbial Growth

• To determine the size of a bacterial population in a specimen, cell counting techniques are used

─ Often there are too many cells per ml or gram of specimen…

◦ A small proportion of the specimen (a dilution) is counted

◦ The number of cells in the original specimen can be calculated based on the count in the small dilution

• Plate Counts: Perform serial dilutions of a sample

• How many cells are in 1 mL of original culture?

Direct Measurements of Microbial Growth:

Viable Cell Count

Figure 6.15, top portion

10-1DF:

DF=1

10-2 10-3 10-4 10-5

• Inoculate one agar plate with each serial dilution

Figure 6.16

Direct Measurements of Microbial Growth:

Viable Count

• After incubation, count colonies on plates that have 30-300 colonies (CFUs)

Figure 6.15

Direct Measurements of Microbial Growth:

Viable Count

• Filtration

─ Ideal when microbial density is low in a sample

Direct Measurements of Microbial Growth

Figure 6.17a, b

Direct Measurements of Microbial Growth

Figure 6.19

Disadvantages: -Likely to count dead cells-Motile cells can be difficult to count