Microbial GrowthMicrobial GrowthMicrobial Growth

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Dr. Carmen RexachDr. Carmen Rexach

MicrobiologyMicrobiology

Mt San Antonio CollegeMt San Antonio College

Microbial growth• Definition

– Increase in the number of cells– Increase in microbial mass

• Requires specific physical and chemical conditions

Physical requirementsTemperaturepHOsmotic pressure

Minimum, optimum, maximum

Classifications by temperature

• Psychrophiles• Psychrotrophs• Mesophiles• Thermophiles• Extreme thermophiles

Cold temperature

• Psychrophiles– [-10 to -18oC (optimum = -15)– Example: ocean floor

• Psychrotrophs– 0 to 30oC (optimum = 20)– Example: grow in refrigerator – freezing stops microbial growth,

does not necessarily kill bacteria

Moderate temperature

• Mesophiles– 10-48oC (optimum 25-40)– Examples: human pathogens,

spoilage, disease organism

Hot temperature

• Thermophiles– 40-72oC (optimum 50-60)– Example: hot springs

• Extreme thermophiles– 65-110oC – Example: hydrothermal vents– Taq polymerase = enzymes from Thermus aquaticus

pH

• Most organisms require 6.5-7.5 for optimum growth

• Acidophils– Bacteria tolerant to acidity

• Optimum pH in lab– Maintained by adding buffers (peptones,

phosphate salts, etc)

Osmotic pressure• Bacteria 80-90% water• Hypertonic solutions

– crenation• Hypotonic solutions

– Lysis of membrane– Prevented by cell wall

• Halophiles– Require salt for growth (Dead Sea)

• Facultative halophiles– Can grow in salt, not required

Halophiles

Salt lake

Chemical requirements• Carbon• Nitrogen, Sulfur, Phosphorus• Trace Elements• Oxygen

Carbon• All organisms require carbon source

– By definition: all organic molecules contain carbon and hydrogen

• Chemoheterotrophs– Utilize organic carbon sources

• Chemoautotrophs and photoautotrophs– Utilize inorganic carbon = CO2

Nitrogen, sulfur, phosphorus• N

– amino acid formation– Some bacteria fix inorganic nitrogen– Others use organic nitrogen = symbiosis

• S– amino acid synthesis and some vitamins

• P– synthesis of nucleic acids and

phospholipids

Trace elements

Small amount of mineral elementsUsually needed by enzymes as cofactorsInclude

FeCa++

Mg++

Oxygen

• All organisms using oxygen require special enzymes to protect cells from toxic effects of oxygen– Superoxide dismutase– Catalase or peroxidase

• Two categories based on oxygen requirements– Aerobes– Anaerobes

Aerobes• Aerobes = use oxygen• obligate aerobes = require oxygen• How much?

– Microaerophiles = grow in oxygen concentrations lower than air

– Facultative anaerobes = aerobes with the ability to grow in absence of oxygen• Intestinal bacteria, yeasts

Anaerobes

• Aerotolerant anaerobes– Tolerate oxygen– Can’t use it for growth– Many ferment carbohydrates to lactic acid

• Ex: Lactobacillus

• Obligate anaerobes– Killed by oxygen– Do not contain enzymes necessary to detoxify

Culture mediumAny material prepared for growth of bacteria in a labCulture = microbes growing in or on culture mediumAgar used to solidify culture

Types of culture media(start with sterile media)

• Chemically defined• Complex media• Anaerobic growth media/methods• Special techniques• Selective and differential media• Enrichment culture• Pure culture

Culture media• Chemically defined

– Know exact chemical composition• Complex

– Several components– Varies from batch to batch– Ex) nutrient agar, nutrient broth

• Special techniques– Ex) culturing certain bacteria or parasites on

living tissues or cells

Anaerobic growth media and methods

• Special reducing media– Thioglycolate broth– Ties up oxygen

• BBL jars– Removes oxygen chemically– Contains anaerobic indicator (methylene

blue)– Other options: anaerobic glove box

Anaerobic methods

Culture media

• Selective and differential media– Encourages growth of selected microorganisms

• Ex) sabdex– Helps to identify certain organisms

• Ex) CCFA• Blood agar = α, β, γ hemolysis

• Enrichment culture– Mixed culture and want to encourage

preferential growth of one organism

Pure culture

• Techniques work on basis that each colony arises from division of single bacterial cell

• Two techniques– Pour plate– Streak plate

– Goal: to grow isolated colonies

Bacterial division

• Binary fission• Budding• Spore formation

– Actinomycetes (at tip of filaments)• Fragmentation

– Filamentous bacteria

Binary fission

Bacterial cell elongates. Bacterial DNA replicates.

Cell wall and plasma membrane grow inward from both sides, forming a septum. Cross wall forms and two cells are produced identical to the parent cell.

Generation time

• Amount of time it takes for cell to divide or for population to double

• Growth rate– Population growth = change in the number of

cells or cell mass per unit of time• Bacterial cells multiply logarithmically

– Rate varies among different organisms and under different nutritional and genetic influences

Phases of growth

Lag phase

• Getting used to new environment– Lots of metabolism

• No lag phase – if inoculate from exponentially growing culture

• Lag phase – If inoculate from stationary phase due to

depletion of certain nutrients– If move from richer culture to poorer culture

Log phase & stationary phase

• Log phase– Exponential growth– Greatest metabolic activity– Increased susceptibility to adverse conditions

• Stationary phase– Number of microbial deaths equal to new cells

produced– Nutrient depletion, increased wastes, changes

in pH

Death or logarithmic decline

• Deaths exceed new cell formation• Exponential function but slower than

log phase

How to measure microbial growth• Plate counts

– Serial dilutions to keep number of colonies to be counted low

• Other methods– Direct count under microscope using slides with

counting chambers– Electronic cell counters, etc.– Indirect methods

• Turbidity = indicator of increased number• Measure amount of metabolic activity

– Amount of CO2 produced– Dessicate and weigh

Bacterial plate count