Biology 120 lectures for 2nd exam 2012 2012 (part 1 microbial growth)

MICROBIAL GROWTH

AY 2012-2013

Friday, July 27, 2012

DEFINITION OF MICROBIAL GROWTH

• NUMBER OF CELLS

• NOT CELL SIZE

• e.g. Growing microbes = increase in numbers, accumulating colonies


DEFINITION OF MICROBIAL GROWTH

• Note: for coenocytic organisms (multinucleate): growth = increased cell size


FOR YOU TO GROW....


HOW ABOUT THEM?


RECALL MICROBIAL NUTRITION

CARBON SOURCESCARBON SOURCES

Autotrophs CO2 sole or principal biosynthetic carbon source

Heterotrophs Reduced, preformed, organic molecules from other organisms

ENERGY SOURCESENERGY SOURCES

Phototrophs Light

Chemotrophs Oxidation of organic or inorganic compounds

HYDROGEN AND ELECTRON SOURCESHYDROGEN AND ELECTRON SOURCES

Lithotrophs Reduced inorganic molecules

Organotrophs Organic moleculesFriday, July 27, 2012


MAJOR NUTRITIONAL TYPES SOURCES OF ENERGY, HYDROGEN/ELECTRONS AND CARBON

REPRESENTATIVE MICROORGANISMS

PHOTOLITHOTROPHIC AUTOTROPHY

Light energyInorganic hydrogen/electron donorCO2 carbon source

AlgaePurple and green sulfur bacteriaBlue-green algae (cyanobacteria)

PHOTOORGANOTROPHIC HETEROTROPHY

Light energyOrganic hydrogen/electron donorOrganic carbon source (CO2 may also be used)

Purple non-sulfur bacteriaGreen non-sulfur bacteria



MAJOR NUTRITIONAL TYPES SOURCES OF ENERGY, HYDROGEN/ELECTRONS AND CARBON

REPRESENTATIVE MICROORGANISMS

CHEMOLITHOTROPHIC AUTOTROPHY

Chemical energy source (inorganic)Inorganic hydrogen/electron donorCO2 carbon source

Sulfur-oxidizing bacteriaHydrogen bacteriaNitrifying bacteriaIron bacteria

CHEMOORGANOTROPHIC HETEROTROPHY

Chemical energy source (organic)Organic hydrogen/electron donorOrganic carbon source

ProtozoaFungiMost non-photosynthetic bacteria


REQUIREMENTS FOR MICROBIAL GROWTH

•PHYSICAL REQUIREMENTS

• TEMPERATURE

• pH

• OSMOTIC PRESSURE

•CHEMICAL REQUIREMENTS

• CARBON

• NITROGEN, SULFUR & PHOSPHORUS

• TRACE ELEMENTS

• OXYGEN

• ORGANIC GROWTH FACTORS


• “Most microorganisms grow well at temperatures favored by humans”

• 3 primary groups (on the basis of temperature preference)

• psychrophiles (cold-loving)

• mesophiles (moderate-temperature-loving)

• thermophiles (heat-loving)

REQUIREMENTS FOR MICROBIAL GROWTH: TEMPERATURE



MINIMUM, OPTIMUM, MAXIMUM


• Psychrotrophs: grow between 0°C and 20-30°C; cause food spoilage

• Hyperthermophiles

: extreme temperatures (members of the archaea)



• RECALL: pH acidity or alkalinity of a solution

• acidophiles

• neutrophiles

• alkaliphiles

REQUIREMENTS FOR MICROBIAL GROWTH: pH


• Reactions of microorganism in solution based on solute concentration: hypertonic, isotonic, hypotonic

• e.g. based on osmotic pressure requirement: Halophiles (obligate/extreme or facultative)

• Water activity (aw): water that is available for metabolic processes; i.e. water in food which is not bound to food molecules can support the growth of bacteria, yeasts and molds (fungi) or unbound and available water

REQUIREMENTS FOR MICROBIAL GROWTH: OSMOTIC PRESSURE


REQUIREMENTS FOR MICROBIAL GROWTH: OSMOTIC PRESSURE


• one of the most important requirements for microbial groth

• structural backbone of living matter

• e.g. Chemoautotrophs (carbon dioxide) and Chemoheterotrophs (organic materials)

REQUIREMENTS FOR MICROBIAL GROWTH:

CARBON


• ACCESS: amino acids and proteins

• Most bacteria decompose proteins

• Some bacteria use NH4+

or NO3–

• A few bacteria use N2 in nitrogen fixation

REQUIREMENTS FOR MICROBIAL GROWTH: NITROGEN


• ACCESS: amino acids, thiamine and biotin

• Most bacteria decompose proteins

• Some bacteria use SO4

2– or H2S

REQUIREMENTS FOR MICROBIAL GROWTH: SULFUR


• ACCESS: In DNA, RNA, ATP and membranes

• PO43– is a

source of phosphorus

REQUIREMENTS FOR MICROBIAL GROWTH: NITROGEN, SULFUR

AND PHOSPHORUS


• iron, copper, molybdenum, zinc

• essential for the function of co-factors

REQUIREMENTS FOR MICROBIAL GROWTH: TRACE ELEMENTS


REQUIREMENTS FOR MICROBIAL GROWTH: TRACE ELEMENTS

• BIOTIN

• Carboxylation (Leuconostoc)

• CYANOCOBALAMIN or VIT B12

• Molecular rearrangements (Euglena)

• FOLIC ACID

• One-carbon metabolism (Enterococcus)

• PANTOTHENIC ACID

• Fatty acid metabolism (Proteus)

• PYRIDOXINE or VIT B6

• Transamination (Lactobacillus)

• NIACIN

• Precursor of NAD and NADP (Brucella)

• RIBOFLAVIN or VIT B2

• Precursor of FAD and FMN (Caulobacter)

• THIAMINE or VIT B1

• Aldehyde group transfer (Bacillus


• “microbes that use molecular oxygen produce more energy from nutrients than microbes that do not use oxygen”

REQUIREMENTS FOR MICROBIAL GROWTH: OXYGEN


• aerobic bacteria

• anaerobic bacteria

• microaerophilic bacteria



• Microbes can be harmed by toxic forms of oxygen

• singlet oxygen (1O2-): normal molecular oxygen that has been boosted into a higher-energy state; extremely reactive

• hydroxyl radical (OH•): most reactive intermediate form of oxygen formed in cellular cytoplasm by ionizing radiation




• peroxide anion (O22-): toxic; active ingredient in hydrogen peroxide and benzoyl peroxide

• SOLUTION: catalase and peroxidase




• superoxide free radicals (O2-): toxicity is caused by their great instability; they steal an electron from a neighboring molecule, which in turn becomes a free radical, and the cycle continues

• SOLUTION: production of superoxide dismutase (SOD): aerobic, FA and aerotolerant anaerobes

• convert superoxide free radicals to molecular oxygen and hydrogen peroxide



• VITAMINS: Unlike humans, most bacteria can synthesize all their own vitamins and are not dependent on outside sources

• Some bacteria lack the enzymes needed for the synthesis of certain vitamins, amino acids, purines and pyrimidines

REQUIREMENTS FOR MICROBIAL GROWTH: ORGANIC GROWTH

FACTORS


CULTURE MEDIAFriday, July 27, 2012

• nutrient material prepared for the growth of microorganisms in a laboratory

•IMPORTANT TERMS:

• inoculum: microbes introduced into a culture medium

• culture: microbes that grow and multiply in a culture medium

• sterile medium: a pre-requisite = no living microorganisms

CULTURE MEDIA


AGAR

• solidifying agent

• only a few microbes can degrade it

• liquifies at 1000C and solidifies below 400C

• pouring temperature: 500C (prevents injury to microbes)

• used for the preparation of slants, stabs/deeps, plates


TYPES OF CULTURE MEDIA: Chemically-defined Media

• exact chemical composition is known

• mostly for autotrophic bacteria, fastidious bacteria

• Contents: organic growth factors (carbon and energy)


TYPES OF CULTURE MEDIA: Complex Media

• made up of nutrients including extracts from yeasts, meat or plants, or digests of proteins

• exact chemical composition varies from batch to batch

• mostly for heterotrophic bacteria and fungi


TYPES OF CULTURE MEDIA: Anaerobic Growth Media

•“reducing media”

• sodium thioglycollate: chemically combine with dissolved oxygen and deplete the oxygen in the culture medium

• heated first before use to drive off absorbed oxygen


ANAEROBIC CULTURE TECHNIQUES


TYPES OF CULTURE MEDIA: Selective & Differential Media

• Goal: to detect the presence of specific microorganisms associated with disease or poor sanitation

• SELECTIVE: suppress growth of unwanted bacteria and encourage the growth of desired microbes



•Why it can select:

• BSA: Bismuth Sulfite Indicator and Brilliant Green are complementary, inhibiting Gram-positive bacteria and coliforms, allowing Salmonella spp. to grow

• SDA: pH 5.6 where fungi can outgrow bacteria



• Goal: to detect the presence of specific microorganisms associated with disease or poor sanitation

• DIFFERENTIAL: distinguish colonies of desired organisms when grown together with others


TYPES OF CULTURE MEDIA: Differential Media


TYPES OF CULTURE MEDIA: Enrichment Media

• mostly for soil and fecal samples or when desired microbe is injured

• may also be selective

• e.g. MRS agar (deMann, Rogosa and Sharpe agar or Lactobacillus agar)

• e.g. lactose brothFriday, July 27, 2012

PURE CULTURE


PREPARING PURE CULTURE

• Julius Richard Petri (1887)

• Easy to use, stackable (saving space), requirement for plating methods


OBTAINING PURE CULTURES: Streak Plating


PURE VS MIXED CULTURE


CHARACTERIZING COLONIES


CULTURE PRESERVATION


WAYS TO PRESERVE YOUR CULTURE

•subculturing

• mineral oil overlay

• freezing as glycerol stocks

• liquid nitrogen storage

• lyophilization



• subculturing

•mineral oil overlay



• lyophilization



• subculturing


•freezing as glycerol stocks


• lyophilization



• subculturing



•liquid nitrogen storage

• lyophilization



• subculturing




•lyophilization


REVIVAL OF PRESERVED L-DRIED CULTURES

http://www.jcm.riken.jpFriday, July 27, 2012

http://www.jcm.riken.jp

http://www.jcm.riken.jp

GROWTH OF BACTERIAL CULTURES


BACTERIAL DIVISION


OTHER FORMS OF DIVISION BY OTHER MICROBES

Budding = Rhodopseudomonas

Chains of conidiospores carried externally at the tips of the filaments = Actinomycetes Fragmentation of

filaments = Actinomycetes


THE MATHEMATICS OF GROWTH


CELL DIVISION• Generation

time: time required for a microbial population to double

• g = mean generation time

• g = t/nFriday, July 27, 2012

GENERATION TIME

•g = t/n


SAMPLE...

•Given an initial density of 4 x 104

•After 2 hours the cell density became 1 x 106

•Compute for the generation time

•Solution: t = 2

•n = [ log (1 x 106) – log (4 x 104)]/ 0.301; n = 4.65

•Generation time = (t/n); 2/4.65 or 0.43 hours OR 25.8 minutes


GENERATION TIME

MICROORGANISM TEMPERATURE (°C) GENERATION TIME (hours)

Escherichia coli 40 0.35

Bacillus subtilis 40 0.43

Mycobacterium tuberculosis

37 12

Euglena gracilis 25 10.9

Giardia lamblia 37 18

Sacharomyces cerevisiae

30 2


THE GROWTH CURVE


OBTAINING A GROWTH CURVE

• The Growth Curve can be obtained via a Batch Culture

• Microorganisms are cultivated in a liquid medium and grown as a closed system

• Incubated in a closed culture vessel with a single batch of medium and NO fresh medium provided during incubation

• SCENARIO: Nutrient concentration decline and concentrations of waste increase during the incubation period


1. THE LAG PHASE

• No immediate increase in cell mass or cell number

• Cell is synthesizing new components

• Cells retool, replicate their DNA, begin to increase in mass and finally divide


1. THE LAG PHASE

• The necessity of a lag phase:

• Cells may be old and ATP, essential cofactors and ribosomes depleted

• must be synthesized first before growth can begin

• Medium maybe different from the one the microorganism was growing previously

• new enzymes would be needed to use different nutrients

• Microorganisms have been injured and require time to recover


SHORT LAG PHASE

• SHORT LAG PHASE (or even absent)

• Young, vigorously growing exponential phase culture is transferred to fresh medium of same composition


LONG LAG PHASE• LONG LAG PHASE

• Inoculum is from an old culture

• Inoculum is from a refrigerated source

• Inoculation into a chemically-different medium


2. THE LOG/ EXPONENTIAL PHASE

• Microorganisms are growing and dividing at the maximal rate possible given their genetic potential, nature of medium and conditions under which they are growing

• Rate of growth is constant: doubling at regular intervals

• The population is most uniform in terms of chemical and physiological properties

• Why the curve is smooth:

• Because each individual divides at a slightly different moment


3. STATIONARY PHASE

• Population growth ceases and the growth curve becomes horizontal (around 109 cells on the average)

•Why enter the stationary phase:

• Nutrient limitation (slow growth)

• Oxygen limitation

• Accumulation of toxic waste products


4. DEATH PHASE

• Detrimental environmental changes like nutrient depletion and build up of toxic wastes lead to the decline in the number of viable cells

• Usually logarithmic (constant every hour)

• DEATH: no growth and reproduction upon transfer to new medium

• NOTE: Death rate may decrease after the population has been drastically reduced due to resistant cells


DIRECT MEASUREMENT

• Plate counts

• Filtration

• Most Probable Number (MPN)

• Direct Microscopic Count


PLATE COUNTS


RECALL: HOW TO COMPUTE CFU


FILTRATION


MPN


DMC


INDIRECT MEASUREMENTS: ESTIMATING BACTERIAL NUMBERS• Turbidity: spectrophotometry estimates

• Metabolic Activity

• e.g. MBRT for Milk = Class 1. Excellent, not decolorized in 8 hours; Class 2. Good, decolorized in less than 8 hours but not less than 6 hours; Class 3. Fair, decolorized in less than 6 hours but not less than 2 hours; Class 4. Poor, decolorized in less than 2 hours

• Dry Weight: for filamentous molds


NEXT MEETING: MICROBIAL METABOLISM

& PHYSIOLOGY


Biology 120 lectures for 2nd exam 2012 2012 (part 1 microbial growth)

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Transcript of Biology 120 lectures for 2nd exam 2012 2012 (part 1 microbial growth)