MICROBIAL GROWTH and

CULTURE MEDIA
MARY LOU DULLONA-BASA
 MICROBIAL GROWTH
• Refers to an increase in cell number, not in cell size
• Bacteria grow and divide by binary fission
• Two types of asexual reproduction in microbes
– Binary fission
• the process whereby a cell divides asexually to produce two daughter cells
• produce new individuals that are genetically identical to the parent
organism.
• the outcome of cell reproduction is a pair of daughter cells that are
genetically identical to the parent cell. In unicellular organisms, daughter
cells are individuals.
– Budding
• forming a bubble-like growth that enlarges and separates from the parent
cell
 STAGES OF BACTERIAL GROWTH
• When a few bacteria are inoculated into a liquid growth
medium, it is possible to plot a bacterial growth curve that
shows the growth of cells over time
• Bacterial populations follow a sequential series of
growth phases
– LAG PHASE
– LOG PHASE – EXPONENTIAL PHASE
– STATIONARY PHASE
– DEATH PHASE
• Lag Phase
– Bacteria are first introduced into an environment or
media culture
– Bactria are trying to adapt to nutrients
– Preparing to grow in size and synthesize enzymes
– Varies in length
– In some cases can be very short or even absent
• Log (Exponential) Phase
– Also called log phase
– Rate of growth is constant
– Population number of cells undergoing binary
fission doubles at a constant interval called
generation time
– Continue as long as cells have adequate nutrients
and good environment
• Stationary Phase
– If exponential growth continued unchecked,
startling large numbers of cells could arise
– In this stage microbial death is equal to microbial
growth
• Death = growth
• Possible reasons for entry into stationary phase
– Nutrient limitation
– Limited oxygen availability
– Toxic waste accumulation
– Critical population density reached
• Death Phase
– Decline in the number of viable cells
– Cells dying, usually in exponential rate
– Death loss of ability to reproduce
– In some cases, death rate slows down due to accumulation of
resistant cells
– Slower than log phase
 REQUIREMENTS FOR GROWTH
• PHYSICAL REQUIREMENTS
– TEMPERATURE
– pH
– OSMOTIC PRESSURE
– HYDROSTATIC PRESSURE
– MOISTURE RADIATION
• CHEMICAL REQUIREMENTS-NUTRITIONAL
– CARBON
– NITROGEN, SULFUR AND PHOSPHORUS
– VITAMINS
– TRACE ELEMENTS
– OXYGEN
 PHYSICAL REQUIREMENTS
• 1. TEMPERATURE – Microbes are loosely classified into
several groups based on their preferred temperature
ranges
– A. PSYCHROPHILES – Cold-loving, can grow at 0OC
• TRUE PSYCHROPHILES
– sensitive to temperatures over 20OC
– Optimum growth at 15OC or below
– Found in very cold environments (northpole,
ocean depths)
– Seldom cause disease or food spoilage
• B. PSYCHOTROPHS
– Optimum growth at 20 to 30OC
– Responsible for most low temperature food spoilage
• C. MESOPHILES
– Middle-loving
– most bacteria includes most pathogens and common spoilage
organisms
– Best growth between 25 to 40OC
– Optimum temperature commonly 37OC
• D. THERMOPHILES
– Heat-loving
– Optimum growth between 50 – 60OC
– Many cannot grow below 45OC
– Adapted to live in sunlit soil, compost piles, hot springs
– Form extremely heat resistant endospores
• E. HYPERTHERMOPHILES
– Optimum growth at 80OC or higher
– Archaea bacteria
– Most live in volcanic and ocean vents
• 2. pH
– Most bacteria prefer neutral pH(6.5 – 7.5)
– Molds and yeast grow in pH between 5 and 6)
– ACIDITY – inhibits most microbial growth; used in food
preservation
– ALKALINITY – inhibits microbial growth; not used in food
preservation
– Acidic products of bacterial metabolism interfere with
growth
– Buffers can be used to stabilize pH
• ACIDOPHILES
– acid-loving
– Grow at ph 0.1 to 5.4
– Lactobacillus produce lactic acid, tolerates mild acidity

• NEUTROPHILES
– Grow at pH 5.4 to 8.5
– Includes most human pathogens

• ALKALOPHILES
– Alkali-loving
– Grow at pH 7,0 to 12 or higher
• Vibrio cholerae and alkaligenes faecalis
– Optimal pH = 9
• Agrobacterium – grow at pH =12
• 3. Hydrostatic pressure
–  pressure exerted by standing water (ex. lakes,
oceans, etc.)
– some bacteria can only survive in high hydrostatic
pressure environments
• ex. ocean valleys in excess of 7000 meters
– the high pressure - necessary
• to keep their enzymes in the proper 3-D shape
• without it, the enzymes lose their shape and denature
and the cell dies.
• 4. OSMOTIC PRESSURE - TONICITY
– Cells are 80 to 90% water
– HYPERTONIC SOLUTIONS
• High osmotic pressure
• Removes water from cell causing shrinkage from cell membrane
(plasmolysis)
• Used to control spoilage and microbial growth
– Sugar in jelly; salt on meat
– HYPOTONIC SOLUTIONS
• Low osmotic pressure
• Causes water to enter the cell
• Cell wall prevents excessive entry of water
• Microbe may lyse or burst if cell wall is weak
• HALOPHILES
– Requires moderate to large salt concentration
– Ocean water – 3.5%salt
– Most bacteria in the oceans
• EXTREME OR OBLIGATE HALOPHILES
– Require very high salt concentration
– 20 to 30%
– Bacteria in dead sea
• FACULTATIVE HALOPHILES
– Do not require high salt concentrations for growth
– Tolerate 2% salt
• 5. Moisture 
– only the spores of sport-forming bacteria can
exist in a dormant state in a dry environment.
• 6. Radiation
– UV rays and gamma rays can cause mutations in
DNA and even kill microorganisms
– Some bacteria have enzyme systems that can
repair some mutations.
CHEMICAL REQUIREMENTS FOR GROWTH

• 1. CARBON
– Makes 50% of dry weight of cell
– Structural backbone of all organic compounds
– CHEMOHETEROPHS
• Obtain carbon from their energy source- lipids,
proteins and carbohydrates
– CHEMOAUTOTROPHS AND PHOTOAUTOTROPHS
• Obtain carbon from carbon dioxide
• 2. NITROGEN, SULFUR AND PHOSPHORUS
– A. NITROGEN
• Makes up 14% of dry cell weight
• Used to form amino acids, DNA and RNA
• SOURCES OF NITROGEN
– Protein – most bacteria
– Ammonium – found in organic matter
– Nitrogen gas – obtain N directly from atmosphere
» Important nitrogen fixing bacteria
» Lives free in soil or associated with legumes ( peas, beans,
alfalafa, clover etc
– Nitrates – salts that dissociates to give NO3
• B. SULFUR
– Used to form proteins and some vitamins (thiamine and biotin)
– SOURCES
• Protein – most bacteria
• Hydrogen Sulfide
• Sulfates – salts that dissociates to give SO4-2
• C. PHOSPHORUS
– Used to form DNA, RNA, ATP and Phospholipids
– SOURCES
• Inorganic phosphate salts and buffers
• 3. VITAMINS
– Potassium, magnesium, and calcium – required as enzyme
co factors
– CALCIUM – required for cell wall synthesis in gram positive
bacteria
• 4. TRACE ELEMENTS
– Used as enzyme cofactors
– Commonly found in tap water
• Fe, Cu, Md, Zn
• 5. OXYGEN
– Organism that use molecular oxygen produce more energy from
nutrients than anaerobes
– OBLIGATE AEROBES
• Require oxygen to live
• E.g. Pseudomonas – common nosocomial pathogen
• OBLIGATE ANAEROBES
– Can use oxygen and are harmed by the presence of toxic
forms of oxygen
– E.g. Clostridium – causes tetanus and botulism
• FACULTATIVE ANAEROBES
– Can use oxygen but can grow in its absence
– E.g. E. Coli, Staphylococcus, yeasts, intestinal
bacteria
– AEROTOLERANT ANAEROBES
• Can use oxygen but tolerate its presence
• Can break down toxic forms of oxygen
• E.g. Lactobacillus carries out fermentation regardless of oxygen
presence
– MICROAEROPHILES
• Require oxygen but at low concentrations
• Sensitive to toxic forms of oxygen
• E.g. campylobacter
OXYGEN REQUIREMENTS VARY GREATLY

The Effects of Oxygen on the Growth of Various Types of Bacteria

 OXYGEN REQUIREMENTS
• strict or obligate anaerobes – oxygen kills the bacteria;
ex. Clostridium tetani
• strict or obligate aerobes – lack of oxygen kills the bacteria;
ex. Pserdomonas
• facultative anaerobes – can shift their metabolism
(anaerobic if oxygen is absent or aerobic if oxygen is
present); ex. E. coli, Staphylococcus
• aerotolerant – the bacteria don’t use oxygen, but oxygen
doesn’t harm them; ex. Lactobacillus
• microaerophiles – like low oxygen concentrations and higher
carbon dioxide concentrations; ex. Campylobacter
 TOXIC FORMS OF OXYGEN
• 1. SINGLET OXYGEN
– Extremely reactive form of oxygen
– Present in phagocytic cells
• 2. SUPEROXIDE FREE RADICALS
– Extremely toxic and reactive form of oxygen
– All organisms growing in atmospheric oxygen must produce
an enzyme superoxide dismutase (SOD) to get rid of them
– SOD is made by aerobes , facultative aerobe, and
aerotolerant anaerobes
– REACTION
• 3. HYDROGEN PEROXIDE
– Toxic and the active ingredient of
antimicrobials (benzoyl peroxide)
– ENZYMES THAT BREAKDOWN PEROXIDES
• CATALASE
– Breaks H2O2 into H2Oand O2
• PEROXIDASE
– Converts H2O2 into H2O
 CULTURE MEDIA
• CULTURE MEDIUM
– Nutrient material prepared for microbial growth in the laboratory
– REQUIREMENTS
• Must be sterile
• Contain appropriate nutrients
• Must be incubated at appropriate temperature
• CULTURE
– Microbes that grow or multiply in or on a culture medium
• Inoculum: Microbes introduced into medium
• Chemically defined media: Exact chemical composition is known (for research purposes
only)
• Complex media: Extracts and digests of yeasts, meat, or plants, e.g.:
– Nutrient broth
– Nutrient agar
– Blood agar
 TYPES OF CULTURE MEDIA
• 1. SOLID MEDIA
– Nutrient material that contains a solidifying agent(plates, slants,
deeps
– AGAR – most common solidifier, first used by Robert Koch
– UNIQUE PROPERTIES OF AGAR
• Melts above 95OC
• Once melted, does not solidify until it reaches 40OC
• Cannot be degraded by most bacteria
• Polysaccharride made by red algae
• Originally used as food thickener (Angelina Hesse)
• 2. CHEMICALLY DEFINED MEDIA
– Nutrient material whose exact chemical composition
is known
– PROPERTIES
• For chemoheterotrophs – must contain organic
source of carbon and energy – glucose, starch
• May also contain amino acids, vitamins and other
important building blocks required by microbe
• Not widely used
• expensive
• 3. COMPLEX MEDIA
– Nutrient material whose exact chemical composition is not known
– PROPERTIES
• Widely used for heterotrophic bacteria and fungi
• Made of extracts from yeast, meat, plants, protein digest
• Composition may vary slightly from batch to batch
• Energy, C, S, N requirements are primarily met by protein fragments –
peptone
• Vitamins and organic growth factors provided by meat and yeast
extracts
• TWO FORMS OF COMPLEX MEDIA
– Nutrient Broth – liquid media
– Nutrient Agar – solid media
• 4. ANAEROBIC GROWTH MEDIA
– Used to grow anaerobe that might be killed by oxygen
– PROPERTIES
• Reducing media
• Contain ingredients that chemically combine with
oxygen and remove it from medium
– E.g. Sodium thioglycolate
• Tubes are heated shortly before use to drive off oxygen
• Plates must be grown in oxygen free containers
(anaerobic chamber)
• 5. Special Culture Techniques
– Used to grow bacteria with unusual growth requirements
– Bacteria that do not grow on artificial media
• Mycobacterium leprae (leprosy) – grown in armadillos
• Treponema pallidum (syphilis) – grown in rabbit testicles
• Obligate intracellular bacteria (rickettsia and chlamydia) – only
grow in host cells
– Bacteria that require high or low CO2 levels
• Cannophiles – grow better at high CO2 levels and low O2 levels
- similar to environment of intestinal tract,
respiratory tract and other tissues
• 6. SELECTIVE MEDIA
– Used to suppress the growth of unwanted bacteria and encourage
the growth of desired microbes
• SABORAUD’S DECTROSE AGAR
– pH of 5.6 discourages bacterial growth
– Used to isolate fungi
• BRILLIANT GREEN AGAR
– Green dye selectively inhibits gram-positive bacteria
– Used to isolate gram-negative Salmonella
• BISMUTH SULFITE AGAR
– Used to isolate Salmonella typhi
– Inhibits growth of most other bacteria
• 7. DIFFERENTIAL MEDIA
• Used to distinguish coloniesof a desired organism
• BLOOD AGAR
– Used to distinguish bacteria that destroy red
blood cells (hemolysis)
– Hemolysis appears as an area of around
colony
» E.g. Streptococcus pyogenes
• 8. BOTH SELECTIVE AND DIFFERENTIAL MEDIA
– Used both to distinguish colonies of a desired
organism and inhibit the growth of other microbes
• MANNITOL SALT AGAR
– Used to distinguish and select for
Staphylococcus aureus
– High salt (7.5% NaCl) discourage growth of
other organisms
– pH indicator changes color when mannitol is
fermented to acid
• 9. BOTH SELECTIVE AND DIFFERENTIAL MEDIA
– Used both to distinguish colonies of a desired organism
and inhibit growth of other microbes
• MACCONKEY AGAR
– Used to distinguish and select for Salmonella
– Bile salts and crystal violet discourage growth of
gram-positive bacteria
– Lactose plus pH indicator
» Lactose fermenters produce pink or red colonies
» Non fermenters are colorless
• 10. ENRICHMENT CULTURE
– Used to favor the growth of a microbe that
may be found in very small numbers
– Does not necessarily suppress the growth of
other microbes
– Used mainly for fecal and soil samples
– After incubation in enrichment medium,
greater numbers of the organism, increase the
likelihood of positive identification
 CULTURING BACTERIA
• Streak Plate Method
• Pour Plate Method
• Spread Plate Method
 Streak Plate Method

3 or 4
quadrant
methods
 PURE CULTURE
• Contain only one species or strain.
• Most patient specimens and
environmental samples contain
several different kinds of bacteria
• Streak-plate method is commonly used
• Colony formation: A population of cells arising from
a single cell or spore or from a group of attached
cells (also referred to as CFU).
• Only ~1% of all bacteria can be successfully cultured
 OBTAINING PURE CULTURE
• PURE CULTURE
– Contains a single microbial species
– To obtain a pure culture, individual organisms must
ISOLATED
– The most common method of isolation is the STREAK
PLATE in which a sterile loop is inserted into a sample and
streaked onto a plate in a pattern, to obtain individual
colonies
– COLONY
• a group of descendants of an original cell
GROWTH OF BACTERIAL CULTURES
• BACTERIAL DIVISION
– Occurs mainly by binary fission
– A few bacterial species reproduce by BUDDING

• GENERATION TIME
– Time required for a cell to divide
– And its population to double
– Generation time varies considerably
• E. coli divides every 20 minutes
• Most bacteria divide every 1 to 3 hours
• Some bacteria require over 24 hours to divide
MEASURING MICROBIAL GROWTH
• Direct methods of measurement
– Plate Count
– Filtration
– Most Probable Number (MPN)
– Direct Microscopic Count
• Indirect methods of measurement
– Turbidity
– Metabolic activity
– Dry weight
DIRECT METHODS OF MEASUREMENTS
• 1. PLATE COUNT
– Most frequently used method of measuring bacterial population
– HOW? Inoculate plate with a sample and count number of colonies
– ASSUMPTIONS:
• Each colony originates from a single bacterial cell
• Original medium is homogenous
• No cell aggregates are present
– ADVANTAGES
• Measures viable cells
– DISADVANTAGES
• Takes 24hours or more for visible colonies to appear
• Only counts between 25 and 250 colonies are accurate
• Must perform serial dilutions to get appropriate numbers/plate
Viable cell counts: Plate counts: Serial dilutions put on plates
CFUs form colonies
• 2. FILTRATION
– Used to measure small quantities of bacteria
– E.g. Fecal bacteria in a lake or in ocean water
– A large sample (100 ml or more) is filtered to retain
bacteria
– Filter is transferred onto a petri dish
– Incubate and count colonies
• 3. MOST PROBABLE NUMBER (MPN)
– Used mainly to measure bacteria that will not
grow on solid medium
– Dilute a sample repeatedly and inoculate
several broth tubes for each dilution point
– Count the number of positive tubes in each set
– STATISTICAL METHOD
• Determines 95% probability that a bacterial
population falls within a certain range
• 4. DIRECT MICROSCOPIC COUNT
– A specific volume of a bacterial suspension (0.01 ml) is placed
on a microscope slide with a special grid
– Stain is added to visualize bacteria
– Cells are counted and multiplied by a few factor to obtain
concentration
– Advantages
• No incubation time required
– Disadvantages
• Cannot always distinguish between live and dead bacteria
• Motile bacteria are difficult to count
• Requires a high concentration of bacteria (10 million/ml)
INDIRECT METHODS OF MEASUREMENTS
• 1. TURBIDITY
– A bacteria multiply in media, it becomes turbid
– Use a spectrophotometer to determine % transmission or
absorbance
– Multiply by a factor to determine concentration
– Advantages
• No incubation time required
– Disadvantages
• Cannot distinguish between live and dead bacteria
• Rtequires a high concentration of bacteria (10 to 100 million cells/ml
Spectrophotometry to measure
turbidity
OD is function of cell number
• 2. METABOLIC ACTIVITY
– A bacteria multiply in media, they produce certain products
• Carbon dioxide
• Acds
– Measure metabolic products
– Expensive
• 3. DRY WEIGHT
– Bacteria or fungi in liquid are centrifuged
– Resulting cell pellet is weighed
– Doesn’t distinguish live and dead cells
 PRESERVING BACTERIAL CULTURE
• Deep-freezing: Rapid cooling of pure culture
in suspension liquid to –50°to –95°C. Good for
several years.
• Lyophilization (freeze-drying): Frozen (–54° to
–72°C) and dehydrated in a vacuum. Good for
many years.