B.E Pruitt & Jane J.

Stein

Chapter 05
Microbial Growth

PowerPoint® Lecture Slide Presentation

REFERENCES

Tortora GJ, Funke BR, Case CL, 2007, Microbiology

an Introduction, 9th edition, Benjamin Cummings, San
Francisco, CA 94111, USA

Madigan MT, Martinko JM, 2006, Brock Biology of

Microorganisms, 11th edition, Pearson Education Inc.,
USA

Doorne H, 2008, Seminar of Course on Current

Pharmaceutical Microbiology: Methods,
Harmonization and Technology, Faculty of Pharmacy
UBAYA, Indonesia

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 Microbial Growth
• Microbial growth: increase in number of cells,
not the cell size
• The requirement for growth:
• Physical
• Temperature, pH, osmotic pressure
• Chemical
• C, N, S, P, trace element, O2, organic
growth factor

Physical requirement

• Temperature
• Most microorganisms grow well at the temperatures
favored by human
• Temperature (cardinal temperature)
• Minimum growth temperature
• Optimum growth temperature
• Maximum growth temperature
• Typical range of any given microorganism is 30-40
degrees

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Physical requirement

Temperature

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 Temperature

• Psychrophiles: capable of growing at 0°C

• Grup 1: grow at 0°C but has optimum growth at
15°C; found in oceans’ depths
• Grup 2 (psychrotrophs / moderate psychrophiles /
facultative psychrophiles): grow at 0°C, has
optimum growth at 20-30°C and cannot grow
above 40°C; cause refrigerator food spoilage
• Mesophiles: optimum growth at 25-40°C
• Thermophiles: optimum growth at 50-60°C
• Hyperthermophiles / extreme thermophiles: optimum
growth at ≥ 80°C; members of archaea

Food spoilage temperature

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 Physical requirement

• pH (extracellular environment)
• Most organisms show a growth pH range of 2-3
units
• Most natural environments have pH values 5 and 9
• Only few species can grow at pH value < 2 or > 9
• Most bacteria grow between pH 6.5 and 7.5
• Molds and yeasts grow between pH 5 and 6
• Acidophiles: grow in acidic environments (pH < 5.5);
stability of cytoplasmic membrane, e.g. archaea
• Alkalophiles (pH > 9), neutrophiles (pH 6-8)

Physical requirement

• pH
• The intracellular pH usually must remain relatively
close to neutral in order to prevent destruction of
acid- or alkali-labile macromolecules in the cell
• Buffer: peptones and amino acid, KH2PO4 (pH 6-
7.5)
• Buffering system for one to another organisms
may be considerably different

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 Physical requirement
• Osmotic effect
• Water activity (aw): ratio of the vapor pressure of the
air in equilibrium with a substance or solution to the
vapor pressure of pure water
• The values of aw vary between 0 and 1
• When a cell is in environment of low aw, there is a
tendency for water to flow out the cell
• Hypertonic environments, increase salt or sugar,
cause plasmolysis
• Hypotonic environments, decrease salt or sugar,
cause osmotic lysis (plasmoptysis)
• Extreme / obligate halophiles (30% salt) and
facultative halophiles (2-15% salt)

Osmotic effect

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 Water activity in pharmaceutical preparation
Dosage form Aw Single use Potential to Invasiveness Potential for
Multiple use support of the route of patient infection
microbial growth administration
Parenterals 0.99 Single and Moderate to high Very high low
multiple
Inhalation 0.99 Multiple High High High
solutions
Nasal sprays 0.99 Multiple Moderate to high Moderate Moderate to low
Ophthalmic 0.97 Single and Moderate to high High Low
liquids multuple
Topicals 0.97 Multiple Low to moderate Moderate Moderate to low
(lotions/creams)
Oral liquids (aq.) 0.90 Multiple Moderate to high Moderate to Moderate to low
low
Compressed 0.36 Single Very low Very low Very low to none
tablets
Rectal 0.30 Single Very low Low Low to very low
suppositories
Vaginal 0.30 Single Low Moderate to Moderate to low
suppositories low
Aerosol inhalants 0.25 Multiple None High Low

Chemical requirement

• Carbon
• Structural backbone, energy source
• Half of the dry weight is carbon
• Chemoheterotrophs, autotroph

• Nitrogen
• Synthesis amino acids (a.a.), DNA, RNA
• 14% of the dry weight of bacterial cell
• Most bacteria decompose proteins and
reincorporating a.a. and other nitrogen compound
(e.g. NH4+ or NO3−) into newly synthesized proteins

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 Chemical requirement

• Sulfur
• Synthesis amino acids and vitamin (thiamine, biotin)
• Important sources: SO42−, H2S, sulfur-containing
amino acid

• Phosphorus
• In DNA, RNA, ATP, and phospholipids membranes
• PO43− is a source of phosphorus
• S and P together constitute about 4%
• P, Mg, Ca are also used as cofactor for enzymes

Chemical requirement

• Trace Elements
• Inorganic elements required in small amounts
• Usually as enzyme cofactors
• Naturally present in tap water or even distilled water

• Organic Growth Factors

• Essential organic compounds that is unable to be
synthesized by organisms
• Obtained from the environment
• Vitamins, amino acids, purines, pyrimidines
• Non fastidious and fastidious bacteria

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Trace element

Trace element

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Organic growth factor

Chemical requirement

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 Toxic Forms of Oxygen

• O2 is a powerful oxidant and the best electron receptor

for respiration
• O2 is not the poison but certain O2 derivates that toxic
to microorganisms

Enzymes that destroy toxic O2

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 Culture media

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

• Inoculum
microbes are introduced into a
culture medium to initiate growth

• Culture
the microbes that grow and
multiply in or on a culture medium

Criteria must the culture medium meet

• It must content the right nutrient for

the specific microorganisms
• Have a sufficient osmotic balance,
adjusted pH and suitable level of O2
• Sterile, it must initially contain no
living microorganisms
• Growing culture should be incubated
at proper temperature

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 Culture media
• Agar: complex polysaccharide
derived from marine alga
• Used as solidifying agent for
culture media in petri plates
• Generally not metabolized by
microbes
• Liquefies at 100°C
• Solidifies at 40°C
• Chemically Defined Media: exact
chemical composition is known
• Complex Media: extracts and
digests of yeasts, meat, or plants

Culture Media

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 Culture Media

Anaerobic Culture Methods

• Reducing media
• Contain chemicals (thioglycollate) that combine O2
• Heated to drive off O2
• Special anaerobic jar
• Contain sodium bicarbonate and borohydride
• Added with H2O → H and CO2
• Palladium catalyst combines with H and O2 → H2O
• Oxyrase enzyme (reduces O2 → H2O)
• Petri (OxyPlate); self-contained anaerobic chamber

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 Anaerobic Culture Methods

Special culture technique

• Canophiles

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 Obtaining pure culture

• 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 cells
• A colony is often called a colony-forming unit (CFU)

Streak Plate

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 Preserving bacteria cultures

• Deep-freezing: pure culture is placed in a

suspending liquid and quick-frozen at -50°to -95°C
• Lyophilization (freeze-drying): frozen (-54° to
-72°C) and dehydrated in a vacuum (sublimation)
• The microorganisms can be revived at any time by
hydration with a suitable liquid nutrient medium

The Growth of Bacterial Culture

• Binary fission, budding, conidiospores

(actinomycetes), fragmentation of filaments
• Generation time: the time required for a cell to divide
(and its population to double)
• Most of bacteria have a generation time of 1-3 hours,
others require more than 24 hours per generation
• E. coli have a generation time of 20 minutes

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Binary Fission

Binary Fission

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 Growth curve

Generation time calculation

If 100 cells growing for 5 hours produced 1,720,320 cells:

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 Phases of Growth

Phases of Growth

• Lag Phase
• It can last for 1 hour or several days, the cells are
not dormant
• Period of intense metabolic activity (synthesis of
enzymes and various molecules)
• Log or Exponential Growth Phase
• Generation time reaches a constant minimum
• Metabolically active and preferred for industrial
purposes
• Sensitive to adverse conditions (e.g. radiation and
antimicrobial drugs)

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 Phases of Growth

• Stationary Phase or period of equilibrium

• Cryptic growth
• Metabolic activities of surviving cells are slowing
• Exhaustion of nutrients, accumulation of waste
products, changes in pH may all play a role
• Chemostat apparatus; continuous culture
• Death or Logarithmic Decline Phase
• Involution : their morphology changes dramatically,
making them difficult to be identified

• Some species pass through all the phases in few days;

others retain some surviving cells almost indefinitely

Chemostat apparatus

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 Measuring Microbial Growth

• Weight of some component of cell mass (protein,

nucleic acid or dry weight of the cells)
• Population’s total mass (number of the cells in a
milliliter liquid or in a gram solid material)

• Methods:
• Direct measurement
• Direct microscopic count, viable count,
filtration, MPN
• Indirect measurement
• Turbidity, metabolic activity and dry weight

Direct measurements of microbial growth

• Direct Microscopic Count
• Petroff-Hausser cell counter
• Advantage:
• No incubation is required (fast)
• Disadvantages:
• It is needed special attention to count the cells
• Dead cells are not distinguished from living cells
• Precision is difficult to achieve
• Motile bacteria are difficult to be counted
• A rather high concentration of cells is required to
be countable (e.g. 106 bacteria / milliliter)

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 Direct microscopic count

Direct measurement of microbial growth

• Viable count
• Measures the number of viable cells
• Plate count or colony count
• Assume: each viable cell can grow and divide to
yield one colony
• Unit: Colony-forming unit (CFU)
• Serial dilution
• Pour plate (0.1-1.0 ml) and spread plate (0.1 ml or
less)

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Serial dilution
• After incubation, count
colonies on plates that
have 30-300 colonies

Plate count

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 Plate count

• Advantages:
• Only living cells is counted
• Some species can be countable all at once
• Can be used for isolation and identification

• Disadvantages:
• The result does not reflect the actual value; some
cells can closely growth resulting one colony
• Different time, medium and incubation condition will
result a different value
• The microbial must be grown in/on the solid medium;
show dense, distinct and not spreading colonies

Direct measurements of microbial growth

• Filtration method
• Used for very small quantity bacteria
• Usually using 100 ml of sample
• Applied frequently to detection and enumeration
of coliform bacteria

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 Direct Measurements of Microbial Growth

• Most Probable Number (MPN)

• Useful when the microbes being counted will not
grow on solid media or when the growth of
bacteria in a liquid differential medium is used to
identify the microbes
• The greater the number bacteria in a sample, the
more dilution is needed to reduce the density to
the point at which no bacteria are left to grow in
the tubes in a dilution series
• The MPN is only the statement that there is a
95% chance that the bacterial population falls
within a certain range and that the MPN is
statistically the most probable number

Most probable number

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 Most probable number
• Multiple tube
MPN test
• Count
positive
tubes and
compare to
statistical
MPN table

Indirect measurements of microbial growth

• Turbidity measurement
• As bacterial multiply in a liquid medium, the medium
becomes turbid or cloudy with cells
• Spectrophotometer or colorimeter
• Parameter: absorbance or sometimes called optical
density (OD)
• About 106 – 107 cells / milliliter are needed to make
suspension turbid enough to read on a
spectrophotometer

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 Turbidity measurement

Indirect measurements of microbial growth

• Metabolic activity
• The amount of a certain metabolic product (acid,
CO2) is proportional to the number of bacterial
present
• Vitamin bioassay

• Dry weight
• Used for filamentous bacteria or molds
• The molds is removed from the growth medium,
filtered to remove extraneous material, dried in a
desiccator and then weighed

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