Microbial Growth

Growth: an increase in cellular constituents

Most bacteria and some yeasts divide by a
process of binary ssion whereby the cell
enlarges or elongates, then forms a cross-wall
(septum) that separates the cell into two more-
or-less equal compartments each containing a
copy of the genetic material.
Binnary fission and other cell division proceses
bring about an increase in the number of cells in
population
 Bacteria every 2530 minutes under
optimal conditions of laboratory cultivation

Starting from a single cell many bacteria can

achieve concentrations of the order of 109 cells ml-1
or more following overnight incubation in common
liquid media
 Population growth analyzing the growth
curve of a microbial culture
Microorganism liq medium batch culture
closed system
No fresh medium is provided during
incubation nutrient conc and conc of
waste
The growth of microorganisms
reproducing by binary fission can be
plotted log of the number of viable cell
vs incubation time
The growth curve
 Lag phase
No immediate increase in cell number occurs
The cell is synthesizing new components
getting ready to grow
during which time the inoculum adapts its
physiology to that required for growth on the
available nutrients.
 Exponential phase
Microorganisms are growing and dividing
at the maximal rate

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 As growth proceeds nutrients are
consumed and waste materials
accumulate. This has the effect of
reducing the rate of growth (late
logarithmic phase) towards an eventual
halt (stationary phase)
 Stationary phase
This phase is attained by bacteria at a
population level of around 10 9 cells/ml
The total number of viable microorganisms
remain constant
 Microbial population enter the stationary
phase for several reason
Nutrient limitation
The accumulation of toxic waste products

Starvation during the stationary phase will

eventually lead to the death of some of the
cells and adaptation to a dormant state in
others (decline phase)
 Senescence and Death
Death decline, cell death exceeds cells
formed
Nutrient deprivation
The build up of toxic wastes
Patterns of growth such as this occur
within inadequately preserved pharmaceutical
products, in water storage tanks and in industrial
fermentations
 Environmental factors that inuence
growth and survival

Physicochemical factors that affect growth

and survival of bacteria
Temperature
pH
Osmotic pressure
Availability of oxygen

Nutrition and growth

 Temperature
Enzyme-catalyzed reactions
Microbial membranes
 Five class of microorganisms based on
their temperature ranges for growth:
Psychrophiles
Psychrotroph facultative psychrophiles
Mesophiles
Thermophiles
Hyperthermophiles
Growth Rates and
Temperature
 As temperatures rise, chemical and enzymic rx
within the cell proceed more rapidly, and growth
becomes faster until an optimal rate is achieved.
Beyond this temperature certain proteins may
become irreversibly damaged through thermal
lysis, resulting in a rapid loss of cell viability
The majority of bacteria that have medical or
pharmaceutical signicance are mesophiles and
have optimal growth temperatures between
ambient and body temperature (37C)

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 pH
Generally those microorganisms that have
medical or pharmaceutical signicance have pH
growth optima of between 7.4 and 7.6
Drastic variation in cytoplasmic pH
Disrupting the plasma membrane
Inhibiting the activity of enzymes and
membrane transport protein
Changes in the external pH
Ionization of nutrient molecule and thus
reduce their availability to the organism
 Acidophiles : pH 0 5,5
Neutrophiles : pH 5,5 8
Alkalophiles: pH 8 11,5

Accordingly, the pH of a pharmaceutical

preparation may dictate the range of
microorganisms that could potentially
cause its spoilage.
 Osmotic pressure
Osmotic pressure
Plasma membrane selectively permeable
Hypotonic solution water will enter the cell and
cause it to burst
Hypertonic solution water will flow out of the cell
If the cells are placed in a high salt or sugar
solution the cells lose water and cannot grow
In microbes that have cell walls the membrane
shrinks away from the cell wall PLASMOLYSIS
PLASMOLYSIS
 Halophiles:
Requires high levels of sodium chloride,
usually above about 0.2M to grow
Osmotolerant:
Able to grow over wide ranges of water
activity or osmotic conc
 Availability of oxygen
For many aerobic microorganisms oxygen
acts as the terminal electron acceptor in
respiration and is essential for growth
Alternate terminal electron acceptors are
organic molecules whose reduction leads
to the generation of organic acids such as
lactic acid.

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 Availability of oxygen
Strict aerobe requires oxygen
Facultative anaerobe grows in the
presence of oxygen but can without
oxygen
Obligate anaerobes cannot use oxygen
for growth
Aerotolerant anaerobes do not use
oxygen for growth but can tolerate it
Anaerobe jar
 Whats the problem with oxygen?
During normal respiration bacteria produce
oxygen free radicals
Many microorganisms posses enzymes that
afford protection against toxic O2 products
superoxide dismutase and catalase, which
catalyze the destruction of superoxide radical
and hydrogen peroxide
Obligate anaerobes usually have neither SOD or
catalase and are killed by oxygen and toxic by-
products
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 Nutrition and growth
Nutrients: substances used in biosynthesis
and energy release and therefore are
required for microbial growth
Macroelements large amounts
C,H,O,N,S,P
P, K, Ca, Mg
Microelements small amounts
Mn, Zn, Co, Mo, Ni, Cu ---cofactor enzyme

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 Nutrition factor
Heterotroph: organisms that use reduced,
preformed organic molecule as their
carbon dsource
Autotroph: organisms that use CO2 as
their sole or principal source of carbon
the vast majority of organisms of interest
in pharmacy and medicine are described
as chemoheterotrophs
they obtain carbon, nitrogen and energy by
breaking down organic compounds
 Microbial cultivation
The vast majority of microorganisms of
interest in pharmacy and medicine can be
cultivated in the laboratory
Some organisms are parasites and so can
only be grown inside the cells of a host
species which often necessitates
mammalian cell culture facilities and
there are a few (e.g. The organism
responsible for leprosy) that have never
been cultivated outside the living animal.
 Culture media
Culture media is a solid or liquid
preparation used to grow, transport, and
store micoorganism
To be effective, the medium must contain
all the nutrients the microorganismrequires
for growth
 Types of culture media
I. Based on their consistency
a) solid medium
b) liquid medium
c) semi solid medium
II. Based on the constituents/ ingredients
a) simple medium
b) complex medium
c) synthetic or defined medium
d) Special media
Special media
Enriched media
Enrichment media
Selective media
Indicator media
Differential media
Sugar media
Transport media
Media for biochemical reactions

III. Based
on Oxygen requirement
- Aerobic media
- Anaerobic media
Solid media contains 2% agar
Colony morphology,
pigmentation, hemolysis can
be appreciated.
Eg: Nutrient agar, Blood agar

Liquid media no agar.

For inoculum preparation,
Blood culture, for the isolation
of pathogens from a mixture.
Eg: Nutrient broth

Semi solid medium 0.5% agar.

Eg: Motility medium
Simple media / basal media
- Eg: NB, NA
- NB consists of peptone, meat extract,
NaCl,
- NB + 2% agar = Nutrient agar
Complex media
Media other than basal media.
They have added ingredients.
Provide special nutrients

Synthetic or defined media

Media prepared from pure chemical
substances and its exact composition is
known
Eg: peptone water 1% peptone + 0.5%
NaCl in water
Enriched media
Substances like blood, serum, egg are
added to the basal medium.
Used to grow bacteria that are exacting in
their nutritional needs.
Eg: Blood agar, Chocolate agar
Blood agar Chocolate agar
Enrichment media
Liquid media used to isolate
pathogens from a mixed culture.
Media is incorporated with
inhibitory substances to
suppress the unwanted
organism.
Eg:
Selenite F Broth for the
isolation of Salmonella, Shigella
Alkaline Peptone Water for
Vibrio cholerae
Selective media
The inhibitory substance is added to a solid
media.
Eg:
Mac Conkeys medium for gram negative
bacteria
TCBS for V.cholerae
LJ medium M.tuberculosis
Wilson and Blair medium S.typhi
Potassium tellurite medium Diphtheria
bacilli
Mac Conkeys medium TCBS
Potassium Tellurite media LJ media
Indicator media
These media contain an indicator which
changes its colour when a bacterium
grows in them.
Eg:
Blood agar
Mac Conkeys medium
Christensens urease medium
Urease medium
Differential media
A media which has substances
incorporated in it enabling it to distinguish
between bacteria.
Eg: Mac Conkeys medium
Peptone
Lactose
Agar
Neutral red
Taurocholate

Distinguish between lactose fermenters &

non lactose fermenters.
 Lactose fermenters Pink colonies
Non lactose fermenters colourless colonies
Sugar media
Media containing any fermentable
substance.
Eg: glucose, arabinose, lactose, starch etc.
Media consists of 1% of the sugar in
peptone water.
Contain a small tube (Durhams tube) for
the detection of gas by the bacteria.
Transport media
Media used for transporting the
samples.
Delicate organisms may not
survive the time taken for
transporting the specimen
without a transport media.
Eg:
Stuarts medium non nutrient
soft agar gel containing a
reducing agent
Buffered glycerol saline enteric
bacilli
Anaerobic media
These media are used to grow anaerobic
organisms.
Eg: Robertsons cooked meat medium,
Thioglycolate medium.