Microbial Growth

P H A R MA C E U T I C A L M I C R O B I O L O G Y
Outline

THE REQUIREMENTS CULTURING THE GROWTH OF

FOR GROWTH MICROORGANISMS MICROBIAL CULTURES

Microbial Growth : the increase in the number of cells, NOT the size
The
requirements
of Growth
 PHYSICAL REQUIREMENTS (TEMPERATURE, CHEMICAL REQUIREMENTS (CARBON,
PH, OSMOTIC PRESSURE) NITROGEN, SULFUR, PHOSPHORUS, TRACE
ELEMENTS, ORGANIC GROWTH FACTORS,
OXYGEN)

Microbial Growth Requirements

Temperature

MINIMUM OPTIMUM MAXIMUM

TEMPERATURE TEMPERATURE TEMPERATURE
GROWTH GROWTH GROWTH
Optimum Growth
Temperature
Psychroprile (cold loving) : can grow at 0oC,
optimum growth temperature : ±15oC 

Psychrotrophs : can grow at 0oC, optimum growth

temperature : 20-30oC, cause food spoilage

Mesophiles (moderate temperature loving) : 25-

40oC

Thermophiles (heat loving) : 50-60oC

Hyperthermophiles (extreme) : ≥80oC

Temperature
pH
Most bacteria grow between pH 6.5 and
7.5

Molds and yeasts grow between pH 5

and 6

Acidophiles (pH < 5.5)

Alkalinophiles (pH > 9)

Netralophiles
Osmotic
Pressure
Chemical requirement : Carbon

THE STRUCTURAL BACKBONE OF HALF DRY WEIGHT OF BACTERIAL

LIVING MATTER, ENERGY CELL IS CARBON
SOURCES
 Light (photo-) Chemical compounds
(chemo-)
Carbon Dioxide Photoautotrophs Chemoautotrophs
(auto-)
Organic Compounds Photoheterotrophs Chemoheterotrophs
(hetero-)

Carbon source & energy source

Other chemical requirements
• In amino acids & proteins
Nitrogen • Most bacteria decompose proteins into NH4+ or NO3-
• A few bacteria use N2 in nitrogen fixation

• In amino acids, thiamine and biotin

Sulphur • Most bacteria decompose proteins
• Some bacteria use SO42- or H2S

Phosphorus • In DNA, RNA, ATP and membranes

• PO43- is a source of phosphorus
Trace Elements
I n o rg a n i c e l e m e n t s r e q u i r e d i n
small amounts
Usually as enzyme cofactors
C o b a l t , c o p p e r, m a n g a n e s e ,
selenium, zinc, etc
About 0.25% of the dry weight
of bacterial cell
Organic
growth factors
Essential organic compounds that is
unable to be synthesized by
organisms

Obtained from the environment

Vitamins, amino acids, purines,

pyrimidines

Organisms that require a relatively

large number of growth factors :
fastidious
 Element % dry source function

Major
of
weight
Carbon 50 Organic compounds or CO2 Main constituent of cellular

elements,
material

Oxygen 20 H2O, organic compounds, Constituent of cell material and cell

CO2 and O2 water; O2 is electron acceptor in

their source Nitrogen 14 NH3, NO3, organic

aerobic respiration

Constituent of amino acids, nucleic

and function
compounds, N2 acids nucleotides, and coenzymes

in bacterial
Hydrogen 8 H2O, organic compounds, Main constituent of organic
H2 compounds and cell water

Phosphorus 3 Inorganic phosphates (PO4) Constituent of nucleic acids,

cells
nucleotides, phospholipids, LPS,
teichoic acids

Sulfur 1 SO4, H2S, organic sulfur Constituent of cysteine,

compounds methionine, glutathione, several
coenzymes
 Element % dry of source function
weight

Major
Potassium 1 Potassium Main cellular inorganic cation
salt and cofactor for certain enzymes

magnesium 0.5 Magnesium

salt
Inorganic cellular cation, cofacto
r for certain enzymatic reactions
elements,
calcium 0.5 Calcium salt Inorganic cellular cation,
their source,
cofactor for certain enzymes and
a component of endospores and function
iron 0.2 Iron salt Component of cytochromes
and certain nonheme iron
in bacterial
proteins and a cofactor for some
enzymatic reactions cells
Vitamin Coenzyme form Function
p-aminobenzoic acid (PABA) Precursor of the biosynthesis of folic acid
Folic acid Tetrahydrofolate Transfer of one-carbon units and required for
synthesis of thymine, purine bases, serine,
methionine, and panthothenate
Biotin Biotin Biosynthetic reactions that require CO2 fixation

Lipoic acid Lipoamide Transfer of acyl groups in oxidation of keto

acids
Mercaptoethane-sulfonic acid Coenzyme M CH4 production by methanogens

Nicotinic acid NAD (nicotinamide adenine dinucleotide) Electron carrier in dehydrogenation reactions

and NADP

Common vitamins required in the nutrition of certain bacteria

Vitamin Coenzyme form function
Pantothenic acid Coenzyme A and the Acyl Carrier Protein (ACP) Oxidation of keto acids and acyl
group carriers in metabolism
Pyridoxine (B6) Pyridoxal phosphate Transamination, deamination,
decarboxylation and racemation
of amino acids
Riboflavin (B2) FMN (flavin mononucleotide) and FAD (flavin Oxidoreduction reactions
adenine dinucleotide) Oxidoreduction reactions
Thiamine (B1) Thiamine pyrophosphate (TPP) Decarboxylation of keto acids and
transaminase reactions
Vitamin B12 Cobalamine coupled to adenine nucleoside Transfer of methyl groups

Vitamin K Quinones and napthoquinones Electron transport processes

Common vitamins required in the nutrition of certain bacteria

Toxic form of
oxygen
Highly reactive oxygen

O2 is a powerful oxidant and

the best electron receptor for
respiration
Enzyme that
destroy toxic
oxygen
Culturing microorganism
Culturing
microorganism
s
To cultivate or culture microorganisms, a
sample called an INOCULUM (inocula)
is introduced into a collection of nutrient
called a MEDIUM

CULTURE can refer to the act of

cultivating microorganisms or to the
microorganisms that are cultivated.

Characteristics of bacterial colonies:

shape, margin, elevation, size, texture,
and appearance of the colony’s margin
Culture Medium
Liquid medium : Broths

Solid medium : Solidifying agent  agar

Agar
◦ Complex polysaccharide
◦ Used as solidifying agent for culture media in Petri plates, slants, and
deeps
◦ Generally not metabolized by microbes
◦ Liquefies at 100oC
◦ Solidifies ~ 40oC
 a. Nutrient Agar
Complex medium, Selective and b. MacConkey Agar
Differential medium
c. MacConkey Agar
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

 Special culture
technique
Aerotolerant anaerobs,
microaerophiles, capnophiles
Special Culture technique
Obtaining pure cultures
Isolate the specific microorganism (one species or strain) from a sample or
specimen
If sample or specimen are plated out onto the surface of a solid medium,
COLONIES will form that are exact copies of the original organism.
A COLONY : a population of cells arising from a single cell or spore or from
a group of attached cells
The progenitor from which a particular pure culture is derived, termed as
colony-forming unit (CFU)
Isolation method
Selective Enrichment for very small
number of isolated microbe
◦ Streak plate method
◦ Pour plate method
Streak plate
method
Pour plate
method
Preserving
culture
Refrigeration – short period of time

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 microbial culture
Bacterial
division :
Binary fission
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 (<7 hours: ± 20

generation  220  : > 1.000.000 cell; in 24 hours??
Bacterial
Growth
Curve
 Lag phase
• the organisms are adjusting to their environment
• t 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 Phase/ Exponential Growth Phase

• the cell begin to divide, enter a period of growth  logarithmic increase
• Generation time reaches a constant minimum

Phases of • Metabolically active and preferred for industrial purposes

• ensitive to adverse conditions (e.g. radiation and antimicrobial drugs)

Growth Stationary Phase

• new organisms are being produced at the same rate at which they are dying
• Metabolic activities of surviving cells are slowing
• Exhaustion of nutrients, accumulation of waste products, changes in pH may all
play a role

Death Phase
• The number of deaths eventually exceeds the number of new cells formed
• Some species pass through all the phases in few days; others retain some
surviving cells almost indefinitely
 Direct Method Indirect Method

• Viable Plate Counts • Metabolic activity

• Membrane filtration • Dry weight
• Microscopic counts • Turbidity
• Electronic counters
• Most Probable Number (MPN)

Measuring microbial growth

 The most frequently use method

Assume: each live bacterium grows and divides to produce a single colony

The colony not only from a single bacterium, but also from short segment of

Viable Plate chain or bacterial clump  UNIT: COLONY FORMING UNIT (CFU)

If too many colonies are present, some cells are overcrowded and do not develop

Counts
 inaccuracies in the count

Only plates with 25-250 or 30-300 colonies are counted

Serial dilution

Pour plate (0.1-1.0 ml) and spread plate (0.1 ml or less)

Viable plate counts : serial dilution
Viable plate
count
 Used for very small quantity bacteria

At least 100 ml of sample passed through a

thin membrane filter
Membrane
filtration
Applied frequently to detection and
enumeration of coliform bacteria

The colonies formed are distinctive when a

differential nutrient medium is used
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. 10 6 bacteria / milliliter)
Microscopic
counts : Petroff-
Hauser cell
count
(cytometer)
Electronic counters
Coulter counters

Automatically count the number of cells in a measured

volume of liquid

Directly count cells as they interrupt an electrical

current flowing across a narrow tube held in front of
an electronic detector.
Most Probable
Number (MPN)
Most Probable
Number (MPN)
Most Probable
Number (MPN)
Indirect Method
Metabolic activity 
◦ The amount of a certain metabolic
product (acid, CO2) is proportional to
the number of bacterial present

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
Indirect method
Turbidity

As bacterial multiply in a liquid medium, the medium becomes turbid or

cloudy with cells

Instrument: Spectrophotometer or colorimeter

Parameter: absorbance or sometimes called optical density (OD)

About 107 – 108 cells / milliliter are needed to make suspension turbid
enough to read on a spectrophotometer

Not a useful meassure of contamination of liquids by relatively small

numbers of bacteria
Turbidity