MICROBIOLOGY

CULTIVATION OF MICROORGANISMS
Lorenzo Magat, MD
1st Shift | 23 August 2018

TOPIC OUTLINE III. Mechanism of Growth Enhancement

I. Introduction Organic matter
II. Requirements for Growth - A macromolecule formed by anhydride bonds (important for cell
A. Organic Matters wall) between building blocks
B. Inorganic Ions - Requirement for the synthesis of anhydride bonds:
III. Mechanism of Growth o Chemical energy
IV. Sources of Metabolic Energy ▪ Provided by 2 phosphodiester bonds in ATP (adenosine
A. Fermentation triphosphate)
B. Respiration o Energy provided by proton motive force
C. Photosynthesis ▪ Is potential energy that can be derived by passage of a
V. Nutritional Requirements proton across a membrane
A. Carbon Source ▪ It is also an electrochemical gradient with 2 components:
B. Nitrogen Source ● Difference in pH
C. Sulfur Source ● Difference in Ionic charge
D. Other Sources
VI. Factors Affecting Growth IV. Sources of Metabolic Energy
A. pH Fermentation
B. Temperature - process where ATP synthesis is not coupled with electron transfer
C. Aeration but is characterized by substrate phosphorylation, an enzymatic
D. Ion Strength and Osmotic Pressure process in which a pyrophosphate bond is donated directly to ADP
VII. Cultivation Method (adenosine diphosphate) by phosphorylation in metabolic
A. Medium intermediate to form ATP
B. Isolation of Microorganisms from Pure Culture (Pi + ADP → ATP)
- Phosphorylated Intermediates
I. Introduction o inorganic phosphate (Pi) donor
- Cultivation the process of propagating organism by providing the o formed by metabolic rearrangement of fermentable substrate
proper environmental condition (Glucose, Lactose, Arginine)
- Metabolically, accessible forms of nutrients o Glycolysis: Glucose + 2ADP → Lactic acid + 2ATP
- Metabolic energy to synthesize macromolecules for growth and
survival Respiration
- Chemical reduction of an oxidant (electron acceptor) through a
II. Requirements for Growth specific series of electron carriers in the membrane establishing
Organic Matter the proton motive force across the bacterial membrane
- comprises most of the dry weight of microorganism - Reductants (electron donor) may be organic (lactic acid) or
1. Carbon - most important (for physical infrastructure) inorganic (hydrogen gas)
2. Hydrogen o the one oxidized
3. Nitrogen o electron donor
4. Oxygen - Oxidants: Gaseous oxygen (O2), CO2, SO4-2, NO3-1
5. Phosphorus
o the one reduced
6. Sulfur
o electron acceptor
Inorganic Ions Photosynthesis
1. Potassium - The reduction of an oxidant via a specific series of electron carrier
2. Sodium establishes the proton motive force
3. Calcium - The reductant and oxidant are created photochemically by light
4. Chloride energy absorbed by pigments in the membrane
5. Iron - Oxygen is evolved in this process
6. Magnesium - CO2 (oxidant) + H2O (reductant) + light → O2 + organic matter
Mechanisms of Action V. Nutritional Requirements
- Facilitate enzymatic catalyst (Catalysis) Carbon Source
- Maintain chemical gradients across the cell membrane - Autotrophs
(Energetics) o Ability to reduce carbon dioxide at the expense of water
o Creatures that do not require organic nutrients for growth
o Plants and some bacteria
Peter David, Aia Dayrit, Jay Dayrit, Meya De Castro, Tine de Gucena [LE, MG, SG] 1 of 4
 MICROBIOLOGY – CULTIVATION OF MICROORGANISMS

- Chemolithotrophs o Mg2+ and Fe2+ - found in porphyrin derivatives

o Organisms that use an inorganic substrate such as hydrogen o Mg2+ - found in chlorophyll
or thiosulfate as reductant and carbon dioxide as carbon o Fe2+ - found in cytochrome and peroxidases
source o Mg2+ & K+ - essential for ribosome integrity
- Heterotrophs o Ca2+ - Gram positive cell wall component
o Organisms that require organic carbon (in metabolizable form) 3. Siderophores
for growth - Compounds that chelate iron and promote the transport of ions
▪ Naphthalene- may provide all carbon energy required but as a soluble complex
only a few organisms are able to metabolize this 4. Growth Factors
▪ Glucose – can support fermentative respiratory growth of - Is an organic compound which a cell must contain in order to
many organisms grow but cannot be synthesized by their own
o Animals and Bacteria - must be obtained from the environment

Nitrogen Source VI. Factors Affecting Growth

- Nitrogen Hydrogen Ion Concentration (pH)
o Major component of protein and nucleic acids - Neutralophiles
o N2 has a triple bond between, very difficult to break o Grow best at a pH of 6.0 – 8.0
o NH3 (Ammonia) is the end product of all pathways which o Most pathogens grow in this pH
breakdown nitrogen/the most reduced form - Acidophiles
- Nitrogen fixation o Optimum growth at pH 3.0
o Ability of microorganism to assimilate N2 reductively via NH3 - Alkalophiles
(sole Nitrogen source) o Optimum growth pH 10.3
o Requires large amount of metabolic energy and is readily - Regulator of Internal pH
inactivated by oxygen o Pumping protons in and out of the cell by a set of proton
o Unique to Prokaryotes and a few Bacteria transport system in the cytoplasmic membrane
- Ammonification
o Intracellular production of NH3 from the deamination of amino Temperature
acids - Psychrophilic
o Ammonia is introduced into organic matter by biochemical acid o Grow best at low temperatures (15-20ºC)
pathways involving glutamate and glutamine o Psychotropes - optimum living temperature at 20-30ºC but
- Assimilatory Nitrile/Nitrate Reduction grow well at lower temperatures, important cause of food
o Ability to assimilate nitrate (NO3) and nitrite (NO2) reductively by spoilage
conversion of these ions to NH3 - Mesophilic
- Dissimilation o Grow best at 30-37ºC
o Used by microorganisms that use NO2 and NO3 as terminal o Most pathogens grow in this temperature
electron acceptors in respiration
- Denitrification - Thermophilic
o Conversion of NH3 → N2 under anaerobic conditions o Grow best at 50-60ºC
o Anammox reaction- oxidation of ammonia by NO2: NH4 + NO2 - Hyperthermophilic
→ N2 + 2H2O o Grow best above temperature of boiling water
- Thermal stability is expressed in 2 ways:
Sulfur Source 1. Heat
- Sulfur ▪ Shock response
o part of the structure of coenzymes and is found in the cysteinyl ▪ A transient synthesis of a set of “heat-shock proteins”
and methionyl side chains of proteins when exposed to a sudden rise in temperature above the
o elemental form cannot be used by plants and animals growth minimum
o oxidized to sulfate (SO4-2-) ▪ End result – these proteins appear to be unusually heat
o Sulfur source of most microorganisms resistant and stabilize heat sensitive proteins of the cell
o Can be reduced to hydrogen sulfide (H2S) 2. Cold Shock
o toxic to many organisms ▪ Killing of cells by rapid cooling
▪ Example: Rapid cooling of Escherichia coli from 37ºC to
Other Sources 5ºC can kill 90% of the cells
1. Phosphorus (PO4) ▪ Glycerol, Dimethyl sulfoxide
- Is required as a component of ATP, nucleic acids and such
coenzymes as NAD, NADP and flavins, phospholipids, lipid A, Aeration
and cell wall components and proteins - Obligate aerobes
- always assimilated as free inorganic phosphate (Pi) o Require oxygen as hydrogen acceptor
2. Mineral Sources o Ex. Staphylococcus aureus
- Required for enzyme function

Peter David, Aia Dayrit, Jay Dayrit, Meya De Castro, Tine de Gucena [LE, MG, SG] 2 of 4
 MICROBIOLOGY – CULTIVATION OF MICROORGANISMS

- Facultative aerobes Medium

o Can live aerobically or anaerobically - 3 situations maybe encountered in choice of technique and type of
o Ex. Escherichia coli medium
- Obligate anaerobes o Growing cells at a given species
o Require a substance other than oxygen as hydrogen acceptor ▪ Look for a suitable medium that can be devised by
and being sensitive to oxygen inhibition carefully reproducing the condition found in the
o Ex. Clostridium tetani organism’s natural environment
- Microaerophiles ▪ Providing suitable source of nutrients
o require small amounts of oxygen (2-10%) for aerobic o Microbiological Examination of natural materials
respiration, higher amounts are inhibitory ▪ Choose a medium that will give microorganisms chance to
- Aerotolerant anaerobes grow and avoid crowding of colonies
o indifferent to oxygen, they can grow in its presence but do not o Isolation of a Particular Type of Microorganism
use it as hydrogen acceptors ▪ Enrichment culture
- Byproducts of aerobic metabolism ● Medium is prepared so as to duplicate the natural
o natural byproducts are hydrogen peroxide and superoxide environment (“Niche”) of the desired microorganisms
o in the presence of iron, free hydroxyl radicals are formed which ● Ex. Blood agar
can be harmful to the organism ▪ Differential medium
Fe3+/Fe2+ ● Is medium that will cause the colonies of a particular
▪ O2 +H2 O2 → O2 + OH-2 +  OH
type of organism to have a distinctive appearance
o aerobic organisms have special enzymes (peroxidase and
● Example: E. coli appears with green metallic sheen
superoxide dismutase) that protect the cell from harmful
on EMB agar (differentiating lactose and non-lactose
radicals by converting the natural byproducts into more safer
fermenter)
metabolites.
superoxide dismutase
▪ 2O-2 +2H+ → O2 +H2 O2 Isolation of Microorganisms from Pure Culture
peroxidase
▪ 2H2 O2 → 2H2 O+ O2 - Agar
o Exceptions o Ideal gelling agent for most microbiological media
▪ Lactobacillus plantarum are aerotolerant but do not o Acidic polysaccharide extracted from red algae
contain catalase or superoxide dismutase. Here oxygen is - Plating
not reduced and therefore H2O + O2- are not produced. o the ideal gelling agent for most microbiological media is agar
▪ All strict anaerobes lack both superoxide dismutase and o Cells are immobilized therefore each cell will grow into an
catalase isolated colony
▪ Some organisms eg. Peptococcus anaerobius, have o Methods of plating
considerable tolerance to oxygen, because of their ability ▪ Pour plate method
to produce high level of the enzyme NAD oxidase, that ● A suspension of cells is mixed with melted agar at
reduces oxygen to H2O 50ºC and pour in a Petri dish (higher temperature =
death of microorganism)
Ion Strength and Osmotic Pressure ● When the agar solidifies, the cells are immobilize in
- Halophilic – requires high salt concentration the agar and grow into colonies increasing the
- Osmophilic – requires high osmotic pressure probability that the colony came from a single cell
- Most bacteria can tolerate a wide range of external osmotic ▪ Streak-Plate Technique
pressure & ionic strength ● Original suspension of cells can be streaked on an
o Due to the ability to regulate internal osmolality & ion agar plate with a wire loop
concentration ● As streaking continues, fewer and fewer cells are left
o Osmolality – can be regulated by the active transport of K into on the loop
the cell ● Finally the loop may deposit single cells on the agar
o Internal ionic strength - kept constant by compensating ● Note: This method only works if the spreading tool
excretion of positively charged putrescine usually an inoculating loop is resterilized after each
of steps 1 to 4
VII. Cultivation Method
- Problems to be considered
o Choice of suitable medium
o Isolation of a bacterial organism in pure culture

Peter David, Aia Dayrit, Jay Dayrit, Meya De Castro, Tine de Gucena [LE, MG, SG] 3 of 4
 MICROBIOLOGY – CULTIVATION OF MICROORGANISMS

▪ Spread-Plate technique
● A small volume of dilute microbial suspension
containing ca 30-300 cells is transferred to the center
of an agar plate & spread evenly over the surface
with a sterile bent-glass rod
● The dispersed cell develop into isolated colonies
● Can be used to count the microbial population -
because the number of colonies should equal the
number of viable organisms in a sample
▪ Dilution Method
● A much less reliable method - not used unless
plating is impossible to perform
● A suspension is serially diluted & samples of each
dilution are plated - if only a few samples of a
particular dilution exhibit growth, it is presumed that
some of the colonies started from single cells
● Can only be used to isolate the predominant type of
organism in a mixed population

References
- Powerpoint presentation of Dr. Magat
- Instalinotes Microbiology
- Carroll, K.C, Morse, S.A., Mietzner, T., and Miller, S. (2016).
Jawetz, Melnick, & Adelberg’s Medical Microbiology (27e). New
York: Lange.

Peter David, Aia Dayrit, Jay Dayrit, Meya De Castro, Tine de Gucena [LE, MG, SG] 4 of 4