MICROBIOLOGY AND PARASITOLOGY

PBS300 (AY 2021-2022)

Mr. Joseph Valen Roales, RPh

UNIT 5
MICROBIAL NUTRITION AND GROWTH

METABOLISM 1. OBLIGATE AEROBES

controlled chemical reactions w/in cells Requires oxygen bc it serves as final electron acceptor of
ultimate outcome of metabolic activity: reproduction electron transport chains producing most of ATPs

RESULT OF MICROBIAL GROWTH 2. OBLIGATE ANAEROBES

Colony Oxygen is a deadly poison
No enzymes to counteract toxic forms of oxygen
aggregation of cells arising from a single parent cell
3. FACULTATIVE ANAEROBES
Biofilm
grow with or without oxygen, but their ability to use aerobic
collection of microbes living on a surface in a complex respiration pathways enhances their growth near surface
community
4. AEROTOLERANT ANAEROBES
GROWTH REQUIREMENTS Don’t use aerobic metabolism, tolerate oxygen by having
NUTRIENTS: CHEMICAL AND ENERGY REQUIREMENTS some of enzymes that detoxify oxygen’s poisonous forms
Sources of Carbon, Energy, and Electrons
5. MICROAEROPHILES
Require low oxygen levels (2% to 10%)
21% oxygen concentration would kill them
Ex. Helicobacter pylori

Toxic Forms of Oxygen

1. SINGLET OXYGEN (1O2)
molecular oxygen with electrons boosted to a higher
energy state, typically during aerobic metabolism
Phagocytic cells use this to oxidize pathogens
Plants use carotenoids to remove excess energy of this

2. SUPEROXIDE RADICAL (O2-)

form during incomplete reduction of O2 during ETC in
Oxygen Requirements aerobes & metabolism by anaerobes in presence of O2
superoxide dismutases to detoxify it

3. PEROXIDE ANION (O22-)

formed during rx catalyzed by superoxide dismutase (&
other metabolic reactions) contains peroxide anion
peroxide anion makes H2O2 an antimicrobial agent
Catalase converts H2O2 to water and O2
o Production of bubbles = presence of catalase
Peroxidase breaks down H2O2 w/out forming O2, using a
reducing agent such as coenzyme NADH

4. HYDROXYL RADICAL (OH·)

from ionizing radiation & incomplete reduction of H2O2
most reactive of 4 but H2O2 does not accumulate in
aerobic cells so threat of OH· is virtually eliminated

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 Microbiology and Parasitology

Nitrogen Requirements
N is abt 14% of the dry weight of microbial cells
growth-limiting nutrient
o anabolism ceases bc don’t have sufficient
nitrogen to build proteins and nucleotide
Cyanobacteria & Rhizobium reduce N2 to NH3
o Nitrogen fixation

Other Chemical Requirements

1. PHOSPHORUS
Component of phospholipid membrane, DNA, RNA, ATP

2. SULFUR
TEMPERATURE TERMINOLOGIES
sulfur-containing amino acids; proteins & vitamins
MINIMUM GROWTH TEMPERATURE
ex. Thiamine, biotin
lowest temp an organism can conduct metabolism
many microbes can still survive below min temp
3. TRACE ELEMENTS
Req in very small amts
MAXIMUM GROWTH TEMPERATURE
Ex. Selenium
highest temp an organism continues to metabolize
Lithotrophic Photoautotroph: synthesize all metabolic
when temp exceeds, organism’s proteins are permanently
and structural needs from inorganic nutrients
denatured, and it dies
Growth Factors
OPTIMUM GROWTH TEMPERATURE
temp an organism’s metabolic activities produce highest
growth rate

TYPES OF MICROBES BASED ON TEMPERATURE

1. PSYCHROPHILES
Grows best below 15°C & can continue to grow below 0°C
Most die at temperatures above 20°C
Don’t cause human disease bc cannot thrive at body temp

2. PSYCHROTOLERANT / PSYCHROTROPHS
tolerate but don’t grow best in cold
may also infect warm-blooded animals, including humans
Listeria monocytogenes can grow in refrigerated foods

3. MESOPHILES
Grow best in 20°C to ~40°C, can survive at higher & lower
temp
body temp=~37°C, most human pathogens = mesophiles

4. THERMODURIC ORGANISMS
mesophiles w/c can survive brief periods at higher temp
Physical Requirements
5. THERMOPHILES
Temperature
Grow best > 45°C
Proteins denature & lose their shape & function when
hydrogen bonds break 6. HYPERTHERMOPHILES
If temperature is too low Grow best > 80°C
o membranes become rigid & fragile
if temperature is too high
o lipids can become too fluid, & membrane cannot
contain cell or organelle
different temp have different effects on survival & growth
of microbes

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 Microbiology and Parasitology

pH
measure of concentration of H+ a soln (acidity or alkalinity)
orgs are sensitive to changes in acidity bc H + interfere with
hydrogen bonding within proteins and nucleic acids
TYPES OF MICROBES BASED pH
1. NEUTROPHILES
Grow best at neutral pH (pH 6.5 to pH 7.5)
Biofilms
complex, synergistic relationships
primary residence of microorganisms in nature
cause up to 70% of bacterial diseases in industrialized
countries
Cells within biofilms communicate & coordinate with one
another, acting similar to a tissue in a multicellular org

2. ACIDOPHILES
Grow best at acidic habitats

A. OBLIGATE ACIDOPHILES
require acidic environment & die if pH approaches 7.0

B. ACID-TOLERANT
merely survive in acid without preferring it

3. ALKALINOPHILES
QUORUM SENSING
live in alkaline soils and water up to pH 11.5
Biofilms form as a result of
quorum sensing, in which
Physical Effects of Water
microbes respond to density of
1. OSMOTIC PRESSURE
nearby microbes
hypotonic: lysis / swell
microbes secrete quorum-
hypertonic crenation
sensing molecules that act to
communicate # & types of cells
TYPES OF MICROBES BASED ON OSMOTIC PRESSURE
among members of biofilm
OBLIGATE HALOPHILES
> osmotic pressure, 30% salt; burst if placed in lakewater
CULTURING MICROORGANISMS
FACULTATIVE HALOPHILES
TERMINOLOGIES
Can tolerate high salth conc
Inoculum
Staphylococcus aureus (tolerate 20%salt can grow in skin)
sample placed in medium
2. HYDROSTATIC PRESSURE
For every additional 10 m of depth, water pressure Medium
increases 1 atmosphere (atm) collection of nutrients

MICROBE BASED ON HYDROSTATIC PRESSURE Culture

BAROPHILES
microbes that grow from an inoculum
depend on pressure to maintain their 3D functional shapes
act of cultivating microbes or microbes that are cultivated
don’t cause human diseases
Broths
Associations and Biofilms
liquid in media
TYPES OF RELATIONSHIPS
1. ANTAGONISTIC
Colonies
microbe harms or even kills another organism
cultures visible on surface of solid media
2. SYNERGISTIC
members cooperate, each receives benefits that exceed Pure Cultures = axenic
those that would result if each lived by itself cultures composed of cells arising from a single progenitor
each member could live separately
Colony-Forming Unit (CFU)
3. SYMBIOTIC progenitor from a particular pure culture is derived
close nutritional/physical contact, become interdependent may be either a single cell or a group of related cells
members rarely (if ever) live outside relationship

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 Microbiology and Parasitology

TYPES OF SAMPLING 2. Pour Plates

1. ENVIRONMENTAL SPECIMENS
Taken from envi or inaminate objects
2. CLINICAL SPECIMENS
taken from patients & handled in ways that facilitate
examination of microbes or testing for their presence
contain pathogens & normal microbiota (microbes that
does not cause diseases)
CFUs are separated from one another using a series of
dilutions
Agar: gelling agent derived from cell walls of red algae
After agar cools & solidifies, filled dishes = Petri plates
Isolated colonies: colonies that are separate & distinct
from all others—form in plates from CFUs that have been
separated via dilution series

3. Other Isolation Techniques

Micropipettes
Hollow tubes with small diameters can be used to pick up
single cells that are then used to establish axenic cultures

CULTURE MEDIA
1. Defined Media / Synthetic Media
3. STORED SPECIMENS exact chemical composition is known
Cultured organisms in the lab fastidious: orgs req relatively large # of growth factors
o may be used as living assays
CLINICAL SAMPLING
Clinical specimens are often transported in special 2. Complex Media / Nonsynthetic Media
transport media chemically formulated to maintain contain nutrients released by partial chemical breakdown
relative abundance of different microbial species or to of yeast, beef, soy, or proteins, such as casein from milk
maintain an anaerobic environment exact chemical composition is unknown
Nutrient broth, Trypticase soy agar, MacConkey agar,
OBTAINING PURE CULTURES
blood
1. Streak Plates
most commonly used isolation technique in microbe lab 3. Selective Media
substances that either favor
growth of particular microbes or
inhibit the growth of unwanted
ones
Eosin, methylene blue, crystal
violet dyes, & bile salts inhibit
growth of G+ bacteria w/out
adversely affecting most G-
Sabouraud dextrose agar
(inhibits bateria growth,
selective fungi)

MUKSAN, SM 4
 Microbiology and Parasitology

ENRICHMENT CULTURE 5. Anaerobic Media

use a selective medium and are designed to increase very
small numbers of a chosen microbe to observable levels
COLD ENRICHMENT
enrich a culture with cold-tolerant species
Anaerobes can be introduced with a straight inoculating
wire into the anoxic (oxygen-free) depths of solid media to
form a stab culture, but special media called reducing
media provide better anaerobic culturing conditions

4. Differential Media
shows presence of visible changes in the medium or
differences in the appearance of colonies

6. Transport Media
carry clinical specimens of feces, urine, saliva, sputum,
blood, & other bodily fluids to ensure that people are not
infected & that specimens are not contaminated

SPECIAL CULTURE TECHNIQUES

1. Animal and Cell Culture
for growing microbes w/c artificial media are inadequate

2. Low-Oxygen Culture
Carbon dioxide incubators electronically monitor &
control CO2 level
CAPNOPHILES: grow best with high CO2 (3-10%)

PRESERVING CULTURES
1. Deep-freezing
freezing cells at temperatures from -50°C to -95°C

2. Lyophilization
removing water from a frozen culture using an intense
vacuum
can last for decades and are revived by adding lyophilized
cells to liquid culture media

MUKSAN, SM 5
 Microbiology and Parasitology

GROWTH OF MICROBIAL POPULATIONS GENERATION TIME

BINARY FISSION time required for a bacterial cell to grow and divide
time required for a population of cells to double in number

MATHEMATICAL CONSIDERATIONS IN POPULATION

GROWTH
Logarithmic Growth / Exponential Growth

PHASES OF MICROBIAL POPULATION GROWTH

Growth Curve
graph that plots the numbers of organisms in a growing
population over time
difficult or impossible to distinguish numbers in early
generations from baseline
as population grows it becomes impossible to
accommodate the graph on a single page

MUKSAN, SM 6
 Microbiology and Parasitology

Lag Phase MEASURING MICROBIAL REPRODUCTION

cells are adjusting to their new environment Direct Methods Not Requiring Incubation
most cells do not reproduce immediately but instead 1. MICROSCOPIC COUNTS
actively synthesize enzymes to utilize novel nutrients in suitable for stained prokaryotes & large eukaryotes
the medium sample is placed on a cell counter (also called a Petroff-
Hausser counting chamber)
Log Phase
population increases logarithmically
reproductive rate reaches a constant as DNA and protein
syntheses are maximized
more susceptible to antimicrobial drugs that interfere with
metabolism

Stationary Phase
number of dying cells = number of cells being produced,
and the size of the population remains constant

Death Phase / Decline Phase

a population reaches a point at which cells die at a faster
rate than they are produced

CONTINUOUS CULTURE IN A CHEMOSTAT

Chemostat
open system
fresh medium is continuously supplied while an equal
amount of old medium (containing microbes) is removed
maintains a culture in a particular phase, typically log
phase
study of microbial population growth at steady but low
nutrient levels

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 Microbiology and Parasitology

2. ELECTRONIC COUNTERS 2. MEMBRANE FILTRATION

larger cells of yeasts, unicellular algae, & protozoa Use when population is density
Coulter device: directly counts cells as they interrupt an large sample (perhaps as large as several liters) is poured
electrical current flowing across a narrow tube held in front (or drawn under a vacuum) through a membrane filter with
of an electronic detector pores small enough to trap the cells
Flow cytometry: variation of counting w/ Coulter counter
o cytometer uses a light-sensitive detector to
record changes in light transmission through tube
as cells pass
o distinguish among cells that have been
differentially stained with fluorescent dyes or
tagged with fluorescent antibodies
o count bacteria in a solution & even count host
cells that contain fluorescently stained
intracellular parasites

Direct Methods Requiring Incubation

1. SERIAL DILUTION AND VIABLE PLATE COUNTS
serial dilution: stepwise dilution of a liquid culture in
which the dilution factor at each step is constant
viable plate count: count colonies on plates with 25 to
250 colonies & multiply number on each plate by
reciprocal of dilution used to inoculate that plate to
estimate number of bacteria per milliliter of original culture
accuracy of a viable plate count also depends on
o homogeneity of dilutions
o ability of bacteria to grow on medium used
o number of cell deaths
o growth phase of sample population
Thoroughly mixing each dilution, inoculating multiple
plates per dilution, & using log-phase cultures minimize
errors

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 Microbiology and Parasitology

3. MOST PROBABLE NUMBER 2. METABOLIC ACTIVITY

based on fact that the more bacteria are in a sample, the Under standard temperature conditions, rate at which a
more dilutions are required to reduce their number to zero population of cells utilizes nutrients & produces wastes
depends on their number

3. DRY WEIGHT
suitable for broth cultures, but growth cannot be followed
over time because the organisms are killed during the
process

4. MOLECULAR METHODS
isolate unique DNA sequences representing uncultured
prokaryotic species using genetic techniques, s.a
polymerase chain reaction (PCR) and hybridization of
DNA that codes for ribosomal RNA

TYPE OF DESCRIPTION
MICROBES
Neutrophiles Grow best at neutral pH (pH 6.5 to pH 7.5)
Acidophiles Grow best at acidic habitats
Obligate require acidic environment & die if pH
Acidophiles approaches 7.0
Acid-Tolerant merely survive in acid without preferring it
live in alkaline soils and water up to pH
Alkalinophiles
11.5
Obligate > osmotic pressure, 30% salt; burst if
Halophiles placed in lakewater
Can tolerate high salth conc;
Facultative
Staphylococcus aureus (tolerate 20%salt
Halophiles
can grow in skin)
Indirect Methods Hydrostatic For every additional 10 m of depth, water
Pressure pressure increases 1 atmosphere (atm)
1. TURBIDITY depend on pressure to maintain their 3D
estimating growth of a microbial population by measuring Barophiles functional shapes; don’t cause human
changes in turbidity using spectrophotometer diseases
Capnophiles grow best with high CO2 (3-10%)

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 MICROBIOLOGY AND PARASITOLOGY
PBS300 (AY 2021-2022)
Mr. Joseph Valen Roales, RPh

CULTURING MICROORGANISMS
Clinical Sampling
Streak Plates
Obtaining Pure Cultures Pour Plates
Other Isolation Techniques (Micropippettes)
Defined Media / Synthethic Media
Complex Media / Nonsynthethic Media
Selective Media
Culture Media
Differential Media
Anaerobic Media
Transport Media
Animal and Cell Culture
Special Culture Techniques
Low-Oxygen Culture
Deep-freezing
Preserving Cultures
Lyophilization

GROWTH OF MICROBIAL POPULATIONS

Generation Time
Mathematical Logarithmic Growth
Considerations in
Population Growth Exponential Growth
Growth Curve Lag Phase
Phases of Microbial Log Phase
Population Growth Stationary Phase
Death Phase
Continuous Culture in a
Chemostat
Direct Methods Not Microscopic Counts
Requiring Incubation Electronic Counters
Serial Dilution and Viable Plate Counts
Direct Methods
Membrane Filtration
Measuring Microbial Requiring Incubation
Most Probable Number
Reproduction
Turbidity
Metabolic Activity
Indirect Methods
Dry Weight
Molecular Methods

MUKSAN, SM 10