Chapter IV

Microbial Physiology
 Coverage

• Physiology and Nutrition

• Growth and Control
• Sterilization and Anti-Microbial methods
Metabolism
 Metabolism

• Two key players:

enzymes and
adenosine
triphosphate
(ATP)

• ATP is a molecule
that cells use to
manage energy
needs
 Impacts of microbial
metabolism

• Disease and food spoilage

• Nitrogen cycle
• Beverages and food
• Sewage treatment
• Drugs
 Important processes

Carbohydrate Catabolism
• Respiration – glycolysis is followed by Krebs cycle , and
electron transport chain which generates the most ATP
– 38 ATPs can be generated from 1 glucose molecule

• Fermentation – does not require Krebs cycle or ETC, and

produces end products such as lactic acid or ethanol
– 2 ATPs are produced from 1 glucose molecule
 Important processes

Lipid Catabolism
– Lipids are first broken down into component fatty acids and
glycerols by lipases
– Each component can then enter the Krebs cycle

Protein Catabolism
– Proteases and peptidases break down proteins into
component amino acids
– AAs must undergo enzymatic conversion into substances
that can enter the Krebs cycle
 Metabolic Diversity

• Phototrophs – light as energy source

• Chemotrophs – redox of in/organic compounds

• Autotrophs – self-feeders
• Heterotrophs – feed on others

Most medically important organisms are

chemoheterotrophic, because typically, infectious organisms
catabolize substances obtained from the host
Nutritional classification of
organisms
Microbial Nutrition
 Physical Requirements

Temperature
– Psychrophiles – cold-loving
– Mesophiles – moderate temperatures
– Thermophiles – heat-loving

Most bacteria grow within a limited range of temperatures

– Min and max growth temps are only 30°C apart
– Optimum temperature - temperature at which the
species can best grow
 Typical growth rates in
response to temperature
 Physical Requirements

pH
– Most bacteria grow best between pH6.5-7.5
– Few bacteria grow below pH 4

When bacteria are cultured in the lab, they often

produce acids which interfere with their growth
– Chemical buffers such as phosphate salts and
peptones are included
 Physical Requirements

Osmotic pressure
– Microbes obtain nutrients in solution from water

Adaptations
– Extreme halophiles
– Obligate halophiles
– Facultative halophiles – do not require high salt
concentrations but can grow at concentrations up to
2%
 Chemical Requirements
Carbon
– Structural backbone of living matter; needed for all organic
compounds that make up a living cell

Nitrogen
– For forming the amino group of amino acids

Sulfur
– For synthesis of sulfur-containing amino acids and vitamins
such as thiamine and biotin
 Chemical Requirements
Phosphorus
– For the synthesis of nucleic acids and phospholipids of the cell
membrane; also in ATP

Trace Elements
– Essential for some enzymes, sometimes as cofactors
– Includes iron, copper, molybdenum, and zinc

Organic growth factors

– essential compounds not synthesized which are obtained from the
environment
– include enzymes for vitamin synthesis, amino acids, purines, pyrimidines
 Chemical Requirements

Oxygen
– Obligate aerobes
– Facultative anaerobes – can use anaerobic respiration or
fermentation when oxygen is absent, e.g. E. coli and yeasts
– Obligate anaerobes - cannot use molecular oxygen for
energy-yielding reactions, e.g. Clostridium
– Aerotolerant anaerobes – e.g. lactobacilli; they can survive
convert harmful forms of oxygen to O2
– Microaerophiles – can only tolerate oxygen concentrations
lower than air
Culture Media
 Culture Media
• Nutrient material prepared for the growth of microorganisms

• Microbes that are introduced into a culture medium to initiate

growth are called inoculum

• Microbes that grow and multiply in a culture medium is called

culture

• Culture media must be initially sterile

 Agar

• A solidifying agent
• Few microbes can degrade it
• Liquefies at 100°C
• Remains liquid until temperature drops to 40°C

• NOT a nutrient
 Forms of Culture Media

Broth
– liquid

Pellicle: A mass of organisms is

floating on top of the broth.

Turbidity: The organisms appear as a

general cloudiness throughout the
broth .

Sediment: A mass of organisms

appears as a deposit at the bottom of
the tube.
 Forms of Culture Media
Slants – tubes are held at
an angle

Stab tubes / deeps –

bacteria are inoculated by
stabbing the medium

Plates – use Petri dishes;

commonly used in the
culturing, separating, and
counting of microorganisms slant deep
 Types of Culture Media
Chemically-defined media
– medium whose exact chemical composition is known
– must contain organic growth factors that serve as source of
carbon and energy
– Best for autotrophs

Complex media
– made up of nutrients including extracts from yeasts, meat,
plants or digests of proteins from cheese and other sources
– E.g. nutrient broth or nutrient agar
 Types of Culture Media
Reducing media
– contain ingredients such as sodium thioglycolate that chemically
combine with dissolved oxygen and deplete the oxygen in the
culture medium
– when the culture must be grown in Petri plates to observe
individual colonies, special anaerobic jars are used

Selective media
– designed to suppress growth of unwanted bacteria and encourage
the growth of desired microorganisms
– E.g. bismuth sulfite agar that inhibits growth of gram-positive
bacteria
 Types of Culture Media

Differential media
– make it easier to
distinguish
colonies of the
desired organism
from other
colonies growing
on the same
plate
 Mannitol Salt Agar MacConkey Agar
both differential (distinguishes mannitol Differentiates from lactose fermenters (left)
fermenters and non-fermenters) and selective and non-fermenters
(high salt con’c prevents most bacteria except
Staphylococcus spp.)
 Types of Culture Media
Enrichment culture
– selective medium and designed to increase the small numbers of
chosen microbe to observable levels
– Usually liquid and provides nutrients and environmental conditions
that favor the growth of a particular microbe but not others

Special Culture Techniques

– Mycobacterium leprae are grown in armadillos
– Treponema pallidum is grown in rabbits
– Bird eggs and cultures of living cells are used to culture and study
obligate intracellular parasites like chlamydias, rickettsias and
viruses
 Obtaining pure cultures
Most commonly used method is the streak plate method
 Preservation

• Refrigeration for short-term storage

• Deep-freezing - pure culture of microbes is placed in a

suspending liquid and quick-frozen at temperatures
ranging from -50°C to -95°C.

• Lyophilization (freeze-drying) - suspension of microbes is

quickly frozen at temperatures ranging from -54°C to -
72°C, and the water is removed by a high vacuum
 Five “I”s of Culturing Microbes

1. Inoculation: Producing a pure culture

2. Isolation: Colony on media, one kind of microbe, pure culture

3. Incubation: growing microbes under proper conditions

4. Inspection: Observation of characteristics (data)

5. Identification: use of data, correlation, to ID organism to exact species

Microbial Growth
 Microbial Growth

produces more cells thus increases microbial count and

consequently microbial growth

Binary fission is the most common mode of reproduction

Budding is another mode of reproduction

 Generation time
Generation time is the time required for a cell to divide and its
population to double

– varies considerably among organisms and with environmental

conditions such as temperature

– most bacteria have a generation time of 1-3 hours while others

require 24 hours per generation

– If binary fission continues unchecked, an enormous number of

cells will be produced
 Generation Time

Calculation:
2 # 𝑜𝑓 𝑔𝑒𝑛𝑒𝑟𝑎𝑡𝑖𝑜𝑛𝑠

Final concentration:
𝐼𝑛𝑖𝑡𝑖𝑎𝑙 # × 2# 𝑔𝑒𝑛𝑒𝑟𝑎𝑡𝑖𝑜𝑛𝑠

For example, if 5 cells were allowed to divide 9 times,

this would result to: 5 x 29 = 2560 cells
 Generation Time
How to obtain the number of generations:

log # 𝑐𝑒𝑙𝑙𝑠 (𝑒𝑛𝑑) − log # 𝑐𝑒𝑙𝑙𝑠 (𝑠𝑡𝑎𝑟𝑡)

# 𝑜𝑓 𝑔𝑒𝑛𝑒𝑟𝑎𝑡𝑖𝑜𝑛𝑠 =
0.301
*0.301 is used because it is the log of 2 (1 cell divides into two)

Generation time:
𝑚𝑖𝑛
60 𝑋 ℎ𝑟
min 𝑝𝑒𝑟 𝑔𝑒𝑛𝑒𝑟𝑎𝑡𝑖𝑜𝑛 = ℎ𝑟
# 𝑔𝑒𝑛𝑒𝑟𝑎𝑡𝑖𝑜𝑛𝑠
 Generation Time

Calculate the generation time if 100 cells growing for 5 hours

produced 1,720, 320 cells

Log1,720,320 – log 100/0.301 = 14 generations

60 mins/hour x 5 hours
__________________ = 21 minutes/generation
14 generations
 Let’s Try!

Given:
900 of the species were grown.
After 15 hours, 3,276,800 cells were produced.

Calculate the generation time.

 Growth Phases

LAG PHASE
• period of little or no cell division
• last for an hour or several days but cells aren’t dormant
• happens because cells don’t immediately reproduce in a
new medium
• microbial population during this stage is undergoing a
period of intense metabolic activity involving synthesis of
enzymes and various molecules
 Growth Phases

LOG PHASE or EXPONENTIAL GROWTH PHASE

• cells begin to divide and enter a period of growth or
logarithmic increase
• cellular reproduction is most active during this period and
generation time reaches a constant minimum
• during this period, cells are most active metabolically
• microorganisms are particularly sensitive to adverse
conditions such as antibiotics
 Growth Phases

STATIONARY PHASE
• period of equilibrium where growth rate slows
down and the number of microbial death
balances the number of new cells and the
population stabilizes
• metabolic activities of individual surviving cells
also slow down at this stage
 Growth Phases

DEATH PHASE or LOGARITHMIC DECLINE

• Number of cells dying exceeds the cells that are
newly formed
• Continues until the population diminished or few
cells remain
 Direct Measurement of
Microbial Growth

Plate counting
– Measures # of viable cells
– Takes some time, e.g. after 24hrs
– Reported as colony-forming units (CFU)
– Serial dilutions are done to ensure that colonies on the
plate are countable (30-300 colonies per plate)
– After that, pour plate or spread plate is done
 Direct Measurement of
Microbial Growth
Filtration

– used when bacterial quantity is small

– at least 100ml of water is passed through a filter;
– bacteria are filtered out and remain of filter surface
– filter is then transferred to a plate
 Direct Measurement of
Microbial Growth
Most Probable Number (MPN)
– estimates the amount of bacteria present

– 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 useful when microbes being counted will not grow on solid
media; or when the growth of bacteria in liquid differential media
is used to identify microbes
MPN Table
 Direct Measurement of
Microbial Growth
Direct Microscopic Count
– measured volume of a bacterial suspension is placed within a
defined area on a microscope slide, e.g. 0.01ml sample

– Once the number of bacteria has been counted in several different

fields, the average number of bacteria per viewing field can be
calculated

– the number of bacteria in the square centimeter over which the

sample was spread can also be calculated
 Indirect Measurement
Turbidity
– using a spectrophotometer to determine absorbance (used
to plot bacterial growth)
Metabolic activity
– assumes that the amount of a certain metabolic product,
such as acid or CO2 , is in direct proportion to the number
of bacteria present
Dry weight
– the fungus/bacterium is removed from the growth medium,
filtered to remove extraneous material, dried in a desiccator,
and weighed.
Sterilization and
Anti-Microbial Methods
 Methods

Sterilization - the removal or destruction of all living

microorganisms

Disinfection – control of harmful organisms

– Antisepsis (antiseptic) if directed at living tissue

Sanitization - lower microbial counts to safe public

health levels and minimize the chances of disease
transmission
 Aseptic Technique

• To protect your self from contact with bio hazards

• To protect your sample from contamination
• To protect others in the lab
 Aseptic Technique

Aseptic technique is the process of:

– Preventing contamination of a culture with
environmental microbes
– Preventing contamination of yourself or the
environment with the organism in the culture
– Remember everything is contaminated with a
variety of environmental microbes.
 Control of Growth

• Moist heat
– Best for dishes, various equipment; autoclave for media and other
items that can withstand pressure
– Kills vegetative bacterial and fungal pathogens and almost all
viruses within 10 min; less effective on endospores
– Autoclaving - at about 15 psi of pressure (121°C), all vegetative
cells and their endospores are killed in about 15 min

• Pasteurization
– Heat treatment that kills all pathogens and most non-pathogens
– Best for food
 Control of Growth

• Dry heat
– Direct – e.g. in inoculating loops
– Incineration – e.g. for paper cups, dressings
– Hot-air sterilization – empty glassware, etc.

• Filtration
– Separation of bacteria from suspending liquid
– Useful for sterilizing liquids such as vaccines that can
be destroyed by heat
 Control of Growth

• Refrigeration
– Bacteriostatic
– Best for food preservation
• Deep-freezing and freeze-drying
• High pressure
• Desiccation
• Osmotic pressure
• Radiation
 Disinfection

• Unfortunately, few chemical agents achieve

sterility; most of them merely reduce microbial
populations to safe levels or remove vegetative
forms

• No single disinfectant is appropriate for all

circumstances
Disinfection
 Disinfectants
• Phenols and phenolics
– Triclosan is a common example
– Can be put in disinfectant hand soaps
– Disrupts plasma membranes

• Biguanides (chlorhexidine) – skin disinfection, esp. for surgical

hand scrubbing

• Halogens
– Chlorine forms –HOCl which alters cellular components
 Disinfection
• Alcohols
– Protein denaturation
– Useful for thermometers and other instruments

• Heavy metals – enzyme denaturation

• Surface-active agents
– Soaps and detergents for the skin
– Acid-anionic sanitizers are used in food industry
– Cationic detergents – useful for instruments
 Disinfection
• Aldehydes – protein denaturation

• Chemical sterilization
– for sterilization of materials that may be damaged by heat
– Also for sterilizing organic medical implants

• Peroxygens and other oxygen forms

– ozone is a supplement for chlorination
– Hydrogen peroxide is a poor antiseptic but a good
disinfectant