Control of Microbial Growth

In vitro
 Definitions
• Sterilization: destruction of all forms of
microbial life (including endospores).
• Disinfectant: destruction of vegetative
pathogens (does not include endospores).
• Antiseptic: destruction of vegetative
pathogens on living tissue (not endospores).
• -cide: this suffix means, kills
– Ie. Germicide: kills germs
• biocide: kills life
• -stasis or static: inhibits bacterial growth.
– Ie. Bacteriostatic: inhibits growth of bacteria.
• Main targets that are used to control
microbial growth are bacterial cell wall,
plasma membrane, DNA, RNA, and protein
synthesis.
• Sepsis – presence of pathogens in
tissues/blood; asepsis – absence
• Antisepsis – prevention of infection
 Antimicrobial methods
• Ways to destroy or inhibit microbial growth which are categorized as Physical, Chemical or Both
• Effectiveness are affected by the following factors:
– Length of time
– Temperature
– Concentration
– Nature and number of microbes (bioburden)
– Presence of organic load
– Prior cleaning
 Methods of Sterilization
• Osmotic Pressure: beef jerky (addition of salt or sugar).
• Radiation (UV light): UV light causes lethal mutations in
DNA when exposed for long periods of time. It is
frequently used in virology labs to sterilize rooms where
viruses are transferred from old media to fresh media. It is
also used to sterilize equipment such as goggles.
• Filtration: air filters are used in hospitals to keep patients
from contracting disease while there.
– especially important in the burn unit
– Some liquids are heat sensitive and can be damaged if exposed to
the temperatures needed to sterilize it. Filters are used to sterilize
those too.
• Dry heat: means using an open flame to kill
organisms, for example.
– Direct flame
– Incineration
• Moist heat:
– It is able to reach very hot temperatures and is very
effective in killing many organisms and endospores.
– Autoclaves are found in many places. They are used to
sterilize surgical equipment, gowns, towels, media, test
tubes, etc.
• Cold
– Inhibits their growth; lowers their metabolic activities
– Lyophilization
• Dessication
– Drying; many microbes can’t reproduce when they
have been dried
• Some Chemicals: Formaldehyde,
glutaraldehyde
– Most chemicals are not considered sterilizing
agents.
– Formaldehyde and glutaraldehyde are so
effective in killing organisms that they are
included as sterilizing agents.
– They work by inhibiting enzyme function.
– Generally these chemicals are used to disinfect
instruments or materials that may be damaged
by heat. They are also used to preserve tissue
so that it can be studied.
 Types of Disinfectants and
Antiseptics
• Most chemicals have a particular concentration and
length of exposure to microbes, to be most effective.
– For example, isopropyl alcohol is frequently used in the
clinic when administering injections or drawing blood. It is
most effective when the solution is 65-75% alcohol. Some
stores sell 95% alcohol and consumers buy it because they
think that the greater the concentration, the better.
– It evaporates quickly.
– The other challenge with alcohol is that it denatures (or
inactivates) protein but does not remove it very well. So if a
surface is particularly dirty the alcohol just denatures the
protein and leaves it on the surface.
• Triclosan: common antibacterial agent added to
soaps, cutting boards and other products.
– Triclosan is a pretty effective antibacterial agent
(antiseptic) but it is being overused. A decreased
sensitivity is being seen in several microorganisms
including Staphylococcus.
– Triclosan is what is added to antibacterial hand soaps.
It kills the organisms but the soap leaves a residue of
triclosan in the sink. As the organisms are continually
exposed to the triclosan it gives them opportunities to
develop resistance.
– In addition, there is no need, particularly in a home, to
use antibacterial soap. Ordinary hand soap removes the
organisms just as effectively and does not needlessly
use an extra chemical to which microbes are now
growing resistant.
• Chlorhexadine: used for surgical hand scrubs
and pre-operative skin preparation as well as other
uses.
– This chemical damages the plasma membrane and
causes protein denaturation.
– In medium to high concentrations it is very effective
against vegetative pathogens but not against
endospores.
– An advantage to using chlorhexadine is that it has a
very low toxicity. That means that it can be used in
large or amounts as an antiseptic without being toxic.
• Halogens: iodine, chlorine
– Effective against all kinds of bacteria and endospores,
various fungi, and viruses.
– This group interferes with protein synthesis and
folding.
– Chlorine can be used as an antiseptic or a disinfectant
depending upon the form in which it is used.
• It is used to treat drinking water and swimming pools for
example.
• In higher concentrations it is used to disinfect equipment,
bedding, etc.
– Iodine is used as a disinfectant to sterilize cutting
blades, plastic, and rubber items. (It’s exact method of
killing microorganisms is not really understood yet.)
• It is also used as an antiseptic for wound care.
• Tincture of iodine: combination of iodine and alcohol
• An iodophore is a combination of iodine and an organic
molecule that allows iodine to be released slowly.
– The best example I can think of is those tablets they sell in the
pet stores for feeding fish while your on vacation. The longer
the tablet sits in the water, the more the outer coating is exposed
to the water and wears it away. As it is worn away, the food is
gradually released.
• Hydrogen Peroxide: decomposes in the
presence of light, metals, or catalase into
water and oxygen gas.
– That is why it is stored in dark bottles, to
prevent its decomposition.
– It is more stable now than used to be.
– It is highly toxic to cells that don’t have the
enzyme, catalase.
– Bactericidal, virucidal, fungicidal, and can be
sporicidal
• Surface-active agents (surfactants): soaps and
detergents
– Surfactants disrupt cell membranes, more useful in
killing microbes.
– Not an antiseptic but does bind to org. to remove from
skin.
 Characteristics of ideal chemical
antimicrobial agents
• Should have a broad spectrum
• Fast acting
• Not affected by the bioburden
• Non toxic and non corrosive
• Should leave a residual antimicrobial film
• Soluble in water and easy to apply
• Inexpensive
• Odorless
 Temperature and Microbial Growth
• Cardinal temperatures
– minimum
– optimum
– maximum
• Temperature is a major
environmental factor
controlling microbial
growth.

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 Temperature
• Minimum Temperature: Temperature below which
growth ceases, or lowest temperature at which
microbes will grow.
• Optimum Temperature: Temperature at which its
growth rate is the fastest.
• Maximum Temperature: Temperature above
which growth ceases, or highest temperature at
which microbes will grow.

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Classification of Microorganisms by
Temperature Requirements

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 Temperature Classes of Organisms
• Mesophiles ( 20 – 45C)
– Midrange temperature optima
– Found in warm-blooded animals and in terrestrial and
aquatic environments in temperate and tropical latitudes
• Psychrophiles ( 0-20C)
– Cold temperature optima
– Most extreme representatives inhabit permanently cold
environments
• Thermophiles ( 50- 80C)
– Growth temperature optima between 45ºC and 80ºC
• Hyperthermophiles
– Optima greater than 80°C
– These organisms inhabit hot environments including
boiling hot springs, as well as undersea hydrothermal vents
that can have temperatures in excess of 100ºC

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 Classification of organisms based on O 2
utilization

• Obligate (strict) aerobes require O2 in order to grow

• Obligate (strict) anaerobes cannot survive in O 2
• Facultative anaerobes grow better in O2
• Aerotolerant organisms don’t care about O2
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• Microaerophiles require low levels of O2
 Oxygen and Microbial Growth
• Aerobes :
– Obligate : require oxygen to grow
– Facultative : can live with or without oxygen but
grow better with oxygen
– Microaerphiles : require reduced level of oxygen

• Anaerobes :
– Aerotolerant anaerobes : can tolerate oxygen but
grow better without oxygen.
– Obligate : do not require oxygen. Obligate
anaerobes are killed by oxygen
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 Test for Oxygen Requirements of
Microorganisms
Thioglycolate broth :
contains a reducing agent
and provides aerobic and
anaerobic conditions
Which Thio broth shows
obligate aerobe,
anaerobe, aerotolerant,
microaerophile,
facultative?

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 Culture Media: Composition
• Culture media supply the nutritional needs of
microorganisms ( C ,N, Phosphorus, trace elements, etc)
– defined medium : precise amounts of highly purified
chemicals
– complex medium (or undefined) : highly nutritious
substances.
• In clinical microbiology,
– Selective : contains compounds that selectively inhibit
– Differential: contains indicator
– terms that describe media used for the isolation of particular
species or for comparative studies of microorganisms.
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 Types of Media
• Media can be classified on three primary
levels
1. Physical State
2. Chemical Composition
3. Functional Type

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 Physical States of Media
• Liquid Media
• Semisolid
• Solid (Can be converted into a liquid)
• Solid (Cannot be converted into a liquid)

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 Liquid Media
• Water-based solutions
• Do not solidify at temperatures above
freezing / tend to be free flowing
• Includes broths, milks, and infusions
• Measure turbidity
• Example: Nutrient Broth, Methylene Blue
Milk, Thioglycollate Broth
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 Semi-Solid Media
• Exhibits a clot-like consistency at ordinary
room temperature
• Determines motility
• Used to localize a reaction at a specific site.
• Example: Sulfide Indole Motility (SIM) for
hydrogen sulfide production and indole
reaction and motility test.
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 Solid Media
• Firm surface for discrete colony growth
• Advantageous for isolating and culturing
• Two Types
1. Liquefiable (Reversible)
2. Non-liquefiable
• Examples: Gelatin and Agar (Liquefiable)
Cooked Meat Media,
Potato Slices (Non-liquefiable)
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Chemical Composition of Culture Media

1. Synthetic Media
• Chemically defined
• Contain pure organic and inorganic compounds
• Exact formula (little variation)
2. Complex or Non-synthetic Media
• Contains at least one ingredient that is not
chemically definable (extracts from plants and
animals)
• No exact formula / tend to be general and grow a
wide variety of organisms 40
 Selective Media
• Contains one or more agents that inhibit the
growth of a certain microbe and thereby
encourages, or selects, a specific microbe.
• Example: Mannitol Salt Agar [MSA]
encourages the growth of S. aureus. MSA
contain 7.5% NaCl which inhibit the growth
of other Gram +ve bacteria

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Growth of Staphylococcus aureus on
Mannitol Salt Agar results in a color change
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in the media from pink to yellow.
 Differential Media
• Differential shows up as visible changes or
variations in colony size or color, in media color
changes, or in the formation of gas bubbles and
precipitates.
• Example: Spirit Blue Agar to detect the digestion of
fats by lipase enzyme. Positive digestion
(hydrolysis) is indicated by the dark blue color that
develops in the colonies. Blood agar for hemolysis
(α,β,and γ hemolysis), EMB, MacConkey Agar, …
etc. 44
Growth of Staphylococcus aureus on
Manitol Salt Agar results in a color change
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in the media from pink to yellow.
MSA
Eosin-Mehtylene blue agar

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 Enrichment Media
• Is used to encourage the growth of a
particular microorganism in a mixed
culture.
• For fastidious microorganisms
• Ex. Manitol Salt Agar for S. aureus
• Blood agar , chocolate agar

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 Bacterial Colonies on Solid Media

P. aeruginosa (TSA)

S. Marcescens (Mac)
S. Flexneri (Mac)

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Laboratory Culture of Microorganisms
• Microorganisms can be grown in the
laboratory in culture media containing the
nutrients they require.
• Successful cultivation and maintenance of
pure cultures of microorganisms can be
done only if aseptic technique is practiced
to prevent contamination by other
microorganisms.

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 Microbial growth

• Microbes grow via binary fission, resulting in exponential

increases in numbers
• The number of cell arising from a single cell is 2n after n
generations
• Generation time is the time it takes for a single cell to grow
and divide 54
 Growth curve

• During lag phase, cells are recovering from a period of no

growth and are making macromolecules in preparation for
growth
• During log phase cultures are growing maximally
• Stationary phase occurs when nutrients are depleted and wastes
accumulate (Growth rate = death rate)
• During death phase death rate is greater than growth rate
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Methods used to measure microbial growth

• Count colonies on plate or filter (counts

live cells)
• Microscopic counts
• Flow cytometry (FACS)
• Turbitity

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 Viable counts

• Each colony on plate or filter arises from single live cell

• Only counting live cells
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Direct Count
Pour Plate

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Direct Count
Spread or
Streak Plate

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 Microscopic counts

• Need a microscope, special slides, high power

objective lens
• Typically only counting total microbe numbers, but
differential counts can also be done
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Turbitity

• Cells act like large particles

that scatter visible light
• A spectrophotometer sends a
beam of visible light through
a culture and measures how
much light is scattered
• Scales read in either
absorbance or % transmission
• Measures both live and dead
cells

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 Inoculation
• Sample is placed on sterile medium providing
microbes with the appropriate nutrients to
sustain growth.

• Selection of the proper medium and sterility of

all tools and media is important.

• Some microbes may require a live organism or

living tissue as the inoculation medium. 64
 Incubation
• An incubator can be used to adjust the proper
growth conditions of a sample.

• Need to adjust for optimum temperature and gas

content.

• Incubation produces a culture – the visible

growth of the microbe on or in the media
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 Isolation
• The end result of inoculation and incubation is
isolation.

• On solid media we may see separate colonies, and

in broth growth may be indicated by turbidity.

• Sub-culturing for further isolation may be

required.
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 Inspection
• Macroscopically observe cultures to note color,
texture, size of colonies, etc.

• Microscopically observe stained slides of the

culture to assess cell shape, size, and motility.

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 Identification
• Utilize biochemical tests to differentiate the
microbe from similar species and to determine
metabolic activities specific to the microbe.

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