CONTROLLING MICROBIAL GROWTH IN VITRO
LEARNING TARGETS
1. Factors that Affect Microbial Growth
2. Encouraging the Growth of Microbes of in
Vitro
3. Inhibiting the Growth of Microbes in Vitro
FACTORS THAT AFFECT MICROBIAL GROWTH
 Availability of Nutrients
 Moisture
 Temperature
 pH
 Osmotic Pressure and Salinity
 Barometric and Gaseous Atmosphere
AVAILABILITY OF NUTRIENTS
 All living organisms require nutrients to
sustain life.
 Nutrients are energy sources. Organisms
obtain energy by breaking chemical bonds.
MOISTURE
 Water is essential for life. It is needed to
carry out normal metabolic processes.
 Certain microbial stages can survive a
drying process called “desiccation”
(e.g. bacterial endospores and protozoal
cysts
TEMPERATURE
 Every organism has an optimum growth
temperature.
 The temperature (and pH) ranges over
which as organism grows best are largely
determined by its enzymes.
 THERMOPHILES are microorganisms that
grow best at high temperatures.
 MESOPHILES are microbes that grow best
at moderate temperatures (e.g. 37 degrees
Celsius).
 PSYCHROPHILES prefer
temperatures (like deep ocean water.
 Psychrotrophs- a particular group of
pschrophiles, a prefer refrigerator
temperature (4 degrees Celsius.)
 PSYCHRODURIC organisms prefer warm
temperatures, but can endure very cold or
even freezing temperatures.
pH
 pH refers to the acidity alkalinity of a
solution.
 Most microorganisms prefer a neutral or
slightly alkaline growth medium (pH 7.0-
7.4)
 ACIDOPHILES- pH of 2 to 5
 ALKALIPHILES- pH > 8.5
OSMOTIC PRESSURE AND SALINITY
 OSMOTIC PRESSURE is the pressure that
is exerted on a cell membrane by solutions
both inside and outside the cell.
 OSMOSIS is the movement of a solvent,
through a permeable membrane, from a
lower concentration of solutes (dissolved
substances) to a higher concentration of
solutes.
 When the concentration of solutes in the
external environment of a cell is greater
than that of solutes inside the cell, the
solution in which the cell is suspended is
said to be HYPERTONIC.
 PLASMOLYSIS is a condition in which the
cell membrane and cytoplasm of a cell
shrink away from the cell wall; occurs when
bacteria with rigid cell walls are placed into
hypertonic solution.
 When the concentration of solutes outside a
cell is less than that of solutes inside a cell,
the solution in which the cell is suspended is
said to be HYPOTONIC.
 If a bacterial cell is placed into hypotonic
solution, it may not burst because of the
RIGID CELL WALL.
 A solution is said to be ISOTONIC when the
concentration of solutes outside a cell
equals the concentration of solutes inside
the cell.
 Organisms that prefer to live in salty
environments are called HALOPHILIC
organisms; HALODURIC do not prefer salty
environment but capable of surviving it.
cold
BAROMETRIC AND GASEOUS ATMOSPHERE  Mycobacterium tuberculae: every 18 to
24hrs
 Barometric pressure is also known as
 Treponema pallidum- DO NOT grow on
ATMOSPHERIC PRESSURE; refers to the
artificial media. “FASTIDIOUS” – very
pressure within the atmosphere of Earth=
complicated nutritional requirements
14.7psi
 Certain microbes such as PIEZOPHILE
I. CULTURE MEDIA
(Thermococcus piezophilus) are capable of
Used in microbiology laboratories to culture
living in the deepest parts of the ocean and
bacteria are referred to as ARTIFICIAL
in oil wells.
MEDIA or SYNTHETIC MEDIA.
 Microorganisms vary with respect to the
type of CASEOUS ATMOSPHERE that they
CHEMICALLY DEFINE MEDIUM
require.
- Is done in which all
a. OBLIGATE AEROBES- prefer the
ingredients are known
same atmosphere that humans do
 Carbohydrates
b. MICROAEROPHILES- require
reduced concentration of oxygen  Amino Acid
(around 5%)  Salts
c. CAPNOPHILES- grow best in COMPLEX MEDIUM
environments rich on carbon dioxide - Is one which the exact
contents are not known.
ENCOURAGING THE GROWTH OF MICROBES  Vitamins
OF IN VITRO  Minerals
CULTURING OF BACTERIA IN THE  Nutrients
LABORATORY - Categories
 Liquid Media- “broth”
 This has been the way to obtain  Solid Media- pure
BACTERIAL GROWTH in order determine culture, “agar”
which organisms are “normal flora” and CATEGORIES OF CULTURE MEDIA
which bacteria are “bad”, causing disease or 1. ENRICHED MEDIUM is a broth or
infection. medium containing a rich supply of
 CULTURING allows “bad” bacteria to be special nutrients that promotes the
tested for susceptibility to certain antibiotics. growth of fastidious organisms.
 Think of BACTERIAL GROWTH as an a. Chocolate Agar (nutrient agar +
increase in the number of organisms rather powdered hemoglobin) support
than an increase in their size. the growth of Neisseria
 Bacteria reproduce by BINARY FISSION gonorrhea
(one cell divides to become two cells). b. Blood agar (nutrient agar + 5%
BINARY FISSION continues until a colony sheep red blood cells)
is produced. BINARY FISSION continues 2. SELECTIVE MEDIUM are used for the
for as long as there is a sufficient supply of growth of only selected microorganisms
nutrients, water and space. such as Salmonella typhi
 The time it takes for one cell to become two 3. DIFFERENT MEDIUM- allows one to
cells is called the generation time (e.g. , reality differentiate among the various
coli= 20 minutes.) types of organisms that are growing on
 Under IDEAL growth conditions: the medium.
a. Escherichia coli a. Ex. Lactose is added in
b. Vibrio cholerae MacConkey agar, E. Coli is
c. Streptococcus spp. lactose fermenting bacteria
d. Staphylococcus spp. produce pink colonies on
All of this have a generation time of 20 MacConkey plate while on
minutes. lactose fermenting Salmonella
 Pseudomonas and Clostridium: 10 minutes
 typhi produce pale colonies on
MacConkey.
II. INOCULATION OF CULTURE MEDIA
 From Latin word “INOCULARE”
which means “implant or introduce
microorganisms or infectious
material into a culture medium for
growth of microorganism”; are
routinely inoculated with clinical
specimens.
 Inoculation involves adding a portion
of a specimen to the medium.
 Inoculation is accomplished using a
sterile inoculating loop.
III.IMPORTANCE OF USING “ASEPTIC
TECHNIQUE”
 ASEPTIC TECHNIQUE is practiced
when its is necessary to exclude
microbes when inoculating culture
media.
 Unwanted organisms are referred to
as CONTAMINANTS; the growth
medium or plate is said to be
contaminated.
 The sterility of the media must be
maintained before inoculation.
“Avoid touching the surface of the
agar”
ASEPTIC TECHNIQUE IS PRACTICED TO
PREVENT:
1. Microbiology professionals from becoming
infected.
2. Contamination of their work environment,
and
3. Contamination of clinical specimens,
cultures and subcultures.
BSC (Biology Safety Cabinet) – to minimize the
possibility of contamination and protects the
laboratory worker from becoming infected with the
organisms that he or she is working with.
IV. INCUBATION
- After media are inoculated, they
must be placed into an
INCUBATOR which will maintain
the appropriate ATMOSPHERE,
TEMPERATURE and MOISTURE
LEVEL.
3 TYPES OF INCUBATOR IN CLINICAL
MICROBIOLOGY LABORATORY:
1. C02 INCUBATOR- contains 5-10%
CO2
2. NON-CO2 INCUBATOR- contains
room air
3. ANAEROBIC INCUBATOR- the
atmosphere is devoid of O2
BACTERIAL GROWTH POPULATION CURVE
 A POPULATION GROWTH CURVE for any
particular species of bacterium may be
determined by growing a pure culture of the
organism in a liquid medium at a constant
temperature.
 Samples of the culture are collected at fixed
intervals to determine the number of viable
organisms.
 A graph is prepared by plotting the
logarithmic number of viable organism (on
the vertical or y-axis) against the incubation
time (on the horizontal or x-axis).
DEFINITION OF TERMS
1. STERILIZATION is the complete destruction
of all microbes, including cells, spores, and
viruses.
2. DISINFECTION is the destruction or
removal of pathogens from nonliving objects
by physical or chemical methods;
pasteurization is an example of a
disinfection technique.
3. DISINFECTANTS are chemical substances
that eliminate pathogens on inanimate
objects.
4. ANTISEPTICS are solutions used to
disinfectant skin and other living tissues.
THE SUFFIX -CIDE OR -CIDAL REFERS TO
“KILLING”
  GERMICIDAL AGENTS, BIOCIDAL
AGENTS and MICROBICIDAL AGENTS
are chemicals that kill microbes.
 BACTERICIDAL AGENTS are chemicals
that specifically kill bacteria, but not
necessarily bacterial endospores.
 SPORICIDAL AGENTS kill bacterial
endospores.
 FUNGICIDAL AGENTS kill fungi, including
fungal spores.
 ALGICIDAL AGENTS kill algae.
 VIRCIDAL AGENTS destroy viruses.
5. MICROBISTATIC AGENT is a drug or
chemical that inhibits growth and
reproduction of microbes.
6. BACTERIOSTATIC AGENT is one that
specifically inhibits the metabolism and
reproduction of bacteria
7. SEPSIS refers to the presence of
pathogens in blood or tissues, whereas
ASEPSIS means the absence of pathogens.
8. ANTISEPSIS is the prevention of infection
9. LYOPHILIZATION is a process that
combines dehydration (drying) and freezing.
This process is widely used in industry to
preserve foods, antibiotics, microorganisms,
and other biologic materials.
USING PHYSICAL METHOD TO INHIBIT
MICROBIAL GROWTH
PHYSICAL METHODS commonly used in
hospitals, clinics and laboratories to destroy or
control pathogens include heat, the combination of
heat and pressure, desiccation, radiation sonic
disruption and filtration.
HEAT is the most common type of sterilization for
inanimate objects that can withstand high
temperature.
TYPE OF HEAT:
1. Dry Heat (oven, electrical incinerators or
flame)
2. Moist Heat (boiling or autoclave)
TWO FACTORS: TIME and TEMPERATURE
a. Determine the effectiveness of heat
sterilization.
b. The higher the temperature the shorter the
time required to kill the organism.
THERMAL DEATH POINT (TDP) of any species is
the lowest temperature that will kill all of the
organisms in a standardized pure culture within a
specified time.
AUTOCLAVE
- A large metal pressure cooker that uses
steam under pressure tom completely
destroy all microbial life.
- Increased pressure raises the
temperature above the temperature of
boiling water (above 100 degrees
celsius) and forces steam into materials
being sterilized.
- Autoclaving at a pressure of 15 psi at
121.5 degrees celsius for 20 minutes
destroys vegetative microorganism,
bacterial endospores and viruses
- Can use pressure-sensitive tape or spore
strips or solutions as a quality control
measure to ensure proper autoclaving.
OTHER METHODS:
1. DESICCATION the state of drying, refers to
the drying out of a living organism
2. RADIATION energy that comes from a
source and travels through space at the
speed of light; SUN is not dependable
disinfecting agent
3. ULTRASONIC WAVES used means in
cleaning delicate equipment in hospitals,
medical and dental clinics.
4. FILTRATION used to filter or separate cells,
larger viruses, bacteria and other microbes
from liquids or gases
USING CHEMICAL AGENTS TO INHIBIT
MICROBIAL GROWTH
Chemical disinfection refers to the use of
CHEMICAL AGENTS to inhibit the growth of
pathogens, either temporarily or permanently.
DISINFECTANTS are affected by:
 Prior cleaning of the object or surface
 The organic load (e.g. feces, blood, pus)
 The bioburden; types and numbers of
microbes
 Concentration of the disinfectant
 Contact time
  Physical nature of the object being
disinfected
 Temperature and pH

FACTORS TO CONSIDER:
 Should have a broad antimicrobial spectrum
 Fast ing Not affected by the presence of
organic matter
 Nontoxic to human tissues and
noncorrosive
 Should leave a residual antimicrobial film on
surface soluble in water and easy to apply
inexpensive and easy to prepare
 Stable as both a concentrate and a working
solution
 Odorless
ANTISEPTICS
 May safely be used on human tissues.
 Reduce the number of organisms on the
surface of the skin; do not penetrate pores
and hair follicles.
 Antiseptic soaps and scrubbing are used by
healthcare personnel to remove organisms
lodged in pores or folds of the skin.

“Control of microorganisms is essential in order

prevent the transmission of disease and infection,
stop decomposition and spoilage, and prevent
unwanted microbial contamination.”