Chapter 8

Controlling
Microbial Growth
In Vitro
 Factors that Affects
Bacterial Growth
• Availability of nutrients
• Moisture
• Temperature
• pH
• Osmotic pressure and salinity
• Barometric pressure
• Gaseous atmosphere
Availability of Nutrients

• All living organisms require nutrients to

sustain life.
• To survive, appropriate nutrients must
be available.
• Catabolism and anabolism
• Essential nutrients, elements and trace
elements
• About 25 of the 92 naturally occurring
elements are essential to life.
Moisture

• Water is essential to life.

• Cells consist of 70-95% water.
• Water is required to carry out normal
metabolic processes.
• Endospores and cysts can survive
complete drying process (desiccation).
Temperature

• Optimum growth temperature

• Minimum growth temperature
• Maximum growth temperature
• Thermophiles
• Mesophiles
• Psychrophiles
– Psychrotrophs- refrigerator temperature
– Psychroduric organism- can endure
freezing temperature
pH

• Acidity or alkalinity
• Most microorganism prefer a neutral or
slightly alkaline medium (pH 7-7.4)
• Most bacteria grow between pH 6.5 and 7.5
• Molds and yeasts grow between pH 5 and 6
• Acidophiles
• Alkaliphiles
• Vibrio cholerae is the only human pathogen
that grows well above pH 8.
Osmotic Pressure and Salinity
• Osmotic pressure- pressure that is exerted on a
cell membrane by solutions both inside and
outside the cell.
• Osmosis
• Hypertonic
• Crenation
• Plasmolysis
• Desiccation
• Hypotonic
• Hemolysis
• Plasmoptysis
• Isotonic
• Halophilic and haloduric organisms
Plasmolysis
Barometric Pressure

• Most bacteria are not affected by minor

changes in barometric pressure.
• Some thrive at normal atmospheric
pressure (about 14.7 psi).
• Barophiles- thrive deep in the ocean
and in oil wells, where the atmospheric
pressure is high.
Gaseous Atmosphere

• Oxygen (O2)

Obligate Facultative Obligate Aerotolerant

Microaerophiles
aerobes anaerobes anaerobes anaerobes
 Encouraging the Growth of
Microorganism In Vitro
• Gather information in the identification of
any pathogens present.
• Learn more about microorganisms.
• Harvest antibiotics and other microbial
products.
• Test new antimicrobial agents and
produce vaccines.
• Viruses, bacteria, fungi and protozoa,
with emphasis on bacteria.
 Culturing Bacteria in the
Laboratory

• petri dishes
• test tubes
• bunsen burners/alcohol lamps
• wire inoculating loops
• bottles of staining reagents
• incubators
Bacterial Growth

Microbial growth = increase in

number of cells, not cell size
Generation Time

• The time it takes for one cell to become two

cells by binary fission.
– Rapid growers (short GT)
– Slow growers (long GT)
• E. coli, V. cholerae, Staphylococcus and
Streptococcus- 20 mins.
• Pseudomonas and Clostridium- 10 mins.
• M. tuberculosis- 18 to 24 hours
Culturing Bacteria

• Fastidious- with complex nutritional

requirements
• Using culture media
• Obligate intracellular parasites- do
not grow in culture media
• Treponema pallidum and
Mycobacterium leprae
Culture Media

• Artificial media or synthetic media-

they are prepared in the laboratory
• Culture medium- nutrients prepared for
microbial growth
• Inoculation- introduction of microbes
into medium
• Culture- microbes growing in/on culture
medium
 Classification of Culture Media Based on
Whether the Exact Contents are Known

• Chemically defined media- exact

chemical composition is known
• Complex media- exact contents are not
known, from extracts and digests of
yeasts, meat, or plants
Liquid and Solid Media

• Liquid media- or broths are contained

in tubes, referred to as tubed media.
• Solid media- prepared by adding agar
to liquid media and then poured into test
tubes or petri dishes, where the media
solidifies.
– Agar plate
– Agar slant
– Agar butt/deep
Enriched Medium

• Broth or solid medium containing rich

supply of special nutrients that promotes
the growth of fastidious organisms.
• Prepared by adding extra nutrients to a
medium called nutrient agar.
• Blood agar and chocolate agar
• N. gonorrhoeae and H. influenzae
Selective Medium
• Has added inhibitors that discourage the
growth of certain organisms without inhibiting
growth of the organism being sought.
• MacConkey agar- inhibit growth of Gram (+)
bacteria and is selective for Gram (-) bacteria.
• Phenylethyl alcohol agar (PEA) and
colistin-nalidixic acid agar (CNA)- inhibit
growth of Gram (-) bacteria.
• Thayer-Martin agar and Martin-Lewis agar-
selective for N. gonorrhoeae.
• Mannitol salt agar (MSA)- only for salt-
tolerant (haloduric) bacteria
Differential Medium
• Permits the differentiation of organisms that
grow on the medium.
• MacConkey agar- used to differentiate
various Gram (-) bacilli that are isolated from
fecal spcimens.
– Gram (-) bacteria are able to ferment lactose
produces pink colonies, those are unable to
ferment lactose produce colorless colonies.
– Differentiates between LF and NLF Gram (-)
bacteria.
• Mannitol salt agar- used to screen for S.
aureus, pink to yellow.
Remember…
• Various categories of media are not mutually
exclusive.
• Ex: blood agar is enriched and differential
• MacConkey agar and MSA are selective and
differential
• PEA and CNA are enriched and selective
• Thayer-Martin and Martin-Lewis are highly
enriched and highly selective
• Thioglycollate broth (THIO) is a liquid
medium that supports the growth of all
categories of bacteria.
In the history…

• Robert Koch- described his culture

techniques in 1881.
• Fanny/Frau Hesse- suggested the use of
agar.
• Richard Julius Petri- invented the glass
Petri dishes.
• Joseph Lister- the first person to obtain a
pre culture of bacterium (Streptococcus
lactis) in a liquid medium.
Inoculation of Culture Media

• Inoculation- adding a portion of the

specimen to the medium.
• Inoculaton of a solid or plated medium
involves the use of sterile inoculating
loop to apply a portion of the specimen
to the surface of the medium; a process
commonly referred to as “streaking”.
Streaking the Agar Plate
 Importance of Using
“Sterile Technique”

• Necessary to exclude all microorganisms

from a particular area, so that area will be
sterile.
• Media should remain sterile before
inoculation.
• Contaminants- unwanted microorganisms
• Contaminated- if the sample contains
contaminants
Incubation

• After media are inoculated, they must be

incubated, and placed in a chamber
(incubator).
• To culture most human pathogens, the
incubator is set at 35 – 37 o C
• Carbon dioxide incubator – 5 to 10%, is
used to isolate capnophiles
• Non-carbon dioxide incubator – 20 to 21 %
of Oxygen
• Anaerobic incubator
Bacterial Population Counts

• Determine the total number of bacterial

cells in the liquid
• Determine the number of viable cells
• Spectrophotometer
• Viable plate count
– Is used to determine the number of viable
bacteria in a liquid sample such as milk,
water, ground food diluted in water, or broth
culture.
 Spectrophotometer
• Turbidity
Viable Plate Count

• Number of colonies must be multiplied

by the dilution factors.
• If 220 colonies were counted on the
agar plate that had been diluted with a
1.0-ml sample of a 1:10,000 dilution,
there were:
• 220 X 10,000= 2,200,000 bacteria/ml
 Viable Plate Count

• Plate Counts: Perform serial dilutions of

a sample
 Viable Plate Count

• Inoculate Petri
plates from
serial dilutions
 Viable Plate Count
• After incubation, count colonies on plates that have
25-250 colonies.
Bacterial Population Growth Curve

• Determined by growing a pure culture of

the organism in a liquid medium at a
constant temperature.
• Data are plotted on a graphic paper,
plotting the logarithm (log10) of the
number of viable bacteria (y-axis)
against the incubation time (x-axis).
Bacteria Population Growth Curve
Phases of the Growth Curve
• Lag phase- during which the bacteria absorb
nutrients, synthesize enzymes, and prepare
for cell division, the bacteria do not increase in
number.
• Log phase- exponential growth phase;
bacteria multiply so rapidly that the number of
organisms double with each generation time.
• Stationary phase- the number of bacteria
that are dividing equals the number that are
dying; greatest population density.
• Death/decline phase- culture may die
completely
Bacteria Population Growth Curve
Culturing Obligate Intracellular
Pathogens in the Laboratory
Culturing Fungi in the Laboratory

• Brain Heart Infusion Agar

• Sabouraud Dextrose Agar- pH 6.5
selective for fungi
 Culturing Protozoa in the
Laboratory
• Acanthamoeba spp.
• Entamoeba hisolytica
• Balamuthia spp.
• Giardia lamblia
• Leishmania spp.
• Trypanosoma cruzi
• Toxoplasma gondii
• Trichomonas vaginalis
• Naegleria fowleri
 Inhibiting the Growth of
Microorganism In Vitro

• Sterilization
– Dry heat
– Autoclaving (steam under pressure)
– Gas (ex. ethylene glycol)
– Various chemicals (formaldehyde)
– Radiation (UV, gamma rays)
 Disinfection, Pasteurization,
Disinfectants, and Sanitization

• Disinfection- removal of pathogens from

nonliving objects by physical or chemical
methods. Ex. Pasteurization
• Disinfectants- are strong chemical
substances that cannot be used on living
tissue.
• Antisepsis- removal of pathogens from living
tissue
• Sanitization- lower microbial counts on eating
utensils
 Microbial Agents
• Biocidal agents/ Germicidal agents/ Microbicidal
agents- are disinfectants that kill microbes
• Bactericidal agents- disinfectants that specifically kill
bacteria but not necessarily bacterial endospores.
• Sporicidal agents- to kill bacterial endospores
• Fungicidal agents- to kill fungi, including fungal
spores
• Algicidal agents- to kill algae in swimming pools and
hot tubs.
• Viricidal agents- destroy viruses
• Pseudomonicidal agents- Pseudomonas species
• Tuberculocidal agents- kill M. tuberculosis
 Microbistatic Agents
• Microbistatic agent- is drug or
chemical that inhibits growth and
reproduction of microorganism
• Bacteriostatic agents- is one that
specifically inhibits the metabolism and
reproduction of bacteria.
• Lyophilization- is a process that
combines dehydration and freezing.
– To preserve foods, antibiotics, anti-sera,
microorganisms
 Sepsis, Asepsis, Aseptic Technique,
Antisepsis, and Antiseptic Technique
• Sepsis­  refers  to  microbial  contamination  or 
presence of pathogens in blood or tissues
• Asepsis­  is  the  absence  of  significant 
contamination.
• Aseptic  techniques­  prevent  microbial 
contamination of wounds.
– Hand washing, use of sterile gloves, masks, and 
gowns.
– Antisepsis : prevention of infection
– Antiseptic  Technique­  developed  by  Joseph 
Lister, refers to use of antiseptics
Actions of Microbial Control Agents

• Alternation of membrane permeability

• Damage to proteins
• Damage to nucleic acids
 Using Physical Methods to
Inhibit Microbial Growth
• Heat
– Temperature and time
– Thermal death point (TDP)- lowest temperature at
which all cells in a culture are killed in 10 min.
– Thermal death time (TDT)- time to kill all cells in a
culture
– Decimal reduction time (DRT)- Minutes to kill 90%
of a population at a given temperature
– Dry Heat- Oven, 160 to 165 C for 2 hours or 170 to
180 C for 1 hour.
– Incineration- or burning of contaminated
disposable materials
• Moist heat-
denatures
proteins
• Autoclave:
– Large pressure
cooker
– Steam under
pressure
– 15 psi, 121.5C,
20 minutes
 Using Physical Methods
to Inhibit Microbial Growth

• Cold
• Low temperature inhibits microbial
growth
– Refrigeration
– Slow freezing
– Rapid freezing (liquid N)
– Lyophilization (freeze drying)
• Desiccation
– prevents metabolism
• Radiation- damages DNA
– Ionizing radiation (X rays, gamma rays,
electron beams)
– Non-ionizing radiation (UV)
– Ultrasonic waves
– Microwaves kill by heat; not especially
antimicrobial
• Filtration
• Gaseous Atmosphere
– altering the atmosphere in which the
microorganisms are located
– Ex. Gas gangerene
 Using Chemical Agents
to Inhibit Microbial Growth

• Chemical disinfection refers to the use of

chemical agents to inhibit the growth of pathogens,
either temporary or permanent.
Factors to Consider Whenever a
Disinfectant is Used

• prior cleaning
• organic load
• bioburden
• contration of disinfectant
• contact time
• physical nature of the object
• temperature and pH
 Characteristics of an Ideal
Microbial Agent
• broad anti-microbial spectrum
• fast acting (short contact time)
• not affected by the presence of organic matter
• non-toxic and non-corrosive
• leave a residual microbial film
• soluble in water and easy to apply
• inexpensive and easy to prepare
• stable, can be stored for long periods
• odorless
 How do disinfectant kill
microorganisms?

• target and destroy cell membranes

(triclosan, detergents, alcohols,
chlorhexidine and phenolic compounds)
• destroy enzyme and structural enzymes
(hydrogen peroxides, formaldehyde, salt
of heavy metals, formaldehyde and
ethylene oxide)
• attack cell wall or nucleic acids
 Using Chemical Agents to Inhibit
Microbial Growth

• Evaluating a disinfectant
– Use-dilution test
1. Metal rings dipped in test bacteria are dried
2. Dried cultures placed in disinfectant for 10
min at 20°C
3. Rings transferred to culture media to
determine whether bacteria survived
treatment
 Using Chemical Agents to
Inhibit Microbial Growth
• Evaluating a disinfectant
• Disk-diffusion method
Chemical Food Preservatives
• Chemical Food Preservatives
– Organic Acids
• Inhibit metabolism
• Sorbic acid, benzoic acid, calcium propionate
• Control molds and bacteria in foods and
cosmetics
• Nitrite prevents endospore germination
• Antibiotics- nisin and natamycin
prevent spoilage of cheese
Microbial Characteristics and Microbial Control
 FINISHED…

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