Microbial Growth Flashcards
increase in the number of cells or microbial population rather than in the size of individual cells
microbial growth
Factors Affecting Microbial Growth
- biochemical
factors (nutrition) - physical factors
- generation time
- biochemical
factors (nutrition)
– macronutrients
– micronutrients
– vitamins
- physical factors
– pH
– temperature
– oxygen concentration
– moisture
– hydrostatic pressure
– osmotic pressure
– radiation
supplying cells with chemical tools they need
to make monomers of macromolecules that mainly
comprise microbial cells
microbial nutrients
- made up of chemical elements
- extracellular substances that provide the microbial cell
with materials to
➢ build protoplasm
➢ generate energy
nutrients
microorganisms
Bacteria
Archaea
Protozoa
Virus
Algae
Fungi
nutrients required in relatively larger amounts
macronutrients
nutrients required in lesser quantities
Micronutrients
any nutrient material prepared/used for the growth and
cultivation of microorganisms in the laboratory
Culture Medium
Culture Media for?
- for the growth and maintenance of microbial cultures
- to favor the production of particular compounds
- to study microbial action on some constituents of the medium
no solidifying agent
inoculum preparation:
fermentation
nutrient broth
liquid
with 0.1- 0.5%
solidifying agent
Motility test
Sulfur Indole Motility
(SIM) Medium
semi-solid
with 1.5-2.0%
solidifying agent
Colony morphology
observation; hemolysis and
pigmentation characterization
Nutrient
Agar; Blood
Agar
solid
- complex polysaccharide (usually derived from
red algae) - used as solidifying agent for culture media in
Petri plates, slants, and deeps - no nutritive value; generally not metabolized by
microbes - not affected by growth of bacteria
- Liquefies at 100°C
- Solidifies at ~40°C
Agar
Types of Culture Media
(based on chemical composition)
Synthetic or chemically-defined
Complex
are not
chemically-defined
Nutrient Agar,
yeast extract
complex
All components are
chemically-defined
(precise nutrient
composition and
amounts)
Glucose
Inorganic Salt
Phosphate
Synthetic or chemically-defined
- uses: of synthetic medium
- disadvantages:
- uses: important for genetic and specific or
precise studies - disadvantages:
– preparation is time-consuming
– microorganisms grow relatively slow
– prepared only for microorganisms with known
nutritional requirements
- use: complex medium
- advantages:
- use: routine purposes
- advantages:
– easy to prepare
– support rapid growth of most microorganisms
Types of Culture Media
(based on principal purpose, function, or application)
general purpose
differential
selective
enrichment
assay
Can support most
or almost all
types of species
Nutrient Agar,
Tryptic Soy
Agar, Brain
Heart Infusion
Agar
General purpose
Can distinguish
visually one type
of bacterium
from another
Differential
Allows growth of
a specific type of
microorganism only
selective
Used to increase the number
of microorganisms with
unusual physiological
characteristics; contains
special nutrients for
microorganisms of interest
Cellulose agar,
Petroleum broth, Blood
agar
Enrichment
Used for______ of vitamins,
amino acids, antibiotics; may
be used for qualitative or
quantitative production of a
compound by a microorganism
fermentation media,
TSI agar, Vitamin B12
assay medium
Assay
contains selective agents/additives/toxic chemicals
(salts, dyes, antibiotics, and other inhibitors)
– sodium azide, potassium tellurite, thallium acetate (0.1-0.5 g/L), crystal violet (2 mg/L), penicillin (5-50 units/mL)
- extreme pH value or unusual carbon source to favor growth of a particular organism
- i.e. Thayer-Martin agar (Neisseria gonorrhoeae); NA
with penicillin (Gram-negative bacteria); Thiosulfate
selective medium
- identifies microorganisms by the appearance of
their colonies and exploits the ability of a
particular microorganism to change the appearance of the medium - with special reagents like pH indicators and dyes
- i.e. blood agar (Streptococcus species); Mac-
Conkey agar (E. coli and lactose fermenters)
differential medium
contains general nutrients and 5% sheep blood.
It is useful for cultivating fastidious organisms and for determining the hemolytic capabilities of an organism.
Blood agar
Some bacteria produce exoenzymes that lyse red blood cells and degrade hemoglobin;
hemolysins.
breaks down the red blood cells and hemoglobin
completely.
Beta-hemolysin
Beta-hemolysin leaves a clear zone around the bacterial growth. Such results are referred to as
β-hemolysis (beta hemolysis).
partially breaks down the red blood cells and leaves a greenish color behind.
Alpha-hemolysin
The greenish color is caused by the presence of biliverdin, which is a by-product of the breakdown of hemoglobin.
α-hemolysis (alpha hemolysis).
If the organism does not produce hemolysins and does not break down the blood cells, no clearing will occur.
γ-hemolysis (gamma hemolysis).
enrichment contains special nutrient(s) for the microbe of interest
and inhibitory substances to suppress unwanted microorganisms
cellulose, petroleum, blood
- used for the assay of vitamins, amino acids,
antibiotics, etc.; used for qualitative or
quantitative production of such a compound by
a microorganism - of prescribed composition
- i.e. fermentation media, Triple Sugar Iron
Agar, Media for Antibiotic Sensitivity Testing,
Vitamin B12 Assay Medium
Assay Medium
Other Types of
Culture Media
Indicator Medium
Sugar Medium
Transport Medium
Biochemical Reaction Medium
- medium contains an indicator which
changes its color when a bacterium
grows in them - i.e. Blood agar (also a differential
media); Mac Conkey’s medium
Indicator Medium
- medium containing any fermentable substances (i.e.
glucose, arabinose, lactose, starch) - consists of 1% of the sugar in peptone water
- contain a small tube (Durham tube) for the detection
of gas by the bacteria
Sugar Medium
A differential medium that contains 1% lactose,1% sucrose, 0.1% glucose, ferrous sulfate and pH indicator phenol red
performed in gram negative negative bacteria
used to differentiate enteric based on ability to reduce sulfur and ferment carbs.
Triple sugar Iron Test
A differential medium that contains 1% lactose,1% sucrose, 0.1% glucose, ferrous sulfate and pH indicator phenol red
performed in gram negative negative bacteria
used to differentiate enteric based on ability to reduce sulfur and ferment carbs.
Triple sugar Iron Test (TSI)
a.TSI Red/red-
b.control
c. Red/yellow-
d. Yellow/yellow-
e. Red/yellow with H2S
a. no sugar fermentation
c. glucose fermented but lactose and sucrose not fermented
d. glucose fermented. lactose and/or sucrose fermented
medium used for transporting
samples (prevent microbial
proliferation; maintain viability
of microorganisms)
- i.e. Stuart’s medium (non nutrient
soft agar gel containing a reducing
agent); buffered glycerol saline (for
enteric bacilli)
Transport Medium
recommended for the preservation and
transportation of Neisseria species and other fastidious organisms from the
clinic to laboratory.
This medium is a chemically defined, semisolid, non-nutrient medium which
prevent microbial proliferation.
Transport Medium Stuart
The transport medium provides an adequate degree of ______ which can be monitored by means of the redox indicator methylene blue.
anaerobiosis
- medium used to provide additional information for the identification of the bacterium
- i.e. Triple sugar iron agar (sugar fermentation);
SIM Medium (Indole test); Citrate utilization;
Christensens urease medium (Urease test)
Biochemical Reaction Medium
Yellow – Acid
Pink - Alkaline
a. Yellow slant / Yellow butt (A/A) –
b. Pink slant / Yellow butt (K/A) –
c. Pink slant / no colour change (K/K) –
d. Black colour –
( TSI)
e. Gas bubbles or crack in the medium –
f. LF –
g. NLF –
h. H2S -
a. Lactose
fermenters.
b. Non lactose
fermenters
c. non-fermenters
d. H2S production
e. gas production
f. E.coli, Klebsiella
g. Salmonella, Shigella
h. Proteus
- Used to detect indole production by
the organism. - They produce indole from tryptophan
present in peptone water. - After overnight incubation, a few
drops of indole reagent (Kovac’s
reagent) is added. - Positive test is indicated by a pink ring
indole test
- Indole positive –
- Indole negative –
E.coli
Klebsiella, Salmonella
– Positive indole test –
– Negative indole test -
pink ring
yellow ring
SIM medium result:
S
a.black
b.colorless
I
a.Red with kovac’s
b. colorless
M
a. organism growing only in line of inoculation
b. organism appears as haze beyond line of inoculation
S
a.positive for cysteine defulrase production
b. negative or cysteine defulrase production
I
a.positive for tryptophanase production
b.negative for tryptophanase production
M
a. non-motile
b.motile
- Done in Simmon’s Citrate medium.
- To detect the ability of certain
bacteria to utilize citrate as the
sole source of carbon. - Contains Sodium citrate and
bromothymol blue as the indicator. - If citrate is utilized, alkali is
produced which turns the medium to
blue.
– Citrate positive – blue color
– Citrate negative – green color - Positive – Klebsiella
- Negative – E.coli
CITRATE UTILIZATION TEST
- Done in Christensen’s urease
medium. - This test is used to detect
organisms that produce urease. - Urease produced by the
organisms split urea into ammonia
and CO2.
– Urease positive – pink color
– Urease negative – yellow color - Positive – Proteus, Klebsiella
- Negative – E.coli, Salmonella
UREASE TEST
– Urease positive –
– Urease negative –
* Positive –
* Negative –
pink color
yellow color
Proteus, Klebsiella
E.coli, Salmonella
– Citrate positive –
– Citrate negative –
* Positive –
* Negative –
blue color
green color
Klebsiella
E.coli
- These media are used to grow
anaerobic organisms. - e.g: Robertson’s cooked meat
medium, Thioglycolate medium.
Anaerobic media
are anaerobic organism
clostridia
optimum temp. for growth of anaerobic
pH
37’C
7-7.4
most organism produce gas in this medium
saccharolytic species turn meat pink
proteolytic species turn meat black with foul smell
anaerobic media
example of anaerobes attack meat proteolytic
Cl. tetani
example of anaerobes attack carbohydrates in meat saccharolytic
clostridium perfringens
hydrogen ion concentration
* pH values less than 7 – acidic
* pH values greater than 7 - basic
* optimum pH for most bacteria is near
neutrality (pH 7)
* cytoplasm of most bacteria is pH 7
pH
pH range
Acidophiles
Neutrophiles
Alkaliphiles
Acidophiles < pH 5.4
Neutrophiles pH 5.4 - 8.5
Alkaliphiles pH 7.5 – 11.5
– acid-loving organisms
– can be found in acidic lakes, gastrointestinal tract
– most fungi (acid-tolerant; optimum temperature 5 or below)
– some algae, bacteria, and several Archaea
acidophiles
– acid-loving organisms
– can be found in acidic lakes, gastrointestinal tract
– most fungi (acid-tolerant; optimum temperature 5 or below)
– some algae, bacteria, and several Archaea
– high H+ concentration is required to maintain cell membrane
stability
acidophiles
– some algae, bacteria, and several Archaea pH
- Lactobacillus
- Helicobacter pylori
- Acidithiobacillus (sulfur-oxidizing bacteria)
- red alga Cyanidium caldarium, green alga Dunaliella acidophila
- fungi: Aconitum cylatium, Cephalosporium sp., Trichosporon cerebriae
- archaea: Sulfolobus and Thermoplasma, Picrophilus
- Lactobacillus (pH 6)
- Helicobacter pylori (pH 2 or less)
- Acidithiobacillus (sulfur-oxidizing bacteria) (pH <4)
- red alga Cyanidium caldarium, green alga Dunaliella acidophila (pH <1)
- fungi: Aconitum cylatium, Cephalosporium sp., Trichosporon cerebriae
(near pH 0) - archaea: Sulfolobus and Thermoplasma, Picrophilus (negative pH values)
Acidophile examples
-Lactobacillus
-fungi
-Helicobacter pylori
-Acidithiobacillus thiooxidans
-Thermoplasma
-Picrophilus
-Sulfolobus acidocaldarius
most human disease-causing bacteria
(human blood and tissues pH = 7.2 – 7.4)
protozoans and most bacteria (pH 6.5-7.5)
neutrophiles
examples of neutrophiles
-E. coli bacteria in gut
-Balantidium coli (protozoan) in
human large intestines
-Salmonella bacteria on tissue surface
-Staphylococcus skin infection
base-loving organisms
live in soda lakes, high-carbonate soils
i.e. Bacillus, Vibrio cholerae (pH 9), Alcaligenes
faecalis (>pH 9), Agrobacterium (pH 12)
some produce hydrolytic enzymes (proteases and lipases)
alkaliphiles
examples of alkaliphiles
-Vibrio cholerae
-Agrobacterium
-Alcaligenes faecalis
- one of the most, if not the most, important
environmental factors affecting growth and
survival of microorganisms
Temperature
three critical temperatures (affecting enzyme
function) or cardinal temperatures:
-minimum growth temperature
-optimum growth temperature
-maximum growth temperature
lowest temperature
at which cells can divide (a)
* membranes solidify; slow transport process thus
growth could not occur
minimum growth temperature
– temperature at
which cells divide most rapidly (b)
* enzymatic reaction occurring at maximal possible
rate
optimum growth temperature
– highest temperature
at which cells can divide (c)
* protein denaturation, collapse of cell membrane,
cell lysis
maximum growth temperature
Temperature Classes of
Microorganisms
Psychrophiles:
Mesophiles
Thermophiles
Hyperthermophiles
Psychrophiles: <0 to 20’C 15’C
Mesophiles 10 to 48’C 37’C
Thermophiles 40 to 72’C 60’C
Hyperthermophiles 65 to 110’C 80’C
“cold-loving organisms”
grow best at -10 ̊ to 20 ̊C
live mostly in cold water and soil (Arctic and
Antarctic regions) and can cause spoilage of
refrigerated food
psychrophiles
(Sporosarcina globispora, <20 ̊C)
obligate psychrophiles
(Xanthomonas pharmicola,
above or below 20 ̊C)
facultative psychrophiles (Xanthomonas
Chlamydomonas nivalis
snow alga:
(can multiply at -4.4 ̊C)
Listeria monocytogenes
➢ most bacteria including pathogens
➢ most common group of microorganisms
➢ 25 ̊to 40 ̊C
➢ found in warm-blooded animals
➢thermoduric microorganisms (Bacillus,
Micrococcus, Lactococci, Corynebacterium)
mesophiles
can withstand short periods of exposure to high
temperatures; can cause food spoilage
➢thermoduric microorganisms (Bacillus,
Micrococcus, Lactococci, Corynebacterium)
➢ “heat-loving organisms”
➢ 40 ̊ to 72 ̊C
➢compost heaps, hot springs
➢contaminants in dairy products
thermophiles
– temperatures above 37 ̊C
– Geobacillus stearothermophilus (65-75 ̊C)
obligate thermophiles
– can grow both above and below 37 ̊C
– thermophilic sulfur bacteria in runoff troughs of geysers
– Bacillus coagulans (35-50 ̊C), B. licheniformis,
Anoxybacillus spp., Paenibacillus spp., Thermoanaerobacter spp. and Clostridium thermobutyricum /thermopalmarium
facultative thermophiles (moderate thermophiles)
➢ extreme heat-loving organisms
➢ 65 to 110 ̊C
➢ boiling hot springs, geysers, hot-water vents
hyperthermophiles
boiling hot springs, geysers, hot-water vents
- archaeobacteria (deep-sea vents, 115 ̊C)
- Pyrolobus fumarii (“firelobe of the chimney”) – (113 ̊C)
- Thermus aquaticus
reduces growth of
psychrophiles; prevents growth of other
microorganisms (i.e. refrigerator)
refrigeration (4 ̊C)
-30 ̊C (i.e. ultra-low freezer)
long-time storage
high temperatures
prevent bacterial growth (i.e.
pressure cooker)
unsaturated (polyunsaturated) fatty acids in phospholipids
with enzymes functional at low temperatures
active transport occurs well at low temperatures
psychrophiles
saturated fatty acids in phospholipids
heat-stable proteins and enzymes
thermophiles
no fatty acids in their membrane (phytane)
lipid monolayer
hyperthermophiles
bacteria can be divided into: oxygen
aerobes – require oxygen to grow
anaerobes – do not require oxygen to grow
microorganisms can be classified as : oxygen
- obligate aerobes
- obligate anaerobes
- microaerophiles
- facultative anaerobes
- aerotolerant anaerobes
is necessary for aerobic cellular
respiration; oxygen oxidize substrates
to produce energy
oxygen
must have free oxygen for aerobic respiration
Pseudomonas spp.
obligate aerobes
does not require/use oxygen for metabolism
Bacteroides, Clostridium methanogens, Thiomargarita namibiensis
organisms can be found in muds, sediments of lakes, rivers, oceans, marshes, water-logged soils, canned foods, intestinal tracts, sewage treatment systems, anoxic environments
obligate anaerobes
- grow best in the presence of small amount of free oxygen
- Campylobacter (also a capnophile: organism that requires
high carbon dioxide concentration) - Treponema pallidum
microaerophiles
- ordinarily carries aerobic metabolism when oxygen is
present but shifts to anaerobic metabolism when oxygen is
absent - Staphylococcus and E. coli
- have complex enzyme systems
facultative anaerobes
- can survive in the presence of oxygen but do not use it in
their metabolism - Lactobacillus (captures energy by fermentation)
aerotolerant anaerobes
Aerobes
a.Group
b. Relationship to O2
c. Types of
metabolism
d. Example Habitat
Obligate
Facultative
Microaerophilic
obligate
a.Required
b. Aerobic
respiration
c.Micrococcus
luteus
d.Skin, dust
Facultative
a.Not required, but grows better with O2
b. Aerobic, anaerobic, fermentation
c. Escherichia coli
d.Mammalian large intestine
Microaerophilic
a.Required but at levels lower than atmospheric O2
b. Anaerobic respiration
c. Spirillum volutans
d.Lake water
- Use reducing media, containing chemicals (e.g.:
thioglycolate) that combine with O2 - Use anaerobic jar (GasPak)
- Novel method in clinical labs:
Add oxyrase to growth media
OxyPlate (no need for anaerobic jar) - Work in a glove box
- Use candle jars
Anaerobic Culture Methods
essential ingredient of bacterial
protoplasm.
needed by actively metabolizing
cells
water
- Effect of drying varies:
– Treponema pallidum – highly sensitive
– Staphylococcus sp. – can stand for months
– endospore-former bacteria and
xerophiles –resistant to desiccation
- Effect of drying varies:
– Treponema pallidum – highly sensitive
– Staphylococcus sp. – can stand for months
– endospore-former bacteria and
xerophiles –resistant to desiccation
minimum pressure needed to be applied
to a solution to prevent the flow of water across a semi- permeable membrane
osmotic pressure
-most bacteria require an isotonic environment or a hypotonic environment for optimum growth
-have transport systems to regulate movement of
substances
osmotic pressure
osmotic pressure outside > osmotic pressure inside the cell
hyperosmotic environment → plasmolysis
too high osmotic pressure outside cell → water loss → inhibits growth or kill bacterial cells
osmotic pressure
Osmotic Pressure
application: use of salt or sugar as preservative
salting of fish
sugaring of fruits
brining of vegetables
jams, marmalades, preserves, and pickles
Osmotic Pressure
application: use of salt or sugar as preservative
salting of fish
sugaring of fruits
brining of vegetables
jams, marmalades, preserves, and pickles
organisms that can grow at relatively
high salt concentration (up to 10%)
osmotolerant
salt-loving organisms; require relatively
high salt concentrations for growth (i.e. archea require NaCl concentrations of 20 % or higher)
halophiles
effect on salt in cell
a. normal cell in isotonic solution
b.plasmolyzed cell in hypertonic solution
the solute solution of a cell is .85% NaCl
normal cell in isotonic solution
growth of cell is inhibit due to the high concentration of NaCl in the cell
plasmolyzed cell in hypertonic solution
require moderate to large quantities of salt
membrane transport system actively transport
sodium ions out of the cell and concentrate potassium ions in
typically found in the ocean (optimum: 3.5% salt
concentration)
found in exceptionally salty bodies of water (Dead Sea, brine vats)
halophiles
Classification
low halophiles –
mild or moderate halophiles –
extreme halophiles –
1-6% NaCl
6-15% NaCl
15-30% NaCl
- pressure exerted by standing water, in
proportion to its depth - doubles with every 10 meter increase in
depth - i.e. 50-m deep lake – 32x atmospheric
pressure
Hydrostatic Pressure
bacteria that
live at high pressures
– membranes and enzymes (3-D
configuration) require high pressure to
function properly
piezophiles (barophiles)
exhibit optimal reproduction rate at hydrostatic pressure exceeding 10MPa and temp. 2-4’C
psychropiezophiles
the only example is archaeon- pyrococcus yayanosii, exhibit optimal rate at 52MPa and 98’C
Thermoiezophiles
visible light is the source of energy for
photosynthesis (photosynthetic microorganisms)
Radiation/Radiant Energy
radiant energy inspired oraganism
Cyanobacteria
(photosynthetic
bacteria)
Volvox
(photosynthetic
green alga)
- ionizing radiation (gamma rays and UV light)
can cause mutations in DNA and can even kill
microorganisms - some organisms have pigments that screen
radiation and help to prevent DNA damage - other organisms have enzyme systems that
can repair certain kinds of DNA damage
Radiation/Radiant Energy
How do bacteria reproduce?
most bacteria reproduce by binary fission
Binary Fission
- Replication of chromosome
- Cell grow in size (double)
- Septum formation
- Completion of septum with
formation of distinct walls - Cell separation
interval for the formation of two cells from
one cell
generation
interval of time between for two cells to form from one cell
the time required for a bacterium to give rise to 2
daughter cells under optimum conditions
population doubling time
exponential growth
time is variable and dependent on many factors
Generation Time
Generation Time
◼ Escherichia coli –
◼ Staphylococcus aureus-
◼ Mycobacterium tuberculosis -
◼ Treponema pallidum -
Escherichia coli – 20 mins
Staphylococcus aureus- 27-30 mins
Mycobacterium tuberculosis - 792-932 mins
Treponema pallidum -1980 mins
Calculating Generation Times 1
N = N02n
g = t/n
* N = final cell number
* N0 = initial cell number
* n = number of generations that have occurred
during the period of exponential growth
- g = generation time
- t = hours or minutes of exponential growth
Calculating Generation Times2
- n= log N – log N0 = log N – log N0
log2 0.301
=3.3 (log N – log No) - k = ln 2/g = 0.693/g
k = number of generations that occur per unit time in an exponentially growing culture
g = generation time
bacterium in a suitable medium, incubated, growth
follows a definite course
Bacterial Population Growth Cycle
4 phases of bacterial growth curve:
– Lag
– Log or Exponential
– Stationary
– Decline/Death phase
No significant or immediate increase
in cell numbers but there may be an increase in the size of the cell.
Lag phase
dependent in the characteristics of the
bacterial species and conditions in the media
(“old and new”, “rich and poor”)
length
– cells start dividing and their number increases
exponentially–
(Logarithmic) or Exponential phase
– cell division decreases
due to depletion of nutrients & accumulation of
toxic products; inadequate oxygen supply; pH
change
Stationary phase
- population
decreases due to the death of cells
-cells undergo lysis or involution (assume a
variety of unusual shapes)
Decline (Death) Phase
Morphological & Physiological
Alterations During Growth
* Lag phase -
* Log phase –
* Stationary phase –
* Phase of Decline –
- Lag phase – maximum cell size towards the end of lag phase.
- Log phase – smaller cells, stain uniformly
- Stationary phase – irregular staining, sporulation
and production of exotoxins - Phase of Decline –involution forms (with aging)
- processes are either physical or chemical, or a
combination of both - physical methods
– heat treatment, irradiation, filtration, mechanical removal - chemical methods
– antimicrobial chemicals
Controlling Microbial Growth
killing or complete elimination
of all viable microorganisms
– agents – sterilants or sterilizing agents
sterilization
killing or complete elimination
of all viable microorganisms
– agents – sterilants or sterilizing agents
sterilization
elimination or reduction of
pathogens from inanimate objects or surfaces
-agents – disinfectants
(i.e. alcohol, formaldehyde, chlorine)
disinfection
reduction of microbial
populations to levels considered safe by
public health standards
– agents – sanitizers (i.e. iodine, chlorine)
sanitization
prevention of infection in living
tissues using chemicals
– agents – antiseptics (i.e. iodine, alcohol,
hydrogen peroxide)
antisepsis
- one of the most useful methods of microbial control
- reliable, safe, relatively fast, inexpensive
- use to sterilize or decrease microbial number
- moist heat or dry heat
heat
– temperature that kills all bacteria
in a 24-hour old broth culture at
neutral pH in 10 minutes
thermal death point
– time required to kill all bacteria
in a particular culture at a
specified temperature
thermal death time
- efficient penetrating properties
- destroys microorganisms by irreversibly coagulating their proteins
- boiling, pasteurization, pressurized steam
moist heat
- 100oC
- destroys most bacteria
and fungi, inactivates
some viruses - kills vegetative cells and
eukaryotic spores within
10 minutes
boiling
moist heat types
boiling
pasteurization
pressured steam
Tyndallization
- use of brief heat treatment (moderately high temperature) to
reduce the number of spoilage organisms and kill pathogens
(Ex. Salmonella, Mycobacterium) - wine, beer, vinegar, milk, juices
- significantly reduce numbers of heat-sensitive
microorganisms; does not significantly alter quality of food - increases shelf-life of food and protects consumers
pasteurization
– 62.8oC for 30 minutes
- low temperature holding (LTH)
kind of Pasteurization
- low temperature holding (LTH)
- high-temperature-short-time (HTST) method
- high-temperature-short-time (HTST) method
- mechanical pasteurization (non-food)
– milk: 72oC, 15 seconds (flash method)
– ice cream: 82oC, 20 seconds
high-temperature-short-time (HTST) method
– 140oC -150oC (several seconds)
– involves complex cooling process
– boxed juices, coffee creamers
- ultra-high-temperature (UHT) method
- pressure cookers and autoclave
- heat water in an enclosed vessel that achieves
temperatures above 100oC - 15 minutes, 15 psi, 121oC (kills endospores and disrupts
viruses’ nucleic acids) - items that can be penetrated by steam and withstand heat
and moisture (i.e. surgical instruments, microbiological media,
reusable glassware, microbial cultures, biohazards before
disposal)
Pressurized Steam
commercial canning
process uses retort machine
(industrial-
sized autoclave)
- ensure Clostridium
botulinum endospores
are destroyed - commercially sterile –
endospores of some
thermophiles may
survive
Pressurized Steam
- fractional steam sterilization or intermittent sterilization
- for materials that can be destroyed at more than 100 oC
- exposure to 90-100 oC for 30 minutes for 3 consecutive days
Tyndallization
- not as efficient as wet heat (lower penetrating
properties) - require longer times and higher temperatures
- metal objects, glassware
- i.e. oven, open flame (incineration)
dry heat
oxidizes cell components to ashes
Incineration
dry heat types
Incineration
Dry Heat Oven/Hot Air
- oxidizes cell components and irreversibly denature
proteins - Petri dishes and glass pipettes
- 170oC to 180oC for 1 hour
- powders, oils, anhydrous material
Dry Heat Oven/Hot Air
for materials that are heat-sensitive or impractical to treat using heat
refrigeration,
filtration (fluid or air),
irradiation,
high- pressure treatment
- cold temperatures retard microbial growth
(slow rate of enzyme-controlled reactions)
do not achieve sterilization
low temperature
USING PHYSICAL METHODS TO
DESTROY MICROORGANISMS
Heat
Low temperature
Filtration
Drying/Desiccation
Increased Osmotic Pressure
Radiation
– used to prevent food spoilage
refrigeration
– preserve both food and microorganisms
freezing, drying, freeze-drying
- 4 to 5 oC
- limited to few
days because
bacteria and
molds continue
to grow at low
temperatures
Refrigeration
low temperature types
refrigeration
Freezing/Deep Freezing
- 0- -95oC
- used to preserve food in homes and in food industries
- slows the rate of chemical reactions in bacterial cells
Freezing/Deep Freezing
- remove organisms from heat-sensitive fluids
- unpasteurized beer, sterilization of sugar solutions, wine
clarification - filtration units: remove Giardia cysts and bacteria from water
- paper-thin membrane filters (polycarbonate or cellulose
nitrate): have microscopic pores that allow liquid to pass
through while trapping small particles (vacuum or pressure)
– 0.2 μm pore removes bacteria - depth filters: trap material within thick filtration material
(cellulose fibers or diatomaceous earth) that retain
microorganisms and let fluid pass through holes
Fluid Filtration
- high-efficiency particulate air (HEPA): remove from air
nearly all microorganisms with diameter greater than 0.3
μm - hospital rooms, biological safety cabinets, laminar flow
hood
Air Filtration
types of filtration
fluid filtration
air filtration
- used to preserve food (absence of
water inhibits action of enzymes) - endospores survive but do not
produce toxins - minimizes spread of infectious
agents (i.e Treponema) - i.e. peas, beans, raisins
Drying/Desiccation
- lyophilization
- drying of material from frozen state
- for long-term preservation (frozen in alcohol
and dry ice/liquid nitrogen → high vacuum) - i.e. instant coffee, culture preservation
Freeze-drying
high salt/sugar concentration create hyperosmotic
medium drawing water from microorganisms
causes plasmolysis of bacterial cells
Increased Osmotic Pressure
- electromagnetic radiation: radio waves, microwaves, visible
and UV light rays, X rays, gamma rays - ionizing and non-ionizing radiation
- free radical formation or thymine dimer formation
Radiation
types of radiation
Ionizing Radiation
Non-ionizing radiation: Ultraviolet Radiation
- gamma rays, X rays (0.1 to 40 nm), electron accelerators
- causes biological damage directly (destroying DNA, cell
membranes) or indirectly (produce reactive molecules, i.e.
superoxide, hydroxyl free radicals/oxidizing agents) - kills microorganisms (0.3 to 0.4 millirads) and viruses
- bacterial endospores: radiation-resistant microbial forms
- Gram-negative bacteria (Salmonella and Pseudomonas): radiation-
susceptible - sterilize heat-sensitive materials (plastic laboratory and medical
equipment), drugs, packed materials, fruits (200-300 kilorads),
spices and herbs, meat (50-100 kilorads), milk
Ionizing Radiation
- 40 to 390 nm (200 nm)
- damages DNA
- effective in inactivating viruses,
kills fewer bacteria - microbes in air and water,
surfaces - poor penetration power
Non-ionizing radiation: Ultraviolet Radiation
USING CHEMICALS TO
DESTROY MICROORGANISMS
A. Alcohols
B. Aldehydes
C. Phenols/Phenolics
D. Halogens
E. Heavy Metals
F. Sterilizing Gases
G. Surface Active Agents or Surfactants
H. Organic Acids
I. Other Oxidizing Agents
- disinfect and sterilize
- irreversibly react with proteins, DNA, cell membranes
- less reliable than heat; suitable for treating large
surfaces and heat-sensitive items; some are non-toxic;
can be used as preservatives (bacteriostatic)
Chemical Agents
- 60% to 80% ethyl or isopropyl alcohol
- kill vegetative bacteria and fungi
- coagulate enzymes and other essential proteins, damage
lipid membranes - used as antiseptics for degerming or as disinfectants for
treating instruments and surfaces - non-toxic, inexpensive, no residue, evaporates quickly
Alcohols
- glutaraldehyde, formaldehyde, orthophthaldehyde (OPA)
- inactivate proteins and nucleic acids
- 2% alkaline glutaraldehyde solution: widely used liquid
sterilants for treating heat-sensitive medical items - formalin (aqueous 37% formaldehyde): kill most forms
of microorganisms - toxic, irritating vapors, suspected to be carcinogenic
Aldehydes
Phenols/Phenolics
* disrupts cell membrane, denatures proteins and
inactivates enzymes
* phenol, cresol, xylenol, triclosan
Phenols/Phenolics
- oxidation of cell constituents
- iodine, chlorine
Halogens
- denatures enzymes and essential proteins
- i.e. silver nitrate (prevents ophthalmic gonorrhoeae); copper
sulfate (algicide); silver sulfadiazine (used on burns);
merthiolate (disinfects skin mucous membranes)
Heavy Metals
- denatures proteins
- i.e. ethylene oxide, ozone, chlorine dioxide
- for heat-sensitive items (catheters, plastic Petri dishes)
Sterilizing Gases
- soaps and acid-anionic detergents: mechanical removal
of microorganisms - cationic detergents: disrupt cell membrane and denature proteins
Surface Active Agents or
Surfactants
- inhibit microbial metabolism
- sorbic acid, benzoic acid, calcium propoionate
- widely used in foods/cosmetics
Organic Acids
- inhibit microbial metabolism
- sorbic acid, benzoic acid, calcium propoionate
- widely used in foods/cosmetics
Organic Acids
- oxidation of cell components
- i.e. hydrogen peroxide
Other Oxidizing Agents
