Download Microbial growth control and nutrition

MICROBIOLOGIA GENERALE
Microbial growth control
and nutrition
The control of microbial growth is
necessary in many practical situations,
and significant advances in agriculture,
medicine, and food science have been
made through study of this area of
microbiology.
What does sterilization mean?
•Sterilization is the complete destruction or
elimination of all viable organisms (in or on
an object being sterilized).
•There are no degrees of sterilization: an
object is either sterile or not.
•Sterilization procedures involve the use of
heat, radiation or chemicals, or physical
removal of cells.
Microbial growth control and
nutrition:
the control of microorganisms
by physical factors
Methods of Sterilization: Heat
•Most important and widely used.
•For sterilization always consider type of heat,
time of application and temperature
•Endospores are considered the most
thermoduric of all cells so their destruction
guarantees sterility.
The effect of temperature on the viability of a mesophilic bacterium
The relationship between the temperature and the rate of
killing as indicated by the D for two different microorganisms
D value: the time (min) at a given temperature, for a population of a
bacterium or endospore to fall by 90% (by one unit on the log10 scale)
Microorganism
D (min) at 100° C, D100
Bacillus cereus
0.8
Bacillus megaterium
2.1
Bacillus anthracis
5
Bacillus licheniformis
24.1
Clostridium botulinum
300
Bacillus stearothermophilus
459
Botulinum cook = 12 times the D for C. botulinum at that temperature
D values: food pathogens and environmental effects
Microorganism
Source
D (min)
Clostridium botulinum
endospores
D121=0.204
Clostridium perfringens
cell culture
D90=3-5
Salmonella spp.
chicken
D60=0.40
Staphylococcus aureus
chicken
D60=5-6
Staphylococcus aureus
turkey
D60=15
Staphylococcus aureus
cell culture
D60=2.0-2.5
Botulinum cook = 12 times the D for C. botulinum at that temperature
Heat sterilization
•Incineration: burns organisms and physically destroys
them. Used for needles , inoculating wires, glassware, etc. and
objects not destroyed in the incineration process.
•Autoclaving (steam under pressure): 121o for 15
minutes (1 atm pressure). Good for sterilizing almost any thing,
but heat-labile substances will be denatured or destroyed.
•Boiling: 100o for 30 minutes. Kills everything except some
endospores. To kill endospores, and therefore sterilize the
solution, very long or intermittent boiling is required.
•Dry heat (hot air oven): 160o/2hours or 170o/1hour.
Used for glassware, metal, and objects that won't melt.
Recommended use of heat to control bacterial growth
Treatment
T °C
Effectiveness
>500o
Vaporizes organic material on nonflammable
surfaces but may destroy many substances
Boiling
100o
30 minutes of boiling kills microbial pathogens
and vegetative forms of bacteria but may not
kill bacterial endospores
Intermittent boiling
100o
Three 30-minute intervals of boiling, followed
by periods of cooling kills bacterial endospores
121o/15 min
Kills all forms of life including bacterial
endospores. The substance being sterilized must
be maintained at the effective T
160o/2
For materials that must remain dry and which are
not destroyed at T between 121o and 170o
(glassware, metal, not plastic or rubber items)
Incineration
Autoclave (steam
under pressure)
Dry heat (hot air
oven)
Pasteurization
(batch method)
Pasteurization
(flash method)
h
63o/30
min
72o/15 sec
Kills most vegetative bacterial cells including
pathogens such as streptococci, staphylococci
and Mycobacterium tuberculosis
Effect on bacterial cells similar to batch method;
for milk, this method is more conducive to industry
and has fewer undesirable effects on quality
Autoclave
Use of the autoclave for sterilization
Methods of Sterilization: Irradiation
•Irradiation: usually destroys or distorts
nucleic acids.
•Ultraviolet light is usually used (commonly
used to sterilize the surfaces of objects).
•Gamma-rays are used to sterilize plasticwares
Relationship between the
survival fraction and the
radiation dose
Methods of Sterilization: Filtration
•Filtration: involves the physical removal
(exclusion) of all cells in a liquid or gas.
•It is especially important to sterilize solutions
which would be denatured by heat (e.g.
antibiotics, injectable drugs, amino acids,
vitamins, etc.)
Filter sterilization
Pore sizes: 0,2 mm – 0,4 mm
Membrane filter
Nucleopore filter
Bacteria and algae trapped
on a Nucleopore filter.
Leptospira interrogans
(0.1 x 20 mM) trapped
on a Nucleopore filter.
Filter system for large volumes
Filter system for small volumes:
syringe filters
Microbial growth control and
nutrition:
the control of microorganisms
by chemical factors
Antimicrobial agents
Antimicrobial agents are chemicals that
kill or inhibit the growth microorganisms.
•Antimicrobial agents include chemical
preservatives and antiseptics, as well as
drugs used in the treatment of infectious
diseases.
•Antimicrobial agents may be of natural or
synthetic origin, and they may have a static
or cidal effect on microorganisms.
Three types of action of
antimicrobial agents.
At the time indicated by the arrow, a growthinhibitory concentration was added to the
exponentially growing culture.
Antiseptics
• Antiseptics: microbicidal agents harmless
enough to be applied to the skin and
mucous membranes.
• They should not be taken internally.
•
•
•
•
•
Mercurials
Silver nitrate
Iodine solution
Alcohols
Detergents
Disinfectants
• Disinfectants: Agents that kill microorganisms,
but not necessarily their spores.
• Not safe for application to living tissues.
• They are used on inanimate objects such as
tables, floors, utensils, etc.
• Chlorine, hypochlorites & chlorine
• Copper sulfate.
• Quaternary ammonium compounds.
Disinfectants and Antiseptics
• Disinfectants and antiseptics are distinguished
on the basis of whether they are safe for
application to mucous membranes.
• Often, safety depends on the concentration of
the compound.
• For example, sodium hypochlorite (chlorine), as
added to water is safe for drinking, but "chlorox"
(5% hypochlorite), an excellent disinfectant, is
hardly safe to drink.
Common antiseptics and disinfectants
Common antiseptics and disinfectants
Chemical
Ethanol (50-70%)
Isopropanol (50-70%)
Formaldehyde (8%)
Action
Denatures proteins and
solubilizes lipids
Denatures proteins and
solubilizes lipids
Reacts with NH2, SH
and COOH groups
Tincture of Iodine (2% in
70% alcohol)
Inactivates proteins
Chlorine (Cl2) gas
Forms HClO, a strong
oxidizing agent
Silver nitrate (AgNO3)
Mercuric chloride
Detergents (e.g. quaternary
ammonium compounds)
Phenolic compounds (e.g.
lysol, hexylresorcinol,
hexachlorophene)
Ethylene oxide gas
Uses
Antiseptic used on skin
Antiseptic used on skin
Disinfectant, kills endospores
Antiseptic used on skin
Disinfect drinking water; general
disinfectant
Antiseptic and used in the eyes
Precipitates proteins
of newborns
Inactivates proteins by
Disinfectant, occasionally used
reacting with SH groups as an antiseptic on skin
Disrupts cell
Skin antiseptics and
membranes
disinfectants
Denature proteins and
disrupt cell membranes
Antiseptics at low
concentrations; disinfectants at
high concentrations
Alkylating agent
Disinfectant used to sterilize
heat-sensitive objects (plastics)
Microbial growth control and
nutrition:
cultivation of microorganisms
Photoautothroph
Chemioautothroph
Chemioheterothroph
Elemental and macromolecular composition of a bacterial cell
Major elements, their sources and functions in bacterial cells.
Element
% dry weight
Source
C
50
organic compounds or CO2
O
20
H2O, organic compounds, CO2, and O2
N
14
NH3, NO3, organic compounds, N2
H
8
H2O, organic compounds, H2
P
3
inorganic phosphates (PO4)
S
1
SO4, H2S, So, organic compounds
K
1
Potassium salts
Mg
0.5
Magnesium salts
Ca
0.5
Calcium salts
Fe
0.2
Iron salts
• Media for bacteria must include source of C, N,
P, S, 4 of the 6 major nutrients as well as
micronutrients.
• The micronutrients are usually present as trace
contaminants in water, on glassware, or in
chemicals used to make media.
• However certain minerals (Ca, Mg, Fe, K) are
needed in slightly higher concentrations, and
may be added.
• Media can be liquid or solid. They can be used
for different purposes
Liquid media
• Easiest to prepare and use.
• Good for growing quantities
of microbes needed for
analysis or experiments.
• Unless inoculated with pure
culture, cannot separate
different organisms.
Solid media
• Usually made by adding
agar, a seaweed extract, to
appropriate liquid. 1.5% agar
is standard for plates.
•Agar melts at 80-90°C, will
remain liquid until temperature
cools to 40-42 °C.
•Very few microbes can
degrade agar, so it is normally
not a source of C, and acts as
inert gelling medium.
Solid media
• Usually made by adding
agar, a seaweed extract, to
appropriate liquid. 1.5% agar
is standard for plates.
•Agar melts at 80-90°C, will
remain liquid until temperature
cools to 40-42 °C.
•Very few microbes can
degrade agar, so it is normally
not a source of C, and acts as
inert gelling medium.
Growth on liquid media
Growth on solid media
Synthetic or Defined Media
• They are usually relatively simple media.
• All components are known.
• Useful for experimental situations such as to
select a microbe that can obtain all its
nitrogen from atmospheric N2.
• You would prepare synthetic medium with
sources of C, P, and S, but no N source.
Microorganisms would be unable to grow
unless they can fix nitrogen from air.
Minimal medium for the growth of Bacillus megaterium.
An example of a chemically-defined medium for growth
of a heterotrophic bacterium
Component
Amount
Function of component
sucrose
10.0 g
C and energy source
K2HPO4
2.5 g
pH buffer; P and K source
KH2PO4
2.5 g
pH buffer; P and K source
(NH4)2HPO4
1.0 g
pH buffer; N and P source
MgSO4 7H2O
0.20 g
S and Mg++ source
FeSO4 7H2O
0.01 g
Fe++ source
MnSO4 H2O
0.007 g
Mn++ Source
water
985 ml
pH 7.0
Defined medium (also an enrichment medium) for the growth
of Thiobacillus thiooxidans, a lithoautotrophic bacterium
Component
Amount
Function of component
NH4Cl
0.52 g
N source
KH2PO4
0.28 g
P and K source
MgSO4 7H2O
0.25 g
S and Mg++ source
CaCl2 2H2O
0.07 g
Ca++ source
Elemental Sulfur
1.56 g
Energy source
C02
5%*
C source
water
1000 ml
pH 3.0
Defined medium M63 for the growth of Escherichia coli
DH10B strain SW102, a recombineering lab strain
Component
Amount
Function of component
(NH4)2SO4
2g
N source
KH2PO4
13,6 g
P and K source
FeSO4 7H2O
0.5 mg
Fe++ and S source
D-biotin
1 mg
L-leucine
45 mg
D-galactose/glycerol
MgSO4 7H2O
Agar
water
0.2%
1 mM
15 g/l
1000 ml
C source and energy source
S and Mg++ source
pH 7.0
Complex media
• The composition of these media is not
completely known.
• Often made from inexpensive organic
materials such as:
– slaughterhouse wastes (tryptic digests
called tryptone, trypticase, etc.)
– soybeans
– yeast wastes from brewing (rich source
of vitamins)
– animal blood, etc.
Complex medium (nutrient broth) for the growth of fastidious
bacteria
Component Amount
Function of component
Beef extract 1.5 g
Source of vitamins and other growth factors
Yeast
extract
3.0 g
Source of vitamins and other growth factors
Peptone
6.0 g
C and energy source, N, P and S source
Glucose
1.0 g
C and energy source
Agar
15.0 g
Inert solidifying agent
water
1000 ml
pH 6.6
Selective media
• Selective Media: media favors the growth of
one or more microbes.
• Example: bile salts inhibit growth of most
gram-positive bacteria and some gramnegative bacteria, but enteric bacteria
adapted to life in animal gut can grow well.
• Include bile salts in some media such as
EMB, MacConkey agar to select for enterics.
Differential media
•Differential Media: allows distinguishing
between different bacteria that grow.
•Ex: MacConkey agar has color indicator that
distinguishes presence of acid.
Bacteria that ferment a particular sugar (e.g.,
glucose in culture media) will produce acid
wastes on plates, turn pH indicator red.
Bacteria that cannot ferment the same sugar
will grow but not affect pH, so colonies remain
white.
•It is possible to design a medium that is both
selective and differential.
Growth of Escherichia coli on differential media
MacConkey agar medium for the growth of enteric bacteria
Component
Amount
Function of component
Peptone
Bile salts
20 g
1.5 g
Lactose
Sodium chloride
Neutral red
Crystal violet
10 g
5g
0.03 g
0.001 g
C and energy source
Inhibit growth of most grampositive bacteria
C and energy source
Agar
water
13,5 g
1000 ml
pH 7.1
Na and Cl source
pH indicator
inhibit growth of most grampositive bacteria
Inert solidifying agent
MacConkey agar is a widely-used culture medium
which is both selective AND differential.
Escherichia coli
Gram Negative: growth
Lactose Fermentation:
positive, (pink colonies)
Salmonella typhimurium
Gram Negative: growth
Lactose Fermentation:
neg.,(colorless colonies)
Mannitol salt agar is both a selective AND differential growth
medium. It is used to differentiate pathogenic Staphylococcus
species from nonpathogenic members of the genus Micrococcus
Staphylococcus aures
Staphylococcus epidermidis
positive = acid end products turn the phenol red pH indicator from red to yellow
negative = prenol red remains red
Blood
agar is used both as an enriched medium for growing fastidious
.
bacteria and as a differential medium. Exotoxins called hemolysins cause
lysis of the redblood cells. The degree of the hemolysis is an especially useful
tool for identification of many of the Gram positive cocci.
1. Beta hemolysis completely lyse the red blood cells and hemoglobin;
this results in complete clearing around colonies.
2. Alpha hemolysis refers to the partial lysis of RBC's and hemoglobin and
produces a greenish discoloration of the blood agar around the colonies.
3. No hemolysis, called gamma hemolysis results in no change of the
medium
b
a
g