Environmental Microbiology -Laboratory Manual- prepared for Environmental Microbiology
Environmental Microbiology
-Laboratory Manualprepared for Environmental Microbiology
III
Microorganism Growth
Factors Controlling Growth
E N V I R O N M E N TA L M A N A G E M E N T T E C H N O L O G Y
FA C U LT Y O F C I V I L A N D E N V I R O N M E N TA L E N G I N E E R I N G
ITB, 2010
anindrya.pahlawan@ymail.com
MICROBIAL GROWTH
The capacity to grow, and ultimately to multiply, is one
of the most fundamental characteristics of living cells
(Posten & Coney).
GROWTH
• In biophysics cells are open systems far from a
thermodynamic equilibrium, exchange material and
energy with their environment, and especially exhibit a
large outfow of entropy.
• In chemical engineering growth is referred to as an
increasing amount of biocatalyst
• Mathematical growth are restricted to a couple of
equations employing hyperbolic and exponential terms.
• Growth is usually considered as an increase of cell
material expressed in terms of mass or cell number.
Ref: Posten & Coney
Microbial Nutrition
• Why is nutrition important?
▫ The hundreds of chemical compounds
present inside a living cell are formed
from nutrients.
Macronutrients : elements required in fairly
large amounts
Micronutrients : metals and organic
compounds needed in very small amounts
This slide is taken from : MICR 300 : Microbiology, California State of Unive
Main Macronutrients
• Carbon (C, 50% of dry weight) and nitrogen
(N, 12% of dry weight)
• Autotrophs are able to build all of their cellular
organic molecules from carbon dioxide
• Nitrogen mainly incorporated in proteins,
nucleic acids
• Most Bacteria can use NH3 and many can also
use NO3• Nitrogen fxers can utilize atmospheric nitrogen
(N2)
This slide is taken from : MICR 300 : Microbiology, California State of Unive
Other Macronutrients
• Phosphate (P), sulfur (S), potassium (K),
magnesium (Mg), calcium (Ca), sodium (Na),
iron (Fe)
• Iron plays a major role in cellular respiration,
being a key component of cytochromes and
iron-sulfur proteins involved in electron
transport.
• Siderophores : Iron-binding agents that
cells produce to obtain iron from various
insoluble minerals.
This slide is taken from : MICR 300 : Microbiology, California State of Unive
This slide is taken from : MICR 300 : Microbiology, California State of Unive
Micronutrients
Need very little amount
but critical to cell
function.
Often used as enzyme
cofactors
This slide is taken from : MICR 300 : Microbiology, California State of Unive
Growth factors
This slide is taken from : MICR 300 : Microbiology, California State of Unive
Culture Media: Composition
• Culture media supply the nutritional
needs of microorganisms
▫ defned medium : precise amounts of highly
purifed chemicals
▫ complex medium(or undefned) : highly
nutritious substances.
• Inclinical microbilogy,
▫ Selective : contains compunds that selectively
inhibit
▫ Diferential: contains indicator
▫ terms that describe media used for the isolation
of particular species or for comparative studies of
microorganisms.
This slide is taken from : MICR 300 : Microbiology, California State of Unive
S
e
C
m
S
N
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iy
m
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Media
Culture Media: Physical Properties
• Liquid
▫ Bouillon or broth
• Solid
▫ Addition of a gelling agent (typically 1% agar) to
liquid media
▫ Immobilize cells, allowing them to grow and form
visible, isolated masses called colonies (Figure
5.2).
• Semisolid
▫ Reduced amount of agar added
▫ Allows motile microorganism to spread
This slide is taken from : MICR 300 : Microbiology, California State of Unive
Bacterial Colonies on Solid Media
P. aeruginosa (TSA)
S. marcescens (Mac)
S. flexneri (Mac)
This slide is taken from : MICR 300 : Microbiology, California State of Unive
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.
This slide is taken from : MICR 300 : Microbiology, California State of Unive
Cell Growth and Binary Fission
• Microbial growth
involves an increase
in the number of
cells.
• Growth of most
microorganisms
occurs by the
process of binary
fssion
This slide is taken from : MICR 300 : Microbiology, California State of Unive
Microbial Growth
Peptidoglycan layer
This slide is taken from : MICR 300 : Microbiology, California State of Unive
Microbial Growth Pattern
• Microbial
populations show a
characteristic type
of growth pattern
called exponential
growth, which is
best seen by
plotting the number
of cells over time on
a semi- logarithmic
graph.
This slide is taken from : MICR 300 : Microbiology, California State of Unive
Growth Curve
• Microorganisms show a characteristic growth
pattern (Figure 6.8) when inoculated into a
fresh culture medium.
This slide is taken from : MICR 300 : Microbiology, California State of Unive
Measuring Microbial Growth
• Growth is measured by the change in the
number of cells over time.
▫ Cell counts done microscopically measure the
total number of cells in a population
▫ whereas viable cell counts (plate counts)
measure only the living, reproducing population.
This slide is taken from : MICR 300 : Microbiology, California State of Unive
Total Cell Count
Viable Cell Count : Determination of Colony
Forming Units
Environmental Efects on Bacterial
Growth
• Temperature
• pH
• Osmotic pressure
• Oxygen classes
This slide is taken from : MICR 300 : Microbiology, California State of Unive
Temperature and Microbial Growth
• Cardinal temperatures
▫ minimum
▫ optimum
▫ maximum
• Temperature is a major
environmental factor
controlling microbial
growth.
This slide is taken from : MICR 300 : Microbiology, California State of Unive
Classifcation of Microorganisms by Temperature
Requirements
This slide is taken from : MICR 300 : Microbiology, California State of Unive
This slide is taken from : MICR 300 : Microbiology, California State of Unive
pH and Microbial Growth
• The acidity or alkalinity of an environment can
greatly afect microbial growth.
• Most organisms grow best between pH 6 and 8, but
some organisms have evolved to grow best at low or
high pH. The internal pH of a cell must stay
relatively close to neutral even though the external
pH is highly acidic or basic.
▫ Acidophiles : organisms that grow best at low pH
▫ Alkaliphiles : organismsa that grow best at high pH
This slide is taken from : MICR 300 : Microbiology, California State of Unive
This slide is taken from : MICR 300 : Microbiology, California State of Unive
Osmotic Efects on Microbial Growth
• Osmotic pressure depends on the surrounding
solute concentration and water availability
• Water availability is generally expressed in
physical terms such as water activity
• Water activity is the ratio of the vapor
pressure of the air in equilibrium with a
substance or solution to the vapor pressure of
pure water.
This slide is taken from : MICR 300 : Microbiology, California State of Unive
This slide is taken from : MICR 300 : Microbiology, California State of Unive
Halophiles and Related Organisms
• In nature, osmotic efects are of interest mainly in
habitats with high salt environments that have
reduced water availability
• Halophiles : have evolved to grow best at
reduced water potential, and some (extreme
halophiles) even require high levels of salts for
growth.
• Halotolerant : can tolerate some reduction in the
water activity of their environment but generally
grow best in the absence of the added solute
• Xerophiles : are able to grow in very dry
environments
This slide is taken from : MICR 300 : Microbiology, California State of Unive
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
This slide is taken from : MICR 300 : Microbiology, California State of Unive
Factors Controlling Microbial
Growth
ys
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s
Physical Methods
46
Heat
Heat: Is the most practical, eficient, and
inexpensive method of sterilization of those
inanimate objects and materials that can
withstand high temperatures.
Two factors, temperature and time, determine
the efectiveness of heat for sterilization.
Sterilization is the process whereby all viable
microbes including spores are removed or
killed.
Nabeel Al-Mawajdeh RN.MCS
47
Heat (2)
a.
Dry: At 160 to 165° C for 2 hours or at 170 to 180°C for 1 hour.
- Red heat and incineration (burning): Direct exposure of material to
fame till it becomes red. E.g., culture loops.
- Direct faming: Passing material over fame many times without
reaching redness. E.g., faming mouths of bottles, slides, fasks and
culture tubes.
- Hot air oven: Supplied with a fan from inside to distribute hot air in
all chamber. It has temperature thermostat. It is used widely in
hospitals, clinics, and laboratories. E.g., test tubes, glass pipettes,
scissors, blades.
b.
Moist: Heat applied in the presence of moisture, as in boiling or
steaming, is safer and more efective than dry heat, and can be
accomplished at a lower temperature for 30 min.; thus, it is less
destructive to many materials. Moist heat causes proteins to coagulate
(as occurs when eggs are hard boiled)
- Boiling and steaming: At 100° C. clean articles made of metal and glass,
such as syringes, needles may be disinfected by boiling for 30 minutes.
Boiling is not always efective because heat- resistance bacterial
endospores, mycobacteria and viruses may be present.
- Autoclaving (steam under pressure):
An autoclave is like a large metal pressure cooker that uses steam under
pressure to completely destroy all microbial life.
48
Cold
2. Cold: Most microbes are not killed by cold
temperatures and freezing, but their
metabolic activities are slowed, greatly
inhibiting their growth.
Nabeel Al-Mawajdeh RN.MCS
49
Radiation
Radiation:
a. Nonionizing radiation:
- ultraviolet (UV) rays (has low degree of penetration): A
ultraviolet lamp (germicidal lamp) is useful for reducing
the number of microorganisms in the air and on
surfaces.
They do, however, penetrate cell and, thus, can cause
damage to DNA. When this occurs, genes may be so
severely damaged that the cell dies. Many biologic
materials, such as toxins, and vaccines, are sterilized with
UV rays.
b. Ionizing radiation:
- X-rays and gamma rays (has high degree of
penetration): Are used in industry for sterilization of
plastic catheters, syringes, surgical equipments,
preparation of vaccines…etc.
Nabeel Al-Mawajdeh RN.MCS
50
Filtration
4. Filtration: Is the passage of a liquid or gas
through a flter with pores small enough to
allow microbes to pass. This method used for
sterilization of serum, hormones, antibiotic
solution.
Nabeel Al-Mawajdeh RN.MCS
51
Gases
5. Gases (ethylene oxide, propylene oxide…etc):
Suitable for plastics, hormones, surgical
dressing, all antibiotics and thermo labile
powder.
Nabeel Al-Mawajdeh RN.MCS
Chemical Agents
53
Antiseptics
1. Antiseptics: chemicals inhibit the growth or
kill microbes on living tissues like human skin
and mucus membranes.
Nabeel Al-Mawajdeh RN.MCS
54
Disinfectants
1. Disinfectants: chemicals inhibit the growth or kill
microbes.
- Factors that determine the efectiveness of any
disinfectant:
a. Time.
b. Temperature.
c. Concentration.
d. Type and number of microbes.
e. Presence of spores.
f. Presence of proteins in feces, blood, vomitus, pus.
Nabeel Al-Mawajdeh RN.MCS
55
Disinfectants (2)
Characteristics of good disinfectant:
Rapid action, easy to use.
Wide range of action.
Good penetration.
Capability of mixing with water.
Activity in organic matter (like blood, feces, vomit)
Resistance to decomposition.
Nonstaining and noncorrosive.
Odorless.
Stable in various temperature and light.
Cheep.
Nabeel Al-Mawajdeh RN.MCS
56
Disinfectants (3)
- Kinds of Disinfectants:
a. Alcohol: 70% to disinfect skin and thermometer.
b. Phenolics:
5% phenol is useful for disinfection of stool, sputum.
0.25% - 0.5% phenol for preservation of sera and
vaccines.
c. Chlorine:
for sterilization of water supplies (1% in million) after
the treatment of water by precipitation or fltration
for removal of organic matters since they can not act
eficiency in the presence of organic matter.
d. Iodine (povidone):
for skin disinfection (available as a tincture ,2% iodine
with 70% alcohol).
e. H2O2 (hydrogene peroxide)
3 – 6 % for wounds, ulcers, and mouth wash.
f. Formaldehyde: for rubber, leather, shoes, books, and blankets.
Nabeel Al-Mawajdeh RN.MCS
57
CHEMOTHEURAPEUTICS
USING ANTI MICROBIAL AGENTS TO CONTROL MICROBIAL GROWTH IN VIVO
Nabeel Al-Mawajdeh RN.MCS
58
Using Chemotherapeutic agents
- Chemotherapeutic agent is any chemical
(drug) used to treat an infectious disease, either
by inhibiting or killing pathogens in vivo.
a. Antifungal agents are used to treat fungal
diseases.
b. Antiprotozoal agents are used to treat protozoal
diseases.
c. Antiviral agents are used to treat viral diseases.
d. Antibiotics
Nabeel Al-Mawajdeh RN.MCS
59
Using Chemotherapeutic agents
(Cont’d)
d. Antibiotics are substances produced by
microorganisms (usually a soil organism) that
efective in killing or inhibiting the growth of
other microorganisms. Some antibiotics (e.g.,
penicillin and cephalosporin) are produced by
molds, whereas others (e.g., tetracycline,
erythromycin, and chloramphenicol) are
produced by bacteria.
Many antibiotics have been chemically modifed
to kill a wider variety of pathogens or reduce
side efects; these modifed antibiotics are called
semisynthetic antibiotics.
Nabeel Al-Mawajdeh RN.MCS
60
Ideal Qualities Of Antimicrobial
Agents
1. Antimicrobial agents should have selective
toxicity
for microorganisms. E.g., it can kill or inhibit
the
growth of a microorganism in concentrations
that
are not harmful to the cells of the host.
Therapeutic index=
large amount of antimicrobial can be given
without harm
Lowest dose that can kill microorganism
The higher the therapeutic index the better the
antimicrobial.
61
Ideal Qualities Of Antimicrobial Agents
(Cont’d)
2.
3.
4.
5.
6.
Should Kill or inhibit the growth of
pathogens.
Cause no damage to the host.
Be stable when stored in solid or liquid
form.
Remain in specifc tissues in the body long
enough
to be efective.
Kill the pathogens before the mutate and
become
resistant to it. Unfortunately, most
antimicrobial
agents have some side efects, produce
allergic
reactions, or permit development of
resistant mutant pathogens.
64
Mechanisms of Resistance to
Antimicrobial Agents
1.
2.
3.
The organism produces enzymes that destroy
the drug. E.g., production of beta – lactamases
that destroys penicillin.
The organism changes its permeability to the
drug, by modifcation of protein in the outer
cell membranes, thus impairing its active
transport into the cell e.g., resistance to
polymyxins.
The organism develops an altered receptor site
for the drug e.g., resistance to aminoglycosides
is associated with alteration of a specifc
protein in the 30s subunit of the bacterial
ribosome that serves as a binding site in
susceptible organisms.
Nabeel Al-Mawajdeh RN.MCS
65
Mechanisms of Resistance to Antimicrobial
Agents (Cont’d)
4.
The organism develops an altered metabolic
pathway that bypasses the reaction inhibited by
the drug e.g., sulphonamide- resistant bacteria
acquire the ability to use performed folic acid
with no need for extracellular PABA (paminobenzoic acid)
Nabeel Al-Mawajdeh RN.MCS
67
Clinical Use of Antibiotics
1.
2.
3.
4.
Antibiotics should not be given for trivial
infections.
Treatment should be based on a clear clinical
and bacteriological diagnosis. According to
results of antibiotic sensitivity testing in vitro.
Antibiotics for systemic treatment should be
given in full therapeutic doses for adequate
period.
Combined therapy with two or more antibiotics
is required in some conditions. E.g,
a. Serious resistant infections e.g., infective endocarditis or
meningitis.
b. In treatment of tuberculosis 2 or 3 drugs are given by
lowering the dose of each to decrease toxic efects of drugs.
c. Severe mixed infections e.g., peritonitis following
perforation of the colon.
Nabeel Al-Mawajdeh RN.MCS
Exp.8 -Temperature
a. Efect of temperature on bacteria and yeast cells
b. Efect of temperature on vegetative and spore cells
of bacteria, yeast, and fungi
8.A. Efect of temperature on bacteria and yeast
cells
Durham Tube
• Durham tubes are used in
microbiology to detect
production of gas by
microorganisms.
• This small tube is initially
flled with the solution in
which the microorganism
is to be grown. If gas is
produced after
inoculation and
incubation, a visible gas
bubble will be trapped
inside the small tube.
8.B. Efect of temperature on
vegetative and spore cells of bacteria,
yeast, and fungi
m.o
4
Broth
tube
Hot
water
80 ˚C
Cold
water
Strea
k in 4
gluco
se
broth
Room
temp
eratu
re 4
days
Gram
&
spore
staini
ng
u
tr
it
io
n
m
b
.o
r
o
t
h
p
H
3
5
7
9
Exp.9 - pH
spectrophotometer
d
a
m
g
.o
a
r
pl
a
t
e
Exp.10 - Oxygen Requirements
Exp.11 –Osmotic Pressure
sl
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e,
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y
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e
Exp.12 Chemotherapeutic
Agent
B
D
A
C
Exp. 13 Disinfectant and
Antiseptic
a. Alcohol efectivity evaluation
b. Antiseptic evaluation using blotter
c. Efectivity of handwashing using soap
13.A. Alcohol efectivity
evaluation
• A = left thumb without
•
B
D
A
C
•
•
•
alcohol
B = left thumb without
alcohol
C = right thumb
without alcohol
D = right thumb with
alcohol
Incubate the dish
z
t
er
e,
s,
in
i
c
m
m
m
u
.o
er
b
se
a
bl
t
o
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at
t
er
o
s
d
in
al
r
c
ai
o
n
h
ol
13.B. Antiseptic evaluation using
blotter
13.C. Efectivity of hand washing using
soap
Unwashed Hand
Washed Hand
Once again, safety
frst !
RATIONAL USE OF
MEDICINE Use
Antibiotics Wisely.
-Laboratory Manualprepared for Environmental Microbiology
III
Microorganism Growth
Factors Controlling Growth
E N V I R O N M E N TA L M A N A G E M E N T T E C H N O L O G Y
FA C U LT Y O F C I V I L A N D E N V I R O N M E N TA L E N G I N E E R I N G
ITB, 2010
anindrya.pahlawan@ymail.com
MICROBIAL GROWTH
The capacity to grow, and ultimately to multiply, is one
of the most fundamental characteristics of living cells
(Posten & Coney).
GROWTH
• In biophysics cells are open systems far from a
thermodynamic equilibrium, exchange material and
energy with their environment, and especially exhibit a
large outfow of entropy.
• In chemical engineering growth is referred to as an
increasing amount of biocatalyst
• Mathematical growth are restricted to a couple of
equations employing hyperbolic and exponential terms.
• Growth is usually considered as an increase of cell
material expressed in terms of mass or cell number.
Ref: Posten & Coney
Microbial Nutrition
• Why is nutrition important?
▫ The hundreds of chemical compounds
present inside a living cell are formed
from nutrients.
Macronutrients : elements required in fairly
large amounts
Micronutrients : metals and organic
compounds needed in very small amounts
This slide is taken from : MICR 300 : Microbiology, California State of Unive
Main Macronutrients
• Carbon (C, 50% of dry weight) and nitrogen
(N, 12% of dry weight)
• Autotrophs are able to build all of their cellular
organic molecules from carbon dioxide
• Nitrogen mainly incorporated in proteins,
nucleic acids
• Most Bacteria can use NH3 and many can also
use NO3• Nitrogen fxers can utilize atmospheric nitrogen
(N2)
This slide is taken from : MICR 300 : Microbiology, California State of Unive
Other Macronutrients
• Phosphate (P), sulfur (S), potassium (K),
magnesium (Mg), calcium (Ca), sodium (Na),
iron (Fe)
• Iron plays a major role in cellular respiration,
being a key component of cytochromes and
iron-sulfur proteins involved in electron
transport.
• Siderophores : Iron-binding agents that
cells produce to obtain iron from various
insoluble minerals.
This slide is taken from : MICR 300 : Microbiology, California State of Unive
This slide is taken from : MICR 300 : Microbiology, California State of Unive
Micronutrients
Need very little amount
but critical to cell
function.
Often used as enzyme
cofactors
This slide is taken from : MICR 300 : Microbiology, California State of Unive
Growth factors
This slide is taken from : MICR 300 : Microbiology, California State of Unive
Culture Media: Composition
• Culture media supply the nutritional
needs of microorganisms
▫ defned medium : precise amounts of highly
purifed chemicals
▫ complex medium(or undefned) : highly
nutritious substances.
• Inclinical microbilogy,
▫ Selective : contains compunds that selectively
inhibit
▫ Diferential: contains indicator
▫ terms that describe media used for the isolation
of particular species or for comparative studies of
microorganisms.
This slide is taken from : MICR 300 : Microbiology, California State of Unive
S
e
C
m
S
N
o
iy
m
a
n
s
p
t
yt
u
o
n
h
si
r
et
ti
a
ti
h
ol
e
c
n
ti
c
Media
Culture Media: Physical Properties
• Liquid
▫ Bouillon or broth
• Solid
▫ Addition of a gelling agent (typically 1% agar) to
liquid media
▫ Immobilize cells, allowing them to grow and form
visible, isolated masses called colonies (Figure
5.2).
• Semisolid
▫ Reduced amount of agar added
▫ Allows motile microorganism to spread
This slide is taken from : MICR 300 : Microbiology, California State of Unive
Bacterial Colonies on Solid Media
P. aeruginosa (TSA)
S. marcescens (Mac)
S. flexneri (Mac)
This slide is taken from : MICR 300 : Microbiology, California State of Unive
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.
This slide is taken from : MICR 300 : Microbiology, California State of Unive
Cell Growth and Binary Fission
• Microbial growth
involves an increase
in the number of
cells.
• Growth of most
microorganisms
occurs by the
process of binary
fssion
This slide is taken from : MICR 300 : Microbiology, California State of Unive
Microbial Growth
Peptidoglycan layer
This slide is taken from : MICR 300 : Microbiology, California State of Unive
Microbial Growth Pattern
• Microbial
populations show a
characteristic type
of growth pattern
called exponential
growth, which is
best seen by
plotting the number
of cells over time on
a semi- logarithmic
graph.
This slide is taken from : MICR 300 : Microbiology, California State of Unive
Growth Curve
• Microorganisms show a characteristic growth
pattern (Figure 6.8) when inoculated into a
fresh culture medium.
This slide is taken from : MICR 300 : Microbiology, California State of Unive
Measuring Microbial Growth
• Growth is measured by the change in the
number of cells over time.
▫ Cell counts done microscopically measure the
total number of cells in a population
▫ whereas viable cell counts (plate counts)
measure only the living, reproducing population.
This slide is taken from : MICR 300 : Microbiology, California State of Unive
Total Cell Count
Viable Cell Count : Determination of Colony
Forming Units
Environmental Efects on Bacterial
Growth
• Temperature
• pH
• Osmotic pressure
• Oxygen classes
This slide is taken from : MICR 300 : Microbiology, California State of Unive
Temperature and Microbial Growth
• Cardinal temperatures
▫ minimum
▫ optimum
▫ maximum
• Temperature is a major
environmental factor
controlling microbial
growth.
This slide is taken from : MICR 300 : Microbiology, California State of Unive
Classifcation of Microorganisms by Temperature
Requirements
This slide is taken from : MICR 300 : Microbiology, California State of Unive
This slide is taken from : MICR 300 : Microbiology, California State of Unive
pH and Microbial Growth
• The acidity or alkalinity of an environment can
greatly afect microbial growth.
• Most organisms grow best between pH 6 and 8, but
some organisms have evolved to grow best at low or
high pH. The internal pH of a cell must stay
relatively close to neutral even though the external
pH is highly acidic or basic.
▫ Acidophiles : organisms that grow best at low pH
▫ Alkaliphiles : organismsa that grow best at high pH
This slide is taken from : MICR 300 : Microbiology, California State of Unive
This slide is taken from : MICR 300 : Microbiology, California State of Unive
Osmotic Efects on Microbial Growth
• Osmotic pressure depends on the surrounding
solute concentration and water availability
• Water availability is generally expressed in
physical terms such as water activity
• Water activity is the ratio of the vapor
pressure of the air in equilibrium with a
substance or solution to the vapor pressure of
pure water.
This slide is taken from : MICR 300 : Microbiology, California State of Unive
This slide is taken from : MICR 300 : Microbiology, California State of Unive
Halophiles and Related Organisms
• In nature, osmotic efects are of interest mainly in
habitats with high salt environments that have
reduced water availability
• Halophiles : have evolved to grow best at
reduced water potential, and some (extreme
halophiles) even require high levels of salts for
growth.
• Halotolerant : can tolerate some reduction in the
water activity of their environment but generally
grow best in the absence of the added solute
• Xerophiles : are able to grow in very dry
environments
This slide is taken from : MICR 300 : Microbiology, California State of Unive
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
This slide is taken from : MICR 300 : Microbiology, California State of Unive
Factors Controlling Microbial
Growth
ys
nt
e
ro
m
ic
al
ic
l
M
al
et
A
h
g
o
e
nt
d
s
Physical Methods
46
Heat
Heat: Is the most practical, eficient, and
inexpensive method of sterilization of those
inanimate objects and materials that can
withstand high temperatures.
Two factors, temperature and time, determine
the efectiveness of heat for sterilization.
Sterilization is the process whereby all viable
microbes including spores are removed or
killed.
Nabeel Al-Mawajdeh RN.MCS
47
Heat (2)
a.
Dry: At 160 to 165° C for 2 hours or at 170 to 180°C for 1 hour.
- Red heat and incineration (burning): Direct exposure of material to
fame till it becomes red. E.g., culture loops.
- Direct faming: Passing material over fame many times without
reaching redness. E.g., faming mouths of bottles, slides, fasks and
culture tubes.
- Hot air oven: Supplied with a fan from inside to distribute hot air in
all chamber. It has temperature thermostat. It is used widely in
hospitals, clinics, and laboratories. E.g., test tubes, glass pipettes,
scissors, blades.
b.
Moist: Heat applied in the presence of moisture, as in boiling or
steaming, is safer and more efective than dry heat, and can be
accomplished at a lower temperature for 30 min.; thus, it is less
destructive to many materials. Moist heat causes proteins to coagulate
(as occurs when eggs are hard boiled)
- Boiling and steaming: At 100° C. clean articles made of metal and glass,
such as syringes, needles may be disinfected by boiling for 30 minutes.
Boiling is not always efective because heat- resistance bacterial
endospores, mycobacteria and viruses may be present.
- Autoclaving (steam under pressure):
An autoclave is like a large metal pressure cooker that uses steam under
pressure to completely destroy all microbial life.
48
Cold
2. Cold: Most microbes are not killed by cold
temperatures and freezing, but their
metabolic activities are slowed, greatly
inhibiting their growth.
Nabeel Al-Mawajdeh RN.MCS
49
Radiation
Radiation:
a. Nonionizing radiation:
- ultraviolet (UV) rays (has low degree of penetration): A
ultraviolet lamp (germicidal lamp) is useful for reducing
the number of microorganisms in the air and on
surfaces.
They do, however, penetrate cell and, thus, can cause
damage to DNA. When this occurs, genes may be so
severely damaged that the cell dies. Many biologic
materials, such as toxins, and vaccines, are sterilized with
UV rays.
b. Ionizing radiation:
- X-rays and gamma rays (has high degree of
penetration): Are used in industry for sterilization of
plastic catheters, syringes, surgical equipments,
preparation of vaccines…etc.
Nabeel Al-Mawajdeh RN.MCS
50
Filtration
4. Filtration: Is the passage of a liquid or gas
through a flter with pores small enough to
allow microbes to pass. This method used for
sterilization of serum, hormones, antibiotic
solution.
Nabeel Al-Mawajdeh RN.MCS
51
Gases
5. Gases (ethylene oxide, propylene oxide…etc):
Suitable for plastics, hormones, surgical
dressing, all antibiotics and thermo labile
powder.
Nabeel Al-Mawajdeh RN.MCS
Chemical Agents
53
Antiseptics
1. Antiseptics: chemicals inhibit the growth or
kill microbes on living tissues like human skin
and mucus membranes.
Nabeel Al-Mawajdeh RN.MCS
54
Disinfectants
1. Disinfectants: chemicals inhibit the growth or kill
microbes.
- Factors that determine the efectiveness of any
disinfectant:
a. Time.
b. Temperature.
c. Concentration.
d. Type and number of microbes.
e. Presence of spores.
f. Presence of proteins in feces, blood, vomitus, pus.
Nabeel Al-Mawajdeh RN.MCS
55
Disinfectants (2)
Characteristics of good disinfectant:
Rapid action, easy to use.
Wide range of action.
Good penetration.
Capability of mixing with water.
Activity in organic matter (like blood, feces, vomit)
Resistance to decomposition.
Nonstaining and noncorrosive.
Odorless.
Stable in various temperature and light.
Cheep.
Nabeel Al-Mawajdeh RN.MCS
56
Disinfectants (3)
- Kinds of Disinfectants:
a. Alcohol: 70% to disinfect skin and thermometer.
b. Phenolics:
5% phenol is useful for disinfection of stool, sputum.
0.25% - 0.5% phenol for preservation of sera and
vaccines.
c. Chlorine:
for sterilization of water supplies (1% in million) after
the treatment of water by precipitation or fltration
for removal of organic matters since they can not act
eficiency in the presence of organic matter.
d. Iodine (povidone):
for skin disinfection (available as a tincture ,2% iodine
with 70% alcohol).
e. H2O2 (hydrogene peroxide)
3 – 6 % for wounds, ulcers, and mouth wash.
f. Formaldehyde: for rubber, leather, shoes, books, and blankets.
Nabeel Al-Mawajdeh RN.MCS
57
CHEMOTHEURAPEUTICS
USING ANTI MICROBIAL AGENTS TO CONTROL MICROBIAL GROWTH IN VIVO
Nabeel Al-Mawajdeh RN.MCS
58
Using Chemotherapeutic agents
- Chemotherapeutic agent is any chemical
(drug) used to treat an infectious disease, either
by inhibiting or killing pathogens in vivo.
a. Antifungal agents are used to treat fungal
diseases.
b. Antiprotozoal agents are used to treat protozoal
diseases.
c. Antiviral agents are used to treat viral diseases.
d. Antibiotics
Nabeel Al-Mawajdeh RN.MCS
59
Using Chemotherapeutic agents
(Cont’d)
d. Antibiotics are substances produced by
microorganisms (usually a soil organism) that
efective in killing or inhibiting the growth of
other microorganisms. Some antibiotics (e.g.,
penicillin and cephalosporin) are produced by
molds, whereas others (e.g., tetracycline,
erythromycin, and chloramphenicol) are
produced by bacteria.
Many antibiotics have been chemically modifed
to kill a wider variety of pathogens or reduce
side efects; these modifed antibiotics are called
semisynthetic antibiotics.
Nabeel Al-Mawajdeh RN.MCS
60
Ideal Qualities Of Antimicrobial
Agents
1. Antimicrobial agents should have selective
toxicity
for microorganisms. E.g., it can kill or inhibit
the
growth of a microorganism in concentrations
that
are not harmful to the cells of the host.
Therapeutic index=
large amount of antimicrobial can be given
without harm
Lowest dose that can kill microorganism
The higher the therapeutic index the better the
antimicrobial.
61
Ideal Qualities Of Antimicrobial Agents
(Cont’d)
2.
3.
4.
5.
6.
Should Kill or inhibit the growth of
pathogens.
Cause no damage to the host.
Be stable when stored in solid or liquid
form.
Remain in specifc tissues in the body long
enough
to be efective.
Kill the pathogens before the mutate and
become
resistant to it. Unfortunately, most
antimicrobial
agents have some side efects, produce
allergic
reactions, or permit development of
resistant mutant pathogens.
64
Mechanisms of Resistance to
Antimicrobial Agents
1.
2.
3.
The organism produces enzymes that destroy
the drug. E.g., production of beta – lactamases
that destroys penicillin.
The organism changes its permeability to the
drug, by modifcation of protein in the outer
cell membranes, thus impairing its active
transport into the cell e.g., resistance to
polymyxins.
The organism develops an altered receptor site
for the drug e.g., resistance to aminoglycosides
is associated with alteration of a specifc
protein in the 30s subunit of the bacterial
ribosome that serves as a binding site in
susceptible organisms.
Nabeel Al-Mawajdeh RN.MCS
65
Mechanisms of Resistance to Antimicrobial
Agents (Cont’d)
4.
The organism develops an altered metabolic
pathway that bypasses the reaction inhibited by
the drug e.g., sulphonamide- resistant bacteria
acquire the ability to use performed folic acid
with no need for extracellular PABA (paminobenzoic acid)
Nabeel Al-Mawajdeh RN.MCS
67
Clinical Use of Antibiotics
1.
2.
3.
4.
Antibiotics should not be given for trivial
infections.
Treatment should be based on a clear clinical
and bacteriological diagnosis. According to
results of antibiotic sensitivity testing in vitro.
Antibiotics for systemic treatment should be
given in full therapeutic doses for adequate
period.
Combined therapy with two or more antibiotics
is required in some conditions. E.g,
a. Serious resistant infections e.g., infective endocarditis or
meningitis.
b. In treatment of tuberculosis 2 or 3 drugs are given by
lowering the dose of each to decrease toxic efects of drugs.
c. Severe mixed infections e.g., peritonitis following
perforation of the colon.
Nabeel Al-Mawajdeh RN.MCS
Exp.8 -Temperature
a. Efect of temperature on bacteria and yeast cells
b. Efect of temperature on vegetative and spore cells
of bacteria, yeast, and fungi
8.A. Efect of temperature on bacteria and yeast
cells
Durham Tube
• Durham tubes are used in
microbiology to detect
production of gas by
microorganisms.
• This small tube is initially
flled with the solution in
which the microorganism
is to be grown. If gas is
produced after
inoculation and
incubation, a visible gas
bubble will be trapped
inside the small tube.
8.B. Efect of temperature on
vegetative and spore cells of bacteria,
yeast, and fungi
m.o
4
Broth
tube
Hot
water
80 ˚C
Cold
water
Strea
k in 4
gluco
se
broth
Room
temp
eratu
re 4
days
Gram
&
spore
staini
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b
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p
H
3
5
7
9
Exp.9 - pH
spectrophotometer
d
a
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g
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a
r
pl
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e
Exp.10 - Oxygen Requirements
Exp.11 –Osmotic Pressure
sl
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Exp.12 Chemotherapeutic
Agent
B
D
A
C
Exp. 13 Disinfectant and
Antiseptic
a. Alcohol efectivity evaluation
b. Antiseptic evaluation using blotter
c. Efectivity of handwashing using soap
13.A. Alcohol efectivity
evaluation
• A = left thumb without
•
B
D
A
C
•
•
•
alcohol
B = left thumb without
alcohol
C = right thumb
without alcohol
D = right thumb with
alcohol
Incubate the dish
z
t
er
e,
s,
in
i
c
m
m
m
u
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b
se
a
bl
t
o
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at
t
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d
in
al
r
c
ai
o
n
h
ol
13.B. Antiseptic evaluation using
blotter
13.C. Efectivity of hand washing using
soap
Unwashed Hand
Washed Hand
Once again, safety
frst !
RATIONAL USE OF
MEDICINE Use
Antibiotics Wisely.