Environmental Microbiology
-Laboratory Manualprepared for Environmental Microbiology
III
Microorganism Growth
Factors Controlling Growth
ENVIRONMENTAL MANAGEMENT TECHNOLOGY
FACULTY OF CIVIL AND ENVIRONMENTAL ENGINEERING
ITB, 2010
anindrya.pahlawan@ymail.com
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 outflow 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 University
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 fixers can utilize atmospheric nitrogen (N2)
This slide is taken from : MICR 300 : Microbiology, California State of University
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 University
This slide is taken from : MICR 300 : Microbiology, California State of University
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 University
Growth factors
This slide is taken from : MICR 300 : Microbiology, California State of University
Culture Media: Composition
• Culture media supply the nutritional needs of
microorganisms
▫ defined medium : precise amounts of highly purified
chemicals
▫ complex medium(or undefined) : highly nutritious
substances.
• Inclinical microbilogy,
▫ Selective : contains compunds that selectively inhibit
▫ Differential: 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 University
Media
Composition
Use
Natural
General
Semisynthetic
Selective
Synthetic
Differential
Enriched
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 University
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 University
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 University
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
fission
This slide is taken from : MICR 300 : Microbiology, California State of University
Microbial Growth
Peptidoglycan layer
This slide is taken from : MICR 300 : Microbiology, California State of University
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 semilogarithmic graph.
This slide is taken from : MICR 300 : Microbiology, California State of University
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 University
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 University
Total Cell Count
Viable Cell Count : Determination of Colony Forming Units
Environmental Effects on Bacterial Growth
• Temperature
• pH
• Osmotic pressure
• Oxygen classes
This slide is taken from : MICR 300 : Microbiology, California State of University
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 University
Classification of Microorganisms by Temperature Requirements
This slide is taken from : MICR 300 : Microbiology, California State of University
This slide is taken from : MICR 300 : Microbiology, California State of University
pH and Microbial Growth
• The acidity or alkalinity of an environment can greatly affect
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 University
This slide is taken from : MICR 300 : Microbiology, California State of University
Osmotic Effects 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 University
This slide is taken from : MICR 300 : Microbiology, California State of University
Halophiles and Related Organisms
• In nature, osmotic effects 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 University
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 University
Temperature
(heat, cold)
Radiation
Physical Methods
Filtration
Gas
Control Methods
Antiseptics
Chemical Agents
Disinfectants
Chemotherapeutics (in
vivo)
46
Heat
Heat: Is the most practical, efficient, and inexpensive
method of sterilization of those inanimate objects
and materials that can withstand high temperatures.
Two factors, temperature and time, determine the
effectiveness 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 flame till it
becomes red. E.g., culture loops.
- Direct flaming: Passing material over flame many times without reaching redness.
E.g., flaming mouths of bottles, slides, flasks 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 effective 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 effective
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
filter 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
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 effectiveness 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 filtration
for removal of organic matters since they can not act
efficiency 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
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 effective 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 modified to kill a
wider variety of pathogens or reduce side effects; these
modified 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.
Nabeel Al-Mawajdeh RN.MCS
61
Ideal Qualities Of Antimicrobial Agents (Cont’d)
2.
3.
4.
5.
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 specific tissues in the body long enough
to be effective.
6. Kill the pathogens before the mutate and become
resistant to it. Unfortunately, most antimicrobial
agents have some side effects, produce allergic
reactions, or permit development of resistant mutant
pathogens.
Nabeel Al-Mawajdeh RN.MCS
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
modification 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 specific 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 (p- aminobenzoic 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 effects of drugs.
c. Severe mixed infections e.g., peritonitis following perforation of the colon.
Nabeel Al-Mawajdeh RN.MCS
a. Effect of temperature on bacteria and yeast cells
b. Effect of temperature on vegetative and spore cells of bacteria,
yeast, and fungi
8.A. Effect of temperature on bacteria and yeast cells
Incubate in
4 ˚C
agar plate
Streak
plate
Incubate in
20/25 ˚ C
Incubate in
37 ˚ C
Incubate in
44 ˚ C
m.o.
culture
Incubate in
4 ˚C
broth tube
+ Durham
tube
Inoculatio
n
Incubate in
20/25 ˚ C
Incubate in
37 ˚ C
Incubate in
44 ˚ C
Durham Tube
• Durham tubes are used in
microbiology to detect
production of gas by
microorganisms.
• This small tube is initially filled
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. Effect of temperature on vegetative and
spore cells of bacteria, yeast, and fungi
m.o.
culture
4 Broth
tube
Hot
water 80
˚C
Cold
water
Streak in
4
glucose
broth
Room
tempera
ture 4
days
Gram &
spore
staining
Exp.9 - pH
Nutrition broth
pH 3
Inoculate E. coli
24-48 jam
incubate 37 ˚C
Nutrition broth
pH 5
Inoculate
Alcaligenes
faecalis
24-48 jam
incubate 37 ˚C
Nutrition broth
pH 7
Inoculate
S.aureus
24-48 jam
incubate 37 ˚C
Nutrition broth
pH 9
Inoculate
S.cereviceae
room temp. 4872 hours
m.o. culture
spectrophotometer
Exp.10 - Oxygen Requirements
divided agar
plate
Streak plate
m.o. culture
divided agar
plate
Streak plate
24-48 hours
incubate 37
˚C
anaerob jar
24-48 hours
incubate 37
˚C
Exp.11 –Osmotic Pressure
slant agar NaCl
0,5 %
Inoculate
24-48 hours
incubate 37 ˚C
slant agar NaCl 5
%
Inoculate
24-48 hours
incubate 37 ˚C
slant agar NaCl
10 %
Inoculate
24-48 hours
incubate 37 ˚C
slant agar NaCl
15 %
inoculate
24-48 hours
incubate 37 ˚C
m.o. culture
Exp.12 Chemotherapeutic Agent
agar plate,
incubate to
drain
swab
Press
immersed
bloaters in
plate
24-48 hours
incubate 37
˚C
Using
tweezers,
immerse
bloaters in
penicillin
m.o. culture
Using
tweezers,
immerse
bloaters in
tetracycline
Using
tweezers,
immerse
bloaters in
streptomycin
B
D
A
C
a. Alcohol effectivity evaluation
b. Antiseptic evaluation using blotter
c. Effectivity of handwashing using soap
13.A. Alcohol effectivity evaluation
B
D
A
C
• A = left thumb without
alcohol
• B = left thumb without
alcohol
• C = right thumb without
alcohol
• D = right thumb with
alcohol
• Incubate the dish
13.B. Antiseptic evaluation using blotter
agar plate,
incubate to
drain
m.o. culture
Using
tweezers,
immerse
bloaters in
alcohol
swab
Press
immersed
bloaters in
plate
24-48 hours
incubate 37
˚C
13.C. Effectivity of hand washing using soap
Unwashed Hand
Washed Hand