Environmental Microbiology -Laboratory Manual- prepared for
Environmental Microbiology III Microorganism Growth Factors
Controlling Growth [email protected] ENVIRONMENTAL
MANAGEMENT TECHNOLOGY FACULTY OF CIVIL AND ENVIRONMENTAL
ENGINEERING ITB, 2010
Slide 2
The capacity to grow, and ultimately to multiply, is one of the
most fundamental characteristics of living cells (Posten &
Coney).
Slide 3
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
Slide 4
Microbial Nutrition Why is nutrition important? The hundreds of
chemical compounds present inside a living cell are formed from
nutrients. This slide is taken from : MICR 300 : Microbiology,
California State of University Macronutrients : elements required
in fairly large amounts Micronutrients : metals and organic
compounds needed in very small amounts
Slide 5
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 NH 3
and many can also use NO 3 - Nitrogen fixers can utilize
atmospheric nitrogen (N 2 ) This slide is taken from : MICR 300 :
Microbiology, California State of University
Slide 6
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
Slide 7
Representative Siderophore Ferric enterobactin Aquachelin This
slide is taken from : MICR 300 : Microbiology, California State of
University
Slide 8
Slide 9
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
Slide 10
Growth factors This slide is taken from : MICR 300 :
Microbiology, California State of University
Slide 11
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
Slide 12
Media Composition Natural Semi- synthetic Synthetic Use
GeneralSelectiveDifferentialEnriched
Slide 13
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
Slide 14
Bacterial Colonies on Solid Media S. marcescens (Mac) P.
aeruginosa (TSA) S. flexneri (Mac) This slide is taken from : MICR
300 : Microbiology, California State of University
Slide 15
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
Slide 16
Microbial Growth Binary fission This slide is taken from : MICR
300 : Microbiology, California State of University
Slide 17
Microbial growth involves an increase in the number of cells.
Growth of most microorganisms occurs by the process of binary
fission Cell Growth and Binary Fission This slide is taken from :
MICR 300 : Microbiology, California State of University
Slide 18
Microbial Growth Peptidoglycan layer This slide is taken from :
MICR 300 : Microbiology, California State of University
Slide 19
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.
Microbial Growth Pattern This slide is taken from : MICR 300 :
Microbiology, California State of University
Slide 20
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
Slide 21
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
Slide 22
Total Cell Count
Slide 23
Viable Cell Count : Determination of Colony Forming Units
Slide 24
Serial Dilution of Cells This slide is taken from : MICR 300 :
Microbiology, California State of University
Slide 25
Indirect Cell Number Measurement : Turbidity Turbidity
measurements are an indirect but very rapid and useful method of
measuring microbial growth (Figure 6.12). However, to relate a
direct cell count to a turbidity value, a standard curve must first
be established. This slide is taken from : MICR 300 : Microbiology,
California State of University
Slide 26
Turbidity Measurements of Microbial Growth This slide is taken
from : MICR 300 : Microbiology, California State of University
Slide 27
Environmental Effects on Bacterial Growth Temperature pH
Osmotic pressure Oxygen classes This slide is taken from : MICR 300
: Microbiology, California State of University
Slide 28
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
Slide 29
Classification of Microorganisms by Temperature Requirements
This slide is taken from : MICR 300 : Microbiology, California
State of University
Slide 30
Temperature Classes of Organisms Mesophiles Midrange
temperature optima Found in warm-blooded animals and in terrestrial
and aquatic environments in temperate and tropical latitudes
Psychrophiles Cold temperature optima Most extreme representatives
inhabit permanently cold environments Thermophiles Growth
temperature optima between 45C and 80C Hyperthermophiles Optima
greater than 80C These organisms inhabit hot environments including
boiling hot springs, as well as undersea hydrothermal vents that
can have temperatures in excess of 100C This slide is taken from :
MICR 300 : Microbiology, California State of University
Slide 31
Heat-Stable Macromolecules Thermophiles and hyperthermophiles
produce heat-stable macromolecules, such as Taq polymerase, which
is used to automate the repetitive steps in the polymerase chain
reaction (PCR) technique. This slide is taken from : MICR 300 :
Microbiology, California State of University
Slide 32
Slide 33
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
Slide 34
Slide 35
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
Slide 36
Slide 37
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
Slide 38
Slide 39
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
Slide 40
This slide is taken from : MICR 300 : Microbiology, California
State of University
Slide 41
Test for Oxygen Requirements of Microorganisms Thioglycolate
broth : contains a reducing agent and provides aerobic and
anaerobic conditions a)Aerobic b)Anaerobic c)Facultative
d)Microaerophil e)Aerotolerant This slide is taken from : MICR 300
: Microbiology, California State of University
Slide 42
Toxic Forms of Oxygen and Detoxifying EnzymesHydrogenperoxide
Superoxide This slide is taken from : MICR 300 : Microbiology,
California State of University
Slide 43
Slide 44
Control MethodsPhysical Methods Temperature (heat, cold)
RadiationFiltrationGasChemical AgentsAntisepticsDisinfectants
Chemotherapeutics (in vivo)
Slide 45
Slide 46
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
Slide 47
47 Heat (2) a.Dry: At 160 to 165 C for 2 hours or at 170 to
180C 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. Nabeel Al-Mawajdeh RN.MCS
Slide 48
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
Slide 49
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
vaccinesetc. Nabeel Al-Mawajdeh RN.MCS
Slide 50
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
Slide 51
51 Gases 5.Gases (ethylene oxide, propylene oxideetc): Suitable
for plastics, hormones, surgical dressing, all antibiotics and
thermo labile powder. Nabeel Al-Mawajdeh RN.MCS
Slide 52
Slide 53
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
Slide 54
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
Slide 55
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
Slide 56
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
Slide 57
USING ANTI MICROBIAL AGENTS TO CONTROL MICROBIAL GROWTH IN VIVO
Nabeel Al-Mawajdeh RN.MCS 57
Slide 58
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
Slide 59
59 Using Chemotherapeutic agents (Contd) 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
Slide 60
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
Slide 61
61 Ideal Qualities Of Antimicrobial Agents (Contd) 2.Should
Kill or inhibit the growth of pathogens. 3.Cause no damage to the
host. 4.Be stable when stored in solid or liquid form. 5.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
Slide 62
62 Mechanisms Of Action Of Antimicrobial Agents 1.Inhibition of
cell wall synthesis. Due to its unique structure and function, the
bacterial cell wall is an ideal point of attack by selective toxic
agents. Some antibiotics e.g., penicillin, cephalosporins,
interfere with cell wall synthesis and cause bacteriolysis.
2.Inhibition of cytoplasmic membrane function. Some antibiotics
cause disruption of the membrane and leakage of cellular proteins
and nucleotides leading to cell death. E.g., nystatin, polymyxins.
Nabeel Al-Mawajdeh RN.MCS
Slide 63
63 3.Inhibition of nucleic acid synthesis or replication (DNA
or RNA). e.g., rifampicin, metronidazole, nalidixic. 4.Inhibition
of protein synthesis. Many antimicrobial agents block protein
synthesis by acting on the 30s or 50s subunits of the bacterial
ribosome. E.g., chloramphenicol, tetracycline, erythromycin,
gentamycin, streptomycin, lincomycin. Nabeel Al-Mawajdeh RN.MCS
Mechanisms Of Action Of Antimicrobial Agents (Contd)
Slide 64
64 Mechanisms of Resistance to Antimicrobial Agents 1.The
organism produces enzymes that destroy the drug. E.g., production
of beta lactamases that destroys penicillin. 2.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. 3.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
Slide 65
65 Mechanisms of Resistance to Antimicrobial Agents (Contd)
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
Slide 66
66 Antiviral Drugs 1.Amantadine hydrochloride: it inhibits the
penetration of the virus into the susceptible cells. It is used
usually in treatment of influenza A virus infection. 2.Idoxuridine:
it inhibits the replication of DNA viruses. It is severely
cytotoxic, used topically for treatment of herpes simplex
infection. 3.Methisazone: it blocks replication of poxviruses.
4.Interferon: used systemic and topical as a ''broad spectrum''
antiviral agent. Nabeel Al-Mawajdeh RN.MCS
Slide 67
67 Clinical Use of Antibiotics 1.Antibiotics should not be
given for trivial infections. 2.Treatment should be based on a
clear clinical and bacteriological diagnosis. According to results
of antibiotic sensitivity testing in vitro. 3.Antibiotics for
systemic treatment should be given in full therapeutic doses for
adequate period. 4.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
Slide 68
68 Complications of Antibacterial Chemotherapy 1. Development
of drug resistance. This can be due to inadequate dosage, prolonged
treatment and the abuse of antibiotics without in vitro
susceptibility testing. 2. Drug toxicity. This can be due to
overdosage, or prolonged. E.g., tetracyclines inhibit growth and
development of bones and teeth in the developing fetus and infant,
chloramphenicol may cause depression of the bone marrow, aspirin
lead to deafness. Voltarin may cause renal failure. 3. Allergy. May
occur if sensitivity test was not done before administering an
antibiotic. 4. Superinfection. This may lead destruction of human
indigenous microflora of the mouth, vagina, and intestine Nabeel
Al-Mawajdeh RN.MCS
Slide 69
a.Effect of temperature on bacteria and yeast cells b.Effect of
temperature on vegetative and spore cells of bacteria, yeast, and
fungi
Slide 70
8.A. Effect of temperature on bacteria and yeast cells m.o.
culture agar plate Streak plate Incubate in 4 C Incubate in 20/25 C
Incubate in 37 C Incubate in 44 C broth tube + Durham tube
Inoculatio n Incubate in 4 C Incubate in 20/25 C Incubate in 37 C
Incubate in 44 C
Slide 71
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.
Slide 72
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
Slide 73
Exp.9 - pH m.o. culture 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. 48- 72 hours
Slide 74
spectrophotometer
Slide 75
Exp.10 - Oxygen Requirements m.o. culture divided agar plate
Streak plate 24-48 hours incubate 37 C divided agar plate Streak
plateanaerob jar 24-48 hours incubate 37 C
Slide 76
m.o. culture 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 Exp.11 Osmotic
Pressure
Slide 77
Exp.12 Chemotherapeutic Agent m.o. culture agar plate, incubate
to drain swab Press immersed bloaters in plate 24-48 hours incubate
37 C Using tweezers, immerse bloaters in penicillin Using tweezers,
immerse bloaters in tetracycline Using tweezers, immerse bloaters
in streptomycin A B C D
Slide 78
a.Alcohol effectivity evaluation b.Antiseptic evaluation using
blotter c.Effectivity of handwashing using soap
Slide 79
13.A. Alcohol effectivity evaluation A = left thumb without
alcohol B = left thumb without alcohol C = right thumb without
alcohol D = right thumb with alcohol Incubate the dish A B C D
Slide 80
13.B. Antiseptic evaluation using blotter m.o. culture agar
plate, incubate to drain swab Press immersed bloaters in plate
24-48 hours incubate 37 C Using tweezers, immerse bloaters in
alcohol
Slide 81
13.C. Effectivity of hand washing using soap Washed
HandUnwashed Hand