This week

 Chapter 7 Coccus

 Chapter 3 Disinfection & Sterilization

 Exp. 8 Effects of physical, chemical and biological

factors on the growth of bacteria
 Chapter 3

Disinfection & Sterilization

 Outline
 Definitions
 Disinfection, Sterilization;
 Asepsis, aseptic technique;
 Bacteriostasis, Pasteurization;
 Questions
 What agents contribute to the control of microbes?
 How are dry heat and moist heat used to control
microorganisms?
 Why is moist heat more effective than dry heat?
 What is the mechanism of UV light’s killing
bacteria?
 What factors influence the effectiveness of
antimicrobial agents?
Terms

 Sterilization
 Disinfection
Disinfectant
 Antisepsis
Antiseptic
 Asepsis
Aseptic technique
 Bacteriostasis
Bacteriostat
Terms
 Sterilization:
A process that destroys or eliminates all forms of microbial life, including
highly resistant bacterial spores, from an object or environment.
-- The criterion of sterilization is killing bacterial spores.

 Disinfection:
A process that eliminates many or all pathogenic microorganisms, except
bacterial spores, from an object or an environment.

Disinfectants:
Antimicrobial agents that destroy microorganisms on inanimate objects.

 Antisepsis:
The disinfection of living tissues such as skin, mucosa and wounds.
Antiseptic (pl. Antiseptics):
Antimicrobial agents that destroy microorganisms on living tissues.
Definitions
 Asepsis:
the absence of microorganisms.
Aseptic technique: the measures taken to ensure that living
microorganisms are absent.

 Bacteriostasis:
The inhibition of growth, but not the killing, of bacteria.
Bacteriostats: the agents used for bacteriostasis.
e.g., antibiotics, sodium azide, thimerosal
Antimicrobial methods

 Physical---t, radiation, filtration, desiccation

 Chemical
 Biological
I. Physical methods

 Temperature
 Radiation
 Filtration
Temperature
 High Temperature
Dry heat
Incineration
Flaming
Hot-air sterilization
Infrared rays

Moist heat
 Incineration

 Dry heat
Flaming

Dry hot-air sterilization

Condition: 160℃ - 180℃, 1-2hrs
Applications: glassware, porcelains
metal objects

Infrared rays visible light (0.4-0.7μm) UV light

(0.7-1000 m)
 Moist heat
Boiling
Flowing steam
100℃ water vapor for 15-30min;
Intermittent sterilization
Flowing steam →RT (or 37 ℃), overnight;
→Flowing steam → RT (or 37 ℃), overnight;
→Flowing steam
Pasteurization
Autoclaving (Steam sterilization under high
pressure)
Pasteurization
a process that utilizes mild heat to destroy vegetative forms
in liquid or in cheese without altering the liquid's palatability.
Condition: 62℃ 30min or 71.7℃(72℃) 15sec
Significance: kills vegetative pathogens
--- from infected cattle (M.bovis);
--- from human environmental contamination (M.tuberculosis);
Applications: milk, beer, wine, cheese, etc.
Autoclaving: (Steam sterilization under high pressure)
-- Conditions: 15 lb/in2 (1.05kg/cm2), 121℃, 15-30 minutes
-- Significance: kills both spores and vegetative microbes
-- Application: heat-tolerate materials

Autoclave
Comparison
Dry heat and Moist heat, which is better?
Dry heat Moist heat
160 ℃ 120 min 120 ℃ 15 min
170 ℃ 60 min 126 ℃ 10 min
180 ℃ 30 min 134 ℃ 3 min

Moist heat is generally more effective than dry heat.

Reasons
- Hot steam more easily denature proteins.
- Hot steam is of strong penetration.
- Hot steam could send out potential heat.
Low temperature?

Refrigeration: most microbes do not reproduce at 0-7℃

Deep freezing: microbes may survive but don’t grow in
subzero temperature
Lyophilization: freeze-dry

-20℃ ~ -80℃
Dry ice, -80℃

-196℃
Physical antimicrobial methods

 Temperature
 Radiation
 Filtration
Radiation --- cause DNA damage;
depend on:
a) Wavelength
b) Intensity
c) Duration
x-ray, α-ray, β-ray, γ-ray
damage DNA and generate peroxides;
--Ionizes water creating highly reactive hydroxyl radicals
high penetration (γ-rays and x-rays)
UV light
Microwaves: kill bacteria indirectly by heat; not very effective
 Ultraviolet radiation (UV, 240nm-300nm)
The most effective wavelengths: 265nm
Mechanism: destroy bacteria by forming thymine dimer
Characteristics:
poor penetration
damage eyes, skin
Applications:
Air disinfection
Surface disinfection
Filtration
 Mechanism -- mechanically removes microorganisms
 Pore size: 0.45m, 0.2m
 Application – air, liquids, serum, toxin, antibiotics that can
not tolerate heat..
Other physical Methods of Microbial Control

 Desiccation --- absence of water prevents

metabolism and can inhibit growth, but some
remain viable
 Osmotic pressure
hypertonic: causes plasmolysis
molds and yeast are more resistant
Antimicrobial methods

 Physical disinfection
 Chemical disinfection
 Biological disinfection
II. Chemical disinfection
disinfectants, antiseptics

 Mechanism of action
-- Destroy cell membranes;
e.g., surfactants/detergents →dissolve lipid bilayer
-- Denature bacterial proteins;
e.g., enzymes → interfere bacterial metabolism
-- Damage genetic materials;
e.g., formaldehyde → DNA, RNA
 Commonly used chemical agents

Phenol
Soaps and detergents
Alcohols 70-75% (medical use)
Denature proteins, dissolve lipids
Heavy metals: 0.5%-1% Silver nitrate
Chlorine
Iodine / iodophor
Aldehydes: 37% formaldehyde (Formalin)
Dyes: crystal violet
III. Biological methods

 Antibiotic
 Bacteriocin
e.g., colicin
 Bacteriophage
virulent phage
 Herbs
Factors influencing antimicrobial activity
 the concentration and kind of an disinfectant;
 the length of exposure to the agent;
 the temperature and pH at which the agent is used;
 the number and kinds of microorganisms;
 the organic matters bearing the microorganism;
Summary of this chapter
 Terms
Disinfection, Sterilization, Asepsis, Aseptic technique,
Bacteriostasis, Pasteurization
 High temperature are used to inhibit microbes’ growth.
 Moist heat is more effective than dry heat.
 UV light is suitable for surface disinfection and air
disinfection.
 Many factors influence the efficacy of antimicrobial
agents.
Exp. Effects of physical, chemical and
biological agents on bacteria growth
 Effect of moist heat;
 Effect of UV light;
 Effect of chemicals and biological agents
  Effect of moist heat 4 students / group

Bacteria E.coil (broth) (shake it) B.subtilis (slant)

Temperature 100℃ RT 100℃ RT

Time 5min 30min Control 5min 30min Control

Tube number 1# 2# 3# 4# 5# 6#
 Effect of UV light
Bacterial strain： S.aureus
Inoculation method：Streaking plate (with cotton stick)
Condition： half open
expose under UV light for 30min

mark
UV 30min
UV
 Effect of chemical and biological agents
Materials: paper disk
1. 1% crystal violet Bring with me
2. 2% mercurochrome tomorrow
3. 2% iodine
4. garlic
5. paper disk of penicillin
6. paper disk of streptomycin
Species：
S.aureus 1 agar plate
E.coli 1 agar plate Mark Manipulation

1 1
6 2 6 2
E S
5 3 5 3
4 4
Exp. 8 Effects of physical, chemical and
biological methods on bacteria

 Part 1. Effect of moist heat on bacteria;

E.coli, B.subtilis
 Part 2. Effect of UV light on bacteria;
S.aureus
 Part 3. Effect of chemical & biological agents on bacteria;
E.coli, S.aureus
Moist heat
 E.coli
1#: 100℃, 5 min
2#: 100℃, 30 min
3#: RT, Control

 B.subtilis
4#: 100℃, 5 min
5#: 100℃, 30 min
6#: RT, Control
 Effect of moist heat on bacteria
Record:

Strains 100℃, 5 min 100℃, 30 min Control

E. coli

B. subtilis

 Clear: “-” or Turbidity: “+”

 pellicle / Turbidity / Sediment growth
 UV radiation
Describe result:
 Covered part
 irradiated part

UV
Effect of chemicals and biological agents

 Measure inhibition zone

diameter or 2 radius
 Effect of chemical and biological agents

Inhibition zone diameter (mm) >15mm 10~15mm <10mm 0mm

Extremely Medium Slight

susceptibility degree Resistant
sensitive sensitive sensitive

Record:
Diameter of Inhibition Zone (mm)

Cultures 1%
2%
Crystal Penicillin Streptomycin 2%Iodine Garlic
Mercurochrome
violet
E.coli
S.aureus
Susceptibility
degree
Discussion outline of lab report:
 Briefly describe the result of the experiment;
 Mechanism that moist heat affect bacterial growth;
 Mechanism of action for UV light;
 Characteristics or disadvantages of UV light bactericidal effect;
 Mechanism of action that chemical agents and antibiotics inhibit
bacterial growth;
 Explain the effects of the same reagent on the growth of different
species of bacteria;
 Explain the effects of different reagents on the growth of the same kind
of bacteria;
 Factors affecting the experimental results should be analyzed;