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Laboratory of Microbiology

Medical Faculty UB 2010


Mid-1800s Ignatz Semmelweis (Hungarian physician)
& Joseph Lister (English physician)
thought to develop some microbial control practices
for medical procedures
hand washing with chlorine of lime (CaCl2) and
aseptic surgery techniques to prevent microbial
contamination of surgical wounds
the nosocomial infection be decreased
(until that time, 10% death of surgical cases and
25% death of delivering mother)
LEARNING OBJ ECTIVES

1. DESCRIBE THE BENEFITS of CONTROL OF THE
MICROBIAL GROWTH
2. DEFINE THE KEY TERMS RELATED TO MICROBIAL
CONTROL
3. DESCRIBE THE CONDITIONS INFLUENCING THE
MICROBIAL CONTROL
4. DESCRIBE THE METHODS OF MICROBIAL CONTROL


Clinical application:
to prevent the spread of infection in population or
hospital

Microbiology Laboratory:
to prevent microbial contamination of appliance
and culture medium from the microbe which we do
not wish
Industrial product:
to prevent the decomposition of drugs, foods
or to sterilize medical appliance

Why we learn the control of microbial growth ?
PRINCIPLES OF MICROBIAL CONTROL
The key terms related to microbial control:
sterilization, disinfection,
antisepsis, asepsis,
bacteriostatic, bactericide,
germicide, degerming,
and sanitation
Sterilization: is the destruction of all forms of microbial life,
including endospores, which are the most
resistant form
Heating is the common methods used for sterilization
In reality, the heat treatment required to ensure absolute
sterility would unnecessarily
e.g. canned food is subjected only to enough heat to
destroy the endospores of Clostridium botulinum, which
can produce a deadly toxin Commercial sterilization
The endospores of a number of thermophilic bacteria,
capable of causing food spoilage but not human disease,
are considerably more resistant to heat than C. botulinum.
If present, they will survive, but their survival is usually of
no practical consequence; and they will not grow at normal
food storage temperatures.

Complete sterilization is often not required
ex. a drinking glass or a fork in a restaurant requires only
enough microbial control to prevent the transmission
possibly pathogenic microbes from one person to another

Disinfection: control directed at destroying harmful microorg
as vegetative form (non-endospore forming)
- disinfection might use of chemicals, ultraviolet radiation,
boiling water
- in practice, the term which is most commonly applied for
a chemical (a disinfectans) using to treat an inanimate
surface or substance
- when the treatment is directed at living tissues, it is called
antisepsis, and the chemical is then called antiseptic

- the same chemical might be called a disinfectant for one
use and an antiseptic for another use
- many chemicals suitable for swabbing a table top would
be too harsh to use on living tissue
Sepsis means decay or putrid indicates bacterial
contamination
Aseptic techniques are important in surgery to minimize
contamination from the instruments, operating personnel,
and the patient
Degerming (or degermation):
the process which mostly results in the mechanical
removal of microbes in a limited area
Sanitization: the process is intended to lower microbial counts
to safe levels (public health) and minimizes
of disease transmission from one to another
Bacteriostatic: a treatment that only inhibits the growth and
multiplication of bacteria. When the agent is
removed, growth may be resumed
suffix: -static means to stop or to steady
-cide means killing
THE CONDITIONS INFLUENCING
MICROBIAL CONTROL
1. The number of microbes
The more microbes there to begin with, it will take the longer time
2. Microbial characteristics
- endospores are difficult to kill
- vegetative microbes exhibit considerable variation in their
susceptibility to physical or chemical controls
3. Environmental influences
- the presence of organic matter such as blood, saliva, or feces
often inhibits the action of chemical antimicrobials
- A suspending medium that contains fats or protein tends to protect
bacteria, which will have a higher survival rate
- pH: heat is more efficient when the microbes are under acidic
conditions
4. Time of exposure
- in heat treatments, a lower temperature will compensate for
a longer exposure
- irradiation effects are very dependent on time
- chemical antimicrobials often require extended exposure for
more resistant microbes or endospores to be affected

Notes:
- Many disinfectans and antiseptics tend to have a greater effect on
Gram-positive than Gram-negative bacteria
ex. Pseudomonads, which are common in the environment, are
unusually resistant to chemical activity and will even grow actively
in some disinfectans and antiseptics pseudomonads are very
troublesome in hospital setting
- Because disinfectans activity is due to temperature-dependent chemical
reactions, disinfectans tend to work somewhat better in warm solutions
METHODS OF MICROBIAL CONTROL
When selecting methods of microbial control, consideration
must be given to effects on things other than the microbes
ex. certain vitamins or antibiotics in a solution might be
inactivated by heat
Many laboratory or hospital materials, such as rubber and
latex tubing, are damaged by repeated heating
Economic considerations: it may be less expensive to use
presterilized/ disposible plasticware than to repeatedly
reuse and resterilize glassware
I. PHYSICAL METHODS
1. Moist heat
Death of the microorganisms primarily caused by coagulation
of their proteins, which is caused by breakage of the
hydrogen bonds that hold the proteins in their three
dimentional structure
Boiling (100
o
C)
- kills vegetative forms of bacterial pathogens, almost all
virus and fungi and their spores about 10 minutes
- hepatitis vi can survive up to 30 minutes, and bacterial
endospores have resisted to boiling for more than 20
hours boiling is therefore not a reliable sterilization
procedure
is the preferred methods of sterilization, unless the material
to be sterilized can be damaged by heat or moisture
to sterilize: culture media, instrument, dressing, solutions,
syringes, intravenous equipment, glassware, and numerous
other items that can withstand high temp and pressure
unlike sterilizing aqueous solutions, in order to sterilize
the surface of a solid, steam must actually contact it
aluminium foil is impervious to steam!
products that do not permit penetration by moisture, such
as mineral oil or petroleum jelly do not sterilized by this
method
Autoclaving (121
o
C for 15 minutes)
Pasteurization (63
o
C for 30 minutes)
come from Louis Pasteur that found a practical method to
prevent the spoilage of beer and wine
is mild heating, which is sufficient to kill the organisms that
caused the particular spoilage problem without seriously
damaging the taste of the product
milk was first pasteurized to eliminate the tuberculosis
bacterium. Many relatively heat-resistant (thermophilic)
bacteria survive to pasteurization, but these are unlikely
to cause disease
almost all pathogenic viruses are inactivated
HTST (high temperature short-time) pasteurization
(72
o
C for 15 seconds):
lower total bacterial counts, so the milk keeps well
under refrigeration
UHT (ultra high temperature) treatments
(140
o
C for less than one second):
milk can be sterilized, so that it can be stored without
refrigeration
Tyndalization (63
o
C for 30 minutes) during 3 days
successively to give opportunity of endospores become
vegetative (germination) kills both vegetatives
and spores
2. Dry Heat Sterilization
- microorganism kills by oxidation effects
1. Flaming
- overcoming appliance above fire (bunsen)
- scalpel, pinset, washbasin
2. Red heat (heating to a red glow)
- inoculating loops (ose) in microbiology laboratory

Incineration: an effective method to sterilize and dispose of
contaminated paper cups, bags, and dressing
3. Hot air sterilization (170
o
C for 2 hours)
- items which will be sterilized are placed in an oven
- to sterilize: glassware, oily solution, fat, powder, metal equip

Alert: sharp pointed metal appliance will be blunt, soldering
will be released, textile or cotton will be burnt

3. Filtration
is the passage of a liquid or gas through a screenlike material
with pores small enough to retain microorganism
to sterilize: culture media, enzymes, vaccine, antibiotic
solutions (heat-sensitive materials)
HEPA (High Efficiency Particulate Air) filters:
in rooms occupied by burned patients lower the numbers
of airborne microbes (retain almost all microorganisms
larger than about 0.3 m in diameter)

4. Radiation
radiation has various effects on cells, depending on its
wavelength, intensity, and duration
two types of sterilizing radiation: ionizing and non-ionizing
Ionizing radiation
- such as gamma rays, X rays
- has a shorter wavelength (less than about 1 nm)
carries much more energy and high penetrating effect
- the principal effect:
ionization of water, which forms highly reactive hydroxyl
radicals react with DNA
- to sterilize: pharmaceutical equipments, disposable dental
and medical supplies, such as plastic syringes, surgical
gloves, suturing materials, and catheters
Non-ionizing radiation

e.g. ultra violet (UV) radiation
has a wavelength longer (> 1 nm)
UV light damages the DNA of exposed cells by causing
bonds to form between adjacent thymine in DNA chains
(thymine dimers) inhibit correct replication of the
DNA during reproduction of the cell
the UV wavelengths most effective for killing microorg
are about 260 nm specifically absorbed by cellular DNA
iused to control microbes in the air, to disinfect vaccines and
other medical products
Disadvantages:
1. UV light is not very penetrating, so the miccroorganism
must be directly exposed to the rays.
Microorg protected by solids, and such coverings as
paper, glass, and textiles are not affected.
2. UV light can damage human eyes, and prolonged
exposure can cause burns and skin cancer in humans

5. Low Temperature
the effect on microorganisms depends on the particular
microbe and the intensity of the application
ex. at temperatures of ordinary refrigerators (0-7
o
C), the
metabolic rate of most microbes (pathogenic bacteria) is
so reduced that they cannot reproduce toxin (bacterio-
static effect)
Alert: psychotroph grow slowly at refrigerator temp and
will alter the appearance and taste of foods

6. Dessication
- a condition that is absence of water
- microorg cannot grow but can remain viable for years
- Lyophilization: dessicating and freezing methods to
preserve microorganism (in laboratory)
- the resistance of vegetative cells to dessication varies with
species and environment
ex. Go bacterium can withstand dryness for only about an
hour, but the tuberculosis bacterium can remain viable
for month.
Bacterium much more resistant if it is embedded in
mucus, pus, or feces.
Alert: dust, clothing, bedding, and dressings might contain infectious
microbes in dried mucus, urine, pus and feces (in hospital setting)
6. Osmotic Pressure
- high concentrations of salts and sugars have a preserve
effect create a hypertonic environment (osmotic pressure)
that causes water to leave the microbial cell make
the plasma membrane shrink away from the cell
wall (plasmolysis)
- is used in preservation of foods (ex. cure meats, preserved
fruits)
Alert: molds and yeasts are much more capable of
growing in materials with high osmotic pressures

II. CHEMICAL METHODS
o are used to control the growth of microbes on both living
tissue and inanimate objects
o most of chemical agents merely reduce microbial
populations to save levels or remove vegetative forms
o we can learn a great deal about a disinfectans properties
by reading the label.

By reading the label of disinfectant, learn about :
what groups of microorganisms will be affected
the concentration of a disinfectans affects its
action, so it should always be diluted exactly as
specified by the manufacturer
pH of the medium often has a great effect on
activity
an area might need to be scrubbed and rinsed
before the disinfectant is applied
to be effective, might need to be left on
a surface for several hours

1. Chemicals which can damage plasma membrane
- Phenolics : CRESOL, HEXACHLOROPHEN
- Biguanides : CHLORHEXIDINE
- Alcohols : ETHANOL (60-90%), ISOPROPANOL
- Surface active agents : QUATERNARY AMMONIUM
- Alkylating agent : ETHYLEN OXIDE, PROPYLEN OXIDE
- Aldehyde : FORMALDEHYD, GLUTARALDEHYDE
2. Chemicals which can damage enzyme
- Oxidators : IODINE, CHLORINE, HYDROGEN PEROXIDE
- Heavy metal: MERCURI CHLORIDE, SILVER, ZINC
3. Chemicals which react with the functional protein
Types of Disinfectans
1. You need some sterilized clothes. Which method do
you choose to sterilize the clothes? Why?
2. If pasteurization does not achieve sterile condition,
why milk is treated by pasteurization?
Discuss about these questions
Phenolics
Biguanides
Halogens
Alcohols
Heavy metal compounds
Surface active agents (Quaternary ammonium)
Aldehyde
Gaseous chemosterilizers
Peroxygens (Oxidizing Agents)

Types of Disinfectans
Phenolic compounds
- derivatives of phenol
- antimicrobial activity by injuring plasma membranes,
inactivating enzymes, and denaturating proteins
- remain active in the presence of organic compound, stable,
they persist for long periods after application
suitable agents for disinfecting pus, saliva, and feces
- ex. Cresol a good surface disinfectans
Hexachlorophen:
to control staphylococcal & streptococcal in hospital
nurseries (excessive use can lead to neurological
damage)

Halogen
- particularly Iodine and Chlorine, both alone and as
ionorganic or anorganic compounds
- Iodine (I
2
):
one of the oldest and most effective antiseptics
effective against all kind of bacteria, endospores,
various fungi and some viruses
mechanism of activity:
- combines with amino acid Tyrosine
- oxidizes the sulfhydryl (-SH) groups
a common component of many enzyme and
protein
available as a tincture (alcohol solution), and
iodophor (combination of Iodine + organic molecule
povidone). Iodophor do not stain and less irritative


- Chlorine (Cl
2
):
its germicidal action is caused by the hypochlorous
acid (HOCl) that forms when chlorine is added to
water:
Cl
2
+ H
2
O H
+
+ Cl
-
+ HOCl
chlorine water hydrogen chloride hypochlorous
ion ion acid

HOCl is a strong oxidizing agent, and diffuses as
rapidly as water through the cell because of neutral
in electrical charge
a liquid form of compressed chlorine gas is used for
disinfecting drinking water, swimming pools,
and sewage

- is a member of biguanide group
- used to microbial control on skin and mucous membranes
- has low toxicity, however contact with eyes cause damage
- effective against most vegetative bacteria and fungi,
certain enveloped viruses but is not sporocidal
- its killing effect is related to plasma membrane damage
Chlorhexidine
Alcohols
- effective to kill bacteria, fungi, but not endopsores and
non-enveloped viruses