6 Microbial Control

8/11/2019 6 Microbial Control

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Laboratory of MicrobiologyMedical Faculty UB 2010


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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) andaseptic 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)


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LEARNING OBJECTIVES

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


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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 contro l of m icrobial grow th ?


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PRINCIPLES OF MICROBIAL CONTROL

The key terms related to microbial control:

sterilization, disinfection,

antisepsis, asepsis,bacteriostatic, bactericide,

germicide, degerming,

and sanitation


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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 absolutesterility 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


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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


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Disinfection:control directed at destroying harmful microorgas 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 treatmentis directedat living tissues, it is called

antisepsis, and the chemical is then calledantiseptic


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- 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

Sepsismeans decay or putrid indicates bacterialcontamination

Aseptic techniquesare important in surgery to minimize

contamination from the instruments, operating personnel,

and the patient


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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

tosafe 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 resumedsuffix: -static means to stop or to steady

-cide means killing


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THE CONDITIONS INFLUENCING

MICROBIAL CONTROL

1. The number of microbesThe more microb esthere to begin with, it will take the longer time

2. Microbial characteristics

- endosporesare difficult to kill- vegetat ive mic robesexhibit considerable variationin their

susceptibility to physical or chemical controls

3. Environmental influences- the presence of organic mattersuch as blood, saliva, or feces

often inhibits the action of chemical antimicrobials- A suspending medium that contains fats or pro teintends to protect

bacteria, which will have a higher survival rate

- pH:heat is more efficient when the microbes are under acidic

conditions


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4. Time of exposure- in heattreatments, a lower temperature will compensate for

a longer exposure- irradiationeffects 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 verytroublesome in hospital setting

- Because disinfectans activity is due to temperature-dependent chemical

reactions, disinfectans tend to work somewhat better in warm solutions


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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 beinactivated 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 usepresterilized/ disposible plasticware than to repeatedly

reuse and resterilize glassware

I. PHYSICAL METHODS


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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 (100oC)

- 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 bacterialendospores have resisted to boiling for more than 20

hours boiling is therefore not a reliable sterilization

procedure


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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 pressureunlike 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, suchas mineral oil or petroleum jelly do not sterilized by this

method

Autoclaving (121oC for 15 minutes)


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Pasteurization (63oC for 30 minutes)

come from Louis Pasteur that found a practical method toprevent 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


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HTST (high temperature short-time) pasteurization

(72oC for 15 seconds):lower total bacterial counts, so the milk keeps well

under refrigeration

UHT (ultra high temperature) treatments

(140oC for less than one second):milk can be sterilized, so that it can be stored without

refrigeration

Tyndalization (63oC for 30 minutes)during 3 days

successively to give opportunity of endospores become

vegetative (germination) kills both vegetatives

and spores


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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(170o

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 poin ted metal appl iance wil l be blunt, sold er ingwi l l be released, text i le or c otton w i l l be burnt


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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)


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4. Radiation

radiation has various effects on cells, depending on itswavelength, intensity, and duration

two types of sterilizing radiation: ionizingand non- ioniz ing

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 hydroxylradicals react with DNA

- to sterilize: pharmaceutical equipments, disposable dental

and medical supplies, such as plastic syringes, surgical

gloves, suturing materials, and catheters


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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 tocontrol microbes in the air, to disinfect vaccines and

other medical products


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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


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5. Low Temperature

the effect on microorganisms depends on the particular

microbe and the intensity of the application

ex. at temperatures of ordinary refrigerators (0-7oC), the

metabolic rate of most microbes (pathogenic bacteria) isso 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


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6. Dessication

- a condition that is absence of water- microorg cannot grow but can remain viablefor years

- Lyophilization:dessicating and freezing methods to

preserve microorganism (in laboratory)

- the resistance of vegetative cells to dessication varies with

species and environmentex. Gobacterium 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 inmucus, pus, or feces.

Alert :dust, clothing, bedding, and dressings might contain infectiousmicrobes in dried mucus, urine, pus and feces (in hos pita l sett ing)


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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


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II. CHEMICAL METHODS

oare used to control the growth of microbes on both living

tissue and inanimate objects

omost of chemical agents merely reduce microbial

populations to save levels or remove vegetative formsowe can learn a great deal about a disinfectans properties

by reading the label.


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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 asspecified by the manufacturer

pH of the medium often has a great effect onactivity

an area might need to be scrubbed and rinsed

before the disinfectant is applied

to be effective, might need to be left ona surface for several hours


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1. Chem icals 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. Chem icals w hich can damageenzyme

-Oxidators: IODINE, CHLORINE, HYDROGEN PEROXIDE

- Heavy metal:MERCURI CHLORIDE, SILVER, ZINC

3. Chem icals wh ich react wi th the func t ional protein

Types of Disinfectans


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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


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Phenolics

Biguanides

Halogens

Alcohols

Heavy metal compounds

Surface active agents (Quaternary ammonium)

Aldehyde

Gaseous chemosterilizers

Peroxygens (Oxidizing Agents)

Types of Disinfectans


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Phenol ic 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 neurologicaldamage)


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Halogen

- particularly Iodine and Chlorine, both alone and as

ionorganic or anorganic compounds- Iodine (I2):

one of the oldest and most effective antiseptics

effective against all kind of bacteria, endospores,

various fungi and some virusesmechanism 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


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- Chlorine (Cl2):

its germicidal action is caused by the hypochlorous

acid (HOCl) that forms when chlorine is added towater:

Cl2 + H2O 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 fordisinfecting drinking water, swimming pools,

and sewage


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- 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

Chlorhexid ine

Alcoho ls

-effective to kill bacteria, fungi, but not endopsores and

non-enveloped viruses

6 Microbial Control

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