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Medical Microbiology & Immunology 7

th

ED:84-87p.

I.Basic Bacteriology 13.Sterilization&Disinfection

Sterilisation and Disinfection

In the ancient time:

 for wound injury: wine, concentrated solution of alcohol
 using balsam for dead people

 1847 Semmelweis Ignac: before the discovery of the bacteria, he used chlor for wash up

 against puerperal fever

The number of the disease had been reduced from 11.4% to 1.27%

Definitions

 Sterilisation: the process of killing or removal all of the organisms in a preparation

(including bacterial spores)
Sterile = treated objects are free of all living organisms

 Disinfection: treatment which reduces the number of potentially pathogenic microorganism
in an environment
(this process does not destroy all micro-organisms)

Disinfectant: a chemical substance used to kill micro-organisms on non-living surfaces
(too toxic to be applied directly to tissues)

Antiseptic agent: chemicals used to reduce number of potentially pathogenic
microorganisms on living surfaces (skin, mucous membrane)

Important for effective disinfection

- for how long we use the agent
- at what concentration we use the agent

There is a formula: C

n

X t = K

C: is the drug concentration
t: time required to kill
K: constant
n: constant

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Methods

STERILISATION
(1) Methods of sterilisation

(A) Physical methods

(i) Heat:acts by denaturing cell proteins and nucleic acids, disrupting cell membranes

 Dry heat

 to burn the infected material (inoculation loop, baby diapers…)
 Hot air chamber (with circulating hot air)

 140

o

C for 3 hours, or 160

o

C for 2 hours, or 180

o

C for 1 hour

 petri dishes, glass equipment, pipette, rubber goods...

 Moist heat: autoclaving

 at 121

o

C, +1 atm for 30 minutes, or at 134

o

C, +2 atm for 3

minutes (“flash autoclave”)

(ii)

-radiation (industrially only)

(B) Chemical methods

(i)

ethylene oxide gas (ETO) sterilisation
Toxic for human being! (after the sterilization procedure we have to wait 24
hours)
 surgical instruments, rubber gloves, plastic instruments, metallic
instruments

(ii)

plasma sterilisation

 hydrogen peroxide (H

2

O

2

) in the plasma state, at 50

o

C, in vacuum

 free radicals

(iii)

liquid compounds or solutions (glutardialdehyde, iodine + potassium iodide
+ formaldehyde, etc.

 for immersing cat-gut, surgical bandages, etc. for days (out-of-date

method)

(C) Membrane filtration

Filtration (virus can go through) - glass filter

 used to sterilize temperature-sensitive fluids
 membranes are made up of cellulose acetate/nitrate
 pore sizes are variable - from 0.1um to 0.45 um = common pore sizes
 contaminated solutions are filtered through this membrane and small particles

(including bacteria) are trapped on the filter

- absorption

-

pore size membrane

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Parameters of inactivation of prions
-

autoclaving + 1 atm; for 1 hour

-

5% hypochlorite solution

-

1,0 M sodium hydroxide

(2) Control of the process of sterilisation

(A)Control of the physical parameters of the equipment

 thermometers, manometers

(B) Control of the outcome of the process

(i)

Chemical methods

 adhesive paper slips impregnated with dyes that change

colour if exposed to appropriate “dose” of heat, or chemical agent, to be
attached to the surface of the package to be sterilised, or small tubes that
contain similar dyes, to be placed next to the packages.

(ii)

Biological methods

 placing spores of Bacillus stearothermophilus (very

resistant) packed into a wax-impregnated paper bag into the sterilising
equipment, then inoculating content of the bags onto blood-agar plates after the
complete process of sterilisation, finally after a 24-hour incubation at 37

o

C,

testing for colonies of the bacterium.

(3) Control of the sterility of drugs administered parenterally

 Sampling is carried out strictly aseptically.
 Sample is inoculated onto the culture media as follows:

 For bacterial contamination thioglycolate medium is used, incubation is done

parallel under aerobic and anaerobic conditions at 37

o

C for 7 days.

 For fungal contamination, fluid fungal culture medium containing glucose, and

casein with a pH 5.3-5.7 is used, incubation is carried out at 22-25

o

C for 7 days.

 “Harmless probe” = biological activity test

inoculation into the rat, rabbit , etc. => to test pyrogenic substance; toxic
substance
“rabbit pyrogen test”; LAL-test

 Pyrogenic agents are relatively heat resistant bacterial endotoxins (derived from the

cell-wall of dead Gram-negative bacteria) that can cause fever in recipients of
intravenous solutions sterilised by heat. To make intravenous solutions “pyrogen-
free”, they are filtered prior sterilisation.

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DISINFECTION
(1) Methods of disinfection

(A) Physical

(i)

Heat (Note: reduction of germ count only!)

 Pasteurisation (of milk): consists of heating the fluid to 62

o

C for 30 minutes,

followed by a rapid cooling.

! Mycobacterium tuberculosis, spores will survive

 Tyndallisation (fractionated heat treatment): consists of heating to 85

o

C for

30 minutes with subsequent incubation at 37

o

C in repeated cycles several

times

 Boiling: at 100

o

C, under normal air pressure for 20-30 minutes

(ii)

Radiation: UV-light (at a wave-length of 253.7

m)

(B) Chemical
Chemicals vary greatly in their ability to kill microorganisms.

The agent should be:

- bactericide, fungicide, virucide, sporocide, parasiticide

- Alcohols- they act as protein denaturants. (ethylalcohol, isopropyl alcohol)
- Phenol - protein denaturation
- Heavy metal ions - protein denaturation at high concentration

 but also toxic to human

tissues

 for skin low concentration  they act by combining with sulfhydryl groups

- Oxidising agents (hydrogen-peroxide, iodine, hypochlorite, chlorine

 oxidise free

sulfhydryl groups

- Alkylating agents:

- formaldehyde

 37% aqua. sol.  formalin

- ethylene oxide

-

Detergents: surface active agents

Chemicals vary greatly in their ability to kill microorganisms.
A qualitative measure of this variation is expressed as the phenol coefficient (PK), which
is the ration of the concentration of phenol to the concentration of the agent required to
cause the same amount of killing under the standard condition of test.

The highest dilution of the examined disinfectant which contains viable
bacteria after 5 minutes but does not contain viable bacteria after 10 minutes of
incubation time

PK=___________________________________________________________________

The highest dilution of the phenol which contains viable bacteria after 5

minutes but does not contain viable bacteria after 10 minutes of incubation time

Procedure:

1. make a dilution from the phenol & the examined disinfectant with distilled water
2. take an equal amount of bacteria into each tube
3. make an inoculation from each tube (equal amount should be taken out) after 5 & 10

minutes onto the surface of the culture media

4. 24-48 hours of incubation ( aerobic, anaerobic)
5. evaluation