PRACTICAL LABORATORY 2

Decontamination methods used in microbiology laboratory and medical practice

Objectives:
1. To define the terms: sterile, sterilization, septic/aseptic, disinfection, antisepsy,
preservation, etc.; basic and important definitions;
2. To know how to choose the decontamination methods according to the level of
their efficiency and depending on the risk conditions;
3. Mechanical decontamination;
4. Physical methods of decontamination - dry and moist heat sterilization
(principle, indications, contraindications, parameters, steps, quality control),
thermic shock, etc.
5. Chemical decontamination.

1. Terms
✓ Contamination = simple presence of microorganisms (mainly bacteria) on a
surface.
✓ Colonization = presence and multiplication of bacteria on a surface, whithout
reaction from the host.
✓ Septic = contaminated with pathogenic microbes (lifeless surfaces) or infected
(living surfaces).
✓ Aseptic = lack of pathogenic microbes.
✓ Asepsy = methods used to avoid contamination.
✓ Antiseptic = antimicrobial substance for living surfaces.
✓ Antisepsy = the destruction or removal of the vegetative form of
microorganisms, but not necessarly the spores, from the living surfaces.
✓ Disinfectant = antimicrobial substance for lifeless surfaces.
✓ Disinfection = the destruction or removal of the vegetative form of
microorganisms, but not necessarly the spores, from the lifeless surfaces.
✓ Sterile = lack of any viable microorganims, including spores.
✓ Sterilization = the destruction or removal of all the microorgnisms, including
the spores.
✓ Preservation = the prevention of microorganisms multiplication in food or
pharmaceutical products.
Note: - cid, defines killing of microorganism
- static, defines stopping of multiplication of a microorgansim
Factors that can influence the efficiency of decontamination:
- the decontaminant concentration and time of action - the time of killing varies
inversely proportional to the decontaminant concentration;
- the environment in which the decontaminant works (organic substances, medium
turbidity, water hardness and pH can diminish the decontaminant efficiency);
- the microorganisms concentration (for the same concentration of an antimicrobial
agent, the time of killing increases directly proportional to the microbial concentration);
- the microorganisms resistance (active metabolic bacteria are more susceptible than
dormant bacteria) - decreasing order of resistance: bacterial spores> mycobacteria>
nude viruses> gram-negative bacteria> fungi> gram positive bacteria> enveloped
viruses.

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 1. The choice of decontamination methods depending on the risk conditions
(risk of infection due to contamination)

Risk Exemples Level of Decontami-

condition decontamination nation
methods
Critical -Medical tools that High Sterilization
conditions make contact with the (mandatory)
(high risk) internal body:
needles, surgical
tools, suture materials,
venous/arterial lines
(catheters), injectable
and perfusion
solutions;
-Culture media,
containers and
laboratory
instruments.
Semi- -Medical tools that High (if it’s Sterilization
critical make contact with possible)
conditions intact mucosa:
(medium endoscope,
risk) laryngoscope,
cystoscope, artificial
ventilation
equipement, medical
thermometer, tongue
depressor, etc.;
Medium Disinfection
-Medical tools
– high level
contaminated with
blood or other body
fluids.
Non- -Medical tools that Low Disinfection
critical make contact with -medium/low
conditions intact skin: level
(low risk) stetoscope, medical
thermometer, Antisepsy
electrodes, medical
tensiometer cuff; Mechanical
-Hands of medical decontaminat
staff. ion

The decontamination agents:

▪ Mechanical: washing with water and soap and hand rubbing;
▪ Physical:
- dry/moist heat;
- thermic shock;

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 - radiation (ionizing/non-ionizing);
- ultrasounds;
- filtration;
▪ Chemical: antimicrobial substances.

2. Mechanical Decontamination
Hand Washing - the previous PL notions related to this subject are reviewed.

3. Physical methods of decontamination

A. THE HEAT
I. DRY HEAT STERILIZATION
a. Sterilization by burning in flame
- Burning to red - microbiological loop, platinum wire;
- Flambation – the edges of the tubes and vials, the rod of the loop;
- Incineration – contaminated materials, biowaste.
b. Dry heat sterilization – oven /poupinel
The oven is a double-isolated metallic box with electrical heating resistors, a
thermostat that allows keeping the temperature to a constant and programmed value
and a fan that ensures the uniformization of the inside air temperature.

 Principle: the dry heat is killing the microorganisms by protein oxidation.

 Indications:
- glassware (tubes, pipettes);
- porcelaine objects (mojar);
- powders (talc, drugs);
- mineral oils.
Contraindications:
- surgical metalic instruments (knifes, scissors, etc.);
- watery solutions;
- rubber/plastic instruments;
- syringe from metal and glass;
- cotton or syntetic materials;
- contaminated materials from laboratory (biowaste).
 Parameters:
- temperature = 180 ° C;
- total time of sterilization = t equalising temperature + t killing of microbes
≈ nature, shape, = constant (25 min)
volume of materials
= variable
 Steps:
- preparing the materials for sterilization (desinfection, washing, drying,
packing);
- the arrangement of the materials and quality controls in the oven (spaced);
- connecting the oven to an electric source and setting the parameters;
- sterilization (time of sterilization is measured from the moment the air
temperature reaches the sterilization temperature);
- coolling of materials;
- checking the quality controls;
- labeling, packing and storage of materials.

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  The quality control:
• Physical: thermometer, stopwatch;
• Chemical: adhesive bands with chemical substances - the colour will change
only if the two physical parameters were correct;
• Biological: papers bands with spores of Bacillus atrophaeus – after
sterilization, the bands are incubated in a culture medium at 37oC, for 48 hours;
if the culture medium remains clear = efficient sterilization.
The physical and chemical quality controls should be done with each
sterilization cycle, and the biological control, daily.

II. MOIST HEAT (desinfection/sterilization - depending on the temperature)

a. Sterilization – steam under pressure (autoclave)

Autoclave = vertical or horizontal shaft box with resistant metal walls and a hermetic
sealing system in which the water vapor compresses to the pressure required for
sterilization.
 Principle: the moist heat is killing the microbes by protein coagulation and is
more damaging than dry heat.
 Indications:
- solutions (culture media);
- labs glassware with special utilisation (cell cultures);
- biowaste (microbial cultures, biological fluids);
- surgical cotton material;
- rubber/plastic objects and instruments;
- metalic instruments.
 Parameters:
- presssure 0,5 atm 1 atm 2 atm
- temperature 115ºC 121ºC 134ºC
- time

time of sterilization = t equalising temperature + t killing of microbes

≈ nature, shape, = constant*
volume of materials
= variable
*18min, at 1 atm; 3-4 min, at 2 atm
 Steps:
- preparing the materials;
- check the water level inside the autoclave;
- the arrangement of the materials and quality controls;
- closing the autoclave,connecting to an electric source and setting the
parameters;
- opening the tap for the removing of the air with the increasing of the pressure
(then closing the tap);
- sterilization (time of sterilization is measured from the moment the air
temperature reaches the sterilization temperature);
- opening the autoclave tap and decreasing the presure;
- oppening the lid of autoclave (when pressure is 0);
- drying of materials (with the lid onpened – the vapor will stop the dust
deposit);
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 - checking the quality controls;
- labeling, packing and storage of materials (for 24 h – if the materials were
packed in perforated metallic containers; 2 months – if the materials were
packed in sealed paper-plastic bags).
 The quality control:
• Physical: thermometer, stopwatch, manometer;
• Chemical:
▪ adhesive bands with chemical substances - the colour will change only
if the three physical parameters were correct;
▪ Bowie-Dick test – shows the elimination of residual air and effective
penetration of vapor in sterilized material;
• Biological: culture media with spores of Bacillus stearothermophilus stored
in vials containing culture medium and a pH indicator; after sterilization, the
vials are incubated at 56oC, for 48 hours; if the culture medium remains clear
= efficient sterilization. If not, after spore germination, bacteria in vegetative
form multiply, disturb the medium (that become turbid), and ferment of glucose
from the culture medium, is indicated by pH (acid values).
The physical and chemical quality controls (adhesive bands) should be done
with each sterilization cycle, and the Bowie-Dick test and biological control,
daily.

b. Tyndalisation (fractionate sterilization)

 Indications: thermolabile substances - food, culture media supplemented with serum
(ex. Löffler media) or egg (ex. Löwenstein-Jensen media).
 Parameters: - temperature: 56-100ºC
 time: 30-60 minutes for 3-8 consecutive days
 Principle: between exposures at 56-100ºC, the products are maintained at room
temperature, which allows spores to germinate (resulting the vegetative forms); then,
this vegetative forms will be destroyed in the following thermal exposure.

c. Boiling - disinfection method

 kills vegetative forms of bacteria, almost all viruses and fungi in about 10 minutes,
hepatitis B virus in about 30 minutes;
 Indications: Decontamination of drinking water, baby clothes; steam iron destroys
bacterial vegetative forms in 5-10 seconds.
 Parameters: 30 minutes, 100 ° C.

d. Pasteurization - a method of preserving liquid foods using the combination of moist

heat (60 oC - 135 oC) and low temperatures (4oC). Thermic shock destroys vegetative
forms, but not bacterial spores.

B. THE COLD
- Refrigeration (0oC – 7oC) - bacteriostatic effect – preservation method of microbial
cultures, biological fluids, food, drugs, etc.
- Freezing - slow -20 oC has –cid effect (ice crystals formation and saline hyper-
concentration);
- rapid -80°C in a protective medium (glycerin broth) can ensure long-las-
ting maintenance of microorganisms;
- cold shock has -cid effect.

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 C. RADIATION
• Non-ionizing radiation (UV)
- Affects the replication of cellular DNA;
- Poor penetration - reduction of microbial load in the air and disinfection of flat
surfaces;
- UV lamps - surgical rooms, aseptic rooms, work surfaces in the laboratory.
• Ionizing radiation (X and gamma rays)
- Significant penetration - industrial sterilization of single use syringes, surgical gloves,
suture materials, drugs;
- Sterilization control - spores of Bacillus pumilus.

D. ULTRASOUNDS -cid effect – dental/surgical instruments disinfection;

E. FILTRATION
- is a method of air and thermolabile substances sterilization (culture media, drugs);
- uses cellulose acetate membranes (0.025 μm pores) ;
- High Efficiency Particulate Air Filters (HEPA) - used for air sterilization in anti-
epidemic safety rooms, surgery rooms, in patient rooms with aerogenic diseases (aer
transmission), immunodeficient patients, burned patients.

4. Chemical agents (sterilization, disinfection or antisepsy)

Chemical agents classification - according to the mechanism of action:

- protein denaturing agents (acids, alkali, alcohols);
- blocking agents of free chemical groups of enzymes (peroxides, heavy metals, aldehydes,
ethylene oxide, halogens);
- agents that damage cell membranes (phenols, detergents);
- agents that damage nucleic acids (aniline derivatives, acridine).

There are differences in antimicrobial efficiency, in activity in the presence of organic

substances (clean/ dirty) and in toxicity.
• Ethylene oxide
Use: sterilization – instruments used in assisted ventilation, plastic objects, heart valves,
catheters, electronic equipment.

• Aldehydes (formaldehyde, glutaraldehyde)

Use: disinfection, sterilization (sporicid at high concentrations).

• Oxidizing agents (hydrogen peroxide, ozone, potassium permanganate, peracetic acid)

Use: disinfectant, antiseptic, medical equipment sterilization.

• Salts of heavy metals

- Silver nitrate (1%) - antiseptic - ophthalmic ointment for newborns (Neisseria
gonorrhoeae); cream for burns;
- Mercury salts – bacteriostatic.
• Halogens

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Use: disinfectants, antiseptics - destroys vegetative forms of bacteria, fungi, some bacterial
endospores and numerous viruses.
Chlorine - is used to treat drinking water;
Chlorine-based compounds - disinfection of plumbing;
Iodine - alcoholic solution, tincture, iodophores – antiseptic;
Bromine - Antimicrobial agent used for effective disinfection of hydromassage
bathtubs, evaporates more slowly than chlorine at high temperatures;
Fluoride - an antibacterial agent used in disinfection of drinking water and as an
antiseptic in toothpaste.

• Phenol and phenolic derivatives (chlorhexidine)

Use: disinfectant - do not destroy viruses and bacterial spores.

• Alcohol - alcohol solutions: 70% ethanol, 60% propanol, 70% isopropanol

Use: antiseptic - bactericidal (including mycobacteria), fungicide, virulicide (for enveloped
viruses only); is not effective against bacterial endospores.
Attention: Pure alcohol is not an effective antimicrobial agent because protein denaturation
requires the presence of water.

• Surfactants (soaps and detergents)

Use: antiseptics and disinfectants - destroys bacteria, fungi, enveloped viruses. They are not
effective against nude viruses or bacterial endospores.

Rules on proper use of antiseptics and disinfectants:

1. Work solutions are prepared in the moment of use, in sterile containers.
2. When choosing concentrations, the clean/dirty condition of the objects will be taken into
account.
3. For hand decontamination, the antiseptic solution will always be poured on the hands
(putting the hands in solution leads to rapid inactivation of the antimicrobial effect).
4. Do not overload containers with disinfectant solutions to avoid contaminating leaks.
5. Floating objects will be filled and immersed completely in the solution or, if the container
has a lid, this will be overturned repeatedly.
6. Protein-rich materials are not introduced into disinfectant solutions, but are collected
separately for sterilization at autoclave.
7. Do not dilute the disinfectant by leaking liquids. For the centrifuge tube supernatant, use a
separate concentrated solution with a funnel to hold contaminated aerosols.
8. Do not leave the materials for more than 24 hours in the disinfectant solution.
9. The solution is not reused because it is possible to select and multiply resistant bacteria.
10. Unused work solutions 24 hours will be thrown away.