LABORATORY TECHNIQUES AND BIOSAFTY

MAP7107
ASEPTIC TECHNIQUES IN LABORATORY PREPARATION
AND ANALYSIS

ORGANIZER; Dr. HILDA IKIRIZA

CHEMUTOS TONNY

2023/MSc/002/PS
ASEPTIC TECHNIQUES IN LABOARTORY
PREPARATION AND ANALYSIS
• TERMINOLOGY
1. Decontamination; The destruction, removal , or
reduction of undesirable microbes
2. Sterilization; The removal or destruction of all viable
microbes
3. Disinfection ; Destruction of vegetative pathogens on
inanimate objects
4. Sanitization ; Cleansing technique that removes
microorganisms and debris from inanimate surfaces
5. Degermation ;
6. Asepsis; Techniques that prevent the entry of
microorganisms into sterile tissue
 STERILIZATION
 Sterilization is a process that destroys or
removes all viable microorganisms , including
viruses.
 Any material that has been subjected to this
process is said to be sterile
 Its important to note that it is either sterile or
not sterile , its an absolute term.
 Control methods that sterilize are generally
reserved for inanimate objects
 How sterilizing agents work
• The cellular targets of physical and chemical
agents are
1. Cell wall
2. Cell membranes
3. Protein and DNA synthesis
4. Protein structure and function
 Different methods of sterilization
PHYSICAL CHEMICAL
• Dry heat • Ethylene oxide
• moist heat • Vaporized hydrogen
• Radiation peroxide
• Filtration • Aldehydes
• Chlorine dioxide
 Factors that must be considered during
decontamination
1. The number of microorganisms
2. The nature of microorganisms in the
population
3. The mode of action of the agent
4. The presence of solvents , interfering organic
matter
5. The concentration (dosage, intensity)
6. The PH of the medium
 Sterilization by moist heat
1. Autoclaving ( sterilization with steam under pressure)
 Autoclaves have an enclosed metal chamber with an
airtight door on one end and racks to hold materials.
Its construction includes a network of valves ,
pressure and temperature guages, and ducts for
regulating and conducting the steam into the
chamber.
 Sterilization is achieved when the steam condenses
against the objects in the chamber and gradually
raises the temperature
 Cont’
• Most efficient pressure –temperature
combination for achieving sterilization is 15
pounds per square inch (psi) , which yields 121oC
• The duration of the process is adjusted according
to the bulkiness of the items in the load and how
full the chamber is
• The range of holding time varies from 10
minutes for light loads to 40 minutes for heavy
or bulky ones ; the average time is 20 minutes
Examples of laboratory materials that can be
sterilized by autoclaving
Autoclaved Not compatible
• Glass ware • Acids, bases and organic
• Tissue culture flasks solvents
• Pipette tips • Flammable , radiactive
• Media solution substances
• Water • Liquids in tightly closed
containers
• Gloves
• Surgical instruments
• Eppendorf tubes
 Cont’
2. Sterilization with non pressurized steam
 Selected substances that cannot withstand the
high temperature of the autoclave can be
subjected to intermittent sterilization , also
called Tyndallization.
 This technique requires a chamber to hold the
materials and a reservoir for boiling water.
 Items in the chamber are exposed to free-
flowing steam for 30 to 60 minutes
 Cont’
• The temperature in steam is not sufficient to
reliably kill spores , so a single exposure will
not suffice .
• On the assumption that surviving spores will
germinate into less resistant vegetative cells,
the items are incubated at 37oC for 24 hours,
and then again subjected to steam treatment.
• The cycle is repeated for 3 days in a row
Dry Heat (Hot air and incineration)
• The temperatures and times employed in dry heat vary
according to the particular method , but generally
greater than with moist heat
• Incineration in a flame or electric heating coil is the most
rigorous of all heat treatments
• The flame of a Bunsen burner reaches 1870oC at its
hottest point, and the furnaces/incinerators operate at
temperatures of 800 to 6500oC .
• Direct exposure to such intense heat ignites and reduces
microbes and other substances to ashes and gas.
 Cont’

• Incineration of microbial
samples on inoculating
loops and needles using a
bunsen burner is a very
common practice in the
microbiology laboratory.
• This method is fast and
effective but limited to
metals and heat resistant
glassware
 Cont’
 The hot air oven
• The hot air oven provides another means of dry heat sterilization.
• The dry oven is usually electric (occasionally gas) and has coils
that radiate heat within an enclosed compartment
• Heated , circulated air transfers its heat to the materials in the
oven
• A cycle takes 12 minutes to 4 hours to complete and involves a
temperature of 150oC to 180 oC
• Its good for sterilizing glassware, metals, powder, oils
• Poor choice for liquids , rubber, plastics
• Its time consuming
 Sterilization by radiation
• Radiation is defined as energy emitted from atomic
activities and dispersed at high velocity through
matter or space .
• It is characterized by a range of wavelengths known
as the electromagnetic spectrum
• The types of radiation suitable for microbial control
are gamma rays, X rays, and ultraviolet radiation.
• Ultraviolet radiation is usually aimed more at
disinfection than at sterilization.
• Ionizing radiations are effective sterilants
 Mode of action of ionizing radiations
 Ionizing radiation is the type of radiation that ejects
electrons from an atom; it causes ions to form
 Gamma rays, X rays and high-speed electrons
(cathode rays) are all ionizing in their effect
 One of the most sensitive targets for ionizing
radiation is DNA, which will undergo mutations on a
broad scale.
 Secondary lethal effects appear to be chemical
changes in organelles and the production of toxic
substances
 Cont’
• Ionizing radiation is highly effective alternative for sterilizing
materials that are sensitive to heat or chemicals.
• Devices that emit ionizing rays include gamma ray machines
containing radioactive cobalt, X ray machines and cathode
ray machines.
• Items are placed in these machines and irradiated for a
short time with a carefully chosen dosage
• The dosage of radiation is measured in grays . Depending
on the application , exposure ranges from 5 to 50 kilograys
• The most penetrating of the radiations is gamma rays,
followed by x-rays and least is cathode rays
 Cont’
• Gamma radiation has been approved for use in over 40
countries for the preservation of food.
• Irradiation is used in situations where heat sterilization would
be inappropriate because of undesirable effects.
• This mainly relates to drugs , vaccines, medical instruments
especially plastics, fruits, vegetables, poultry ,meat and other
delicate materials can be irradiated without harming them.
• Its main advantage include speed, high penetration power
and absence of heat
• Its main disadvantages are potential dangers from factory
exposure to radiation and possible damage to some materials
 Sterilization by filtration
• Filtration is an effective method to remove microbes from
air and liquids.
• In practice , a fluid is strained through a filter with openings
large enough for the fluid to pass through but too small for
microorganisms to pass through
• Most modern microbiological filters are thin membranes of
cellulose acetate , polycarbonate and a variety of plastic
containers (nylon, Teflon) whose pore size can be carefully
controlled and standardized
• Ordinarily substances such as charcoal, diatomaceous earth,
or unglazed porcelain are also used in some applications
Most filters are perforated
by very precise uniform
pores
The pore diameter vary
from coarse (8μm) to
ultrafine (0.02 μm),
permitting selection of the
minimum particle size to
be trapped.
Those with even small
pore diameters will retain
viruses and large proteins ,
permitting true sterilization
Application of filtration sterilization
• Sterilization by filtration is used to prepare liquids that cannot
withstand heat, including serum and other blood products ,
vaccines ,drugs, enzymes and media
• Filtration has been employed in sterilizing milk and beer without
altering their flavour
• It has the disadvantage of not removing soluble molecules
(toxins) that cause disease
• Filtration is a very effective means of removing airbone
contaminants that are a common source of infection and
spoilage
• High efficiency particulate air(HEPA) filters are widely used to
provide a flow of sterile air to hospital rooms and laboratories
 Chemical agents in Sterilization
1. Ethylene oxide (ETO)
 ETO and its relative propylene oxide (PO) are colorless gases
at room temperature .
 ETO is very explosive in air , a feature that can be eliminated
by combining it with a high percentage of carbon dioxide or
fluorocarbon
 ETO is a very strong alkylating agent , and it reacts vigorously
with functional groups of DNA and proteins .
 Through this actions , it blocks both DNA replication and
Enzymatic actions.
 ETO is one of a very few gases generally accepted for chemical
sterilization because it is a sporicide
 Cont’
• A specially designed ETO sterilizer is equiped with a
chamber , gas ports , and controls for temperature ,
pressure and humidity
• Ethylene oxide is rather penetrating but relatively
slow-acting , requiring from 90 minutes to 3 hours
• Some items absorb ETO residues and must be aerated
with sterile air for several hours after exposure to
remove as much residual gas as possible
• ETO is used as a sterilant for some foods and plastic
items
 Cont’
• For all its effectiveness , ETO has some
unfortunate features .
1. Its explosiveness makes it dangerous to
handle
2. It can damage the lungs , eyes and mucous
membranes if contacted directly
3. It is rated as carcinogen by the FDA, USA
government
 Cont’
2. Chlorine dioxide
 Despite the name , chlorine dioxide works in a
completely different way from other microbicidal
chlorine compounds .
 It’s a strong alkylating agent, which disrupts proteins and
is effective against vegetative bacteria , fungi, viruses and
endospores
 Chlorine dioxide has mainly been used for the treatment
of drinking water , medical waste,decontamination of
rooms , buildings and large spaces or objects.
3. Betapropiolactone(BPL)
 Is a substance somewhat similar to ETO in
applications. It is rapidly microbicidal when
used as an aerosol or liquid to sterilize bone
and arterial grafts, and to inactivate viruses in
vaccines.
4 . Aldehyde sterilants
• Organic substances bearing a –CHO functional
group ( a strong reducing group) on the terminal
carbon are called aldehydes.
• The three aldehydes used most often in microbial
control are glutaraldehyde, ortho-phthaldehyde
and formaldehyde.
• Its only glutaraldehyde of the three which is
sporicidal qualifying it to be a sterilant. The two are
disinfectants.
• Glutaraldehyde is ayellow liquid with a mild odor
• The molecule’s two aldehyde two aldehyde groups favor the
formation of polymers.
• The mechanism of activity involves cross-linking protein molecules on
the cell surface .
• In this process , amino acids are alkylated, meaning that a hydrogen
atom on an amino acid is replaced by the glutaraldehyde molecule
itself .
• This irreversibly disrupts the activity of enzymes and other proteins
within cells
• Glutaraldehyde is rapid and broad spectrum ,
• It is one of the few chemicals that can be used to sterilize delicate
reusable medical and dental instruments
 Cont’
• It kills spores in 3 hours and fungi and vegetative bacteria in
a few minutes
• Viruses ,including the most resistant forms , appear to be
inactivated after relatively short exposure
• A special device known as chemiclave vaporizes the solution
under pressure to sterilize delicate instruments without heat
• Glutaraldehyde retains its potency even in the presence of
organic matter, is non corrosive ,does not damage plastics
and is less toxic or irritating than formaline
• Its principle disadvantage is that it is some what unstable ,
especially with increased pH and temperature
 Cont’
5. Vapourised hydrogen peroxide
 Hydrogen peroxide is a colorless , caustic liquid that decomposes in
the presence of light, metals or catalase into water and oxygen gas
 The germicidal effects of hydrogen peroxide are due to the direct
and indirect actions of oxygen.
 Oxygen forms hydroxyl free radicals (-OH), which , like the
superoxide radical are highly toxic and reactive to cells.
 Hydrogen peroxide is bactericidal ,virucidal, fungicidal and in
higher concentration sporicidal thus sterilant .
 A vaporized form of hydrogen peroxide is effective as an
environmental sterilant used in decontaminating large spaces and
objects.
 Disinfection
• This refers to the use of a physical process or a
chemical agent (a disinfectant ) to destroy
vegetative pathogens but not bacterial
endospores
• It is important to note that disinfectants are
normally used only on inanimate objects
because at the concentration required to be
effective , they can be toxic to living tissue
 Disinfection methods
1. Boiling water
 A simple boiling water bath or chamber can quickly
decontaminate items in the clinic , home and laboratory.
 Because a single processing at 100oC will not kill all resistant
cells, this method can be relied on only for disinfection and
not for sterilization.
 Exposing the materials to boiling water for 30 minutes will kill
most non spore forming pathogens , including resistant
species such as the tubercle bacillus and staphylococci
 The greatest disadvantage with this method is that the items
can be easily recontaminated when removed from the water
 Cont’
2. Pasteurization; Disinfection of beverages
 Fresh beverages such as milk, fruit juices , beer, and wine are easily
contaminated during collection and processing
 Because microbes have the potential to spoil these foods or cause
illness, heat is frequently used to reduce the microbial load and destroy
pathogens
 Pasturization is a technique of applying heat to consumable liquids to kill
potential agents of infection and spoilage , while at the same time
retaining the liquids flavor
 A widely used pasteurization technique is the flash method , that
exposes the liquid to heat exchangers at 71.6oC for 15 seconds.
 This method does not appreciably change flavor and nutrient content ,
and effective against some resistant pathogens such as mycobacterium
and coxiella
 Cont’
 Although these treatment inactivate most viruses and destroy
the vegetative stages of 97% to 99% of fungi and bacteria,
they do not kill endospores and thermoduric
microbes( mostly non pathogenic lactobacilli , micrococci, and
yeast
 Many commercial dairies process untrapasteurized milk using
ultrahigh temperature (UHT) -134 oC for 2 to 5 seconds
 The primary targets of pasteurization are non-spore forming
pathogens ; salmonella species , campylobacter jejuni, listeria
monocytogenes , brucella species, coxiella burnetti,
mycobacterium bovis, M. tuberculosis and several enteric
viruses.
A small flash
pasteurizer
 Cont’
3. Nonionizing radiation ; Ultraviolet Rays
 Ultraviolet (UV) radiation ranges in wavelength from
approximately 100nm to 400nm.
 It is most lethal from 240nm to 280nm(with a peak at 260nm)
 The source of UV rays is the germicidal lamp which generates
radiation at 254nm
 Owing to its lower energy state , UV is not as penetrating as
ionizing radiation
 Because UV radiation passes readily through air, slightly
through liquids , and poorly through solids, the object to be
disinfected must be directly exposed to it for full effect
 Cont’
• As UV radiation passes through a cell , it is initially absorbed by DNA.
Specific molecule damage occurs on the pyrimidine bases which
form abnormal linkages with each other called pyrimidine dimmers
• These bonds occur between adjacent bases on the same DNA strand
and interfere with normal DNA replication and transcription .
• The results are inhibition of growth and cellular death.
• UV radiation also disrupts cells by generating toxic photochemical
products called free radicals . These highly reactive molecules
interfere with essential cell processes by binding to DNA , RNA and
Proteins .
• UV rays are a powerful tool for destroying fungal cells and spores ,
bacterial vegetative cells , protozoa and viruses. Bacterial
endospores are more resistant.
• UV disinfection of air has proved effective in reducing
postoperative infections , preventing the transmission of
infections by respiratory droplets and curtailing the growth
of microbes in food processing plants and slaughter houses
• UV irradiastion o=f liquids requires special equipment to
spread the liquid into a thin, flowing film that is exposed
directly to a lamp. This method can be used to treat water
and to purify other liquids as an alternative to heat
• The surfaces of solid, non porous materials such as walls
and floors as well as drugs, nuts, meat etc have been
succefully disinfected with UV
 Cont ‘
• One major disadvantage of UV is its poor powers
of penetration through solid materials such as
glass , metal ,cloth , plastic and even paper
• Another drawback is the damaging effect of
overexposure on human tissues , including
sunburn ,retinal damage , cancer and skin
wrinkling
• These harmful effects occur only if a person is
directly exposed to the UV rays , but necessitate
placing of barriers to cut down this possibility
 Chemical disinfectants ( Germicides)
• Phenol and its derivatives
 First adopted by Joseph lister in 1867as a surgical germicide
 Phenol was the dominant antimicrobial chemical until other
Phenolics with fewer toxic and irritatting effects were developed
 Solutions of phenol are now used only in cetrtain limited cases ,
but remains one standard against which other phenolic
diinfectants are rated
 Substances chemically related to phenol are often refered to us
phenolics
 Phenolics consist of one or more aromatc carbon rings with
added functional groups.
 Cont’
 Among the most common phenolics are alkylated
phenols(cresols)
 Chlorinated phenols( triclosan)
 Bisphenols
 In high concentrations , phenolics are cellular poisons , rapidly
disrupting cell walls and membranes and precipitating proteins
 In lower concentration they inactivate certain critical enzymes
 Phenolics destroy vegetative bacteria , fungi and many viruses
but not sporicidal
 Their sustained activity in presence of organic matter and their
detergent actions contribute to their usefulness
 Their toxicity make them questionable choice as antiseptics
 Cont’
• Chlorhexidine
 The compound chlorhexidine (hibiclens, hibitane) is a complex
organic base containing chlorine and two phenolic rings
 Its mode of action targets cell membranes by lowering the surface
tension and causing the denaturation of proteins
 At moderate to high concentrations its bactericidal for both gram-
positive and gram-negative bacteria but inactive against spores
 Its effects on viruses and fungi vary.
 It posses distinct advantages over many other antiseptics because of
its mildness , low toxicity , and rapid action, and it is not absorbed
into deeper tissues to any extent
 It is the antiseptic cleanser of choice to control Staphylococcus MRSA
and Acinetobacter outbreaks in hospitals
 Cont’
• Hydrogen peroxide
Versatile use as an antiseptic , including skin and
wound cleansing , bedsore care , and mouth
washing
Disinfectant for soft contact lenses , surgical
implants , plastics , bedding , and room interiors
3% Hydrogen peroxide –most common form-is
useful in treating infections by anaerobic bacteria
because of the lethal effects of the oxygen released.
 Cont’
• Chlorine and its compounds
 Chlorine has been used for disinfection and antisepsis for approximately 200 years
 The major forms used in microbial control are liquid and gaseous chlorine ,
hypochlorites, chloramines.
 In solution , these compounds combine with water and release hypochlorous acid
(HClO)
 Hypochlorous acid oxidises the sulhydryl (S-H) groups on amino acid cysteine and
breakes disulphide (S-S) bridges on numerous enzymes
 The resulting denaturation of of the enzymes is permanent and suspends
metabolic reactions
 It also demages the structure of DNA, RNA and fatty acids.
 Death of almost all microorganisms usually occurs within 30 minutes
 Endospores may require several hours
 Chlorine compounds are less effective and relatively unstable if exposed to light ,
alkaline pH, and excess organic matter
 Cont’
• Iodine and its compounds
 Iodine (I2) is a pungent blue-black element that forms a
brown-colored solution when dissolved in water.
 It rapidly penetrates the cells of microorganisms, where it
disturbs a variety of metabolic functions by interfering with
the disulfide bonds of proteins
 Iodine is less susceptible to inactivation by organic material
 All classes of microorganisms can be killed if proper exposure
times and iodine concentrations are used
 Medium-level disinfection for plastic instruments,
thermometers , contaminated water
 Cont’
• Alcohols
 Alcohols are colorless hydrocarbons with one or more-OH functional
groups
 Of several alcohols available , only ethyl alcohol and isopropyl are
suitable for microbial control
 Methyl alcohol is not particularly microbicidal , and more complex
alcohols are either poorly soluble in water or too toxic for routine use
 Alcohols are employed alone in aqueous solutions or as solvents for
tinctures (with iodine , for example)
 Alcohols mechanisms of action depend in part upon its concentration
 Concentrations of 50% and higher dissolve membrane lipids , disrupts
cell surface tension and compromise membrane integrity
 Cont’
 Alcohols that has entered the proptoplasm denatures proteins
through coagulation but only in alcohol-water solutions of 50% to
95%
 Because water is needed for proteins to coagulate , alcohols
greater microbicidal acticvity at 70% concentration than at 100%
 Absolute alcohol dissolves cell membranes and inhibits cell
growth but is generally not a protein coagulant
 Alcohols can destroy resistant vegetative forms including
tuberculosis bacteria and fungal spores
 Alcohols tend to inactivate enveloped viruses more readily than
nonenveloped virues because of the surfactant effect on the
envelope.
 Cont’
• Heavy metal compounds
 Various forms of the metallicc elements mercury , silver,gold,copper,
aresenic and zinc have been applied in microbila control over several
centuries
 From the list , only prep containing mercury and silver still have any
significance as germicides.
 The higher molecular weight metals can be very toxic even in minute
quantities( parts per million)
 This property of having antimicrobial effects in exceedingly small
amounts is called oligodynamic
 Heavy metal germicides contain either an inorganic or organic metal
salts and they cme inform of tinctures,ointements, aqueous solutions
or soaps
 Cont’
 Mercury ,silver and most other metals exert micobicidal effects by binding
onto functional groups of proteins and inactivating them, rapidly bringing
metabolism to a standstill
 This mode of action can destroy many types of microbes including
vegetative bacteria, fungal cells and spores , algae , protozoa and viruses
 A newer use of metals is their incorporation into medical supplies and
hoaspital facilities
 Silver is now added to catheters ,IV lines , and prostheses to prevent
biofilm formations and infections .
 Copper is added to metal parts of lavatory fixtures, doors, and doorknobs.
 It destroys the most problematic drug-resistant pathogens such as MRSA,
as well as Clostridium difficile vegetative cells, influenza virus , and gram-
negative opportunitists
 cont’
• The main drawbacks to heavy metals are;
1. Metals are very toxic to humans if ingested,
inhaled or absorbed through the skin., even in
small quantities , for the same reasons that
they are toxic to microbial cells
2. They commonly cause allergic reactions
3. Large quantities of biological fluids and wastes
neutralize their actions
4. Microbes can develop resistance to metals
 Cont’
• Acids and alkalis
 Conditions of very low or high PH can destroy or inhibit microbial cells , but they
are limited in applications due to corrosive caustic and hazardous nature
 Alkaline solutions are good for cleaning and some types of disinfection because
they inactivate proteins
 Ammonium hydroxide is a major component of detergents and cleansers , and a
strong sodium hydroxide solution is one of the few substances that reliable
destroys prions.
 Organic acids are widely used in food preservation because they prevent spore
germination and bacterial and fungal growth and because they are generally
regarded as safe to eat.
 Acetic acid is a pickling agent that inhibits bacterial growth ; propionic acid is
commonly incorporated into breads and cakes to retard molds
 Lactic acid is added to sauerkraut and olives to prevent growth of anaerobic
bacteria (especially clostridia); benzoic acid and sorbic acids are added to beverages
, syrups and margarine to inhibit yeasts
 Sanitization
 Sanitization refers to any cleansing technique that removes
debris , soil, microorganism, and toxins from inanimate surfaces
and in this way reduces the potential for infection and spoilage .
 Degermation refers to the cleansing technique that removes
microorganisms and debris from living tissue. This process usually
involves scrubbing the skin or immersing it in chemicals or both .
 Microorganism are removed both mechanically and indirectly by
emulsifying the oils that coat the skin and provides a habitat for
growth
 Examples of degerming procedures include; the surgical hand
scrub, the application of alcohol wipes , and the cleansing of
wounds with germicidal soap and water
 Cont’
• The common sanitizers employed are soaps and detergents because of their
surfactant properties
 Soaps are alkaline compounds made by combining the fatty acids in oils with
sodium or potassium salts
 Soaps alone are weak microbicides and they destroy only highly sensitive forms
such as the agents of gonorrhea, meningitis, and syphilis
 Pseudomonas is so resistant to soap that various species grow abundantly in
soap dishes.
 Soaps function primarily as cleansing agents and sanitizers in industry , home
and the medical settings
 Combining germicides such as chlorhexidine or iodophor with soaps produces
highly active antiseptics, degermers, and disintfectant
 Vigorously brushing the hands with germicidal saop over a 15-second period
can remove dirt,oil, and surface contaminats as well as some superficial resident
microbes, but is unable to remove those deeply entrenched in the skin
 Cont’
 Detergents are polar molecules that act as surfactants .
• Much more effective are positively charged detergents , particularly the
quaternary ammonium compounds (quats)
• The activity of cationic detergents arises from the attachment of four
organic radicals to a central positively charged nitrogen atom . At least
one of the radicals is along-chain hydrocarbon surfactant that disrupts
the cell membranes of sensitive microbes . After a few minutes of
contact , the cells burst and die.
• When used at high enough concentrations , quats are effective against
some gram-positive bacteria , viruses and fungi and algae
• Drawbacks include their ineffectiveness against tuberculosis bacterium ,
hepatitis virus, pseudomonas and spores at any concentration
• They also require alkaline conditions to function
THANKS FOR THE
ATTENTION