BASIC LABORATORY PROCEDURE

AND SAFETY REGULATION IN

MICROBIOLOGY LABOARTORY
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CLINICAL MICROBIOLOGY

 A branch of Microbiology that deals with the study of

microorganisms and their role in human health and
disease.

 It mainly concerned with the diagnosis, treatment,

prevension of various infectous disease.

 There are broad range of microbes from Prokaryotes to

Eukaryotes but Bacteria, Virus, Fungi and Parasites are
the main disease causing microorganisms. 2
HISTORY

 Antonie van Leeuwenhoek was the first who observed

bacteria using a single lense microscope.
 Edward Jenner developed first vaccine, the smallpox
vaccine.
 Louis Pasteur known as Father of Microbiology for his
contributions sterilization techniques, pasteurization of
milk, germ theory of disease also developed many
vaccine etc.
 Robert Koch introduce solid media, hanging drop
method, koch's postulate etc.
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BACTERIA

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VIRUS

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STANDARD MICROBIOLOGY PRACTICES (SMPS)

Generally defined as the basic hygiene practices that apply to

maintain all the laboratories that deals with microorganisms.
 Eating, drinking, use of cosmetics, gum tobacco products are
strictly prohibited in the lab.
 Work surface should be disinfected at the beginning and
completion of lab work.
 Hand should be washed by using chemical disinfectants
after handling infectious material.
 Laboratory aprons and gloves must be worn for all
procedures involving direct contact with infected material.
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 Hair longer that shoulder length should be tied back, hand
should be kept away from face.
 Mouth pipetting of specimen should be avoided.
 All contaminated wastes should be decontaminated (by
autoclave) before disposal.
 Laboratory doors should be kept closed when work is
progress.
 Persons who are at increased risk of acquiring infection
(children, pregnant woman or immunodeficient person)
should not be allowed in lab.
 Exposed wounds should be covered and protected.
 All spills accidents and potential exposures to infectious
material must be reported immediately to the laboratory
superior. 7
 RISK GROUP OF MICROORGANISMS
Risk Work Description Examples
Group/ Bio Area
Safety
Level
Group 1/ Open • Low risk microbes E.coli,
BSL 1 bench • that are unlikely to cause Lactobasillus sp,
disease Bacillus subtilis,
• Mainly non pathogenic. S. cerevisiae
(yeast)
Group 2/ Biosafety • Moderate risk group Staphylococcus
BSL 2 cabinet • Can cause disease in aureus,
human but those are Strptocossus sp.,
treatable or preventable. Clostridium sp.,
• Potential hazards if Herpes virus,
percutaneous injury,
ingestion, mucous
membrane exposure occurs. 8
Risk Work Description Examples
Group/ Bio Area
Safety
Level
Group 3/ Class 3 • High risk Y pestis, Micoba
BSL 3 Biosafety • Indigenous or exotic agents terium
cabinet with potential aerosol tuberculosis,
transmission. Rickettsia sp.,
• Treatment & vaccine exist.

Group 4/ Isolation • High risk or life threatening Ebola virus,

BSL 4 suit microbes Small pox virus,
• cause deadly disease. Hepatitis A ,
• Person to person Hepatitis B.
transmission occur.

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BSL 2
BSL 3

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BSL 4
CONTROL OF MICROORGANISMS
 Sterilization : is the process of complete destruction or
removal of all living cells of viable microbes including
spores from an article, body surface or medium.
Examples- Moist Heat sterilization by autoclave.

 Disinfection: the destruction or removal of vegetative

pathogen but not bacterial endospores. Usually used in
inanimate objects .
Example- Formalin, Phenol, Cresol, Lysol 70% alcohol.

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CONTROL OF MICROORGANISMS
 Asepsis: destruction or inhibition of microbes on a living
tissue.
Example- Alcohol, Boric acid, Iodine, Formaldehyde.
 Sanitization: reduction of pathogenic microbial load or
population to a level at which they considered safe by
public health standard. Closely related to disinfection.

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VEGETATIVE CELL & ENDOSPORE

Vegetative
Cell
Vegetative cell Endospore
Containing
Sporulation Endospore

Free
Endospore 13
 Sterilization

Physical Chemical
Method Method

Gas
Dry Heat Sterilization

Moist Heat

Sterilization by
Radiation
Disinfectants
Filtration
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PHYSICAL METHODS OF STERILIZATION

 Dry Heat: Moisture is essential for the growth of bacteria.

Drying therefore has deleterious effect on bacteria. Dry heat
kill the microbes by denaturing outer protein. Various
employed methods are
i) Flaming

ii) Incineration

iii) Hot air oven

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In Flaming Articles are held or
passed through the Bunsen burner
or spirit lamp either for long time
or short time.

• Shorter Time exposure without

allowing Red Hot done for
fragile items like mouth of test
tube.

• Longer Time exposure in flame

or Red Hot is done for
Inoculating Loop, Needle etc.

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HOT AIR OVEN

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Hot Air Oven

 Most widely used method in dry heat sterilization.

 It is electrically heated and is fitted with a fan to ensure adequate

distribution of hot air in the chamber.

 Also fitted with a thermostat which maintain the chamber air at

chosen temperature.

 160ºC for 1 or 2 hour.

 Use to sterilize glassware like glass syringes, petri plates, forceps,

test tubes, pipettes.
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 Media can not be sterilized by this method.
MOIST HEAT STERILIZATION
 It kills microorganisms by denaturing their proteins and
enzymes to lose their three dimensional functional structure.
 Moist heat can disrupt the cell membrane of microbial cell.

 It can penetrate materials much more rapidly than dry heat

because water molecule conduct heat better than air.
 This method is used for heat sensitive materials through
which steam is permeable.
 This can be employed by the following ways

At temperature 100ºC
At temperature below 100ºC
At temperature above100ºC
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AT 100ºC TEMPERATURE
 Boiling at 100ºC for 10 to 15 minute may kill most of the
vegetative forms but not the spores. Boiling used for
disinfection of drinking water and objects that are not
harmed by water.
 Steaming- Koch’s or Arnold steam sterilizer are useful for
those media which are decomposed at high temperature of
autoclave.
 The articles are kept on a perforated tray through which
steam can pass.
 They exposed to steam at atmospheric pressure for 90
minutes.
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 Tyndallization- This process involves steaming at 100ºC
for 20-45 minutes for 3 consecutive days.

 Named by its inventor John Tyndall

 First exposure kills vegetative forms. The spores are

converted into vegetative forms and killed during
subsequent heating.

 It is used for food and for sterilization of gelatin and egg

serum based media which are damaged at higher
temperature.
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BELOW 100ºC TEMPERATURE
 Pasteurization- This method is developed by Louis Pasteur
to reduce the problem of wine spoilage caused by
microorganisms.
 It is used to control microorganisms and extent shelf life of
beverages like fruit juice, beer and dairy product such as
milk at less than 100ºc temperature.
 It can destroy the vegetative cells but not spores.

 There are three methods of pasteurization available

i) Holder Method- Conventional low temperature, Liquid is

maintained at 62 ºC for 30 minutes then cooled to 39ºC.
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ii) Flash method- Products are held at high temperature, short
time (HTST) that is 72ºC for 15 seconds followed by rapid
cooling to 13ºC or lower.
iii) Ultra pasteurization- Heating for 138ºC for 2 second
followed by rapid cooling.

 Water bath- It is used for disinfection of serum, body fluid

and vaccines.
 Bacterial vaccines are disinfected

at 60ºC for 1 hour.

 Serum or heat labile body fluid can

be disinfected at 56ºC for one hour.

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 Inspissation (fraction sterilization)- It is a process of
heating an article on 3 successive days at 80-85ºC for 30
minutes by a special instrument called inspissator.
 The first exposure killed all vegetative forms and in the
intervals between the heating the remaining spores
germinate into vegetative forms which are then killed on
subsequent heating.
 Use- sterilization of egg and serum base media such as L-J
medium, Loeffler’s serum slope which get destroyed at
higher temperature.
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ABOVE 100ºC TEMPERATURE
 Autoclave- developed by Chamberland in 1884.
 121ºC temperature and 15 pound pressure for 15-20 minutes
is preferable.
 It uses steam to sterilize equipment and objects by the
destruction of microbes.
 When water is heated in a closed container, saturated steam
is produced under pressure.
 According to Boyle's law, when volume of the steam is kept
constant, the temperature is directly proportional to
pressure.
 The air initially present in the chamber is forced out until the
chamber is filled with saturated steam and outlets are closed.
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 Use- particularly useful for media containing water
that cannot be sterilized by dry heat, surgical
instruments, culture media, plastic tubes, pipette
tips, glassware, biohazard waste etc.

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Vertical Type (small volume capacity)

Horizontal Autoclave (large Volume capacity)

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RADIATION
Two types of radiation are available

 Ionizing Radiation-
X-rays, gamma rays (Cobalt 60 source)

 Non-Ionizing Radiation-
Ultraviolet radiation

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 Non-Ionizing Radiation- Ultraviolet radiation
 It is quite lethal but do not penetrate glass, dirt films, water
hence their use is restricted.
 Recommended dose is 250-300 nm wavelength, given for 30
minutes.
 UV radiation cause thymine-thymine dimerization of DNA,
preventing replication and transcription.
 This is used in Biological safety cabinets and ceilings of
rooms to sterilize the air and exposed surface and for water
treatment.
 UV rays burns the skin and damages eye hence the area
should be closed and UV lamps must be switched off
immediately after use.
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 Ionizing Radiation-
X-rays, gamma rays (Cobalt 60 source) cosmic rays.
 It causes breakage of DNA without temperature rise
hence it called cold sterilization.
 It can destroy bacterial spores and vegetative cell but
not always effective for virus.
 Ionizing radiation has high penetrating power.
 Gamma radiation is used for sterilization of food,
disposable rubber or plastic syringes, infusion set,
catheters etc. 32
FILTRATION
 It is an excellent way to reduce the microbial population in
solution of heat labile (heat sensitive).
 Used to sterilize various liquid like vaccine antibiotics,
toxin, serum as well as purification of air.
 Rather than directly destroying microbes filter simply
removes them.
 Two types of filters are mainly used depth and membrane
filter.

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 Depth Filter – are porous filters that retain particles
throughout the depth of the filter, rather than just on the
surface.
 It consist of fibrous or granular material that have been
bonded into thick layer filled with twisting channels of
small diameter.
 The solution containing microorganisms is sucked through
this layer under vaccume and microbial cells are moved by
physical screening or entrapment and by adsorption to the
surface of the filter material.
 Examples- Berkefeld filters (made up of diatomaceous
earth), Chamberland filter (unglazed porcelain), Seitz filter
(Asbestos filter),
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Berkefeld Filter Membrane Filter

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 Membrane filter - most widely used for filtration of
bacteria.

 They are porous (0.2 µm 0.1mm thick)

and retain all the particles vegetative
cell on the surface that are larger than
their pore size.

 The liquid is pulled or forced through the filter with a

vacuum and collected in previously sterilized container.
 Membrane filter can be made up of cellulose acetate,
cellulose nitrate, polycarbonate or other synthetic material.
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 Air can also be filtered to remove microorganisms.
 Two common examples are N-95 disposable mask and
cotton plugs on culture vessels that let air in but keep
microbes out.
 N-95 exclude 95% of particles that are larger than 0.3 µm.

 Another example is Biological safety cabinet which employ

High efficiency particulate air (HEPA) filter removes
99.97% of particles 0.3 µm or larger
sterilize the air within the cabinet.
 It can also removes viruses that

are 0.1 µm and smaller.

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HEPA filter in Biological safety cabinet
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CHEMICAL AGENTS OF STERILIZATION

 Chemical agents are more often employed in

disinfection and antisepsis.

 It is also use to prevent microbial growth in food

and in treatment of infectious disease.

 Examples- Alcohols, Phenolics, Aldehydes,

Halogens, Heavy metals.

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 Alcohols- Most widely used disinfectant and antiseptics.
 They are bactericidal, fungicidal but not sporicidal but some
enveloped viruses (e.g. HIV)are also destroyed.
 Example- The most popular alcohol germicides are ethanol
and isopropanol, both are used in 70-80 % concentration.
 They act by denaturing proteins and dissolving membrane
lipids.
 Ethyl alcohol is used as surgical spirit as antiseptics.
 Isopropyl alcohol use to disinfect clinical thermometer and
small instrument by soaking 10-15 minutes. 40
 Aldehyde- Formaldehyde, glutaraldehyde are the commonly
used aldehyde.
 Sporicidal used as chemical sterilant.
 They combine with nucleic acids, protein to inactivate them ,
probably by cross linking and alkylating molecules.
 Glutaraldehyde- It is less toxic and corrosive than
formaldehyde.
 Used to sterilize hospital and laboratory equipment.
 2% concentration usually disinfect object within 20 minutes
but require 12 hours to destroy all spores.
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 Formaldehyde dissolved in water or alcohol before use.
Formalin or formol (40% formaldehyde) most widely used
preparation.

 It is best used for-

1) preservation of anatomical specimen

2) formaldehyde gas is used for fumigation

3)preparation of toxoid from toxin

 Formaldehyde is toxic and irritant when inhaled, as well as

corrosive to the metals.
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 Phenolics- Phenol was the first widely used antiseptic and
disinfectant.
 In 1867 Joseph Lister employed it to reduce the risk of
infection during surgery.
 Phenol and phenolics such as cresol, Lysol, xylenol
orthophenylphenol are used as disinfectant in hospitals and
laboratories.
 Phenolics act by denaturing protein and disrupting cell
membranes in presence of organic compound. And effective
for Mycobacterium tuberculosis.
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 Althought phenolics are toxic and irritant to skin but
certain phenolics are less irritant widely used as
antiseptics.

 Chlorhexidine active ingredient of savlon.

 Chloroxylenol active ingredient of dettol.

 The newer derivative tricolsan is often used in hand

sanitizer due to effective blockage of bacterial fatty acid
synthesis.

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 Halogen- contain five elements fluorine, chlorine,
bromine, iodine, astatine.

 Among them Iodine and chlorine have antimicrobial

activity.

 They exist in free state and form salt with sodium and
other metals.

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 Iodine- It is used as a skin antiseptic and kills microbes by
oxidizing cell constituents and iodinating cell proteins.
 Tincture of iodine- It is a preparation of iodine (2%) in a
water-ethanol solution of potassium iodine
 It is an effective antiseptic, but can cause skin allergy and a
yellow stain is left.
 Iodophore- prepared by complexing iodine with an organic
carrier such as providine.
 Water soluble, stable, non-staining and release iodine slowly
to minimize skin burns, irritation.
 Used as preoperative antiseptics as well as disinfectant in
laboratory.
 Some popular brands are Wescodyne for skin, laboratory
disinfection and Betadine for wounds.
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 Chlorine-It is the most commonly available disinfectant.
 It is used

1) for municipal water supplies and swimming pools

2) also employed in the dairy and food industries.
3) as laboratory disinfectant.
4) as bleaching agent to remove the stain from clothes
 Preparation- available as chlorine gas, sodium hypochlorite,
calcium hypochlorite.
 Mechanism- Hypochlorous acid cause oxidation of cellular
materials and destruction of vegetative bacteria and fungi,
but not spores.

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 Disadvantage-

1) Carcinogenic chlorine reacts with organic compounds to

form carcinogenic trihalomethanes

2) Sodium hypochlorite is corrosive and should be handled

cautiously

3) They are not active against Giardia and Cryptosporidium

4) organic matter interferes with its action, hence excess chlorine

always added to water to ensure microbial destruction.

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 Heavy metal- Salts of heavy metals such as mercury,
silver, arsenic, zinc and copper were widely used in the past
as germicides.
 Heavy metals combine with bacterial cell proteins and
inactivate them. They are more bacteriostatic than
bactericidal.
 Copper sulfate is an effective fungicide in lakes and
swimming pools
 Silver sulfadiazine is used on burn surface

 Mercury salts were known antiseptics but are not used

now.

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 Hydrogen peroxide – It is a strong oxidizer, is used as high
level disinfectant as well as chemical sterilant.
 Mode of action- It breaks and liberates toxic free hydroxyl
radicals which are the active ingradient and attack
membrane lipid, DNA, and other cellular components.
 3-6 % concentration is effective, while catalase producing
organism and spores require higher concentration.
 Used to disinfect soft contact lenses, ventilator. Vaporized
H202 is used for plasma sterilization.
 It is environmentally safe neither carcinogenic not
mutagenic
 It removes microbes from equipment.

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