Biosafety in the Mycobacteriology

Laboratory
June 2011

Natalia Shubladze, TB lab Consultant

Lynee Galley, Senior Program Specialist, International
Affairs, LabCap
 Part III

Disinfectants
 Disinfectants
A chemical or mixture of chemicals used to kill
microorganisms
– Applied to inanimate surfaces, objects or liquids
– Essential for routine procedures working with patient
specimens and bacterial isolates

In the TB lab, disinfectants must be capable of

killing TB
Commercial products should be labeled as
tuberculocidal

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3 Main Types of Disinfectants

Phenol-based

Hypochlorite (bleach)

Alcohol

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Phenol-based Disinfectants
 Main uses for phenol-based disinfectants:
– In discard containers that are used for:
o Decanting contaminated liquids
o Discarding contaminated supplies
– Surface decontamination
– Clean up of spills – both major and minor, inside and outside
BSC

 For phenol-based disinfectants to be effective

– Disinfectant must be left in contact with contaminated
material for at least 15 minutes to ensure killing

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 Commercially-available Phenolic
Disinfectants

 Many commercial
disinfectants use phenol
derivatives
 Some phenolic derivatives are
inactivated by water hardness and Phenol-based
should be diluted to the appropriate disinfectant
concentration using de-ionized
water

 Check to ensure the product is Tuberculocidal

labeled tuberculocidal

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 Chemical Safety: Phenol crystals

 Phenol is corrosive and highly irritating to

the skin, eyes, and mucous membranes
– Gloves must be worn when handling
phenol crystals
– Spills must be carefully cleaned up

 Two main uses for phenol crystals in the TB

lab
– To prepare a disinfectant solution at
a concentration of 2 - 5%
– As an ingredient in carbol fuchsin for
ZN staining for AFB

 Phenol disinfectant solutions should be

prepared daily to ensure efficient killing of
TB (labeled with date of preparation) 7
 Hypochlorite (Bleach)
 Main uses for hypochlorite (bleach)
– In discard containers that are used for:
• Decanting contaminated liquids
• Discarding contaminated supplies
– Surface decontamination

 For hypochlorite disinfectants to be effective in

killing TB
– Disinfectant must be left in contact with contaminated material
for at least 15 minutes
– Diluted solution must be prepared daily because escape of
chlorine gas lessens disinfectant activity
– Final chlorine concentration should not fall below 0.5%
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Concentration of chlorine in bleach
 Check concentration
of total chlorine on
bleach container
 Undiluted commercial
bleach usually is 4 to Commercial
Bleach (5%)
5% chlorine
 Alternatively, a
commercial strip can
be used to check
chlorine concentration

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 Hypochlorite (Bleach) is Corrosive

 Bleach, even in diluted solutions, is irritating to

the skin and VERY corrosive to metal and
plastic surfaces (such as BSCs, centrifuges,
pipettors)

 If used to disinfect metal and plastic surfaces:

– Wash the surface with water or 70% alcohol
after the bleach has disinfected the area (for at
least 15 minutes)

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Chemical Safety: Hypochlorite
(Bleach)

HEALTH HAZARD: Chlorine gas released

from bleach is extremely toxic
Containers containing bleach MUST NOT
be autoclaved
Bleach must always be stored in well
ventilated areas

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 Alcohol
Alcohol used as a disinfectant
– Surface decontamination on both skin and work
surfaces
– Denatured ethanol diluted and used as 70% solution
Safe for use on metal (BSCs, centrifuges, other
equipment)
Major advantage: will not corrode or leave a
residue on treated items
• High evaporation rate: An absorbent surface
that is sprayed with 70% Alcohol has
disinfection activity for only 20 minutes
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 Chemical Safety: Alcohols
 Proper uses for alcohol in the TB lab
 As a surface disinfectant (70%)
 Spirit lamp
 Decolorizing AFB stained smears

 Limit volume stored in the lab since accidental ingestion of

denatured alcohol can be lethal.
! Fire in BSC- Bunsen
Caution: Alcohols are volatile and burner ignited 70%
flammable alcohol being used for
– Volatile – should be stored in cleaning
proper containers to avoid
evaporation
– Flammable – must not be used
near open flames
– Bottles with alcohol containing
solutions must be clearly labeled
to avoid autoclaving 13
 Glutaraldehydes

 Glutaraldehyde is a pungent
colorless oily liquid with the formula
CH2(CH2CHO)2
 A glutaraldehyde solution of 0.1% to
1.0% concentration may be used for
system disinfection and as a
preservative for long term storage.
 Product claims include: "Inactivates
viruses and bacteria. Dries the wart
surface. Stains the area treated
brown, but will not harm the
surrounding skin."
 Chemical safety: Glutaraldehydes

 As a strong disinfectant,
glutaraldehyde is toxic and
can cause severe eye,
nose, throat and lung
irritation, along with
headaches, drowsiness
and dizziness.
 It is a main source of
occupational asthma
among health care
providers.
 Chemical Safety: Acids

• Concentrated HCl or H2SO4 used for

preparation of Acid-Alcohol AFB staining
decolorizers

• Concentrated acids are extremely caustic to

skin and eyes, and corrosive to surfaces

• Wear gloves and goggles when handling acids

• Avoid breathing toxic fumes

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 Safe Method for Diluting Acids
NEVER ADD WATER TO ACID ALWAYS ADD ACID TO WATER

WATER
ACID

ACID WATER

Results in excessive heat, foaming, Still need to pour slowly to avoid

splashing! excessive heat build-up.
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Remember SAFETY FIRST!
• Skills
• Accountability
• Facility
• Equipment
• Training
• YOU
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 Literature
• CDC/NIH Biosafety in Microbiological and Biomedical Laboratories, 5th
Edition. (2007) http://www.cdc.gov/od/ohs/biosfty/bmbl5/bmbl5toc.htm
• Laboratory biosafety manual. 3rd edition, 2004. WHO.
http://www.who.int/csr/resources/publications/biosafety/en/
• Biosafety7.pdf Biosafety recommendations for the contained use of M.
tuberculosis complex isolates in industrialised countries. P Herman et al, 2006.
Scientific Institute of Public Health, P Herman et al, 2006. Scientific Institute of
Public Health, Brussels, Belgium.
http://www.biosecurite.be/CU/PDF/Mtub_Final_DL.pdf
• Interim Laboratory Biosafety Guidance for Extensively Drug-Resistant (XDR)
M. tuberculosis strains.
http://www.cdc.gov/tb/XDRTB/BiosafetyGuidance_xdrtb.htm
• Laboratory Biosafety Levels for TB Diagnostics (African Center for Integrated
Laboratory Training)
• Thomas M. Shinnick, Ph.D. Mycobacteriology Laboratory Branch Division of
Tuberculosis Elimination Biosafety in the Mycobacteriology Laboratory