Module 6
Processing the specimens
and inoculation on
solid and liquid media
1
Learning objectives
At the end of this module you will be able to:
 work properly within a BSC;
 process specimens from sterile and non sterile sites,
according to protocols, for TB culture;
 homogenize and decontaminate respiratory specimens
by:
– Petroff modified method,
– NALC-NaOH method;
 inoculate cultures (solid and/or liquid media);
 incubate inoculated media under proper conditions.
2
Content outline
• Working within a BSC
• Homogenization and decontamination
procedures:
– sodium hydroxide (modified Petroff)
– N-acetyl-L-cysteine-sodium hydroxide (NALC)
• Alternative protocols for processing different
specimens
• Culture tube inoculation
• Culture tube incubation
• Quality assurance
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Work in a BSC (1)
• Prepare a written checklist of materials needed.
• Place absorbent paper towelling on the work
surface.
• DO NOT block the grille.
• Perform all operations at least 10 cm (4 inches)
from the front grille on the work surface.
• Place all materials and aerosol-generating
equipment away from the front grille.
4
Example of a written checklist
•
•
•
•
•
•
•
Centrifuge tubes and
Pipettes (n)
culture tubes should be
Centrifuge tubes (n)
clearly labelled in order
Media (n)
to avoid mismatching of
patients.
Loops (n)
Distilled water (ml)
Decontaminating solution (ml)
Slides (n)
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Work in a BSC (2)
• Pipette-discard containers should contain an
appropriate disinfectant.
• Do not keep several tubes or bottles open at
the same time.
• Do not keep a sterile reagent open while
processing specimens.
• All infectious material must be discarded in
an autoclave bag. The bag is transported to
the autoclave at the end of work.
6
Work in a BSC –
practices to be avoided
• Do not tape autoclavable disposal bags to
the outside of the cabinet .
• Do not keep the flame of the Bunsen burner
continuously alight during operations in the
BSC.
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Work in a BSC (3)
Acceptable
Unacceptable
Avoid overcrowding the work surface of the BSC.
8
Homogenization and
decontamination
9
Homogenization and
decontamination – why?
• To free the bacilli from the mucus, cells or tissue
in which they may be embedded.
• Most clinical specimens, especially sputum, are
contaminated,
• Organisms of the normal flora would rapidly
multiply in the culture medium before the
tubercle bacilli started to grow.
• To concentrate TB bacilli from specimens.
10
Remember…
• Process only one specimen at a time.
• Do not keep several containers or centrifuge
tubes open at the same time in the BSC.
• Use aliquots of decontamination solutions (1 vial
of decontaminant per specimen).
• Use a fresh pipette at every step to avoid transfer
of bacilli from one specimen to another.
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Specimens for TB culture
Contaminated clinical
specimens:
– sputum
– urine
– other specimens from
sites contaminated by
normal flora
Specimens from sterile
sites:
– bone marrow
– body fluids
– biopsies, tissues and
surgical specimens
– cerebrospinal fluid.
DECONTAMINATION
12
Caution
Digesting/decontaminating agents are toxic to tubercle
bacilli to some extent. Therefore:
•Follow digestion/decontamination procedure precisely.
•Acceptable contamination rate (solid) = 3–5%
• Acceptable contamination rate (liquid) = 5–10%
Contamination rates lower than those listed above may
indicate too harsh a decontamination procedure ‒ with
the possible consequence of false-negative cultures.
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Digestion and decontamination
procedures – good practice
Sputum specimens
1. To minimize the risk of cross-contamination, do not open more than
one tube at a time.
2. Carefully label all the tubes before starting to process the samples.
3. Work in batches corresponding to one centrifuge load (e.g. 8
specimens at a time).
4. Always digest/decontaminate the whole specimen.
5. Pour out the specimen gently from the container into the centrifuge
tube, to avoid spilling.
6. Never transfer the sample before labelling the tube.
If possible, digest/decontaminate the sample in the same
container as was used for collection.
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Other specimens – gastric lavage
Specimens obtained by gastric lavage should be
processed within 4 hours of collection.
MIND that collection has to be performed in a tube containing 100 mg
of sodium bicarbonate
Procedure:
•Centrifuge the total volume at 3000g for 15 minutes.
•Discard the supernatant and resuspend the sediment in
5 ml sterile distilled water.
•Add an equal volume of NALC‒NaOH and proceed as
for sputum.
•Inoculate the sediment immediately onto culture
medium.
15
Other specimens – urine
• Do not pool the specimens.
• Centrifuge the total volume at 3000g for 15
minutes.
• Discard the supernatant fluid .
• Proceed as for gastric lavage.
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Other specimens – tissue
• Lymph nodes, biopsies, clots and other
surgically resected tissue should be cut into
small pieces with a sterile scalpel or scissors.
• Homogenize the specimen in a sterile
porcelain mortar or preferably in a small nonreusable tissue grinder, using 2–5 ml sterile
saline.
• Inoculate the suspension onto culture media.
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Other specimens
Mucopurulent materials
• Handle as for sputum when volume is 10 ml or less.
• Handle as for mucoid gastric lavage when volume is more
than 10 ml.
Fluid materials
• If collected aseptically, centrifuge and inoculate sediment
directly onto culture medium (preferably liquid medium).
• If not aseptically collected:
– handle as for sputum when volume is 10 ml or less;
– handle as for gastric lavage fluid when volume exceeds
10 ml.
• To maximize the recovery rate, the entire volume of CSF
or other aseptically collected fluid should be cultured
(preferably in liquid medium).
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Other specimens
Sterile, no decontamination:
–
–
–
–
spinal, synovial or other internal body fluids;
bone marrow;
pus from cold abscesses;
surgically resected specimens (excluding
autopsy material);
– material obtained from pleura, liver and lymph
nodes as well as biopsies (if not connected to
potentially contaminated regions).
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Processing chart
Kudoh
Petroff
Sputum
If preservative
added
NALC
Sterile
Other specimens
Non sterile
Solid, Ogawa
Solid, LJ
Liquid
Centrifuge and inoculate
NO decontamination
Centrifuge if necessary,
then Petroff or NALC
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Main processing methods for
sputum
•
•
•
Sodium hydroxide (modified Petroff)
method.
Simple culture method (modified Kudoh)
N-Acetyl-L-cysteine-sodium hydroxide
(NALC‒NaOH) method.
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Sodium hydroxide (modified Petroff)
method – advantages
•
•
•
•
•
Simple.
Inexpensive reagents, easy to obtain.
Effective control of contaminants.
One hour.
Sterilized NaOH solution can be kept for
several weeks.
22
Sodium hydroxide (modified
Petroff) method – disadvantages
• Specimen exposure times must be strictly
followed.
• The procedure kills up to 60% of tubercle
bacilli in clinical specimens.
• Not suitable for liquid media.
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Sodium hydroxide (modified Petroff)
method – procedure
1.
To x ml of sputum, add x ml of 4% NaOH.
2.
Tighten cap of container and shake to digest.
3.
Let stand for 15 min at room temperature
(20–25 ºC).
4.
Fill the tube to within 2 cm of the top with
phosphate buffer.
5.
Centrifuge at 3000g for 15 minutes.
6.
Carefully pour off the supernatant into a
discard can containing an appropriate
disinfectant and resuspend the pellet
7.
Using a pipette (not a loop), inoculate deposit
onto two slopes of LJ medium (and one of LJ
with pyruvate – optional); inoculate each
slope with 0.2 ml
8.
Prepare the slides for smear microscopy and
store the sediment at 4 ºC or −20 ºC. 24
ADD X ml 4% NaOH
NALC‒NaOH method – advantages
1. Kills only about 30% of tubercle bacilli, allowing
more positive cultures than other methods.
2. Time required to perform the procedure: singlespecimen processing takes approximately 40
minutes; processing 20 specimens would take
approximately 60 minutes.
3. Suitable for liquid media.
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NALC‒NaOH method –
disadvantages
1. Acetyl-cysteine loses activity: it needs to be
made fresh daily.
2. Ready-to-use NALC-NAOH solutions are
commercially available, but at higher cost
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NALC‒NaOH method – procedure
ADD
XmlXNALC-NaOH
ml NALC-NaOH
1. To x ml of sputum, add x ml of NALCNaOH.
2. Tighten cap of container and shake to
digest.
3. Let stand for 15 minutes at room
temperature, with occasional shaking.
4. Add sterile distilled water or phosphate
buffer to make up to approx. 50 ml.
5. Centrifuge at 3000g for 15 minutes.
6. Decant the supernatant and resuspend
the sediment.
7. Inoculate 0.2 ml onto culture medium
(liquid or solid).
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Trisodium phosphate method – procedure
ADDtrisodium
X ml Trisodium
Xml
phosphate
phosphate
solution
1. To x ml of sample, add x ml of
trisodium phosphate solution.
2. Tighten cap of container and shake to
digest.
3. Let stand for 12–18 hours at room
temperature.
4. Fill the tube to within 2 cm of the top
with sterile water (e.g. the 50-ml mark
on the tube) and vortex-mix.
5. Centrifuge at 3000g for 15 minutes.
6. Discard the supernatant.
7. Inoculate 0.2 ml onto solid culture
medium.
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Simple culture method (modified Kudoh)
To x ml of sputum add x ml of 4% NaOH
Tighten cap of container and shake to
digest for 20 seconds
Let stand for 15 minutes at room temperature
XADD
ml 4%
X mlNaOH
4% NaOH
Inoculate deposit on to two slopes of acid
Ogawa medium
Use a pipette (not a loop), inoculate each slope
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with 0.1ml
Simple culture method (modified Kudoh)
Advantages
Because the method does not require
centrifugation, it
- minimizes the manipulation of the samples,
- reduces the execution time of the culture
compared to the Petroff method
- facilitates the qualification of professionals
involved in culture of TB bacilli
Disadvantages
Reduced sensitivity compared to the centrifugation
methods
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1% CPC–2% NaCl method – procedure
Xml
1%CPC
ADD
X mlcontaining
1% CPC
specimen
1. To x ml of sputum, add x ml of 1%
CPC and 2% NaCl. Transport to a
processing laboratory.
2. Allow liquefaction (48–72 hours).
3. Fill the tube to within 2 cm of the top
with sterile distilled water (to reduce
viscosity).
4. Tighten cap and mix well.
5. Centrifuge at 3000g for 15 minutes.
6. Discard the supernatant.
7. Resuspend the sediment in the
remaining sterile distilled water.
8. Inoculate 0.2 ml onto solid culture
medium.
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Remember!
Store the remaining sediment
refrigerated or frozen in a 1.5–2-ml
sterile screw-cap vial, properly labelled.
It may be used later on as a back-up
specimen, in case of contamination or
culture failure for any reason.
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Inoculation and incubation
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Inoculation – solid media
• Use sterile Pasteur pipettes and
carefully inoculate a volume of 0.1–0.2
ml of sediment.
• Two tubes should be inoculated from
each sample.
• LJ with pyruvate and no glycerol could
be added in settings in which M. bovis
is frequently isolated.
• Prepare the slides for smear
microscopy and store the sediment at
4 ºC or −20 ºC.
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Inoculation – liquid media
• Remember : liquid media need to be
supplemented before inoculation.
• Inoculate 0.5–1 ml of sediment into the tube
using a disposable pipette.
• Tighten the caps.
• Mix by inversion.
Use aseptic techniques!
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Inoculation of liquid culture media –
key points
• Minimize the production of aerosols:
– open the caps of liquid media slowly;
– avoid vigorous shaking of the specimen;
– do not expel the last drop from the pipette.
• Use aseptic techniques and sterile material to avoid
contamination.
• Inoculate liquid media first to reduce the chances of carryover of any contaminants.
• Prepare smears for staining after all media have been
inoculated.
• Inoculate the media with clinical specimens as soon as
possible.
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Inoculation work-flow
1. Liquid media
2. Solid media
3. Smear
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Incubation
•
•
•
•
•
Place inoculated cultures in the incubator as soon as possible.
Incubator temperature should be 35–37 ºC.
Monitor the incubator temperature regularly.
Solid media:
– Keep the caps loose for the first 48 hours of incubation to let
the inoculum to dry
– tighten culture caps of solid media after 48 hours of culture to
avoid dehydration of the medium;
– incubate solid cultures in slanted position for at least 1 week;
– incubate for 8 weeks before reporting a culture as negative.
Liquid media:
– incubate for up to 6 weeks before reporting a culture as
negative.
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True and false exercise
1. M. tuberculosis is never affected by the
decontamination protocol.
2. Performing decontamination in the collection
container may reduce the risk of errors in the
laboratory.
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Module review: take-home messages
 Process clinical specimens as soon as possible.
 Correct labelling of tubes avoids the “patient/culture
mismatch”.
 Different decontamination protocols apply to different
samples.
 Too small an inoculum can lead to a false-negative
result.
 Aseptic technique is important to avoid contamination.
 Prepare smears for staining from the processed
sediments after all media have been inoculated.
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Self-assessment
• What are the advantages and disadvantages
of the Petroff/NALC digestion and
decontamination procedures?
• What safety and procedural precautions
should be taken while processing specimens?
• Describe the most important steps of the
decontamination process.
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