Module 6
Processing the specimens and
inoculation on solid and liquid media
Purpose
To provide you with the knowledge and skills for proper
processing of specimens for Mycobacterium tuberculosis culture
and for the inoculation of cultures on solid and liquid media.
Prerequisite
modules
Modules 2, 3 and 5
Module time
2 hours 15 minutes plus 2 hours’ practice in the laboratory.
Learning
objectives
At the end of this module, you will be able to:
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work properly within a BSC;
process specimens from sterile and non sterile sites for TB
culture;
homogenize and decontaminate respiratory specimens by:
Petroff modified method
NALC‒NaOH method,
Simple culture method (modified Kudoh method);
inoculate cultures (solid and/or liquid media);
incubate inoculated media under proper conditions.
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Culture tube labelling procedure
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Processing specimens for culture within a BSC
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Homogenization and decontamination procedures:
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Content outline
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sodium hydroxide (modified Petroff)
N-acetyl-L-cysteine-sodium hydroxide (NALC)
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Alternative protocols for processing different specimens
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Culture tube inoculation
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Culture tube incubation
Exercises
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Practical activity in the laboratory: Processing the
specimens.
Annexes
6.1 Protocol for processing specimens for culture
6.2 Preparation of reagents for specimen processing for culture
IMPORTANT POINTS ABOUT SPECIMEN PROCESSING PROCEDURES
 Process clinical specimens as soon as possible after collection.
 Properly label the media to be inoculated to avoid any mix-up of specimens.
 Minimize aerosol production by opening caps of specimen containers slowly, avoiding
vigorous shaking of specimens, and never expelling the last drop from the pipette.
 Process only one specimen at a time; do not keep several containers or centrifuge tubes
open at one time in the BSC; use aliquots of decontamination solutions (1 vial of
decontaminant per 1 specimen); change pipette at every step to avoid transfer of bacilli from
one specimen to another.
 Aseptic technique is important to avoid contamination.
 Prepare smears for staining after all media have been inoculated.
CULTURE TUBE LABELLING PROCEDURE
Labelling tubes is a simple but important step in the process of performing TB culture.
Inappropriately labelled culture tubes are responsible for a high number of laboratory errors leading
to “culture/patients exchange”: a person without active TB disease may receive a false-positive
result and therefore be treated for TB, while diagnosis of a “real” TB case may be delayed. This
kind of error may have dramatic repercussions for patients’ health status and social life and for the
health economy.
Every tube that lacks a clearly readable/interpretable number or name should be discarded.
Each tube or bottle should be labelled, before inoculation, either with a permanent marker directly
on the side wall or with a sticker (or piece of tape) applied to the tube. The following data should be
recorded:
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TB laboratory register number (or internal laboratory number);
inoculation date.
To permit easy reading of cultures on solid media, labelling should not obscure the seeding surface
of the slant.
WORKING WITHIN A BSC
Good specimen handling is essential both for safe working and for obtaining good results.
In particular, it is important to carry out all the manipulations within the biological safety cabinet
(BSC). However, while the BSC can protect the operator from airborne infection, it offers no
protection against the consequences of spillage or poor techniques.
To minimize disruption of the air barrier by movement in and out of the BSC, it is important that all
necessary supplied are to hand. Prepare a written check list of materials needed for TB culture,
placing the materials required for immediate work in the BSC and storing extra supplies outside the
cabinet.
The work surface, interior walls and interior window surface of the BSC should be wiped with an
appropriate disinfectant and then with sterile water. Ensure that the front grille is not blocked with
laboratory notes, discarded plastic wrappers, pipetting devices, etc. and carry out all operations at
least 10 cm from the front grille on the work surface.
After placing hands/arms inside the cabinet, wait 60 seconds before manipulating materials; this
allows the BSC to stabilize and to remove surface microbial contaminants.
Place absorbent paper towelling on the work surface. To avoid laboratory cross-contamination,
arrange materials to allow work to flow from a clean to a contaminated area across the work
surface. Position materials and supplies so as to limit the movement of dirty items over clean
ones.
Use only horizontal pipette discard trays containing an appropriate disinfectant and use proper
microbiological techniques to avoid splatter and aerosols. Do not hold opened tubes or bottles and
recap or cover them as soon as possible. This will reduce the chance for cross-contamination.
Do not use large open flames in the BSC: these create turbulence which disrupts the pattern of air
supplied to the work surface. Special Bunsen burners for use in BSCs are recommended.
Use an appropriate liquid disinfectant in a discard pan to decontaminate materials before removal
from the BSC. Introduce items into the pan carefully to minimize splatter and allow sufficient
contact time before removal.
Surface-decontaminate all containers and equipment before removal from the BSC. At the end
of the work day (and always before HEPA filters are changed or internal repair work is done)
decontaminate the work surface and the side and back of the BSC, and the interior of the glass
window.
Never:
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tape autoclavable disposal bags to the outside of the cabinet;
place pipette collection containers upright in the BSC or on the floor outside the cabinet.
HOMOGENIZATION AND DECONTAMINATION
Most ‒ but not all ‒ specimens are contaminated (see below) and must therefore be subjected to
rigorous decontamination procedures that liquefy the organic debris and eliminate the unwanted
normal flora. Normal flora would rapidly overgrow the entire surface of the medium and consume it
before the TB bacilli started to grow. Specimens must be homogenized to free the bacilli from the
mucus, cells or tissue in which they may be embedded.
Contaminated specimens
Sterile specimens
Sputum
Pus from cold abscess
Gastric lavage
Bone marrow
Spinal, synovial or other cavitary body
fluids
Biopsy, lymph node, tissue and surgical
resected specimen
Swabs or laryngeal aspirates
Urine
Bronchial lavage, aspirate, brushing
Skin
Autoptic materials
Uterine mucosa
Digesting/decontaminating agents are to some extent toxic to tubercle bacilli; to minimize the
number of dead mycobacteria, the digestion/decontamination procedure must therefore be
followed precisely. A proportion of cultures will be contaminated by other organisms: a
contamination rate 3-5% is acceptable on solid media. Cultures in liquid media may show higher
contamination rates (5-10%). However, if specimens (especially sputum) take several days to
reach the laboratory, the contamination rate may be higher. A contamination rate that approaches
0 indicates that the decontamination procedure was too harsh.
DIGESTION AND DECONTAMINATION PROCEDURES
Three methods are widely used for the digestion and decontamination of contaminated specimens
the sodium hydroxide (modified Petroff) method, the simple culture method (modified Kudoh
method) ,and the N-acetyl-L-cysteine-sodium hydroxide (NALC-NaOH) method. Each has
advantages and disadvantages.
Sodium hydroxide (modified Petroff) method
The sodium hydroxide method is simple to perform, provides good control of contaminants and
uses reagents that are inexpensive. The NaOH solution will last for several weeks after
sterilization. Processing a single specimen takes 1 hour. The exposure time should be strictly
followed: the procedure kills up to 60% of TB bacilli in the clinical specimen, so over-exposure is
likely to result in false-negative results.
The sodium hydroxide method is not suitable for liquid media; if acid Ogawa medium is used, the
neutralization step is unnecessary.
Procedure
For preparation of reagents see Annexes 6.1 and 6.2.
To minimize the chances of error and the risk of (cross-)contamination and to ensure that there is
sufficient time to complete the procedure properly, no more than 6-8 specimens should be
processed at a time. The capacity of the centrifuge (probably 8 tubes) sets a convenient limit.
1. If sputum has not already been collected in centrifuge tubes, select sterile plastic centrifuge
tubes (one for each specimen).
2. With a permanent marker, write the number of the specimen on the wall of the tube (not on the
cap).
3. Write the number of each specimen and the inoculation date on two tubes of media.
4. Pour sputum into the centrifuge tube
5. To x ml of sputum, add x ml of 4% NaOH solution.
6. Tighten the cap of the tube and shake to digest.
7. Allow the tube to stand for 15 minutes at room temperature.
8. Fill the tube to within 2 cm of the top with phosphate buffer.
9. Centrifuge at 3000g for 15 minutes.
10. Decant the supernatant.
11. Inoculate onto culture medium
N-Acetyl-L-cysteine‒sodium hydroxide (NALC-NaOH) method
The NALC method provides more positive cultures than other methods as it kills only about 30% of
the tubercle bacilli in clinical specimens. Time needed for processing a single specimen is
approximately 40 minutes, while 20 specimens would take approximately 60 minutes.
This method is suitable for culture on liquid media.
The disadvantages of the method are that NALC loses activity and must therefore be made fresh
every day.
Commercially prepare solutions are available, but expensive.
After centrifugation, the sediment must be resuspended in a 1:10 dilution with buffer (or water)
to reduce the concentration of any toxic components that may inhibit the growth of TB bacilli.
Procedure
For preparation of reagents see Annexes 6.1 and 6.2.
1. If sputum has not already been collected in centrifuge tubes, select sterile plastic centrifuge
tubes (one for each specimen).
2. With a permanent marker, write the number of the specimen on the wall of the tube (not on the
cap).
3. Write the number of each specimen and the inoculation date on two tubes of media.
4. Pour sputum into the centrifuge tube
5. To x ml of sputum, add x ml of NALC/NaOH
6. Tighten cap of container and vortex-mix for not more than 20 seconds.
7. Invert the tube so that the NALC/NaOH solution comes into contact with the entire inner
surface of the tube. Avoid excessive agitation.
8. Allow the tube to stand for 15 minutes at room temperature with occasional shaking.
9. Fill the tube to within 2 cm of the top with 0.067 mol/litre phosphate buffer (pH 6.8).
10. Mix the contents by inverting the tube.
11. Centrifuge at 3000g for 15 minutes.
12. Decant supernatant and resuspend the sediment.
13. Inoculate onto culture medium.
ALTERNATIVE PROTOCOLS FOR PROCESSING DIFFERENT SPECIMENS
Decontamination using the 5% oxalic method.
The 5% oxalic acid method is suitable for sputum or respiratory samples from patients who are
likely to be colonized with Pseudomonas aeruginosa, such as those with cystic fibrosis and
bronchitis. For reagents and detailed procedure, see Annexes 6.1 and 6.2.
Decontamination using the trisodium phosphate method
Interestingly, the timing of this process is not critical for viability of tubercle bacilli. For reagents and
detailed procedure, see Annexes 6.1 and 6.2.
Decontamination using CPC/NaCl method
The CPC/NaCl method -a soft decontamination method -is a means of digesting and
decontaminating specimens in transit (>24 hours). Further decontamination with the Petroff
method must not be performed.
Mycobacteria remain viable for 7 days after sputum collection in the solution. CPC (cetyl-pyridinium
cloryde) is used to decontaminate the specimen while NaCl effects liquefaction. CPC is
bacteriostatic for mycobacteria and this effect is not neutralized in the digestion process. Two
centrifugation steps are needed for subsequent removal of CPC and sediments should thus be
inoculated onto egg-based media only, not into liquid media. CPC may interfere with the
fluorescent staining of the smear. For reagents and detailed procedure, see Annexes 6.1 and 6.2.
Precautions during processing of CPC containing samples:
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Sufficient time should be allowed for complete liquefaction of sputum with CPC. Process only
those that have been homogenized by CPC. If homogenization is delayed beyond 5 days, as
might happen when the ambient temperature is < 25 °C, incubate the samples at 37 °C until
the homogenization is complete before processing.
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Specimen should be processed according to collection date (older samples first).
Simple culture method (modified Kudoh method)
The simple culture method (modified Kudoh method) is not technically demanding (no
centrifugation, no neutralization) but less sensitive than processing methods that use
centrifugation.
1. Add equal or 2 volumes of 4% NaOH to the sputum specimen in a screw-capped container.
2. Vortex-mix for not more than 20 seconds.
3. Keep at room temperature for 15 minutes for decontamination.
4. Inoculate the decontaminated specimen on to two slopes of modified Ogawa medium with 0.1
ml or two drops of solution
Sputum
Sputum specimens should not be pooled because of the risk of cross-contamination. Always
digest/decontaminate the whole specimen , do not attempt to select portions of the specimen as is
done for direct microscopy. Gently decant from the specimen container into the centrifuge tube. If
the specimen is too viscous to pour, an equal volume of digestant/decontaminant could be added
to the sputum in the specimen container and the mixture poured carefully into the appropriate
centrifuge tube.
Gastric lavage
As acidity can kill mycobacteria in the specimen, gastric lavage specimens must be processed
within 4 hours. The gastric aspirate should be collected in a tube containing 100 mg of sodium
bicarbonate for neutralization and be transported immediately to the laboratory. Proceed as for
sputum.
Note: If the specimen is watery, centrifuge at 3000g for 20 minutes, pour off the supernatant,
resuspend the sediment in 5 ml of sterile distilled water and proceed as for sputum.
Urine
Urine is one of the most common contaminated materials. Handle as for gastric lavage.
Laryngeal swabs
Swabs yield little material, so as much of the material as possible must be collected and not
wasted.
1. Swabs must be cultured on the day that they are received, using sterile precautions.
2. Use sterile forceps to transfer the swab to a sterile centrifuge tube.
3. Add 2 ml of sterile distilled water.
4. Decontaminate according to the Petroff or NaOH‒NALC method.
5. Using sterile forceps, remove the swab from the tube.
6. Fill the tube to within 2 cm of the top with 0.067 mol/litre phosphate buffer (pH 6.8). Mix the
contents by inverting the tube.
7. Centrifuge at 3000g for 15 minutes.
8. Carefully pour off the supernatant into a waste can containing 5% phenol or other disinfectant.
9. Inoculate deposit on two slopes of LJ medium (and one slope of LJ with pyruvate if needed).
Tissue
Mortars, pestles and tissue grinders must be cleaned and sterilised thoroughly to prevent falsepositive results or contamination due to organisms left over from previous specimens
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Lymph nodes, biopsies and other surgically resected tissue should be cut into small pieces with
a sterile scalpel or scissors.
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Homogenize the specimen in a sterile porcelain mortar or tissue grinder, using 5 ml sterile
saline and a small quantity of sterilized sand.
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Inoculate the suspension onto culture media.
Processing of other body fluids
Mucopurulent materials
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Handle as for sputum when volume is 10 ml or less.
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Handle as for mucoid gastric lavage when volume exceeds 10 ml.
Fluid materials
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If fluids have been collected aseptically, centrifuge and inoculate sediment directly onto culture
media, preferably in liquid medium.
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If not aseptically collected:
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handle as for sputum when volume is 10 ml or less;
handle as for fluid gastric lavage when volume exceeds 10 ml.
Warning: To maximize the recovery rate, the entire CSF volume (or other small volume of
aseptically collected fluid) should be cultured, preferably in liquid medium.
Materials that should not be decontaminated are:
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spinal, synovial or other cavitary body fluids;
bone marrow;
pus from cold abscesses;
surgically resected specimens (excluding autopsy material);
material obtained from pleural, liver and lymph nodes as well as biopsies (if not fistulized).
The processing chart shown in Figure 1 may help in the decision process when dealing with
different specimens for culture purpose.
Figure 1. 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
20
.
REMEMBER!
Two options: Sediments should be kept for one week in the refrigerator and redocontaminated if the inoculated cultures show signs of contamination or frozen(20°C) in a screw-cap sterile 1,5-2 ml vial properly labelled.
It may be useful for later use.
KEY MESSAGES
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Contaminated materials must be decontaminated
Non-contaminated materials must be concentrated, but NOT decontaminated
CULTURE TUBE INOCULATION
Solid media
In media purchased ready-to-use, condensed moisture is frequently observed in the culture slants.
Up to 0.5‒0.7 ml of liquid MAY be present in the angle between the slant and the glass wall. This
condensed moisture should be removed with a sterile Pasteur pipette before inoculation of the tube
is attempted. The presence of moisture is less frequently observed with home-made media.
Each slant should be inoculated with 0.2-0.4ml (2-4 drops or 4 loopfuls) of the centrifuged
sediment. Pipettes or loops(wire or disposable) can be used for primary culture seeding, although
plastic Pasteur pipettes are highly recommended. The inoculum should be distributed over the
entire surface of the slant.
Use one pipette or one disposable loop for each specimen (or sterilize the wire loop after each
specimen) to avoid cross-contamination.
At least two slopes of LJ medium per specimen should be inoculated with0.2 ml each of the
sediment. In areas where M. bovis may be a problem, an additional slope containing pyruvate
should be added.
A common fault in inoculation is the use of a too small an inoculum, which can lead to a falsenegative result.
In the upper part of the slant the medium is thin and dehydrates readily; if mycobacteria are
seeded only on this upper section, they might not grow again leading to a false-negative result.
Liquid media
Inoculation on liquid media should be performed under rigorous sterile conditions to avoid the risk
of contamination. Liquid media are more susceptible to contamination than solid media and
therefore need to be supplemented with a mixture of specific antibiotics to kill the contaminants.
Each properly labelled liquid culture tube should be inoculated with 1 ml of sediment; the sediment
must be deposited under the surface of the medium keeping the tube tilted at an angle of45°.
The tube is then returned to a vertical position, leaving the inoculum below the surface of the liquid.
Inoculation of liquid culture media ‒ important points
1. Minimize aerosol production by opening the caps of liquid media slowly, avoiding vigorous
shaking of the specimen and avoiding the expulsion of the last drop from the pipette.
2. The use of aseptic techniques and sterile material is important to avoid contamination.
3. While processing fluid specimen inoculate liquid media first to reduce the chances of carry-over
of any contaminants from contaminated solid media.
4. Prepare a smear from sediment after all media have been inoculated, using the same pipette.
5. Inoculate the media with sediments as soon as possible.
CULTURE TUBES INCUBATION
All cultures should be incubated at 35-37 °C: always check the temperature indicator before
incubating the cultures. The cultures should be incubated until growth is observed or discarded as
negative after 8 weeks (6 weeks if liquid media are used).
Inoculated solid media should be incubated in a slanted position for at least 1 week to ensure an
even distribution of the inoculum. Thereafter, if space is needed in the incubator, tubes could be
placed upright. Tops should be tightened to minimize evaporation with consequent drying of media.
SOURCE MATERIAL
1.
Health Protection Agency. Investigation of specimens for Mycobacteria species. London,
Evaluations and Standards Laboratory, 2006 (National Standard Method, BSOP40 Issue 5).
2.
Class II (laminar flow) biohazard cabinetry. Ann Arbor, Michigan, NSF International, 2002
(NSF/ANSI 49-2002).
3.
Biological containment cabinets (Class I and II): installation and field testing. Toronto,
Canadian Standards Association, 1995 (CSA Standard Z316.3-95).
4.
Richmond JY, McKinney RW, eds. Primary containment for biohazards: selection, installation
and use of biological safety cabinets, 2nd ed. Washington, DC, U. S. Department of Health
and Human Services, 2000.
5.
Drobniewski FA et al. Recommended standards for modern tuberculosis laboratory services
in Europe. European Respiratory Journal, 2006, 28:903.
6.
Narvaiz de Kantor I et al. Laboratory services in tuberculosis control. Part III: Culture. Geneva,
World Health Organization, 1998 (WHO/TB/98.258): pp 48‒52 and 79‒84.
7.
Kudoh S, Kudoh T. A simple technique for culturing tubercle bacilli. Bulletin of the World
Health Organization, 1974, 51:71-82.
8.
http://www.who.int/tb/dots/r_and_r_forms/en/index.html
KEY MESSAGES
 Process clinical specimens as soon as possible.
 Correct labelling of tubes avoids the “patient/culture exchange”.
 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.
Module 6: Review
Find out how much you have learned by answering these questions.
What are the advantages and disadvantages of the two digestion and
decontamination procedures?
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What are the safety and procedural precautions to be taken while processing
specimens?
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Describe the most important passages of the decontamination process.
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