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Module 6 Processing the specimens and inoculation on solid and liquid media

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Title: Module 6 Processing the specimens and inoculation on solid and liquid media


1
Module 6 Processing the specimens and
inoculation on solid and liquid media
2
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.

3
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

4
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.

5
Example of a written checklist
  • Pipettes (n)
  • Centrifuge tubes (n)
  • Media (n)
  • Loops (n)
  • Distilled water (ml)
  • Decontaminating solution (ml)
  • Slides (n)

Centrifuge tubes and culture tubes should be
clearly labelled in order to avoid mismatching of
patients.
6
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.

7
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.

8
Work in a BSC (3)
Acceptable Unacceptable
Avoid overcrowding the work surface of the BSC.
9
Homogenization and decontamination
10
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.

11
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.

12
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
13
Caution
  • Digesting/decontaminating agents are toxic to
    tubercle bacilli to some extent. Therefore
  • Follow digestion/decontamination procedure
    precisely.
  • Acceptable contamination rate (solid) 35
  • Acceptable contamination rate (liquid) 510
  • Contamination rates lower than those listed above
    may indicate too harsh a decontamination
    procedure ? with the possible consequence of
    false-negative cultures.

14
Digestion and decontamination procedures good
practice
  • Sputum specimens
  • To minimize the risk of cross-contamination, do
    not open more than one tube at a time.
  • Carefully label all the tubes before starting to
    process the samples.
  • Work in batches corresponding to one centrifuge
    load (e.g. 8 specimens at a time).
  • Always digest/decontaminate the whole specimen.
  • Pour out the specimen gently from the container
    into the centrifuge tube, to avoid spilling.
  • Never transfer the sample before labelling the
    tube.

If possible, digest/decontaminate the sample in
the same container as was used for collection.
15
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.

16
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.

17
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 non-reusable
    tissue grinder, using 25 ml sterile saline.
  • Inoculate the suspension onto culture media.

18
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).

19
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).

20
Processing chart
Kudoh
Solid, Ogawa
Petroff
Solid, LJ
Sputum
NALC
Liquid
If preservative added
Centrifuge and inoculate NO decontamination
Sterile
Other specimens
Non sterile
Centrifuge if necessary, then Petroff or NALC
21
Main processing methods for sputum
  • Sodium hydroxide (modified Petroff) method.
  • Simple culture method (modified Kudoh)
  • N-Acetyl-L-cysteine-sodium hydroxide (NALC?NaOH)
    method.

22
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.

23
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.

24
Sodium hydroxide (modified Petroff) method
procedure
  • To x ml of sputum, add x ml of 4 NaOH.
  • Tighten cap of container and shake to digest.
  • Let stand for 15 min at room temperature (2025
    ºC).
  • Fill the tube to within 2 cm of the top with
    phosphate buffer.
  • Centrifuge at 3000g for 15 minutes.
  • Carefully pour off the supernatant into a discard
    can containing an appropriate disinfectant and
    resuspend the pellet
  • 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
  • Prepare the slides for smear microscopy and store
    the sediment at 4 ºC or -20 ºC.

ADD X ml 4 NaOH
25
NALC?NaOH method advantages
  • Kills only about 30  of tubercle bacilli,
    allowing more positive cultures than other
    methods.
  • Time required to perform the procedure
    single-specimen processing takes approximately 40
    minutes processing 20 specimens would take
    approximately 60 minutes.
  • Suitable for liquid media.

26
NALC?NaOH method disadvantages
  • Acetyl-cysteine loses activity it needs to be
    made fresh daily.
  • Ready-to-use NALC-NAOH solutions are commercially
    available, but at higher cost

27
NALC?NaOH method procedure
  • To x ml of sputum, add x ml of NALC-NaOH.
  • Tighten cap of container and shake to digest.
  • Let stand for 15 minutes at room temperature,
    with occasional shaking.
  • Add sterile distilled water or phosphate buffer
    to make up to approx. 50 ml.
  • Centrifuge at 3000g for 15 minutes.
  • Decant the supernatant and resuspend the
    sediment.
  • Inoculate 0.2 ml onto culture medium (liquid or
    solid).

Xml NALC-NaOH
ADD X ml NALC-NaOH
28
Trisodium phosphate method procedure
  • To x ml of sample, add x ml of trisodium
    phosphate solution.
  • Tighten cap of container and shake to digest.
  • Let stand for 1218 hours at room temperature.
  • 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.
  • Centrifuge at 3000g for 15 minutes.
  • Discard the supernatant.
  • Inoculate 0.2 ml onto solid culture medium.

ADD X ml Trisodium phosphate
Xml trisodium phosphate solution
29
Simple culture method (modified Kudoh)
X ml 4 NaOH
ADD X ml 4 NaOH
30
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 R
educed sensitivity compared to the centrifugation
methods
31
1 CPC2 NaCl method procedure
  • To x ml of sputum, add x ml of 1 CPC and 2
    NaCl. Transport to a processing laboratory.
  • Allow liquefaction (4872 hours).
  • Fill the tube to within 2 cm of the top with
    sterile distilled water (to reduce viscosity).
  • Tighten cap and mix well.
  • Centrifuge at 3000g for 15 minutes.
  • Discard the supernatant.
  • Resuspend the sediment in the remaining sterile
    distilled water.
  • Inoculate 0.2 ml onto solid culture medium.

Xml 1CPC containing specimen
ADD X ml 1 CPC
32
Remember!
33
Inoculation and incubation
34
Inoculation solid media
  • Use sterile Pasteur pipettes and carefully
    inoculate a volume of 0.10.2 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.

35
Inoculation liquid media
  • Remember liquid media need to be supplemented
    before inoculation.
  • Inoculate 0.51 ml of sediment into the tube
    using a disposable pipette.
  • Tighten the caps.
  • Mix by inversion.

Use aseptic techniques!
36
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 carry-over of any contaminants.
  • Prepare smears for staining after all media have
    been inoculated.
  • Inoculate the media with clinical specimens as
    soon as possible.

37
Inoculation work-flow
1. Liquid media
2. Solid media
3. Smear
38
Incubation
  • Place inoculated cultures in the incubator as
    soon as possible.
  • Incubator temperature should be 3537 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.

39
True and false exercise
  • M. tuberculosis is never affected by the
    decontamination protocol.
  • Performing decontamination in the collection
    container may reduce the risk of errors in the
    laboratory.

40
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.

41
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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