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 3

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 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. 5

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. 7

Work in a BSC (3) AcceptableUnacceptable 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. 11

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. 13

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. Digestion and decontamination procedures – good practice If possible, digest/decontaminate the sample in the same container as was used for collection. 14

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. 16

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 2–5 ml sterile saline. Inoculate the suspension onto culture media. 17

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). 18

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). 19

Sputum Petroff NALC Solid, Ogawa Liquid Other specimens Centrifuge and inoculate NO decontamination Sterile Non sterileCentrifuge if necessary, then Petroff or NALC Processing chart If preservative added Kudoh Solid, LJ 20

Main processing methods for sputum Sodium hydroxide (modified Petroff) method. Simple culture method (modified Kudoh) N-Acetyl-L-cysteine-sodium hydroxide (NALC ‒ NaOH) method. 21

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

Specimen exposure times must be strictly followed. The procedure kills up to 60% of tubercle bacilli in clinical specimens. Not suitable for liquid media. Sodium hydroxide (modified Petroff) method – disadvantages 23

Sodium hydroxide (modified Petroff) method – procedure ADD X ml 4% NaOH 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

1.Kills only about 30% of tubercle bacilli, allowing more positive cultures than other methods. 2.Time required to perform the procedure: single- specimen processing takes approximately 40 minutes; processing 20 specimens would take approximately 60 minutes. 3.Suitable for liquid media. NALC ‒ NaOH method – advantages 25

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 NALC ‒ NaOH method – disadvantages 26

NALC ‒ NaOH method – procedure Xml NALC-NaOHADD X ml NALC-NaOH 1.To x ml of sputum, add x ml of NALC- NaOH. 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). 27

Trisodium phosphate method – procedure Xml trisodium phosphate solution ADD X ml Trisodium phosphate 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. 28

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 X ml 4% NaOH Let stand for 15 minutes at room temperature Inoculate deposit on to two slopes of acid Ogawa medium Use a pipette (not a loop), inoculate each slope with 0.1ml ADD X ml 4% NaOH 29

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 30

1% CPC–2% NaCl method – procedure Xml 1%CPC containing specimen ADD X ml 1% CPC 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. 31

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. 32

Inoculation and incubation 33

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. 34

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! 35

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. 36

Inoculation work-flow 1. Liquid media 2. Solid media 3. Smear 37

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. 38

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. 39

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. 40

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. 41