Sputum from patients with tuberculosis infection often contains solid parti- cles of material from the lungs and this material should be selected for culture, whenever it is found. However, as tuberculous sputum is coughed up through the throat and mouth, contamination with the normal flora of the pharynx is inevitable. The contaminating bacteria must be killed if the Löwenstein– Jensen culture media are not to become overgrown. A concentration- digestion-decontamination procedure of any specimen collected from a site where there are normal flora is therefore recommended. The following three procedures are widely used:
— Sodium hydroxide (NaOH) (Petroff);
— N-acetyl-l-cysteine–sodium hydroxide (NALC–NaOH); and — Zephiran–trisodium phosphate
Sodium hydroxide procedure (Petroff )
This procedure liquifies the sometimes mucoid sputum while destroying the contaminating organisms. However, sodium hydroxide is also toxic for mycobacteria, and care must be taken when using the method to ensure that: — the final concentration of NaOH does not exceed 2%;
— the tubercle bacilli are not exposed to sodium hydroxide for more than 30 minutes, including centrifugation time.
1. Mix equal volumes of sputum and 4% sodium hydroxide 40 g/l (previ- ously sterilized by autoclaving) in a sterile, leak-proof, 50-ml glass bottle or jar, or plastic conical centrifuge tube.
2. Incubate at room temperature (25–30 ∞C) for 15 minutes, shaking the mixture carefully every 5 minutes using a mechanical shaker. In hot cli- mates some cooling may be needed or the reaction time may be reduced to 10–15 minutes.
3. Centrifuge immediately or dilute the mixture to the 50-ml mark with dis- tilled water or phosphate buffer (pH 6.8) to stop the action of the NaOH. 4. After 15 minutes, centrifuge the specimen at 3000 g for 15 minutes. Discard the supernatant carefully into a splash-proof container filled with a suit- able disinfectant (phenol- or glutaraldehyde-based). Neutralize the sedi- ment drop by drop with a 2-mol/l HCl solution containing 2% of phenol red, combined with shaking, until the colour changes persistently from red to yellow. Alternatively add one drop of indicator solution and then add HCl drop by drop while shaking continuously.
BACTERIOLOGICAL INVESTIGATIONS
A 5. If the media is to be inoculated immediately, suspend the neutralized
deposit in 1–2 ml of sterile 0.85% NaCl or sterile distilled water. Otherwise, suspend the sediment in 1–2 ml of sterile bovine albumin fraction V. N-acetyl-L-cysteine–sodium hydroxide procedure
A lower concentration of NaOH in the presence of a mucolytic agent like N- acetyl-l-cysteine (NALC) is less aggressive against tubercle bacilli. Neither the incubation time nor the temperature are as crucial as in the NaOH procedure. However, the short shelf-life of no more than 24 hours of the NALC–NaOH working solution requires daily preparation.
1. Combine equal volumes of sodium citrate solution (29 g sodium citrate dihydrate per litre of distilled water) and 4% sodium hydroxide (40 g/l), and autoclave the mixture. The solution may be stored at room temperature.
2. Just prior to use, add 0.5 g NALC to 100 ml of NaOH–sodium citrate solution.
3. Depending on the number of specimens that must be decontaminated, prepare 2.5 g of NALC in 500 ml of NaOH–sodium citrate solution or 5 g NALC in 1000 ml of NaOH–sodium citrate solution. After 24 hours the reagent must be discarded.
4. Add an equal volume of NALC–NaOH working solution to the specimen in a sterile, leak-proof, 50-ml glass bottle or jar, or plastic conical centrifuge tube. Securely tighten the screw-cap, invert the tube and shake it gently for no longer than 30 seconds.
5. Let the tube stand for 15 minutes at room temperature (20–25 ∞C).
6. Dilute the mixture to the 50-ml mark with distilled water or with 67 mmol/l phosphate buffer (pH 6.8) to stop the action of the NaOH. Discard the supernatant carefully into a splash-proof container filled with a suitable disinfectant (phenol- or glutaraldehyde-based).
7. If the media is to be inoculated immediately, suspend the deposit in 1–2 ml of sterile 0.85% NaCl or sterile distilled water. Otherwise, suspend the sediment in 1–2 ml of sterile bovine albumin fraction V.
Zephiran–trisodium phosphate procedure
Mycobacteria can withstand prolonged treatment with this more gentle pro- cedure. Therefore, incubation time and temperature are not critical.
1. Prepare 1 kg of trisodium phosphate (Na3PO4◊12H2O) in 4 litres of hot
distilled water, add 7.5 ml of 17% benzalkonium chloride (Zephiran), mix well, and store at room temperature.
2. Mix an equal volume of sputum (up to 10 ml) with the Zephiran–trisodium phosphate solution in a 50-ml sterile, leak-proof centrifugation glass bottle. Tighten the cap and vigorously shake the mixture manually or using a mechanical shaker for 30 minutes.
3. Leave the mixture to stand for an additional 30 minutes.
4. Centrifuge at 3000 g for 15 minutes. Discard the supernatant carefully into a splash-proof container filled with a suitable disinfectant (phenol- or glutaraldehyde-based), and re-suspend the sediment in 20 ml of neutraliz- ing phosphate buffer, pH 6.61.
1 Preparation of neutralizing phosphate buffer 67 mmol/l.
Stock solutions:
A. Dissolve 9.47 g of anhydrous disodium phosphate in 1 litre of distilled water. B. Dissolve 9.07 g of anhydrous monopotassium phosphate in 1 litre of distilled water. For pH 6.8 buffers: mix 50 ml of stock solution A with 50 ml of stock solution B. For pH 6.6 buffers. mix 37.5 ml of stock solution A with 62.5 ml of stock solution B. Check the pH. Add solution A to raise the pH, or solution B to lower the pH as necessary.
5. Centrifuge once again at 3000 g for 15 minutes. Discard the supernatant and inoculate the sediment onto the media.
Culture
1. Inoculate 3 drops (about 0.1 ml) of the sediment onto at least three plates of Löwenstein–Jensen medium or equivalent.
2. Determine the contamination rate of the incubated media regularly and record the number of contaminated plates.
The rate of contamination should be 3–5%. Excessive contamination (over 5%) of the Löwenstein–Jensen cultures usually indicates that the decontamination procedure was not effective enough. Contamination rates of <3% suggest that the decontamination procedure was too vigorous and mycobacteria present in the samples may fail to grow.