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DECONTAMINATION REAGENTS FOR THE RECOVERY OF MYCOBACTERIA FROM SPUTUM

Cat. no. U20 TB Base Digestant, 500ml Polypropylene Bottle, 500ml 1 bottle /box
Cat. no. U22 TB Base Digestant, 125ml Polyethylene Bottle, 100ml 12 bottles/box
Cat. no. X45 TB Base Digestant, 2oz. Polyethylene Bottle, 50ml 25 bottles/box
Cat. no. X46 TB Base Digestant, Red, 2oz. Polyethylene Bottle, 50ml 25 bottles/box
Cat. no. Z60 N-Acetyl-L-Cysteine, 16x100mm Tube, 250mg 20 tubes/box
Cat. no. U10 Phosphate Buffer, 500ml Polycarbonate Bottle, 500ml 1 bottle/box
Cat. no. U192 Phosphate Buffer, 1L Polycarbonate Bottle, 1000ml 10 bottles/box
Cat. no. X43 Phosphate Buffer, 2oz. Polypropylene Bottle, 40ml 25 bottles/box
Cat. no. X31 Phosphate Buffer, 2oz. Polypropylene Bottle, 30ml 25 bottles/box
Cat. no. Z81 Bovine Serum Albumin, 13x100mm Tube, 3ml 20 tubes/box
Cat. no. Z161 N-Acetyl-L-Cysteine, 16x100mm Tube, 2.5gm 20 tubes/box

INTENDED USE

Sputum decontamination reagents are used to break down mucous components of sputum and other clinical specimens and to decontaminate the specimen of normal flora in order to allow slower growing mycobacteria to grow.

SUMMARY

The recovery of mycobacteria from sputum or other mucous containing specimens contaminated with other organisms is difficult, since mycobacteria generally grow much slower than other bacterial species. Decontamination and digestion of the mucous components kills contaminating normal flora and allows slower growing mycobacteria to grow. Sodium hydroxide (NaOH), in the TB Base Digestant, acts as an emulsifier and a decontaminant, breaking down mucoid material and inhibiting the growth of contaminants. N-Acetyl-L-Cysteine (NALC), when combined with NaOH, facilitates decontamination by further digesting mucopurulent specimens which allows the NaOH to penetrate. Sodium citrate, in the TB Base Digestant, aids in the liquification by binding heavy metals, thus stabilizing NALC and allowing it to work properly. Phosphate Buffer lowers the specific gravity of the specimen and gently neutralizes the specimen after decontamination. Bovine Serum Albumin is added to the sediment after centrifugation to enhance the growth of mycobacteria. Bovine Serum Albumin also assists in adhering the sediment material to the slide or solid media and increases the volume of material for culture.

REAGENT FORMULA

TB Base Digestant
NaOH (4% Solution) 50%
Sodium Citrate (2.94% Solution) 50%

Final pH 13.5 +/- 0.5 at 25ºC.

TB Base Digestant, Red (Cat. no. X46) is the same formula as above but with the addition of a pH indicator.

NALC
N-Acetyl-L-Cysteine Amount per tube dependent upon catalog number


Phosphate Buffer (CDC Formula)
Disodium Phosphate 2.37gm
Monopotassium Phosphate 2.27gm
Deionized Water 500.0ml

Final pH 6.8 +/- 0.2 at 25ºC.

Bovine Serum Albumin
Bovine Serum Albumin Fraction V 0.20gm
Sodium Chloride 0.85gm
Deionized Water 100.0ml

Final pH 7.2 +/- 0.2 at 25ºC.

STORAGE AND SHELF LIFE

Storage: TB Base Digestant, NALC and Phosphate Buffer are stored at 15-30ºC. Do not use these reagents if they are discolored, have developed a heavy precipitate, or if the expiration date has passed. Do not freeze or overheat.

Bovine Serum Albumin is stored at 2-8ºC. Reagents should not be used if there are any signs of contamination, deterioration or if the expiration has passed.

PRECAUTIONS

PROCEDURE

Specimen Collection: Specimen should be collected in a sterile 50ml screw-cap centrifuge tube or graduated, disposable sputum cup showing the 10ml volume. If a larger volume of sputum is collected, the specimen should be separated into 10ml volumes. Avoid contamination of the specimen with oral or nasal secretions. Transport specimens to the lab without delay. The specimen should be refrigerated if processing will be delayed.

Method of Use: Work within a biological safety cabinet and wear gloves.

1. Prepare digestant solution daily by dissolving 250mg of NALC Reagent (Cat. no. Z60) per 50ml of TB Base Digestant (Cat. no. X45). Prepare only what can be used in 24 hours.

2. Transfer 5-10ml of sputum specimen into a 50ml, aerosol-free, screw-capped centrifuge tube.

3. Add an equal volume of digestant solution to the sputum. Avoid touching the lip of the specimen container with reagent bottles. Tighten caps firmly.

4. Vortex the centrifuge tube until the specimen is liquified. Check for homogeneity by inverting the tube. An extra pinch of NALC crystals may be added to liquify excessively mucoid sputa.

5. Allow the centrifuge tube to sit at room temperature (15-30ºC) for 15 minutes. Do not allow specimen to sit longer than 20 minutes.

6. Fill centrifuge tube within 2cm of the top of the tube with Phosphate Buffer (Cat. no. X43). Swirl the tube to mix. Avoid touching the lip of the specimen container with the reagent bottles.

7. Centrifuge at least 15 minutes at 3600Xg.

8. Wipe the top of the tube with disinfectant.

9. Under a biosafety cabinet, carefully decant the supernatant into a splash proof container containing a cold sterilant. Wipe the lip of the container with disinfectant. Do not allow the disinfectant to enter the tube.

10. Resuspend the sediment in 1-2ml of 0.2% Bovine Serum Albumin (Cat. no. Z81). Swirl to mix. If media will be inoculated immediately, the sediment may be suspended in sterile saline or water.

11. Dilute the suspension 1:10 by adding 0.5ml of suspension from step 10 above to 4.5ml sterile distilled water.

12. The undiluted and dilute sediment suspensions are used to inoculate media for isolation and for susceptibility testing. Place two drops on the surface of each medium. Make a smear by placing one drop of the undiluted specimen with albumin on a slide and allow it to dry thoroughly before staining.

INTERPRETATION OF RESULTS

See listed references or Hardy Diagnostics Technical Information Sheets for the interpretation of growth on various media designed to isolate mycobacteria.

LIMITATIONS

Occasional specimens are so contaminated with resistant bacteria, such as Klebsiella spp. or Pseudomonas spp., that the decontamination process is not effective and the contaminating bacteria will overgrow the slower growing mycobacteria. Sediment material may be redigested using a more alkaline digestion process, or the specimen may be resubmitted and processed using an alternative digestion method. A selective medium, with antibiotics such as Lowenstein Jensen, Selective or Middlebrook 7H11, can be used to decrease the growth of contaminating organisms.

Timing is important during the digestion process. A digestion time of longer than 15 minutes should not be used. Many Mycobacterium spp. are killed by over decontamination.

No more than 10ml of mucopurulent material should be processed in a tube at one time. Sputum specimens should be representative of good sputum samples. Material should not resemble saliva. Never use a preservative or fixative with the specimen.

A pH balance is critical and achieved in the centrifugation step. Timing and speed are important in this step.

If the specimen is excessively mucoid, a few additional crystals of NALC may be added.

Do not reuse NALC. The reconstituted reagent should not be more than 24 hours old, since oxygen exposure will render it ineffective.

If the specimen contains excess blood, the iron in the hemoglobin binds the NALC making it impossible to digest. An alternate digestion method must be considered.

It is recommended that TB Base Digestant and Phosphate Buffer be used in small containers (50ml or less) in order to prevent back-splash contamination. Contamination can occur by touching the rim of the reagent bottle to the rim of the centrifuge tube or when pouring liquid into the centrifuge tubes; as liquid pours out, air and droplets rush back into the container.

MATERIALS REQUIRED BUT NOT PROVIDED

Standard microbiological supplies and equipment such as vortex mixers, biological safety cabinets, centrifuge tubes, slides, media, loops, incinerators, incubators, pasteur pipets, etc., as well as serological and biochemical reagents, are not provided.

User Quality Control

Check for signs of contamination or deterioration.

TB Base Digestant is not a growth medium. The product is tested for its ability to decontaminate.

The following procedure is routinely used for testing at Hardy Diagnostics:

QC Procedure:

1. Prepare a 1ml suspension of Pseudomonas aeruginosa and Staphylococcus aureus. Adjust the turbidity to match that of a 0.5 McFarland opacity standard.

2. Mix 1ml of TB Base Digestant to the suspension and incubate aerobically at 35ºC for ten minutes.

3. Neutralize the suspension by adding 2ml of Phosphate Buffer; vortex and streak 0.01ml onto a Blood Agar plate.

4. Incubate aerobically at 35ºC. Both P. aeruginosa and S. aureus should be partially to completely inhibited after 24 hours of incubation.

Note: The elevated pH is the inhibitory factor in TB Base Digestant, therefore, some pH tolerant organisms may breakthrough (e.g. S. aureus ATCC® 25923), especially during the QC procedure when the process sample is plated on non-selective media.

REFERENCES

1. Jorgensen., et al. Manual of Clinical Microbiology, American Society for Microbiology, Washington, D.C.

2. Tille, P., et al. Bailey and Scott's Diagnostic Microbiology, C.V. Mosby Company, St. Louis, MO.

3. Isenberg, H.D. Clinical Microbiology Procedures Handbook, Vol. I, II & III. American Society for Microbiology, Washington, D.C.

4. Kent P.T., et al. 1985. Public Health Mycobacteriology: A Guide for the Level III Laboratory, U.S. Department of Health and Human Services, Public Health Service, Centers for Disease Control, Atlanta, GA.

ATCC is a registered trademark of the American Type Culture Collection.

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