TB PREP KIT RED™
|Cat. no. Z158||TB Prep Kit Red™||5 tests/kit|
|Each kit contains:|
|X46 - TB Base Digestant Red, 2oz. Polyethylene Bottle, 50ml||5 bottles|
|Z160A - N-Acetyl-L-Cysteine (NALC), 2ml Cryogenic Vial, 250mg||5 vials|
Hardy Diagnostics TB Prep Kit Red™ is used for the digestion and decontamination of clinical specimens suspected to contain mycobacteria, especially Mycobacterium tuberculosis. Sputum decontamination reagents are used to break down mucous components of sputum and to decontaminate the specimen of normal flora in order to allow slower growing mycobacteria to grow. A pH indicator is used to ensure that proper pH is maintained throughout the reaction.
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 without competition from other bacteria.
Sodium hydroxide (NaOH), in the TB Base Digestant Red, acts as an emulsifier and a decontaminant, breaking down mucoid material and inhibiting the growth of contaminants. A pH indicator is used in the TB Base Digestant Red to insure that the proper pH is maintained. TB Base Digestant Red will remain pink during the decontamination steps, and turn clear after the solution has been neutralized by the addition of 1N hydrochloric acid (HCl). 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 Red, 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.
|TB Base Digestant Red (X46):|
|NaOH (4% Solution)||50%|
|Sodium Citrate (2.94% Solution)||50%|
Final pH 13.5 +/- 0.5 at 25ºC.
STORAGE AND SHELF LIFE
Storage: TB Prep Kit Red™ is 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.
Do not use TB Base Digestant Red if the solution is not red or pink. TB Base Digestant Red is especially light sensitive; protect from light sources.
Specimen Collection: Collect 10ml of specimen in a sterile 50ml screw-cap centrifuge tube (Cat. no. 227261) or graduated disposable sputum cup (Cat. no. PC40902005). 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 into the 50ml bottle of TB Base Digestant Red. The resulting solution should be pink. Prepare only what can be used in 24 hours.
2. Transfer 5-10ml of sputum specimen into a 50ml, aerosol-free, screw-capped polypropylene centrifuge tube (aerosol free and graduated, Cat. no. 227261). If more than 10ml of specimen is submitted select 10ml of the most purulent, bloody or mucoid portion.
3. Add an equal volume of digestant solution to the sputum. If the specimen is bloody, do not add more than 8ml of digestant solution. Avoid touching the lip of the specimen container with reagent bottles. Tighten caps firmly. Sample should be pink. If the solution turns clear add digestant solution until the sample is pink.
4. Vortex the centrifuge tube for no more than 30 seconds.
5. Allow the centrifuge tube to sit at room temperature (15-30ºC.) for 15 minutes, to decontaminate the specimen. Do not allow specimen to sit longer than 15 minutes.
6. Fill centrifuge tube within 2cm of the top of the tube with Phosphate Buffer (Cat. no. U10, X31 or X43). Recap the tube tightly, and invert several times until solution is mixed. Avoid touching the lip of the specimen container with the reagent bottle.
7. Centrifuge at least 15 to 20 minutes at 3000Xg.
8. Under a biosafety cabinet, carefully decant the supernatant into a splash proof container containing a cold sterilant (e.g., Amphyl). Wipe the lip of the container with disinfectant. Do not allow the disinfectant to enter the tube.
9. Using a small tipped dropper pipet (e.g., pasteur pipet), titrate with 1N HCl, dropwise, until the color of the sediment changes from red to a persistent yellow. Bloody specimens may remain pink due to residual hemoglobin and can be assumed to be at the proper pH as long as no more than 8ml of digestant solution was used.
10. Resuspend the sediment in 1-2ml of 0.2% Bovine Serum Albumin (Cat. no. Z81), to increase the adhesion of the sample to the slide. Swirl to mix. Use a loop or applicator stick to prepare a slide over a 1 x 2cm area.
11. Dilute the suspension 1:10 by adding 0.5ml of suspension from step 10 above, to 4.5ml sterile distilled water (Cat. no. K187).
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.
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 re-digested 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 (Cat. no. C25) or Middlebrook 7H11 Selective (Cat. no. C38), 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 proper pH must be maintained throughout the reaction. An alkaline pH is critical for decontamination and must be maintained before and during the centrifugation step. Timing and speed are important during centrifugation. A neutral pH after centrifugation is critical to the viability of the mycobacteria, and must be achieved by the addition of the proper amount of Phosphate Buffer and 1N HCl.
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 Red 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 loops, swabs, applicator sticks, pasteur pipets, slides, other culture media, Phosphate Buffer (Cat. no. U10, X31 or X43), Bovine Serum Albumin, 0.2% (Cat. no. Z81), sterile distilled water (Cat. no. K187), sterile saline (Cat. no. K248), 1N HCl, centrifuge tubes (Cat. no. 227261), graduated disposable sputum cups (Cat. no. PC40902005), vortex mixers, biological safety cabinets, incinerators, and incubators, etc., as well as serological and biochemical reagents, are not provided.
- Check for signs of contamination or deterioration.
- TB Base Digestant Red 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:
2. Mix 1ml of TB Base Digestant Red 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 (Cat. no. A10).
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 Red, therefore, some pH tolerant organisms may break through (e.g. S. aureus ATCC® 25923), especially during the QC procedure when the process sample is plated on non-selective media.
USER QUALITY CONTROL
- TB Base Digestant Red (X46) should appear clear, and red-pink in color.
- NALC Powder (Z160A) should appear free-flowing, and white in color.
TB Prep Kit Red™ (Cat. no. Z158).
1.Versalovic, J., 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. 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.