|Cat. no. Z130||MucoGest™ 50, 250mg||20 tubes/box|
|Cat. no. Z131||MucoGest™ 100, 500mg||20 tubes/box|
Hardy Diagnostics MucoGest™ is composed of dithiothreitol, a mucolytic agent recommended for use in the digestion and decontamination procedures of sputum and other clinical specimens for the recovery of mycobacteria and fungi.
MucoGest™ is composed of the mucolytic agent, dithiothreitol. Dithiothreitol serves as a mucolytic agent by disrupting disulfide bonds in the mucus of sputum. Its use as a sputum digestant was described by W.W. Cleland in 1963.(5) The reagent was later evaluated by Reep and Kaplan, who found it to be successful as a mucolytic agent for sputum specimens in the recovery of acid-fast bacilli. Reep and Kaplan also determined dithiothreitol to be useful in liquifying sputa for mycological culture.(10)
Recovery of Mycobacteria:
Although MucoGest™ itself has no inhibitory effect on bacteria, it improves the recovery of acid-fast bacilli by breaking down mucous components of sputum. Specimens intended for the recovery of acid-fast bacilli are first treated with a MucoGest™-TB Base mixture. The specimen is then treated with phosphate buffer and bovine serum albumin.
TB Base Digestant (Cat. no. U22) is composed of sodium hydroxide which serves as a decontaminant by inhibiting the growth of non-acid-fast contaminants.
Phosphate Buffer (Cat. no. U10) lowers the specific gravity, as well as gently neutralizes the specimen after decontamination.
Bovine Serum Albumin (Cat. no. Z81) enhances growth of mycobacteria. It also assists in adhering the sediment material to the slide or solid media and increases the volume of material for culture.
Recovery of Fungi:
MucoGest™ can also be used as the liquifying agent for the recovery of fungi from mucoid specimens. When recovering fungi, however, sodium citrate (Cat. no. U23 or X47), rather than sodium hydroxide (contained within TB Base Digestant), is used as the diluent. Reep and Kaplan found that sodium hydroxide produced fungal toxicity effects on specimens, thereby decreasing the recovery of important pulmonary mycotic disease agents.(10) Use of sodium citrate eliminates deleterious effects on the specimen and allows for the successful recovery of mycological cultures.(10)
Once treated with the MucoGest™-sodium citrate component, specimens are treated with phosphate buffer and bovine serum albumin, respectively.
STORAGE AND SHELF LIFE
Storage: Upon receipt store MucoGest™ at 2-8ºC. Do not use if there are signs of decontamination or if the expiration date has passed. Protect from light, excessive heat, moisture, and freezing.
Specimen Collection: Specimen should be collected according to protocol established by the user laboratory. 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.
For Recovery of Mycobacteria:
1. Add 5ml of TB Base Digestant (2.94% sodium citrate/4% sodium hydroxide) to the MucoGest™ tube. Swirl to dissolve.
2. If preparing MucoGest™ 50, add contents of step 1 (above) to 45ml of TB Base Digestant.*
If preparing MucoGest™ 100, add contents of step 1 (above) to 95ml of TB Base Digestant.*
* Proceed to step 3 below.
For Recovery of Fungi:
1a. Add 5ml 2.94% sodium citrate to a MucoGest™ tube. Swirl to dissolve.
2a. If preparing MucoGest™ 50, add contents of step 1a (above) to 45ml of 2.94% sodium citrate.
Note: Cat. no. X47 fill is 50ml.
If preparing MucoGest™ 100, add contents of step 1a (above) to 95ml of 2.94% sodium citrate.
Note: Cat. no. U23 fill is 100ml.
3. Transfer sputum to a 50ml, aerosol-free, screw-capped centrifuge tube. Transfer no more than 10ml of sputum per 50ml tube. This will ensure that the volume of specimen to be processed never exceeds one-fifth the volume of the tube. When processing the entire specimen, divide the specimen into separate centrifuge tubes and combine the sediments after centrifugation (step 8 below).
4. To the specimen, add a volume of MucoGest™ solution prepared in step 2 and/or step 2a, equivalent to but not more than the amount of the specimen. Avoid touching the lip of the specimen container with reagent bottles. Tighten caps firmly.
5. Vortex specimen until liquified (five to twenty seconds).
6. Allow the vortexed specimen to sit at room temperature (15-30ºC.) for 15 minutes. Do not allow specimen to sit longer than 20 minutes before adding diluent.
7. Add at least 30ml (per 50ml centrifuge tube) of pH 6.8 buffer or water. Tighten cap and swirl to mix.
8. Centrifuge at 3000rpm (1800-2400xg) for 15 minutes.
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. Aseptically aliquot one or two milliliters of 0.2% bovine albumin or sterile distilled water to each sediment. Gently shake by hand to mix. Gentle shaking decreases chance of creating aerosols.
Note: pH adjustment is not necessary.
11. Using a sterile capillary pipette, mix sediment. Additionally, a 1:10 dilution may be cultured when culturing for AFB. 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 or fungi.
MucoGest™ is a liquifying agent and has no inhibitory effect on bacteria.
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. A selective medium with antibiotics, such as Lowenstein Jensen, Selective or Middlebrook 7H11, Selective 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.
It is recommended that TB Base 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.
This product is only a part of the overall identification scheme. It is recommended that biochemical and serological tests be performed on pure cultures for complete identification. For more information, see appropriate references.(1-3)
MATERIALS REQUIRED BUT NOT PROVIDED
Standard microbiological supplies and equipment such as vortex mixer, biological safety cabinet, centrifuge tubes, slides, media, loops, incinerator, incubators, pasteur pipettes, etc., as well as serological and biochemical reagents, are not provided.
Additional supplies that are required but are not provided:
TB Base Digestant (Cat. no. U22) for acid-fast culture digestion-decontamination;
TB Base Digestant is composed of 4% sodium hydroxide / 2.94% sodium citrate.
- 2.94% Sodium Citrate (Cat. no. U23 or X47) for fungal culture digestion.
- Phosphate Buffer, pH 6.8 (Cat. no. U10).
- Bovine Serum Albumin (Cat. no. Z81).
User Quality Control
MucoGest™ should appear as a white, crystalline powder.
MucoGest™ (Cat. no. Z130).
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.
5. Cleland, W.W. 1964. Dithiothreitol: A New Protective Reagent for SH Groups, Biochemistry; Vol. 3, No. 4.
6. Cumitech 3; Practical Quality Control Procedures for the Clinical Microbiology Laboratory, ASM, 1976.
7. Hirsch, S. Roger, et al. 1969. Sputum Liquifying Agents: A Comparative In Vitro Evaluation, J. Lab and Clin. Micro.
8. Kubica, G.P., et al. 1963. Sputum Digesting and Decontamination with N-acetyl-L-cysteine as a Sputum Digestant for the Isolation of Mycobacteria, Amer. Rev. Resp. Dis.; 89:284-286.
9. Reep, B.R. and W. Kaplan. 1972. The Effect of Newer Tubercle Bacillus Digestion and Decontamination Procedures on Fungi Causing Pulmonary Diseases, Mycopathologia and Mycologia Applicata; Vol. 46:325-334.
10. Reep, B.R. and W. Kaplan. 1972. The Use of N-acetyl-L-cysteine and Dithiothreitol to Process Sputa for Mycological and Fluorescent Antibody Examinations, H.L.S.; Vol. 9, No. 2.
11. Vestal, Annie L. 1975. Procedures for the Isolation and Identification of Mycobacteria, CDC, Atlanta.