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Code: SR0233

Directions
Aseptically add the contents of one vial (7.5ml) to 92.5ml of sterile distilled water. Use the working solution immediately or store at 2-8°C for upto 48 hours only.

Description
Sputum generally consists of inflammatory exudate from the lower respiratory tract mixed with saliva.

Mulder¹ recognised the problem of interpreting the significance of growth from sputum and suggested rinsing it in saline before culture to remove the saliva. May² showed that bacteria are often unevenly distributed in the sputum of patients suffering from chronic bronchitis and that single cultures may fail to reveal all the bacterial species present.

The introduction by Rawlins³ of a method for the homogenisation of sputum before culture overcame the variations present in any method that is based on the examination of small proportions of heterogeneous material. It enables the bacteria in the sputum to be distributed evenly throughout the specimen after digestion. Dixon and Muller⁴, in an attempt to distinguish between contaminants and bronchial pathogens, suggested a semi-quantitative analysis by diluting the digested sputum down to10^-4.

Dithiothreitol, Cleland’s Reagent⁵, has been evaluated as a sputum liquefying agent⁶. It was found to be the most effective of a group of agents tested containing a sulphydryl group. A 0.1m solution of dithiothreitol was found to achieve a significantly greater decrease in sputum viscosity than 1.2M N- acetyl cysteine for use prior to sputum culture. The use of dithiothreitol instead of N-acetyl cysteine to digest sputum before decontamination has been shown⁷ to yield a higher number of acid-fast bacilli when smears are stained by the Ziehl-Neelsen method. After culture and incubation for three weeks, it was reported that, in general, the number and size of colonies isolated using dithiothreitol as a liquefying agent was greater than that using N-acetyl cysteine.

Technique
The procedure for the routine liquefaction of sputum is as follows:

1. The sputum is expectorated into a sterile Universal container or other wide mouthed screw-capped bottle
2.  Add approximately 5 times the volume of 0.85% saline and agitate to free the sputum from adherent saliva. Remove the saline with a sterile Pasteur pipette.
3. To the washed sputum, add an equal volume of Sputasol solution
4. Shake the mixture well, place in a 37°C water bath and incubate, with periodic shaking, until liquifaction is complete
5. Inoculate on to a suitable culture medium. For the total cell count, place a drop of the liquefied sputum in a haemocytometer for enumeration. For a differential cell count, fix a dried smear in methyl alcohol and stain with haematoxylin and eosin or with Lieshmann stain.

The working solution of Sputasol will remain stable for at least 48 hours stored at 2-8°C.
An investigation into the survival of respiratory pathogens in specimens that had been stored for 48 hours at 4°C following homogenisation using Sputasol, showed that the organisms remained viable and, when necessary, treated specimens could be successfully re-cultured⁸.

References
1. Mulder J. (1938) Acta. Med. Scand. 94. 98.
2. May J. R. (1952) Lancet 20.12.52. 1206-1207.
3. Rawlins G. A. (1955) J. Med. Lab. Technol. 13. 133-143.
4. Dixon J. M. S. and Miller D. C. (1965) Lancet ii, 1046-1048.
5. Cleland W. W. (1964) Biochemistry 3. 480-482.
6. Hirsch S. R., Zastrow J. E. and Kory R. C. (1969) J. Lab. & Clin. Med. 74. 346-353.
7. Shah R. R. and Dye W. E. (1965) Amer. Rev. Resp. Dis. 94. 454.
8. Could F. K., Freeman R., Hudson S. et al (1996) J. Clin. Pathol. 49. 684-686.