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Novobiocin-Tetrathionate Broth J Clin Pathol: first published as 10.1136/jcp.12.6.568 on 1 November 1959. Downloaded from J. clin. Path. (1959), 12, 568. NOVOBIOCIN-TETRATHIONATE BROTH: A MEDIUM OF IMPROVED SELECTIVITY FOR THE ISOLATION OF SALMONELLAE FROM FAECES BY LEONARD JEFFRIES From the Department of Clinical Pathology, Uriversity College Hospital, London (RECEIVED FOR PUBLICATION APRIL 23, 1959) The selective cultivation of bacteria by the strengths of all solutions were expressed as incorporating chemicals in the media depends on concentrations of pure novobiocin. As the supplying a substance which either stimulates the novobiocin content varied with different batches of growth of the species to be isolated or inhibits the salt a simple calculation was necessary each time a new sample was used. A stock solution containing unwanted species. Tetrathionate is a good novobiocin, 16,000 ug./ml. of water, was stored in selector of Salmonellae in faeces culture, and is the refrigerator. believed to act in both ways. Tetrathionate MacConkey Agar.-Oxoid. reducers, such as Salmonellae, flourish in broth Desoxycholate-citrate Agar. - Southern a of Group containing it, whereas high concentration Laboratory (Lewisham). tetrathionate inhibits many other faecal organisms Indicator Serum Broth.-This was prepared by the copyright. (Knox, Gell, and Pollock, 1943). Unfortunately, method of Stokes however, members of the Proteus group are (1955). powerful tetrathionate reducers, and when they Investigadon are present selection of Salmonellae is much For efficient selection a concentration of novobiocin impaired, consequently the addition of a further is required which will inhibit most, if not all, strains selective inhibitor effective against Proteus of Proteus without jeopardizing the survival of small organisms is desirable. So far no such satis- numbers of Salmonellae. factory substance has been found: brilliant green to Novobiocin.-The sensitivities to Sensitivity http://jcp.bmj.com/ and ox bile (Kauffmann) proved unsuccessful novobiocin of 27 strains of Proteus and of 24 strains (Knox et al., 1942, 1943). of Salmonellae were tested by the paper disc method Antibiotics are becoming increasingly used as and by tube titration. Proteus was always more selective agents in culture media, but most of sensitive than Salmonella (Table I). them are approximately equally active or inactive TABLE I against Salmonellae and Proteus. Novobiocin, NOVOBIOCIN SENSITIVITY TESTS however, inhibits Proteus more than other Tube Titration (Inhi- on September 30, 2021 by guest. Protected Gram-negative bacilli and seemed therefore a bitory Concentration suitable substance for trial. No. of Disc Test of Novobiocin in Strains g./ml. Indicator Organism D e Serum Broth) Materials Tested _____ Sensi-e ta Average Range Tetrathionate Broth.-This was prepared by adding two "oxoid" tablets to 10 ml. of water in a 1 oz. Salmonella species 24 - 24 >2,400 1,600-3,200 to P. morganii 5 5 - 85 25-200 screw-capped bottle and bringing the boil. The P. mirabilis 13 13 - 170 25-400 medium without iodine can be stored at room P. vulgaris. 9 9 - 160 25-400 temperature for one once iodine is added the Paracolon bacilli 5 - 5 month; E. coli 5 - 5 complete medium is unstable. Ps. pyocyanea 4 - 4 Novobiocin-tetrathionate Broth.-Novobiocin solu- tion containing the appropriate concentration in Effects of Concentrations of Novobiocin in 0.1 ml. was added to 10 ml. of the incomplete medium, Tetrathionate Broth upon Growth of Salmonellae.-A 0.2 ml. of the specified iodine solution being added plate viable count (Miles and Misra, 1938) was made before use. on blood agar from an overnight broth culture of Novobiocin Solution.-The sodium salt of Salmonella paratyphi B. Drops from the highest novobiocin (Upjohn of England Ltd.) was used, and dilutions were seeded into bottles containing J Clin Pathol: first published as 10.1136/jcp.12.6.568 on 1 November 1959. Downloaded from NOVOBIOCIN-TETRATHIONA TE BROTH 569 TABLE II RECOVERY OF SALMONELLAE FROM ARTIFICIALLY INFECTED FAECES SUSPENSIONS CONTAINING PROTEUS Minimal Nos. of Salmonellae/ml. of Faeces Suspension Detectable lProteusProteus ~~~~~~~~~~afterCulture in Experi- Salmonella- ment Approximate Tetrathionate Proteus Novobiocn- Proteus Strain No./rml. tetrathsionate Faeces TerathionatentBrothPrsnBot Prsn a Schwarzengrund Morganii (11,229) 5,000 42 x 105 + + + + + 7 + b ,. ,, 266,000 >28 x 103 + + + 28 or less c ,, Vulgaris 5,000 42 or less + + + 42 or less d Typhi-murium Mirabilis (7,406) 5,000 > 140 x 106 +++++ 140 or less + + + + e Paratyphi B 88 x 106 + + + + + 88 x 104 + + f Enteritidis Morganii 5,000 22 x 104 + + + + + 22 x 104 Thompson . 30 or less + + + + + 30 or less h TAnaunn Mirabilis 5,000 34 x 102 + + + ++ 35 or less + Indicates the number of bottles in which Proteus survived. tetrathionate broth and into bottles containing suspension was delivered into each of 5 x 1 oz. different dilutions of novobiocin in tetrathionate screw-capped bottles. Counted numbers of broth. Each concentration of the organism was Salmonellae were then added to give a series of tested in triplicate in each medium. The test was bottles containing faeces, a constant number of repeated with the same strain of Salmonella paratyphi Proteus, and varying numbers of Salmonellae. Each B and also with a strain of Salmonella enteritidis. bottle was shaken thoroughly on a mechanical shaker, As a result of these tests a concentration of and 1 ml. quantities from each bottle were cultured 40 pg./ml. novobiocin in tetrathionate broth appeared in tetrathionate broth with and without novobiocin. to be the most suitable, since there was no inhibition After overnight incubation subcultures were made when growth of about 3 to 30 organisms was on MacConkey agar and two colonies of each type compared in tetrathionate broth and tetrathionate of non-lactose-fermenter were tested for urease copyright. broth plus 40 ,ug./ml. novobiocin. production. Those which proved to be urease negative were tested by slide agglutination, using the Suppression of Proteus by Tetrathionate Containing appropriate Salmonella antiserum. As the properties Novobiocin.-Diluted overnight broth cultures of and colonial appearances of the Salmonella strains four strains of Proteus were seeded into triplicate were well known, final identification by tube bottles of nutrient broth, tetrathionate broth, and agglutination was considered to be superfluous. The novobiocin tetrathionate broth. Novobiocin at a number of each type of non-lactose-fermenting concentration of 40 pg. /ml. completely suppressed colony examined was purposely limited so that the growth of an inoculum of about 70 organisms during chance of isolating Salmonellae should be http://jcp.bmj.com/ incubation overnight. approximately the same as it would be in routine Stability of Novobiocin iD Selective Media.-It examinations of faeces. seemed likely that if novobiocin proved to be a useful Table II shows that Salmonellae representative of selector in tetrathionate broth it might also prove of groups B, C, D, and E can be isolated more success- value in other enrichment media for Salmonellae. Its fully by enrichment in novobiocin-tetrathionate stability was therefore tested in tetrathionate broth, broth than in plain tetrathionate broth when Proteus and is present. Selenite-F medium, in the medium of Rappaport, on September 30, 2021 by guest. Protected Konforti, and Navon (1956). Group A was not investigated because the strain of The drug was stable at a concentration of Salmonella paratyphi A examined proved to be 40 pg. /ml. in all three media for at least 48 hours at inhibited by tetrathionate broth, which confirmed the 370 C. and for at least one month at 40 C. There findings of Banwart and Ayres (1953). was no loss of potency in tetrathionate broth on Comparison of the results of experiments (a) and (b) storage without iodine at room temperature for one shows that the efficiency of novobiocin-tetrathionate month. Novobiocin at a concentration of 40 pg./ml. broth may be unaffected by a 50-fold increase in the tetrathionate broth was used in all subsequent number of Proteus. experiments. In experiments (c), (f), and (g), novobiocin- tetrathionate broth did not detect smaller numbers of Comparison of Tetrathionate and Novobiocin- Salmonellae than plain tetrathionate broth. The only tetrathionate Broths for Recovery of Salmonellae in non-lactose-fermenters recovered from novobiocin- Presence of Proteus from Artificially Infected Faecal tetrathionate broth, however, were Salmonellae, and Suspensions.-An emulsion was prepared in 40 ml. of growths were always heavier than those from water of approximately 5 g. of faeces from a patient tetrathionate broth. who was not receiving antibiotics. A dilution of an Since it was shown in a separate experiment that overnight broth culture of Proteus was added to give small numbers of the Salmonella enteritidis strain approximately 5,000 bacilli/ml., and 5 ml. of this used in experiment (f) survived 40 lAg. /ml. novobiocin J Clin Pathol: first published as 10.1136/jcp.12.6.568 on 1 November 1959. Downloaded from 570 LEONARD JEFFRIES in tetrathionate broth the poor growth of this broth, no Salmonellae were encountered. Novo- Salmonella was not due to a harmful effect of biocin-tetrathionate broth effectively suppressed novobiocin. Proteus when the specimens contained insufficient Incorporation of Novobiocin in Desoxycholate- numbers to give profuse growths on direct cultures. citrate-agar. - Desoxycholate-citrate-agar (D.C.A.) Discusion
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