The assay of vitamin B12. Part VI. Microbiological estimation with a mutant of Escherichia coli by the plate method

Harrison, Eleanor; Lees, K. A.; Wood, Frederick L.

doi:10.1039/an9517600696

Cited by 52 publications

(18 citation statements)

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“…It has been shown by Harrison et al (1951) and confirmed by us that there is a straight line relationship between the diameter of the rings of exhibition and the logarithm of the dose of pure vitamin B12, between the concentrations 0-005 fig./m\. and 0-2 ^g./ml.…”

Section: Dose Response Relationshipmentioning

confidence: 70%

THE DETERMINATION OF VITAMIN B₁₂‐ACTIVITY IN THE ORGANS AND EXCRETA OF SHEEP

Dawbarn

Hine

Remilton

et al. 1954

Aust J Exp Biol Med

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It seems probable from available evidence that vitamin B12 and the closely analogous vitamin Bv2a (or Bi2b) and vitamin Biac all originate in nature only as products of the metabolic activity of certain micro-organisms. The chemical differences between these analogues are minor ones-the cyanogen group, which is attached rather loosely to the cobalt atom in the complex molecule of vitamin B12, is replaced in vitamin Bjoa (or Bjab) by a hydroxyl group, and in vitamin Bi2« by a nitrite radicle; and there is little if any difference between their capacities to provide the nutritional requirements of higher animals and of exacting micro-organisms. These cobalamins (vitamin B12 and its analogues) are readily interconvertible-vitamins Bi2a (Bi2b) and Biac, for example, are converted into vitamin B12 in dilute cyanide solution (Kaczka, Wolf, Kuehl and

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Section: Dose Response Relationshipmentioning

confidence: 70%

THE DETERMINATION OF VITAMIN B₁₂‐ACTIVITY IN THE ORGANS AND EXCRETA OF SHEEP

Dawbarn

Hine

Remilton

et al. 1954

Aust J Exp Biol Med

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“…of vitamin B12/ml. assessed by microbiological assay with an Escherichia coli mutant (Harrison, Lees & Wood, 1951).…”

Section: Methodsmentioning

confidence: 99%

Tracer studies with the B12 vitamins. 2. Biosynthesis of vitamin B12labelled with 60CO and 32P

Smith¹,

Hockenhull²,

Quilter³

1952

Biochemical Journal

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These attempts failed because the livers contained negligible amounts of vitamin B12. We recognized at the time that it would have been preferable to use ruminants, but such animals were not available to us. Subsequent work suggests that we ought to have extracted the faeces rather than the livers of our animals.About the same time we succeeded in preparing very small amounts of radioactive vitamin B12 concentrates by fermentations with Aerobacter aerogenes isolated from hen faeces. More recently we have returned to this problem, using Streptomyces gri8eu; in the meantime the preparation by this method of [6OCo] vitamin B12 with low or moderately high specific activity has been reported (Chaiet, Rosenblum & Woodbury, 1950;. RESULTS Preparation of vitamin B12 containing 60CoTo avoid diminishing the specific activity of the added radioactive cobalt, we used a semi-synthetic medium (Dulaney, 1948) shown to be nearly free from cobalt. We added only about 0-2 p.p.m. of radio-cobalt, thus minimizing any radiation hazard and securing efficient conversion ofcobalt to vitamin B12. The fermentation liquors were worked up to yield a few mg. of crystalline vitamin B12 by a simple procedure described in the Experimental section. The latest fermentation with 60Co at 3-5 mc./mg. has yielded vitamin B12 with a specific activity of 120 ,uc./mg., which is considerably higher than any previously recorded (Rosenblum & Woodbury, 1951). Preparation of vitamin B12 containing 32pIt was of interest to attempt the preparation of vitamin B12 labelled with radioactive phosphorus, since this would be safer for projected human metabolism experiments than vitamin B12 containing radioactive cobalt with its much longer half-life and penetrating gamma radiation. It was foreseen that it would be difficult to prepare material with adequate specific activity because preliminary experiments had shown that Streptomyce8 gri8eus needs a moderately high level of phosphate in the medium, growth and vitamin B12 production being impaired when the total phosphorus level is reduced to about 0-01 %.It was decided to use 10 mc. Of 32p and to aim at preparing about 1 mg. of the radioactive vitamin. It was then almost immaterial what volume of medium was used for the fermentation; the smaller the volume, the higher the specific activity of the resulting vitamin B12 would be, but the greater the proportion of ordinary vitamin B12 that would have to be added.The radioactive phosphate was added aseptically 24 hr. after fermentation started. This was done in the hope that part of the phosphorus in the medium would already have been taken up by the mould and converted into organic phosphorus compounds that would not exchange with the added 32p. This hope was not realized; the specific activity of the product was the same as that from a pilot run with

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“…needle tips (Harrison et al 1951) which the operator strokes over the surface of the incubated assay plate to align the needle tips and the zone edge by means of a diffuse transverse light source. needle tips (Harrison et al 1951) which the operator strokes over the surface of the incubated assay plate to align the needle tips and the zone edge by means of a diffuse transverse light source.…”

Section: Destcn Of Large Plate Assaysmentioning

confidence: 99%

“…In these large plate methods we measure the zones with calipers having 1 in. needle tips (Harrison et al 1951) which the operator strokes over the surface of the incubated assay plate to align the needle tips and the zone edge by means of a diffuse transverse light source. Since the operator cannot see the alignment of the needle tips and the caliper vernier reading a t one and the same time, two completely independent operations are involved and this method of reading, though a little tedious, is regarded as more objective than others tried.…”

Section: Destcn Of Large Plate Assaysmentioning

confidence: 99%

The assay of vitamin B12. Part VI. Microbiological estimation with a mutant of Escherichia coli by the plate method

Cited by 52 publications

References 0 publications

THE DETERMINATION OF VITAMIN B₁₂‐ACTIVITY IN THE ORGANS AND EXCRETA OF SHEEP

THE DETERMINATION OF VITAMIN B₁₂‐ACTIVITY IN THE ORGANS AND EXCRETA OF SHEEP

Tracer studies with the B12 vitamins. 2. Biosynthesis of vitamin B12labelled with 60CO and 32P

Some Applications of Biological Control Methods in the Manufacture of Antibiotics

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The assay of vitamin B12. Part VI. Microbiological estimation with a mutant of Escherichia coli by the plate method

Cited by 52 publications

References 0 publications

THE DETERMINATION OF VITAMIN B12‐ACTIVITY IN THE ORGANS AND EXCRETA OF SHEEP

THE DETERMINATION OF VITAMIN B12‐ACTIVITY IN THE ORGANS AND EXCRETA OF SHEEP

Tracer studies with the B12 vitamins. 2. Biosynthesis of vitamin B12labelled with 60CO and 32P

Some Applications of Biological Control Methods in the Manufacture of Antibiotics

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Product

Resources

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THE DETERMINATION OF VITAMIN B₁₂‐ACTIVITY IN THE ORGANS AND EXCRETA OF SHEEP

THE DETERMINATION OF VITAMIN B₁₂‐ACTIVITY IN THE ORGANS AND EXCRETA OF SHEEP