The survival of bacteria during and after drying

SUMMARY: Sporulation of Bacillus sphaericus, NCTC 7582, in a complex medium was strongly stimulated by growth in mixed culture with a sporing strain of B. cereus and with sporogenous and asporogenous strains of B. subtilis, NCTC 85. There was a similar, but less pronounced, effect with a variety of Gram-positive and Gramnegative organisms. The degree of sporulation of B. sphaericus in these mixed cultures depended on ( a ) an external stimulus from the medium and (b) the previous cultural history of the B. sphaericus inoculum upon which depended its ability to respond to the external stimulus. Sporulation of B. sphaericus in pure culture was strongly stimulated by bicarbonate and ketoglutarate, suggesting that increased carbon dioxide production was the stimulating factor in mixed cultures.The stimulatory effect of one organism upon the growth of another has been observed in bacteria and in fungi (e.g. Hawker, 1939). This report describes a similar phenomenon in the genus Bacillus and suggests a mechanism for the effect. ORGANISMS AND METHODSA culture of Bacillus sphaericus, NCTC 7582, grown for 24 hr. at 37" on casein hydrolysate yeast extract (CCY) agar (Gladstone & Fildes, 1940) was suspended in 50 ml. glucose serum broth (Fry & Greaves, 1951) and freeze-dried in 5 ml. batches. Starting with one of these freeze-dried batches, a series of sporulation tests was made, during which time the organism was maintained by subculturing every 48 hr. on CCY agar, and on potato-extract agar (Robinow, 1951) enriched with 1/10 vol. of CCY medium (Pot. CCY).The strongly sporing strain of Bacillus cereus isolated from air and the sporogenous and asporogenous strains of Bacillus subtilis, NCTC 85, were maintained on CCY agar. The other organisms used were subcultured at least twice in Pot. CCY medium before the mixed culture tests.For the sporulation tests, which were set up in duplicate, 10 ml. batches of liquid Pot. CCY medium in 50 ml. conical flasks were inoculated with 0.5 ml. of a just visibly turbid suspension of organisms and shaken at 37" for periods up to 36 hr. RESULTSThere was rapid growth and remarkably vigorous sporulation of Bacillus cereus and B. subtilis in the test medium, which was selected for this reason. The first signs of sporulation appeared at 14 hr., and at 17 hr. almost every

“…at 37" on casein hydrolysate yeast extract (CCY) agar (Gladstone & Fildes, 1940) was suspended in 50 ml. glucose serum broth (Fry & Greaves, 1951) and freeze-dried in 5 ml. batches.…”

Section: Organisms and Methodsmentioning

confidence: 99%

The Sporulation of Bacillus sphaericus stimulated by Association with other Bacteria: an Effect of Carbon Dioxide

Powell¹,

Hunter²

1955

“…We a q much indebted to Dr R. M. Fry for supplying us with a culture of the paracolon bacillus (D. 201 H) used by Fry & Greaves (1951); also to the Agricultural Research Council for support during this work.…”

Section: R C Valentine and J R G Bradfieldmentioning

confidence: 99%

The Urea Method for Bacterial Viability Counts with the Electron Microscope and its Relation to other Viability Counting Methods

Valentine

Bradfield

1954

SUMMARY: Live rod-shaped bacteria incubated on a medium containing 3 yo urea grow but do not divide. They thus become much longer than dead bacteria lying among them, and the numbers of each kind can easily be counted in an electron microscope. This urea method gives accurate results and has several advantages over colony-counting methods. The significance of various interesting differences in the counts given by the two methods is discussed. Slope cultures at 18 hr. gave live counts of over 95 yo by the urea method.A common need in quantitative bacteriology is to discover what proportion of bacteria in n given sample is alive. The standard procedure is to make counts of the colonies which form when measured volumes of the sample, suitably diluted, are incubated in nutrient media. On the assumption that each colony arises from one live bacterium, the number of the latter in the sample can then be determined. Such counts made before and after some treatment will enable the resulting fall in viability to be measured. In order to find the actual percentage of live cells in a sample a total count of the bacteria in the measured volume must also be made. In studying the survival of bacteria during drying from tiny droplets or thin films, an accurate measurement of the volume of the samples becomes difficult or impossible. To overcome this problem a new method has been developed for determining the proportion of live bacteria in such tiny samples. This paper gives details of the method and typical results of a test of its accuracy.The method is based on the property possessed by urea in concentrations of about 3 yo ( 0 . 5~) of preventing the division of certain bacteria without inhibiting their growth (Wilson, 1906). Hence after incubation of suitable bacteria for a few hours on nutrient agar containing urea, the live cells have grown into giant forms which can readily be distinguished from the shorter dead organisms (Pl. 1, fig. 5 ) . The numbers of live and dead can then easily be counted in an electron microscope. EXPERIMENTALThe action of urea We have no information about the mechanism by which urea exerts its effect. We have, however, investigated its action on the growth of a paracolon bacillus over a range of concentrations of urea from 1 to 10 yo in nutrient agar medium.The bacteria were taken from 18 hr. slope cultures, suspended in broth, and grown on filmed electron microscope specimen grids as described below. 23G. Microb. XI

“…The volume of broth used by these workers has varied from less than 5 to more than 100 times the volume of the water removed. Fry & Greaves (1951) rehydrated their cultures with 100 volumes of Ringer solution, Mazur & Weston (1956) used 2 or 3 drops of a dilute detergent solution for an unspecified amount of dry matter, and Hutton, Hilmoe & Roberts (1951) used an unspecified volume of dilute tryptose solution. Wasserman & Hopkins (1957) rehydrated their preparations with dilute solutions, using c. 0.5 ml./mg.…”

mentioning

confidence: 99%

The Influence of Rehydration on the Viability of Dried Micro-Organisms

Leach

Scott

1959

SulMMARY : F'reeze-dried preparations of certain organisms may suffer considerable mortality during reconstitution to the wet state, For Vibrio metchnikowi the mortality was greatly decreased by rehydration with small volumes of water, or with various volumes of relatively concentrated solutions of some solutes. Within certain limits, slow dropwise addition of water resulted in higher viable counts than did rapid addition of the same volume of water. In preliminary tests Mgcoplasma mycoides and two coliphages were also affected by the volume of water used for rehydration, but no significant effects were observed with nine other bacteria. Comparisons of rehydration at several temperatures showed greatest destruction at 37O for V . metchn i k d , but at 0' for three other bacteria. The importance of rehydration rates and of exposure to hypotonic solutions is discussed.