ON THE NECESSITY OF CULTI- 
 VATING WATER BACTERIA 
 IN AN ATMOSPHERE SAT- 
 URATED WITH MOISTURE 
 
 By GEORGE C. WHIPPLE, S.B. 
 
 Reprinted from Technology Quarterly, Volume XII, No. 4, December, 1899 
 
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George C. Whipple . 
 
 276 
 
 ON THE NECESSITY OF CULTIVATING WATER BAC- 
 TERIA IN AN ATMOSPHERE SATURATED WITH 
 
 MOISTURE . 
 
 By GEORGE C. WHIPPLE, S. B. 
 
 According to present practice the number of bacteria in a sample 
 of water is found by mixing a certain quantity of the water with a 
 certain quantity of sterilized nutrient gelatin and allowing the mixture 
 to solidify in a Petri dish. After a longer or shorter period of incuba- 
 tion at a temperature at or about 20° C., the colonies that have devel- 
 oped upon the gelatin are counted, and from this count the number of 
 bacteria present in the water is determined. Although this method 
 is almost universally used, and although the results obtained are some- 
 times of the greatest moment, there is such a lack of uniformity in 
 the details of the process as ordinarily conducted, that the determina- 
 tions of different observers are seldom comparable unless the methods 
 of procedure are fully described. A standard method of procedure 
 is urgently needed, but cannot be secured until the various factors 
 that influence the result have been analyzed and their magnitude 
 determined. In the course of a series of experiments conducted with 
 this object in view, it was observed that the amount of moisture in 
 the atmosphere of the incubator exercised an important influence upon 
 the number of bacteria that developed. The results of these obser- 
 vations are summarized in this paper. 
 
 Before the use of the Petri dish it was customary to pour the 
 mixed gelatin and water upon a cold glass plate, where it was allowed 
 to spread out and harden. The plate was then covered with a bell-jar 
 and put in the incubator. In order to prevent the gelatin from drying, 
 moist filter paper was put at the bottom of the bell-jar. Thus the bac- 
 teria* developed in a moist atmosphere. With the advent of the Petri 
 dish the matter of moisture seems to have been lost sight of. This 
 dish was provided with a tight fitting cover, and this was supposed to 
 prevent evaporation from the gelatin. It is a fact, however, that the 
 Petri dishes now on the market are not tight, and that often the covers 
 
On the Cultivation of Water Bacteria. 277 
 
 fit the plates very badly. Furthermore, in laboratories where many 
 dishes are in daily use, it is a common thing for dishes and covers to 
 be mismated, and often there is no attempt to mate them. The result 
 is that an appreciable evaporation from the gelatin does take place, 
 and that the amount of evaporation varies with different plates and 
 with different atmospheric conditions. 
 
 The effect which this uncontrolled factor exercises upon the devel- 
 opment of bacteria is shown by the following experiments. Several 
 series of cultures were submitted to varying conditions of moisture, 
 with other conditions remaining constant. The plates were incubated 
 in a moist chamber, in a closed chamber without moisture, in the incu- 
 bator (or the ice chest), and in a desiccator. For the moist chamber a 
 desiccator-jar was used, the sulphuric acid being replaced by water. 
 The closed chamber was a desiccator-jar without sulphuric acid or 
 water. The jars were large enough to hold five plates, and in all 
 cases the figures representing the numbers of bacteria found were 
 obtained from the averages of the five counts. The results are given 
 in the following tables. Table I shows the average number of bac- 
 teria that developed upon each set of plates. In Table II these figures 
 are reduced to percentages of the number that developed in the moist 
 chamber. 
 
 TABLE I. 
 
 
 
 X — 
 
 8 S2 
 
 £ £ 
 ,H 4> 
 
 ■s.P 
 
 Number of Bacteria Per Cubic Centimeter. 
 
 Series. 
 
 Date. 
 
 a a 
 — 1 0 
 
 O W 
 
 T 3 - 
 O S 
 
 •go 
 
 Temperature < 
 cubation. ( 
 tigrade) 
 
 In moist 
 chamber. 
 
 In ice chest. 
 
 In incubator. 
 
 In closed 
 chamber. 
 
 In desiccator. 
 
 A. 
 
 1899. 
 
 July 7. 
 
 96 
 
 16° 
 
 583 
 
 527 
 
 
 
 
 
 465 
 
 B. 
 
 July 7. 
 
 96 
 
 16° 
 
 507 
 
 430 
 
 — 
 
 — 
 
 415 
 
 C. 
 
 July 7. 
 
 96 
 
 16° 
 
 750 
 
 683 
 
 — 
 
 — 
 
 618 
 
 D. 
 
 Oct. 9. 
 
 96 
 
 15° 
 
 450 
 
 438 
 
 — 
 
 395 
 
 342 
 
 E. 
 
 May 20. 
 
 72 
 
 18° 
 
 154 
 
 — 
 
 115 
 
 — 
 
 111 
 
 F. 
 
 Oct. 14. 
 
 72 
 
 20° 
 
 108 
 
 — 
 
 89 
 
 90 
 
 84 
 
 G. 
 
 Oct. 9. 
 
 48 
 
 20° 
 
 423 
 
 — 
 
 413 
 
 354 
 
 324 
 
 H. 
 
 Oct. 17. 
 
 72 
 
 20° 
 
 61 
 
 — 
 
 59 
 
 — 
 
 51 
 
2yS 
 
 George C. Whipple. 
 
 TABLE II. 
 
 
 
 ci ^ 
 
 ■S? 
 3 8 
 
 S fl 
 
 • r " <D 
 
 ■sy. 
 
 Per Cent, which Each Number was of the Number 
 Found in the Moist Chamber. 
 
 Series. 
 
 Date. 
 
 Period of i 
 tion. (H< 
 
 Temperatur 
 
 cubation. 
 
 tigrade.) 
 
 In moist 
 chamber. 
 
 In ice chest. 
 
 In incubator. 
 
 In closed 
 chamber. 
 
 In desiccator. 
 
 A. 
 
 1899 . 
 
 July 7. 
 
 96 
 
 16° 
 
 100 
 
 90 
 
 
 
 
 
 79 
 
 B. 
 
 July 7. 
 
 96 
 
 1 6 ° 
 
 100 
 
 85 
 
 — 
 
 — 
 
 82 
 
 c. 
 
 July 7. 
 
 96 
 
 16° 
 
 100 
 
 91 
 
 — 
 
 — 
 
 82 
 
 D. 
 
 Oct. 9. 
 
 96 
 
 15° 
 
 100 
 
 98 
 
 — 
 
 88 
 
 79 
 
 E. 
 
 May 20. 
 
 72 
 
 18° 
 
 100 
 
 — 
 
 75 
 
 — 
 
 73 
 
 F. 
 
 Oct. 14. 
 
 72 
 
 20 ° 
 
 100 
 
 — 
 
 82 
 
 83 
 
 77 
 
 G. 
 
 Oct. 9. 
 
 48 
 
 20 ° 
 
 100 
 
 — 
 
 98 
 
 83 
 
 77 
 
 H. 
 
 Oct. 17. 
 
 72 
 
 20 ° 
 
 100 
 
 — 
 
 97 
 
 — 
 
 83 
 
 Average. 
 
 
 
 
 100 
 
 91 
 
 88 
 
 85 
 
 78 
 
 From these tables it will be seen that the largest numbers of bac- 
 teria were always obtained from the plates that developed in the moist 
 chamber, and that -the smallest numbers were always obtained from 
 the plates that were kept in the desiccator. If the number that devel- 
 oped in the moist atmosphere be assumed to represent the maximum 
 number obtainable by the method, it follows that only 78 per cent, of 
 the bacteria developed in the desiccator, that 85 per cent, developed 
 in the closed chamber, and that the numbers that developed in the 
 ordinary incubator and in the ice chest varied from 75 per cent, to 
 98 per cent. There was a striking uniformity in the percentages 
 which the numbers in the desiccator were of the numbers in the moist 
 chamber and this was due, no doubt, to the constant atmospheric con- 
 ditions in the two jars. In the closed chamber also the percentages 
 were quite constant. In the ice chest and in the incubator the per- 
 centages were mqre variable. The atmosphere of the ice chest was 
 ordinarily more moistM:han that of the incubator, but was seldom satu- 
 rated on account of ventilation. The amount of moisture in the air 
 of the incubator was generally greater than that of the atmosphere of 
 the laboratory, and varied more or less with the ventilation, the number 
 of plates kept in the incubator, etc. In Series E the relative humidity 
 
On the Cultivation of Water Bacteria. 279 
 
 of the atmosphere in the incubator was estimated at about 60 per cent. 
 In Series F the humidity was determined by hourly readings of a 
 psychrometer and was found to vary between 65 per cent, and 80 per 
 cent., the average being 75 per cent. In Series G the atmosphere of 
 the incubator was kept moist by means of jars of water, and the 
 average humidity was 95 per cent. In Series H the humidity was 
 similarly kept at 98 per cent, of saturation. The relation between 
 the humidity and the development of bacteria is shown by the follow- 
 ing comparison : 
 
 Series. 
 
 Relative Humidity of the Atmosphere 
 of the Incubator in per cent, of 
 Saturation. 
 
 Per cent, which the Number of Bacte- 
 ria that Developed in the Incubator 
 was of the Number that Devel- 
 oped in the Moist Chamber. 
 
 E. 
 
 60 (estimated.) 
 
 75 
 
 F. 
 
 75 
 
 82 
 
 G. 
 
 95 
 
 98 
 
 H. 
 
 98 
 
 97 
 
 The amount of evaporation from gelatin under the conditions 
 described above was determined by weighing the dishes with their 
 contents before and after incubation. It was found that in a satu- 
 rated atmosphere the evaporation was inappreciable ; in the closed 
 chamber it was from 1 to 2 per cent, of the weight of the mixed 
 water and gelatin during 72 hours ; in the ice chest it was about 3 
 per cent. ; in the incubator it was from 3 per cent, to 5 per cent. ; and 
 in the desiccator it was from 10 per cent, to 15 per cent. The 
 amount of evaporation varied greatly with different plates submitted 
 to the same conditions. 
 
 This loss of water by evaporation is sufficient to cause important 
 changes in the composition of the culture media during incubation, 
 and this may be sufficient to affect the development of the bacteria,, 
 as these organisms are very susceptible to slight changes in environ- 
 ment. But the chief reason why increased evaporation is accompa- 
 nied by a decrease in the percentage of bacteria that develop seems 
 to be connected with the supply of oxygen. By successive weighing 
 of the same plates it was found that the rate of evaporation was not 
 constant, but decreased rapidly. For example, during the first hour 
 
28 o 
 
 George C. Whipple. 
 
 of incubation the evaporation from a series of plates proceeded at the 
 rate of .068 gram per hour; after 18 hours the rate of evaporation 
 was .031 gram per hour; after 42 hours it was .012 gram per hour, 
 and after 96 hours it was .002 gram per hour. This retardation of the 
 rate of evaporation appears to be due to a thickening of the gelatin at 
 the surface. If such is the case, the thickened gelatin at the surface 
 might materially reduce the supply of oxygen of the submerged bac- 
 teria and prevent the development of the less vigorous aerobic forms. 
 Indeed, it was observed that the plates in the desiccator and in the 
 moist chamber differed more in the number of submerged colonies 
 than in the number of surface colonies. 
 
 These facts suggested the possibility that the Petri dish itself 
 might serve to prevent the bacteria from getting a sufficient supply 
 of oxygen. The experiment was made, therefore, of cultivating the 
 bacteria in a Petri dish with the ordinary cover replaced by a ground 
 glass top that made an absolutely air tight joint. A series of five 
 cultivations made in this way gave the number of bacteria in a certain 
 sample as 317 per cc., while a series of cultivations of the same water 
 made in the usual manner gave 413 per cc. In the hermetically 
 sealed dish, therefore, only 77 per cent of the bacteria developed. 
 The air of these dishes was then collected over mercury, and the 
 amount of oxygen determined by absorption with pyrogallic acid. 
 It was found that the air of the ordinary dishes contained approxi- 
 mately 1 5 per cent, of oxygen, while the air of the sealed dishes con- 
 tained only 5 per cent. In other words, three-quarters of the original 
 supply of oxygen in the sealed dishes had been used up by the bac- 
 teria during 72 hours, while one-quarter of the original supply had 
 been used up in the ordinary dishes. It was found also that the air 
 in the sealed dishes contained 5 per cent, of C 0 2 , while the air in 
 the ordinary dishes contained but 2 per cent. 
 
 As a matter of interest a series of cultures was made in a jar 
 filled with oxygen, and compared with cultures of the same water 
 made in the incubator. It was found that the average number of 
 colonies that developed in the oxygen was 1 54 per cc., while in the 
 incubator it was 137. The growth in oxygen also took place at a 
 much more rapid rate. 
 
 Finally, one important fact was noted in connection with the cul- 
 tures made in the jars that illustrates the important effect of oxygen. 
 In all the jars the five Petri dishes were piled one on top of the other* 
 
On the Cultivation of Water Bacteria. 
 
 281 
 
 and it was found that almost invariably the plate highest in the jar 
 gave the largest count, and that the lowest plate gave the smallest 
 count. This was particularly striking in the case of the jar filled with 
 oxygen, where the upper plate contained 198 colonies per cc., while 
 the lower plate contained 138. These differences between the upper 
 and lower plates of the jars were greater than the differences observed 
 between plates kept in the incubator. Liquefaction proceeded much 
 more rapidly in the upper plates of the jars. These phenomena 
 seemed to be due to an accumulation of carbon dioxide at the bot- 
 tom of the jars, where either by its own properties or by exclusion 
 of oxygen, it retarded the growth of the bacteria. 
 
 These facts tended to show that the supply of oxygen has a marked 
 effect upon the growth of water bacteria on the gelatin plate, and that 
 whatever tends to reduce the supply of oxygen, whether it be evapo- 
 ration or an air-tight Petri dish or lack of sufficient ventilation, will 
 tend also to reduce the number of bacteria. The practical inference 
 from this is that in order to obtain the greatest possible develop- 
 ment of water bacteria on the gelatin plate, a ventilated dish should 
 be used, and the cultures should be incubated in an atmosphere satu- 
 rated with moisture. 
 
 It has been found that satisfactory ventilation of the Petri dishes 
 may be obtained by grinding several small notches in the edge of the 
 lower plate. If greater ventilation is required, it may be obtained by 
 extending the sides of the lower plate upwards at four points on the 
 circumference so as to form pillars for supporting the cover, thereby 
 allowing a complete circulation of air. It has been found that the 
 bacteria counts obtained by using these ventilated dishes in an atmos- 
 phere saturated with moisture, compare closely with those obtained in 
 open dishes protected from contamination and incubated in a saturated 
 atmosphere, the results of one series of comparisons giving 97 and 95 
 bacteria per cc, respectively, by the two methods. Experiments have 
 shown that there is no danger of these ventilated plates becoming 
 contaminated from the air. 
 
 The cultivation of water bacteria in the moist atmosphere has the 
 further advantage that the growths come to maturity in a shorter time. 
 Liquefaction takes place earlier, but the growth of the deep-seated 
 colonies seems to proceed at a still more rapid rate. A greater pro- 
 portion of the plates are liquefied after 72 hours’ growth, but on the 
 other hand, the percentage increase between the 48-hour count and 
 the 72-hour count is much reduced. 
 
282 
 
 George C. Whipple. 
 
 Incubators used for the cultivation of water bacteria should 
 be well ventilated, and their atmosphere should be kept at or near the 
 point of saturation. They should be provided with wet and dry bulb 
 thermometers, and the relative humidity should not be allowed to fall 
 below 95 per cent. Experience has shown that an atmosphere prac- 
 tically saturated with moisture may be easily maintained. 
 
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