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UNITED STATES DEPARTMENT OF Bye ae oon

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Contribution from Bureau of Animal Industry. A. D. MELVIN, Chief.

Washington, D. C. PROFESSIONAL PAPER June 19, 1917

THE DETERMINATION OF BACTERIA IN ICE CREAM.

By S. Henry Ayers and W. T. Jounson, Jr., of the Dairy Division.

CONTENTS.

Page. Page,

Difficulty of making accurate bacteriological Comparison of incubation at 37°C. for twa 4 De LS 7 a 9 ee 1 daysand at 30° C. for five days............- 12

Method ofsampling and plating the ice cream. 2 | Number of colonies most desirable on Petri Variation in the bacterial content of com- PLATES Re SEAR EIN a tne aa UEC NOS RAR DE cee 13

AMEGera MCE CLeAM sce fee Lk eee tk Se 3 | Variation between duplicate counts from Variation when held in an ice-cream cabinet. 8 same sample... te ae 14 Variation when held in storage...........--- 9 | Interpreting differences! in decal connie 15 ‘Variation in samples taken directly from Summary and conclusions................... 16

HIG SYA STR ce tes eS at ye es cP PR 10

DIFFICULTY OF MAKING ACCURATE BACTERIOLOGICAL ANALYSES.

Statements have been made that the distribution of bacteria in ice cream is markedly uneven, that there is great variability in the bacterial counts of different portions of the same container, and that this variability is so great that any small sample selected for analysis

)

will not represent the whole mass of the ice cream.

It must be remembered that the accuracy of a bacterio- logical analysis can never be so great as that of a chemical analysis. In making bacterial counts we are dealing with living organisms which are distributed in the material under examination. The method of analysis follows the assumption that the bacteria, as | individual cells, are distributed evenly throughout the sample and | that the portion removed for analysis contains a number in exact proportion to the total number in the sample. Having removed a definite part, it must then be placed in a medium suitable for plating in which the individual bacterial cells can multiply and form visible

colonies. The inaccuracy of such a method must be evident at once.

We know that some bacteria are in clumps or chains, and many ‘organisms may then develop into one colony which must be counted |

-asasingle colony. The removal of a quantity of material which will contain the same number of bacteria in suspension as another like

“quantity is known to be impossible. Since we are dealing with ne 95793°—Bull. 563—17—1

2 BULLETIN 563, U. S. DEPARTMENT OF AGRICULTURE.

living organisms, the bacteriological method of analysis must take into account their distribution and development into colonies on the Petri plates. In this part of the method we encounter the difficulty of separating the bacterial cells and distributing them evenly. Their development is more or less influenced by the growth of different kinds of bacteria, one of which may retard the development of those near it.

All these points are recognized by bacteriologists and are men- tioned here merely to call attention to the many difficulties which arise In making accurate bacterial counts and to point out that there must be variations in the result over which the analyst has no definite control. This con- dition has been recognized, consequently duplicate plates are made and results reported from the average counts of both plates. The variation in bacterial counts is particularly important and must be taken into consideration when a study is made of various samples of any material containing bacteria. If these variations are not considered, mistakes are easily made in the study of the distribution of bacteria.

If the bacteria in ice cream are unevenly dis- ia ed tributed, and a bacterial analysis of a sample does not

peienainael (EIve results which will represent the whole mass of the fom gallon cream, this fact will greatly complicate any study of ca ke robe bacteria in the product. Consequently, before starting any further studies on the subject it was con- sidered advisable to carry out some experiments to throw more light on this point.

METHOD OF SAMPLING AND PLATING THE ICE CREAM.

Ice cream from various manufacturers was delivered in 1-gallon cans at the laboratory. As soon as received, the can of cream was removed from the tub, the ice and salt wiped off, the cover removed, and the top layer taken off with a large sterile spoon.

Three samples were taken from the topmost third of the gallon, three from the middle, and three from the bottom third, making a total of nine samples from each gallon, as shown in figure 1. Each sample contained about 30 grams and was removed with asmall sterile scoop and placed in asterile flask. After removing samples from positions 1, 2, and 3, about one-third of the ice cream was removed with a sterile spoon and three more samples taken from positions 4, 5, and 6; similarly the three remaining samples were taken from positions 7, 8, and 9. |

The flasks containing the samples were then placed in water at 40° C. (104° F.) for 15 minutes in order to melt the ice cream, the

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THE DETERMINATION OF BACTERIA IN ICE CREAM, z

melting being hastened by frequent shaking with a circular motion. At the end of the 15-minute period each flask containing the melted ice cream was again shaken 30 times, with a circular motion, in order to mix the sample thoroughly and to shake out as much air as possible.

One cubic centimeter of melted ice cream was then removed from each flask and placed in 99 c. c. of sterile water at a temperature of 40° C. (104° F.). The water in all the dilution bottles was at a temperature of 40° C. (104° F.) in order to keep the fat in a melted ‘condition.

The pipettes were so graduated as to deliver 1 cubic centimeter between two marks. This avoids the necessity of blowing out the pipette or immersing the end in the dilution water, and therefore eliminates the introduction of varying quantities of melted cream which adhere to the pipette. | The dilutions were made in the usual way, using 99 c. c. and 9 ¢. ¢., respectively, of sterile water. Each dilution bottle or tube was shaken 25 times, and great care was taken to measure the quantity accu- rately in the pipettes. Standard beef-infusion agar was used, and sufficient medium was prepared to last through the work; conse- “quently no factor of variation was introduced by the pleting medium. The plates were incubated at 30° C. (86° F.) for a period of five days, after which the duplicate plates were counted.

VARIATION IN THE BACTERIAL CONTENT OF COMMERCIAL ICE CREAM.

VARIATION IN DUPLICATE SAMPLES FROM VARIOUS PARTS OF THE SAME LOT.

In our first experiment twenty-two 1-gallon lots of ice cream were obtained from seven different manufacturers. This cream, as in- tended, was of different flavors, was made in different ways, and included products containing different ingredients and varying percentages of butterfat.

The complete results obtained from a study of these samples are shown in Table I, in which is recorded the percentage of fat in the ice cream from each manufacturer, also the presence or absence of gelatin, the flavor of each lot, the dilution used in plating, the num- ber of colonies found on each of the duplicate plates, and the calcu- lated average number of bacteria in a cubic centimeter of melted ice cream, Where there is a blank space in the number. of colonies on duplicate plates no count could be made on account of ‘‘spreaders,”’

which entirely obscured the colonies. Every count that could be obtained is included in this table of results, and no count was left out as being a “‘freak”’ result.

_ A study of the table shows that the bacterial counts of the nine “samples from as many different positions in each gallon lot of ice “eream check remarkably well with one another.

4

BULLETIN 563, U. S. DEPARTMENT OF AGRICULTURE.

TABLE I.—Bacteria per cubic centimeter in 198 samples from twenty-two 1-gallon lots of

commercial ice cream obtained from different manufacturers.

Number of Plant Lot le | Flavor Dilution, | Colonies on No. : = : 4 duplicate ; | plates. a a ee tag Sea ee att i A ee Brae (Bak ae | ec pie oe a) |S si Al | 1 | A (fat 9.5 per cent, gelatin +)-.. 1} Vanilla...... robes | 243 | 229 2 219 198 3 242 | 243 4 231 | 245 | 5 POS Ue (aes ae | 6 264 | 233 7 PALS yaerea 8: 243 | 245 9 70 i 5) ut ALBA 09 | | | BEE PAAR REE. SONY ' 1 | Chocolate... sine 45] 46 | 2 40 49 3 50 47 4 67 40 5 52 47 6 60 43 Prez 42] 48 8 49 45 | 9 57 60 AV OSIE SS «antl eee, 11 Pages pbc det bn TAB 5G 2 160 150 3 137 155 4 148 | 145 5 140 156 6 170 151 7 153 | 155 8 154 148 9]. 154 | 166 yy See oe Ge eee pee BN 3 | 1 Vaniilas soos Toss 24 26 2 23 25 3 eg ee 4 20 32 5 20 20 6 30 22 7 4c). So: 8 25| 26 | 9 25 27 LAG a SIRO ES a SSS. ae ee 1 | Vanilla..... rhs 64 79 | 2 My rio 3 120} 96 4 109 | 118 } 5 95 118 (agra 108d BLEL 7 138 97 8 Viel eee | 9 | | 131 | 129 | | | DRY Aer a: Mie SERS ES FE AD, 1 |* Vaniiie:!: 2 223 rs1sss | 25 62 2 44, &4 3 54! 46 4 53 47 5 SD: | Sl eee% 6 58 54 7 67 55 8 50 59 9 59 55 TS eer S ica = bade ie jad ds 1 | Chocolate... routoos | 217] 218 2 212] 193 3 ee hefghy. 233 | 217 | 5 204 | 195 5 a 204} 184 bersre 187 | 174 8 203 | 200 8] 192 | 184

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THE DETERMINATION OF BACTERIA IN ICE CREAM, 5

Tasie I.—Bacteria per cubic centimeter in 198 samples from twenty-two 1-gallcn lots of commercial ice cream obtained from different manufacturers—Continued.

Number of Sam- p Average fog Lot. ple Flavor. Dilution. Lae count 2 No. plates. per ¢. ¢. | |

i Ele 1 | Vanilla : 60 64 | 62,000,000

2 | C (fat.9.6 per cent, gelatin +) 3 {OOOO eS 65 | 6470007000

3 73 | 62] 67,500,000

4 70 68 | 69,000, 000

5 64 70 | 67,000,000

6 73 77 | 75,000, 000

7 63 57 | 60,000,000

8 63 72} 67,500,000

9 57 | 721 64,500,000

2 i tet peed | Nicaral lay pc sdss | 286] 290 288, 000

3 | A (fat 9.6 per cent, gelatin +) 5 Scola 307° 000

3 270 | 289 279, 500

| 4 250 | 270 260, 000

5 314 | 250 282, 000

6 251 | 252 251, 500

7 268 | 250 259, 000

8 311 | 287 299, 000

9 261 | 306 283,

Mee ee ni eae ee ye | 1 Peach....... T0000 219 186 202, 500

2 214. os 214, 000

3 222 | 190 , 000

4 192) |e 192, 000

5 212| 194 203, 000

6 197 | 200 198, 500

7 212! 208 210, 000

8 231 | 205 218, 000

9 218 | 224 221,000

Ramet een bara SOT rides Sadie 1 | Chocolate... Lob00 107 104 1 , 055, 000

2 118 99 1,085, 000

3 96 99 975, 000

4 106} 96] 1,010,000

5 108 | 119] 1,135,000

6 123 | 124] 1,235,000

7 102! 97 995, 000

8 124] 123} 1,235,000

9 103} 121 1, 120, 000

+. 5) latin—)... 1} Vanilla... TOO0OD 341 334 33 750, 000

4. | A (fat, 22.5 per cent, gelat ) - Sar ee a 00” 000

3 2840 aes 28, 400, 000

4 294 | 302| 29,800,000

5 284 | 270] 27,700,000

6 301 | 303 | 30,200,000

7 325 | 331 | 32,800,000

8 321 | 329} 32,500,000

9 By 31, 400, 000

» ee hija 5 Sok Ae aie eee 1+ | Peaches. 0.2 todsoo | 321 | 310] 31,500,000

< 2 270 | 305] 28,750,000

3 386 | 372] 37,900,000

4 278 | 305] 29,150,000

5 367 | 356] 36,150,000

6 354 | 357] 35,550,000

7 431 | 466 | 44,850,000

8 ATO: |S eke 47, 000, 000

9 CRY ai 43, 400, 000

aches NN ie a 1 | Chocolate... adooo | 310} 298] 30,400,000

: 2 i a 28, 600, 000

& 3 B51 eee 33, 000, 000 = 5 DPE arab te Mtl LEA ea ha avi a eee (et Sen) MRS male ot Oe

5 293 | 288] 29,050,000

6 284 | 288] 28,600,000

7 328 | 292} 31,000,000

8 286 | 278] 28,200,000

9 275 | 285} 28,000,000

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6

BULLETIN 563, U. S. DEPARTMENT OF AGRICULTURE.

TABLE I.—Bacteria per cubic centimeter in 198 samples from twenty-two 1-gallon lots of commercial ice cream obtained from different manufacturers—Continued.

Number of

EESEEEEEE | ELESELEES | ELESE

Sam- F Average Plan Lot. le Flavor. Dilution. colonies on count No. 5 duplicate rake \ ; plates. Le ai | ; ; | 5 | A (fat, 17 per.cent, gelatin —).. . 1 Vanilaoss. 2. yoint AA Dee ee 440, 006 tei 2 45 | 48 465, 000 Pe 3 46| 49 475, 000 : ACH sc: | 5 £9. | Fes 4 bea | 3 49| 41 450, 000 | 9 26| 41 335, 000 re, PRES MRE ies i an 1] Peach....... Pee Ee eB 485, 000 2 ie 46| 38 420, 000 3 44.2 ute 440, 000 ! ese 5 48). S2e2 - 4 6 42] 44 430, 000 7 41] 46 435,000 | as a5 -|o anu, 480,000 | | 9 rr fs Loe 410,000 i SS Sn ee eee Ee ee CNS Sh te SE Te ee ae 1 | Chocolate... zoubet 142 | 22228 142, 000, 000 | 2 pe er Oe 123, 000, 000 | 3 137 | 107 | 122,000; 000 4 156 | 152 | 154/000; 000 5 120 | 123 | 126,500, 000 | 6 138] 135 | 136,500, 000 7 142 | 130 | 136,000; 000 8 143 | 130 | 136,500, 000 g 145 | 176 | 165,000, 000 6 | A (fat, 9.2 per cent, gelatin+) ... 1 Vanilla... ... 1 144} 178] 16,100,000 Sai 2 ae 13, 200,000 3 17,900, 000 4 20, 000, 000 5 217 250, 000 6 15, 200, 000 | 7 13, 150, 000 | | a8 27, 000, 000 lege 22; 200, 000. | | pilwmectag et oF. 1 102, 000, 000 | 2 97, 000, 000 ao | ese 108, 000,000 6 115,000, 000 { | | ee: 100; 500, 000 | Ree oF ee _.| 1 | Chocolate... 44,000, 000 5 40, 000, 000 | 3 42° 500, 000 Cee MMI ep 5 41, 000, 6 41,000, 7 42° 500, 8 37,000, g 46, 500, i Oe eee 1| Vanilla...... 3 121} 102] 1,115 2 reeee | 714 | 98 joe yes 120| 125| 1,995, 4 126| 106} 1,160, dang 105 | 113] 1,090, ‘i 87| 124| 15055, 7 134] 149| 13415, s 122| 125| 1,235, 9 | 137| 137] 13370, 2S I He | 1 | Chocolate... pa 170| 176] 1,730 2 abede Veet tae 1,610, 3 161 | 183 | 1,720, 4 179| 165] 1,720, | 5 162 | 192] 1,770, 6 em Perec 7 ii re 1/310, | g | 134} 143 | 1,635, g| - Tega ease 1,570, |

THE DETERMINATION OF BACTERIA IN ICE CREAM, T

The maximum and minimum bacterial count of the samples from each gallon of ice cream, together with the per cent of variation, is shown in Table II.

In the samples from Plant No. 1 there was a variation of 300 per cent among the samples from lot D, which showed a minimum count of 20,000 and a maximum of 80,000 per cubic centimeter. This case, as may be noted, is extreme and may be accounted for by the fact that the 80,000 count was obtained from one plate only, the duplicate being covered with spreaders. Reference to the complete results in Table I for this lot shows that among the other samples the colony counts were very nearly alike. A similar explanation holds for lot E from Plant No. 1, in which there was a variation of 81.81 per cent, with a maximum and minimum count of 13,000 and 7,150.

Throughout the rest of the samples the only high percentage of variation was among the samples taken from lot A, Plant No. 6, a variation of 105.32 per cent. Im this case the melted crean® was a thick, viscous mass, which made it difficult to measure accurately in a pipette. Special care was taken in the remaining determinations of bacteria in the ice cream from this manufacturer, with the result that the percentage of variation in bacterial counts was very low.

The general variation among the samples from each gallon of ice cream was from 20 to 30 per cent, which is decidedly low, although at first thought it may seem high.

In Reprint 295 of the Public Health Reports! it is stated that in analyzing duplicate samples of milk the general average variation in each of four laboratories ranged from about 110 to 380 per cent. Just what percentage of variation in duplicate counts is normal to the method of bacterial analysis we shall not attempt to say, but a variation of 20 per cent means only the difference between 100 and 120 colonies on a Petri plate.

The small variation in our results indicates that in the ice cream examined the bacteria were rather evenly distributed and that an analysis of one sample taken in the manner described would show for all practical purposes the bacterial content of any other sample in the 1-gallon lot.

1Conn, H.W. Standards for determining the purity of miik.

8

TaBLe II.— Variation in bacterial content of 9 duplicate samples from each of twenty-two

1-gallon lots of commercial ice cream.

BULLETIN 563, U. S. DEPARTMENT OF AGRICULTURE,

37.03 per cent.

Bacteria per cubic

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ae a centimeter. Pipe avor. lon in No. Lot. bacterial Minimum. | Maximum. | ©°U2t. Per cent. 1} As(fat,(95 per-eent). -:.425- e=2 2-2 Vanilla...... 2,085, 000 2, 530, 000 sept) Fs an ip gale, elated Sn tee Chocolate 445, 000 585, 000 Cd es Sere 8 9 ek. Se eee each... see< 1, 460, 000 1, 600, 000 Edita Sssapipa: Soest Nal ll go Vanilla 20, 000 80, 000 1 ee eee eee: See eee Vanilla... .- 7, 150 13,000 23 ig ot he Re, SR Se ee Vanilla....../ 43,500,000 61, 000, 000 3 5s 9 Sy args Sipe Set oe eae Base Chocolate...| 180,500,000 | 225,000,000 C (fat, 9.6 per cent)...........-- Vanilla...... | 60; 000, 000 75, 000, 000 | | 3 | A (fat, 9.6 per cent)..........--. Vanilla...... 251, 500 307, 000 Bee ee ee ee Cee See ee aS Peach: = 25-2 192, G00 221, 000 e Cee era Ae ee bess ss Chocolate 975, 000 | 1, 235, 000 4| A (fat, 22.5 per cent)......-..:.- Vanilla 27, 000, 000 33, 750, 000 Sea Some ene Be Share aes Peaches ere = 28, 750, 000 47,000, 000 Beret dst teas Vie ee yee ei Chocolate 28,000,000 | 33,000,000 5) |-& Cat, 17:per cent). 2255252222.0- Vanilla. 335, 000 490, 000 2 Bio paee 20 seh teva tapts Bean cee Peach....... 410, 000 500, 000 "ROE ees 0 ae Pe eee er a ee Srey ote Chocolate 122,000,000 | 160,500, 000 6 | A (fat, 9.2 per cent)...<........- Vanilla 13, 150, 000 27,000, 000 Feo Stan LONE Eat Selle aliy bela acl aa Peach. ...... 97,000,000 | 116,000, 000 OP sare beni fests att. Chocolate. . 37,000, 000 46, 500, 000 iD) gel pan aing ae rp RYU CS Vanilla... 1,055, 000 1, 415, 000 pal No rte spe ite ieee Soteaty Sap ake Chocolate 1, 310, 000 1,770,000

VARIATION WHEN HELD IN AN ICE-CREAM CABINET.

It was thought that there might be an uneven distribution of bacteria in ice cream held in an ice-cream cabinet where it is allowed to soften, then is repacked with ice and salt and again hardened. To determine this point, three 1-gallon lots of ice cream were pur- chased from three different manufacturers and held for 11 days in a commercial ice-cream cabinet, such as is used in stores. The ice cream was packed with ice and salt once a day in the regular way. From day to day it softened and again hardened. In Table III it may be seen that even under this extreme condition the highest variation among the samples from each lot was only

highest 1,420,000 per cubic centimeter.

In this case the lowest count was 1,080,000 and the

| THE DETERMINATION OF BACTERIA IN ICE CREAM. 9 | Tapes III.— Variation in bacterial counts of 9 samples of ice cream taken from each of | thrée 1-gallon lots which had been held in a cabinet for 11 days. Number ofcolonies| Average apups Plant | Sample | piution. on duplicate Bounit Variation between lowest and No. No. plates. per c. ¢. highest counts. 1 1 500 44 48 46,000 | Samples 5 and 7, 34.21 per cent. 2 52 43 47, 50! 3 ; 7 RS 46, 000 4 he ete Aa 48, 000 5 SMS Bok Oe 38, 000 6 43 46 44,500 7 Gi cea 51, 000 8 37 50 43, 500 9 gt ale a 43, 000 : 2 1 oa HAKONE Mgt eae Sa ee 1,080,000 | Samples.1 and 8, 37.03 per cent. | 2 ill 125 | 1,180,000 e 3 E52) re Aa GED 1, 120, 000 4 142 134 | 1,380,000 5 132 152 | 1,420,000 6 127 113 | 1,200,000 7 136 149 | 1,425,000 8 130 166 | 1,480,000 9 138 149 | 1,435,000 RS 5 1 Les 62 76 690,000 | Samples 2 and 5, 14.73 per cent. | 2 64 65 | 645, 000 3 76 61] 685, 000 4 ay ae ERD 650, 000 5 iE ge i 740, 000 6 65 77| 710,000 7 66 71 685, 000 8 Gir any: 660, 000 9 70 68 | 690,000 _ Keeping in mind the normal variation in bacterial counts, as indi- _eated in Table II, we do not believe that the results warrant the con- clusion that there is any great uneven distribution of bacteria in ice “cream, even when held under the extreme conditions of this experiment.

& ie

VARIATION WHEN HELD IN STORAGE.

In order to determine the effect of cold storage upon the distribu- tion of bacteria in ice cream, three 1-gallon cans were filled with ice cream from thé same freezer. Of these, one was examined while fresh, one was held in cold storage in a heaienine | room at a plant for one month, and the third was similarly held for two months. The results of this experiment, recorded in Table IV, show that there was no increase in the variation among the sees from each : “gallon lot, even after two months’ storage. The samples as a whole " checked renareably well, showing nothing to indicate any marked uneven distribution of poeta.

10 BULLETIN 563, U. S. DEPARTMENT OF AGRICULTURE. *

TaBLE 1V.— Variation in the bacterial content of samples of ice cream taken from gall lots held in cold storage.

Number of peat

= \Sam- : Average | Variation between acon ee le coro count lowest and highest

Z Oo. per c. c. counts.

Samples 1 and 9,

81.81 per cent. One month....-... 1 | TAS reise cre 11,800 | Samples 7 and 8, | 2 AO |o~ ose 10, 600 15.68 per cent. 2p Wh & be -hy ek ee 11, 200 = at HHS bias eg 11, 400 5]. 110} 112 11,100 Efe neta Sa eee 10, 400 = dos ke Wk fbr 11, 800 Be ae tee 10, y | 117 | 106 11, 150 Two months...... 1 76 77 7,650 | Samples 2 and 9, 2 66 76 7, 100 18.31 per cent. 3 76 76 7, 600 4 92 68 8, 000 5 79 82 8, 050 6 73 72 7,250 7 70 80 7,500 8 75 76 7,500 | 9 76 92 8, 400 |

VARIATION IN SAMPLES TAKEN DIRECTLY FROM FREEZER.

The question of whether a sample of ice cream taken from freezer represents in bacterial content the entire contents of tk freezer is one of considerable importance. To obtain informatic on this point 10 series of from 6 to 10 samples were taken from 4 large commercial freezers in an ice-cream plant. The samples we taken in the following manner: As soon as the cream was frozel and ready to flow into the final containers for hardening, the gat was opened and about 1 pint allowed to flow out. The first sampl of about 50 grams wasthen taken by allowing the partially frozen creal to flow into a sterile salt-mouth bottle. About 1 gallon of ice crean was then allowed to flow out and asecond sample taken. The remain ing samples were secured in a similar way, the last one being takel from the very last portion. All were immediately iced and take to the laboratory, where they were plated in the manner previoush described. The results are shown in Table V. q

‘THE DETERMINATION OF BACTERIA IN ICE CREAM, Toh

| Taste V.—Variation in bacterial content of samples of ice cream taken from the same freezer.

Number of Sam-| colonieson | Average} Variation between

Date samples ie qank : plicate counts | lowest and highest taken. Xo. plates (1/1000 | per ¢. c. counts. c. c. plated). 9-14-14. ........ 1 48 56 52,000 |} Samples 7 and 9, 2 66 47 | ~— 56,500 31.39 per cent. 3 48 46 47,000 4 60 43 51, 500 5 46 56 51,000 ' 6 51 50 50, 500 d 61 52 56, 500 8 52 48 50, 000 9 42 44 43, 000 9-15-14. ........ 1 DOI eceks a8 22,000 | Samples 4 and 5, 2 25 24 24, 500 29.54 per cent. 3 28 22 25, 000 4 29 28 28, 500 5 7 J Ea Nosh rd 22,000 6 23 26 24, 500 9-16-14........- 1 LOE : Sosy! 107,000 | Samples 6 and 7, 2 LOZ eee es 102, 000 7.84 per cent. 3 110 100 | 105,000 451923 118)|22 9 4 118, 5 LOAD ee eas 104, 000 6 LOZ ees 102, 000 7 TO} pe Sa 110, 000 9-17-14 (A)..... 1 58 55 56,500 | Samples 1 and 3, 2 GS} Ieee Li ae 53, 000 28.41 per cent. 3 44 }....... 44, 4 Gy 52, 000 5 53 52 52, 500 6 56 46 51, 000 7 Bot Sov: 53, 000 8 58 53 55, 500 9 5S lsees cee 53, 10 53, 49 51,000 9-17-14 (A)..... 1 B2s0 9s Ore 52,000 | Samples 3 and 6, 2 45 §2 48, 500 25 per cent. 3 53 57 55, 4 Apter 45,000 5 55 39 47,000 6 40 48 44, 7 ab 51 47, 500 8 48 50 49,000 9 42 47 44, 500 10 CW An hs eae 47,000 9-18-14 (B)..... 1 159 155 | 157,000 | Samples 3 and 10, 2 156 157 | 156, 500 35. 87 per cent. 3 167 189 | 178,000 4 164 162 | 163,000 5 126 173 | 149,500 6 149 144 | 146,500 7 157 160 | 158,500 8 157 154 | 155,500 9 130 160 | 145,000 10 IBLE eeoee hs 131, 000 9-18-14 (B)..... 1 186 160 | 173,000 | Samples 3 and 9, 2 LOTE| eerie 157, 000 30.07 per cent. 3 158 227 | 192,500 4 LIQ) isaaccisine 179, 000 5 1CGH ee ose 166, 000 E 6 1S ||Coo aod 185, 000 7 IB loose 2 178, 000 8 EGS] esses 165, 000 9 148) |. 224 sis. 148, 000 10 TGS cae 168, 000 9-19-14..... Rae 1 82 77 79,500 |} Samples 3 and 5, 2 eS llosongeas 83, 000 40.47 per cent. 3 94 83 88, 500 4 85 84 84, 500 5 55 71 63, 000 6 98 68 83, 000 7 Boy ae 82,000 8 83 75 79,

12 BULLETIN 563, U. S. DEPARTMENT OF AGRICULTURE.

It will be seen from the table that the highest variation among the samples taken from any freezer was 40.47 per cent, the minimum count

in that case being 63,000 and the maximum 88,500 bacteria per cubic —

centimeter. The lowest variation was 7.84 per cent, the counts in this instance ranging from 102,000 to 118,000 per cubic centimeter.

It is interesting to note that the two sets of samples marked A were taken on the same day from two different freezers, which were probably filled with the same ‘‘mix.’”’ The same is true of the sets marked B. In each of these cases the samples check well with each

Wt ee

other. From these results it seems evident that the bacterial con-—

tent of one sample from a freezer may be said to represent for prac- tical purposes the bacterial content of the rest of the ice cream in the freezer.

COMPARISON OF INCUBATION OF PLATES AT 37° C. FOR TWO DAYS AND 30° C. FOR FIVE DAYS.

The plates in our experiments were incubated at 30°C. (86° F.) for five days. Since in general practice plates are incubated at 37° C. (98.6° F.) and counts made after 48 hours, it was considered advisable to make a comparison between these two methods of incubation.

A gallon of ice cream was obtained and nine samples taken from nine different positions in the usual manner. Two sets of duplicate plates were made; one set was incubated at 37° C. (98.6° F.)} and counted after 48 Eee The other set was Segre at 30° C. (86°. F.) and counted after five days. The results of this experi- ment (see Table VI) show that the variation among the samples from the same lot of ice cream was 16.89 per cent after the 48-hour count at 37° C. (98.6° F.) and 35.11 per cent after incubation at 30° C. for five days. )

From this experiment it seems evident that incubation at 37° C, (98.6° F.) for 48 hours does not give counts which show any greater variation than those obtained by incubation at 30° C. (86° F.) for

five days. It is interesting to observe, however, that the count —

obtained by incubation for five days at 30° C. (86° F.) is practically double that obtained by incubation at 37° C. (98.6° F.) for 48 hours.

TaBLe VI.— Variation in counts obtained by incubation of plates at 87° C. for two days and 30° C. for five days.

Incubated at 37° C. for 48 hours. Incubated at 30° C. for 5 days. Sam- le Sap eeoe “ Average Variation between saa ‘af Average Variation between 9 | duplicate count lowest and highest aupligate count lowest and highest plates. per c. c. counts. plates. per c. c. counts.

1 77 785, 000 Samples 2 and 9, | 170 | 176 | 1,730,000 Samples 5 and 7, 2 | fh eae 740,000 16. 89 per cent. 161, 142... 1,610, 000 35. 11 per cent. Saute bol. 770, 000 | 161 | 183 | 1,720,000

4 | 76 775, 000 | 179 | 165 | 1,720,000

5 81 820, 000 162 | 192 | 1,770,000

6 75 785, 000 R74 Tees oe 1,740,

7 73 765, 000 ISL ae 1,310, 000

8 SO" fe oe , 000 184 , 635,

9 865, 000 AE Fig Re 1,570,000

; t i 4

THE DETERMINATION OF BACTERIA IN ICE CREAM. 13

THE NUMBER OF COLONIES MOST DESIRABLE ON PETRI PLATES.

When a sample of ice cream is plated it is of course necessary to make several dilutions, since the bacterial content is unknown. It is sometimes a question as to which dilution will give the most accurate count.

Some interesting facts regarding this pot are shown by reference to Table VII. Seven 1-gallon lots of ice cream were obtained, and nine samples from each were plated in the usual manner. Three dilutions were made, but only two are recorded in the table. The aim was to obtain plates with about 200 colonies, as in bacterio- logical work that number is believed to give the most nearly accurate count. The two recorded, therefore, are the counts obtamed from the dilution which gave about 200 colonies per plate and the counts from the next highest dilution.

An examination of the table shows that the variation between samples taken from the same lot of ice cream ranged from 15.10 to 105.32 per cent when the dilution was such that from 100 to 300 colonies were on the plates. The same samples at the next higher dilution, in which the number of colonies was less than 50, varied from 35 to 1,014.28 per cent. In every case in which there was a small number of colonies on the plates the variation between the samples from each gallon lot was decidedly greater than when a lower dilution was used. This fact is by no means new, but it should be kept in mind when interpreting the results obtained from a bacterial analysis of duplicate samples of ice cream.

Tasie VII.—Comparison of bacterial counts obtained from the same samples of ice cream but with different dilutions.

jierated Num- ee ion ion be- Sam-| pay. peariber A Average | tween ns rl Average | tween Lot. pile ree aamlicate count per | lowest | Dilution. an Gite count per | lowest 0. plates. Cyc: ypu plicate (Gh Foe ; 8 plates. nes counts. counts Per ct. Per ct. Ass 1.) se2500 243 | 229} 2,360,000 | 21.34! sos2000 27 | 30} 2,850,000 36. 36 2 219 |} 198 | 2,085,000 22 | 43} 3,250,000 3 242 | 243] 2,425,000 24 | 40} 3,200,000 4 231 | 245 2,280,000 36 | 24} 3,000,000 5 OF Ae 2, 500, 000 36 | 39 3,750,000 6 264 | 233 | 2,485,000 30 | 25} 2,750,000 7 Oe eens 2,300, 000 37 | 28 | 3,250,000 8 243 | 2451 2,440,000 36 | 22 | 2,900,000 9 271 | 235 |. 2,530,000 | 34 | 25 | 2,950,000 B.. 1 | sso 341 334 | 33, 750, 000 25.00 | assd:s00 | 41 | 31 | 36,000,000 2 274 266 | 27,000, 000 31 | 29 | 30,000, 000 3 DSA piecm aie. 28, 400, 000 30 |... -.] 30,000, 000 4 294 | 302 | 29, 800,000 30 |....| 30,000, 000 5 284 | 270 | 27, 700,000 33 | 36 | 34,500,000 6 301 | 303 | 30, 200,000 38 | 29 | 33,500,000 7 325 331 | 32,800, 000 38 | 44 | 41,000, 000 8 Spiele 8} 32, 500, 000 35 | 43 | 39,000, 000 9 STA en 31, 400, 000 35 | 46 | 40,500, 000 (Oasis Li. edoo 286 | 290 288,000 | 22.31 oLooo 42 | 53 475, 000 2, 360 | 254 307, Ate |b aa 410, 000 3 270 | 289 279, 500 40 | 33 365, 000 4 250 | 270 260, 000 31 | 42 365, 000 5 314 250 282, 000 38 | 39 385, 000 6 25t Ae 252 251, 500 30 | 33 315, 000 7 268 | 250 259, 000 32 | 38 350, 000 8 SU 287. 299, 000 32 | 44 380, 000 9 261 306 283, 500 35 | 52 435, 000

14 BULLETIN 563, U. S. DEPARTMENT OF AGRICULTURE.

TaBLE VII.—Comparison of bacterial counts obtained from the same samples of ice cream but with different dilutions—Continued.

2 270 | 305 | 28,750,000 35, 500, 000

3 386 | 372 | 37,900,000 30, 500, 000

4 278 | 305 | 29,150,000 29, 000, 000

5 367 | 356 | 36,150,000 34, 500, 000

6 354 | 357 | 35,550,000 35, 000, 000

7 431 466 | 44, 850,000 35, 000, 000

8 AONce ee 47,000, 000 45, 500, 000

: 9 ASA fe 5. Sk 43, 400, 000 50, 000, 000 EE... 1 202, 500 220, 000 105.71

2 214, 000 250, 000

3 206, 000 235, 000

4 192, 000 175, 000

5 203, 000 210, 000

6 198, 500 360, 000

7 210, 000 220, 000

8 218, 000 280, 000

9 221, 000 290, 000 1 30, 400, 000 | 16,000,000 | 131.25

2 28, 600, 000 28,000, 000 | -

3 33, 000, 000 21, 500, 000

4 29, 050, 000 25, 500, 000

5 28, 600, 000 29, 500, 000

6 31, 000, 000 33, 500, 000

“f 28, 200, 000 37, 000, 000

8 28, 000, 000 29, 500, 000 1 16,100,000 | 105.22 | sastaas 21,000,000 |1, 014, 28.

2 13, 200, 000 32, 000, 000

3 17, 000, 000 10, 500, 000

4 20, 000, 900 22, 000, 000

5 21, 250, 000 | 3, 500, 000

6 15, 200, 000 15, 000, 000

7 13, 150, 000 12, 000, 000

8 27, 000, 000 38, 000, 000

g 22, 200, 000 39, 000, 000

VARIATION BETWEEN DUPLICATE COUNTS FROM SAME SAMPLE AND SAME DILUTION.

As stated, we do not intend to say just what variation should be allowed between duplicate counts or a series of samples of ice cream from the same lot. Table VIII, however, shows some colony counts obtained by making a series of from five to eight plates from the same dilution. In five different samples a variation of from 7 to 26.6 per cent was found. Among the duplicate plates in the exami- nation of other samples of ice cream a variation as high as 41 per cent was found. From figures given by other investigators of the results of bacterial counts of ice cream, variations in counts between duplicate plates as high as 88 per cent have been observed. It is obvious that the factor of variation among plates from the same dilution of ice cream must be taken into consideration when inter- preting the results of the bacterial analysis of duplicate samples or a series of samples from a given mass of ice cream. To this factor of variation must be also added that caused by the difficulty of removing exactly equal quantities of different samples of ice cream in order to make the first dilution.

Oe

\

THE DETERMINATION OF BACTERIA IN ICE CREAM. 15

|: "ABLE VIII.— Colony counts on duplicate plates from the same dilution of a sample of

ine ace cream.

herd

lhe

fe Number of colonies on duplicate plates.

Whew Sample Varia- ba 0. tion.

| 1 2 3 4 5 6 7

[ Sa ro appt

es Percent Ie 1| 243| 229] 225] 208] 232 218 |......[...... 6 |e 2] 450] 460; 510} 470| 480) 470] 500{ 570 26. 6 in SiimozrneAsO2e| vaOsss SOOKe LOU ee|-- eek |oecees 7.0 he EG BOA a Oy PSS PEO ee ecalleee soalsesese 19. 4 te: 5] 144] 152 | PSS LTS a Sil 2 eS ee) | RE 23.6

‘without taking into consideration the total number of bacteria found in each sample. If, for example, it is stated that 1 cubic ‘centimeter of ice cream contains 75,000,000 more bacteria than another sample, the difference of so many millions conveys to the mind a marked disparity in the two samples. If, on the other hand, here is said to be a difference of only 750 or 7,500 bacteria per cubic

centimeter, we should immediately think of the samples as being

Taste 1X.—AHypothetical statement showing variable differences in bacterial counts of _ two samples of ice cream having a fixed ratio between colony counts and a fixed per cent _ of variation.

Colony | Dilution, | Baetoria | Difference |etwveen Per cent. ao fea ee ay 400 \ 750 | 25.00 gC \i alii eee ap ae } 7,500} 25.00 Ete 1 to 1,000....... nui 75,000 | 25.00 378 {hd to 10,000... ry bay we 750,000 | 25.00 370, |S to 100,000... 30° 000° poo |} 7,500,000 | 25. 00 375 |1 to 1,000,000... 300° 000 000 75,000,000 | 25.00 a \1 to 10,000,000 ..|{3> 660) 00” obo 750,000,000} 25.00

, 000, 000 |

Let us consider a hypothetical case, as shown in Table IX. We. will assume that one sample of ice cream shows an average colony “count of 375 and another 300. With a dilution of from 1 to 10, there would be a total count of 3,750 in one sample and 3,000 in ‘the other, a difference of 750 bacteria per cubic centimeter, and a Variation of 25 per cent between counts. If the same colony count were from a dilution of from 1 tc 100 the difference in the bacteria _

‘im the samples would be 7,500, 1 ~ the variation would be still the -

j 16 BULLETIN 563, U. S. DEPARTMENT OF AGRICULTURE.

o> Bee ee Gey

same, or 25 per cent. As the total count becomes still higher, neces- sitating higher dilutions, the difference in bacteria per cubic centi- meter increases until at a dilution of from 1 to 1,000,000 the difference would be 75,000,000 per cubic centimeter. |

A study of this assumed case merely serves to show fhat even though there is a difference of 75,000,000 between two samples of ice cream, there is no greater percentage of variation between the samples than when the difference was only 750 per cubic centimeter. It also shows, however, that a difference between counts expressed in bacteria per cubic centimeter should never be considered by itself but should be interpreted in relation to the total number of bacteria per cubic centimeter in each sample.

SUMMARY AND CONCLUSIONS.

The method of collecting samples and making bacterial counts used in our experiments gives results which indicate that bacteria in commercial ice cream are distributed quite evenly and that an analysis of one sample from a gallon of ice cream gives results which will hold for any other similar sample from the same gallon.

Storage of ice cream for 11 days in a commercial ice-cream cabinet or in a hardening room for a period of two months did not seem to cause an uneven distribution of bacteria.

In a series of from 5 to 10 samples taken directly from a large commercial freezer the bacterial counts on each sample checked within the usual limits of error of bacterial analyses.

No greater variation in bacterial counts between samples was observed when the plates were incubated at 37° C. (98.6° F.) for 48 hours than when incubated at 30° C. (86° F.) for a period of five days.

When dilutions were such that about 200 colonies were present on the plates a lower variation between counts of samples of ice cream was found than when there were 50 or fewer colonies per plate.

The variation between a series of plates made from the same sample and dilution was found to range from 7 to 26.6 percent. Among du- plicate plates a variation as high as 41 per cent was observed. This must be remembered in connection with the fact that the variation found in our experiments between average counts of different samples of ice cream from the same gallon lot ranged, generally speaking, between 20 and 30 per cent. To this variation between duplicate plates or a series of plates from the same dilution must be added the error introduced in removing 1 c.c. portions of ice cream from different samples. |

When interpreting bacterial counts, differences in the number of bacteria per cubic centimeter should never be considered except in relation to the total count of each sample.

WASHINGTON ; GOVERNMENT PRINTING OFFICE : 1917

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