A STU?) " OF SOLED MEDIA WITH ifl'fiRTZCULAR REFERENCE TO ‘E‘ECHNIQUES OF‘ EVALUATING CONS‘TETUENTS Thesis fur the Degree cf M. S, MZCHIGAN STATE. COLLEGE Irving Oliizky 1947 may? C rfim?2 y...9...1 .2: ”.7 392 A STUDY OF BJLID MLDIA fiITfi PA TIJULAR REFLFVEC TO TECHNIQUES OF EVALURTIEJ CQfiSBITUpHTS By _ IRVING ourzxx A TRESIS Submitted to the School of Graduate Studies of Michigan State College of Agriculture and Applied Science in partial fulfillment of the requirements for the degree 0! MASTER OF SCIENCE Department of Bacteriology 1947 BACTERIOLOGY DEPT. V177“? Cl..- ACKN CELLS £431}: I with to express my ainoore thanks for thy very nblo assistance and advice given me by Dr. w. L. Eallmann. The Author 199255 Ifitroduoum.................. 1 Stmiiin on standani Agar for Water Analysis . .. . 7 Shaina on Standard Agar for wiry Products. . . 22 8m................ .....27 Karena”...a...css.caoc.o¢oooz9 INERODUCTIOQ In all bacteriological techniques which use solid nutrient media, the media involved should be those which.are best suited to grow the organisms or organism in question. This is important when the bacteria to be grown are fastidious in nature and especially important 'when the medium is used for purposes of enumerating the numbers of viable organisms in any substrate. There are many instances where the quality of the substrate. such as milk, water, or food is determined by the bacterial count. In these cases the medium used should be one which will most accurately measure the total number of viable organisms in the substance tested. Yet in many cases the nutrient solid medium in use is not the most efficient simply because no concentrated effort has been made to test the comparative value of the medium. The problem of determining the efficiency or the existing media formulae or of new formulae is one of great complexity. The problem is relatively simple when liquid media are being evaluated because here one can use growth curves and generation time as a basis for evaluat~ ion. The method most generally used for solid media is one where the ability of a particular medium to grow out the viable organisms from any source is compared to the ability of another medium to do the same. Since this - 1 - involves the plating method of enumerating organisms the errors involved are those which are inherent in the plat- ing procedure. In 1902 De M. Gage and Adams compared plate counts from various classes of water on agar made with Merck's peptcne and Witte’s peptone. They also compared the relative dev010pment of pure cultures of bacteria on standard gelatine, laurence agar, and Nahrstoff agar.- They used water suspensions from fresh cultures. mater suspensions from cultures which had been kept two days on ice. and water suspensions from thirty day old cultures. They found that Witte’s peptone in agar gave higher counts from water samples. Cook (1916) plated twenty soils on four agars and incubated the plates up to five days, He found that all soils do not behave in the same manner toward the different media. In the last decade a tremendous amount of work was done on evaluation before tryptone-glucose-skimmilk agar was introduced as standard agar for the examination or milk and diary products. (STANDARD METHODS FOR THE Ex- :{INATION OF DAIRY PRODUCTS) (1941) Typical of the work was that done btholts and Martin (1938) on the comparison of ‘ the old standard and new standard agar'ss media for deter- mining the bacterial count in ice cream. Two hundred and seventy-nine samples of vanilla ice cream were plated on .2. the two agars. The authors calculated the geometric average, arithmetic average, and ratio of new standard agar counts to old standard agar counts. The results clearly show the superiority of tryptone-glucose-milk agar to the old standard agar. Abele (1939) as referee on the Committee on standard methods for the examination of milk and dairy products of the A.P.H.A. presented a detailed history of the work done before tryptoneoglucose-milk agar was accepted as stlrfiard agar for dairy products, with twenty-two re- ferences on all aspects of the milk plate count including .ths effect of variation in temperature of incubators, the effect of variation in pH and composition of media. the effect of different plating techniques, etc, He discussed the complexities of using the plating method in evaluating solid nutrient media. Mallmann and Breed (1941) compared the standard agar for water’snalysis with the new standard milk agar for determining bacterial counts in water. A total of six hundred and fiftyofour water samples from various sources were plated on the two agars. The results indicate that the new milk standard agar gives counts comparable with the agar’in use for water*ana1ysis. Leifson (1943), in a study on the preparation and prOperties of bacteriological peptones, prepared three casein peptones and compared them with various commercial .3- peptones. Growth tests were made by preparing a l per cent peptone agar with 0.5 per cent NaCl at pH 7.1-7.3. The agar was poured into petri dishes and the bottom ofthe plate divided into six sections by means of a wax pencil. Twenty-four hour cultures of the bacteria were diluted one leapful to 5 ml. water and one leapful streaked on a section of the plate. Observations were made after 24-48 hours of incubation. Both the size and relative numbers of colonies were recorded. Twentyofour different organisms were tested on the various agars. The findings indicate that with most bacteria the usual 1 per cent concentration of peptone is fur from Optimum as regards the amount of growth obtained. Several experiments using the casein peptones in concen~ 'trations of 0.5 per cent to 10 per cent showed the Optimum concentration of all three of these peptones (casein) to be somewhere in the neighborhood of 8 per cent. However. the Optimum peptone concentration is lower in infusion media than in the media without infusion. Hook and Fabian (1943) studied the influence of the typfi of peptone on the bacterial plate count of raw milk: They prepared various peptones from both animal and vegetable sources and substituted these peptones for the tryptone in Standard Milk Agar. Raw milk samples were plated on the modified agars using Standard Milk Agar*as a control. They observed that some of the peptones from vegetable sources gave higher*plate counts than Bacto-tryp- - 4 - tone but were inferior to Bacto-tryptose. Peptones pre- pared from spleen. heart. and pork were found to be superv ior to Bacte-tryptone in their ability to grow organisms from raw milk. In the following work a study was made on some of the existing methods in use for evaluating solid nurient media. The work was done in two phases. Part I was devoted to studies on Standard Agar for water analysis. Part II to studies on Standard Agar for diary products. In both phases the standard agars now in use were subjected to various modifications and these modified agars were used as a basis for the evaluation studies and also sets means for improv« ing the standard agars now in use. An.attempt was made in this work to adapt the Frost ”little plate” as a means of evaluating plating media. Frost (1915) (1916) described a method of counting viable organisms in milk which on the surface had some advantages over the standard plate count. The method consisted of mixing 0.5 ll. of milk with.0.5 ml. of the nutrient agar which had_previously been melted and cooled to 50°C. One tenth offla milliliter of this mixture was spread over a # sq. cm. area on a clean. sterile slide. The plate was allowed to harden and then incubated in a moist»chamber'at 31° for.4.8 hours. The plate was then dried in an oven - under 100°6. and stained with alcoholic methylene blue. A count was made of the licroscOpic colonies and with the -5- apprOpriate factors the number of organisms in the milk sample could be determined. Frost and other researchers claimed that the "little plate" gave comparable results with the standard plate count. The chief advantage of this method was the savings in laboratory equipment and media. Another advantage was the saving in time as a milk count could be made in 4-8 hours. It was envisioned that the Frost method could be used in evaluation studies as it is relatively simple to measure colony lle on the Plittle plate”. Theoretically r y a medium which is ”nutritionally” better than another ”medium would produce larger colonies at any point of the develcpment of the colony. An experiment was set up in an attempt to utilise this method. Part I Studies on Standard Agar for Water Analysis W8 The first experimental work was done using stand- ard agar for water analysis as a base. (STANDARD MhTHODS FOR THE EXAMINATION OF WATER AND SEHAGE) (1936). This medium contains peptone in a concentration of 0.5 per cent. Darby and Mallmann (1939) in a study on media for coliforn organisms observed that when they varied the Baoto- peptone concentration in a liquid medium. a 2 per cent con- centration of the peptone gave the best growth with Egghgzr 193i; 393; and e. 3 per cent concentration showed a slight inhibitory effect. A comparison of Beets-peptone and Baoto- tryptose was made and much more rapid growth occurred with the Bacto-tryptose. When the concentration of tryptose was altered the highest growth rates were obtained with s 2 per cent and 3 per cent concentration. To see if these same relationships would hold true in a solid medium the follow- ing experiments were set up: To study the effect or the concentration of peptene in the plating media three modified agars were prepared using standard agar as a base and altering the concentration of peptone. The concentrations used were 1 per cent peptone. 1.5 per cent peptone. and 2.0 per cent peptone. Armour Pep- tonum siccun was used. Over's time interval or about two weeks 50 samples of river water were plated on standard agar and the three - 7 - modified peptone agars. The results are tabulated in Table A. The samples were grouped according to the number of colonies found on standard agar. The arithmetic mean for each group and for the total of 50 samples is shown. Using the efficiency of the standard agar as 100 per cent the relative efficiency of the three modified agars was calculated and shown for each group and for the total. In the following roport the modified agar will be referred to as “standard ear” and the modified agars will be called by the concentration and type of protein nutrient used. i.e.. agar where l per cent peptone has been substituted for 0.5 per cent peptone in the standard formula will be called '1 per cent peptone agar". etc. The data for Experiment I indicate that a l per cent concentration of peptone in the plating sodium is the Optimum concentration. The l per cent peptone agar proved to be 26 per cent more efficient than standard car. 10 per cent more efficient than 1.5 per cent peptone agar.= and 65 per cent acre efficient tan 2 per cent peptone agar on the total of so samples. Of the three modified peptone agars only the 2 per cent concentration of peptone gives an agar which is not more efficient than standard ear. The 1 per cent peptone agar was most efficient when the polcny cent in standard agar was between one 299. The same is true of the other two modified agars. Only in this range did the - 3 - an no: US «an I... I: 3: I: 83 8 .58 in an: in :8. 88 I! In. 88 a: . fed. 83 in 3.: 39 as: on. g al.— Sen .= :3 a 88:88 E g ‘3 g 3. 8o. a Is I! a 89-8. .3. ‘3 En la 5 . .3 .8 .3 a: o 818- g is as. 88 .3. 2.. 8. .8 .3 .a. .53 a} use [.4 .34 a: use . 5...... 3a... 3 I35 8.25 slicer-ale in“: lane-reload i u- ! 8 i ‘9" .3 Res no... an n can «suns- deln maze: 33-: , cub-3g. iglflu‘sa‘eg' iillfiiigggtflga 2 per cent peptone agar show greater counts than standard agar. . ' Although it is not shown in Table A. there were 5 samples in the higher plate count ranges where standard agar gave higher counts than any of the three modified agar. This can be attributed to either plating error or to the difference in the flora of these 5 samples. E32631 megs ;; 3 To study the effect of using a different protein hydrolysate nutrient in the plating media, four agars were prepared using Bacto-tryptose as a substitute for Armour peptone. aThe concentrations used were 0.5 per cent to compare with standard agsr'and l, 1.5. and 2.0 per cent. Forty-nine river water samples were plated on the four agars. The results are tabulated in Table B. The samples were grouped according to colony count on 0.5 per cent agar'and the arithmetic mean and per cent efficiency were calculated and.shown as in Table B. Here the counts on 6.5 per cent tryptose agar were used as ioo per cent. It can be seen that the data would indicate that the most efficient of the four modified tryptose agars is the l per cent tryptose agar. This agar was 19 per cent more efficient than 0.5 per cent tryptose agar. 51 per cent more efficient than 1.5 per cent tryptose agar. and 62 per cent more efficient than 2 per cent tryptose agar. 'However in the case of the tryptose agar the l per cent concentration - 1g - Aggfigggsfiilflgfio gzflfitggflfiga missus-na- is most efficient when the colony count is four hundred or over. The efficiency decreases as the colony count gets lower. The efficiency of the 1.5 per cent tryptose. which is in all cases lower than that of 0.5 per cent tryptose agar, decreases as the count increases. Two per cent tryptcse agar acts like the l per cent agar in the respect that its efficiency increases as the colony count increases. It is interesting to note that the increase of the concentration of tryptose in a tryptose agar in no way produces the same magnitude of effect as when the peptcne concentrat- ion was increased in a peptone agar. This is especially apparent in the case of the 1.5 per cent agars. The 1.5 per cent peptcne agar produced higher plate counts than standard 0.5 per cent agar. In the case of the 1.5 per cent tryptose agar the counts were lower than the 0.5 per cent tryptoae sediul. W: To compare 1 per cent peptcne agar with l per cent tryptose agar, 28 river samples were plated on both agars and also on standard agar. The results are tabulated in Table 0.. The efficiency of the two modified agars was calculated on the basis of 100 per cent for the arithmetic mean of the standard agar. Table 0 shows the counts obtained when 28 river water samples were plated on standard agar and the two most - 12 - The Bacterial Count of 28 Samples of River Water as Determined by Plating on Standard Agar and Two Modified Agars Plate Count Standard Agar 1% Peptone Agar 1% TryptoncAgar Plate Count Plate Count 99 520 980 1.150 100 ‘” 1.180 1.0” 101 580 1.050 1,120 102 no 1.050 1,260 103 . 460 1.270 1,020 104 390 680 690 105 .too 660 670 106 370 .630 640 107 I090 750 810 108 430 600 670 109 350 ~ 430 370 110 280 530 ‘We 111 280 560 has 11.2 270 3‘10 320 - 113 sec 500 sec 11h 3&0 1.020 960 . 115 320 900 710 116 310 3‘0 250 117 310 a 360 118 {a 260 119 620 sec 120 150 1&3 £00 121 370 30 a a: :43 a 124 m 500 sec 125 #30 Ass 270 126 330 600 520 Anne- 383 672 627 e on f Effi cine: 175$ 164$ -13- efficient modified agars. The arithmetic average on the counts on the total numbers of samples gives the l per cent peptone agar'an efficiency of 175 per cent compared with 100 per cent of standard agar and 16e per cent of l per cent tryptose agar. All the samples showed higher counts on 1 per cent peptone agar than on standard agar. however. 3 samples showed higher counts on the standard agar than on 1 per cent tryptcse agar and 9 samples had higher colony counts on 1 per cent tryptose agar-than on 1 per cent peptone agar. This very clearly shows the necessity for plating large numbers of samples when the plating method is used to evaluate solid media. W: To determine the effect of the time of incubat- ion on the plate counts of river water using standard agar, the l per cent peptone. and l per cent tryptose agar. five samples were plated and colonies counted at the end of 7. 18, 2‘. and 48 hours. ihble D presents the data showing the effect of time of'incubatian on the colony counts using standard agar and the two best modified agars. Standard agar gave higher counts after 7 hours incubation on all 5 samples. On all samples but one. 1 per cent peptone agar proved its super- ierity at the end of 18. 2e. and 48 hours incubation. Sample 12‘ gave the highest counts on 1 per cent tryptose agar at the end of 24 and #8 hours of incubation. - 14 - The Effect of rise of Incubation on the Colony Count of River Water Plated on Stand- ard Agar’and rue Modified Agsrs 1.} Peptone 1.; Tryptose Agar Agar Plate Count Plate Count 4L 2 2 A50 410 620 I080 970 900 13 93 273 133 £121 2‘ 150 310 200 *8 230 see too 13 263 as: 13 3 #122 24 370 560 430 ‘8 710 1,030 910 13 273 433 3 2 #123 2‘ 270 540 328 48 750 1,. 160 590 1; s 343 7 ‘ 20 1121. 2a 260 m 390 48 810 730 930 W‘ A study was made of the relative efficiency of standard agar. l per cent tryptose agar. and l per cent peptone agar in demonstrating the growth curve of a pure culture of E. coli. g: flask of‘peptcne broth was seeded with.a 24 hour culture of E. egg; and the initial pepulation determined by plating on standard agar and the two modified agars. The broth was incubated at 37° and at the end of 8. 24, and as hours the bacterial pepulation was again de- termined by plating on the three agars. In Table E is tabulated the data obtained when standard agar and the two best modified agars were used to determine the number of organisms in a flask of peptone broth which has been seeded with a pure culture of E. gel}. Figure I is a graphical presentation of the comparative growflh curves obtained by plating on standard agar and l per cent peptone agar. It is interesting to note that on standard ”agar the count remains the same at the end of 2h-and 48 hours. The counts on 1 per cent peptone agar would indicate that the organisms have entered the death phase sometime after 24 hours. The counts on 1 per cent tryptose agar would indicate that the growth phase is still in existence between as and 48 hours. W8 using the Frost little plate technique a comparison‘- was made of Standard Agar and five modified agars. The - 15 - able E The Growth Rate of E, coli in Broth as Determined by Plating on Standard Agar and Two Modified Agars ' Standard 1% Peptone 1% Tryptose Agar Agar Agar ===I=: 4‘0 #86 4A7 118,000,000 1Ao,000,000 139.000.000 24 370,000,000 .h50,000,000 360,000,000 #8 370,000,000 410,000,000 380,000,000 -17.. 2‘. of III-here of bacteria Comparative Growth Curves of E, 0011 in Peptone Broth es Determined by Plating on Standard Agar and 1% Peptone Agar ___Btendard Agar 1S Peptone Agar -18- method used was as follows: A suspension was made in saline of a 21; hour agar slant culture of g, con, The suspension was diluted to a concentration which had previously been determined to give a colony count which was in the proper range for count- ing, Che-half milliliter amounts of this suspension were added to eqml amounts of the six agars which had previously been melted and cooled to 50°C. One-tenth milliliter amounts to the agar-suspension mixture were spread a: four square centimeter areas on clean sterile slides, Five sets were made for each agar, The ”plates” were allowed to harden and then incubated in a moist chamber at 37°C. At intemls of 2, e, 6, 8. and 24 hours one set each of the different agars were removed from the moist chamber and dried, in an even at 80°C, When dry the "plates'I were stained with alcholic methylene blue, washed with water, and dried. The microsccpe used was calibrated for use with three objectives of the microscope. The “little plates” were examined and the colonies counted in 25 to 50 fields. The sise of 25 to 50 colonies was measured with the ocular micrometer, The results are tabulated in Table 1' with the average colony sise in millimeters and the colony count per plate given for the six different agars at the various incubation times, -19.. The Gospariscn of Standard Agar and Plate" Five Modified Agars by the Frost Little Colony Bise (millimeters) ' d '5‘ 1‘ 1'5, e5 7‘ 1‘ 1e” Incubation Poptone Rptone Peptme Tryp ' Tryptosa'hyptose 11-0 m? AG” 591' Agar Agar 101’ #F: v i? ' 2 Eoure .012 .015 .008 . .005 .007 .001 It Hours ‘ .011 .038 .009 .01e .025 .008 6 Hours .096 .056 .011 .m .982 .016 ' s Beers, .10 .110 .015 .088 .099 .015 2‘ 30m ’ .162 ' .181 so” smz e172 e098 Colony (but .5; 1,5: .5} . 11 1.51 Incuba Peptme W086 W6 Tryptose 2 am 21,500 28,800 18,200 18,000 20,300 10,000 1» Hours 21.700 35.000 18,200 19,500 21,000 12,800 6 am 28.500 42,900 22,600 19,700 22,300 14,500 8 30m 28.500 ‘2’” “.000 19.800 22.” 15.100 steam-c 30,500 113,200 41,200 26,0m 22,900 18,700 Of'the six agars tested with the Frost "little plate" method the greatest colony size was produced by the l per cent peptone agar, This same agar also gave the largest colony count of the agars tested, In this ”'respect the results of this experiment agree with the results obtained when the standard plate count was used in the evaluation studies. However, not all the data obtained with the "little Plates” are in agreement with the previous data, The colony counts on the 0.5 per cent peptone agar were at all periods of incubation higher than that on the l per cent tryptose agar, This is in direct contrast to the results obtained in the previous experiments. The difference in substrates might very well account for the lack of agreement of some of the results obtained, 'In one case a pure culture of an organism was used, in the other case the flora of the substrate was quite variable, All the inherent errors present in the standard plate count technique are magnified in the “little plate'e method as the quantity of inoculum used is much smaller. The Frost ”little plates" seem to be of value when colonies are to be measured, but any quantitative work based on colony counts is cpen to the same criticisms that are applicable to the standard plate count, I -21- Part II Studies on Standard Agar for Dairy Products Egpegimegt !§;: In a survey of the literature concerning the evaluation of Tryptone-Glucose-Extract agar’no mention was found of any attempt to increase the concentration of the protein in the accepted formula. The concentration of tryptone in the TGE formula is 0.5 per cent. If the same relationship holds true for the TGE agar as does for the standard agar for water analysis. and increase in the concentration of the tryptone would increase the efficiency of this agar. This was tested in the following experiment: Two batches of Difeo TGE agar were made up and to one was added Bacto-tryptone to produce a final concsn~ tration of l per cent. Fifty samples of silk. including both raw and pasteurized samples. were plated on both agars. fable G presents the data obtained. The plate counts were tabulated both on.a total basis of the 50 samples and on the basis of raw or pasteurized samples. Increasing the concentration of the tryptone in the TGE formula from 0.5 per cent to 1.0 per cent resulted in.a agar’which gave higher colony counts from both raw and pasteurized milk samples. If the average count on TGE is considered 100 per cent then the efficiency of the sodified TGE agar for the 50 milk samples was 123 per cent. For the ..27 raw milk samples the efficiency of the modified agar was -.‘ 276 per cent and for the 23 pasteurized milk samples the -22- W The Comparative Plate Counts Obtained by Plating 50 Milk Samples on T.G.E. Agar and a Modified T.G.E. Agar no.3. Agar 1'.G.E. Agar plus .5? tryptcne Number of Arithmetic Geometric Arithmetic Geometric samples lean mean mean mean 50 376.000 24.000 464.000 29.000 Analysis of Above Data on the hsis of Type of Milk Samples 1.6.32. Agar 'f.G.E. Agra Plus .5% tryptonei Huber of samples Arith. mean Arith. mean 27 (raw milk) 645,000 1.790.000 23 (pasteurized mm) 59.000 . ' 77.000 -23- efficiency was 130 per cent. W: Another modification of TGE agar was tested with 2} milk samples. This modification consisted of increasing the tryptcne concentration to l per cent as was previously done. and also increasing the concentration of beef extract from 0.3 per cent to 0.6 per cent. The milk samples were plated on standard TGE, the modified agar used in Experiment VII, and the new modified agar. The results are shown in Table 3. ‘ The results obtained when the concentration of both tryptone and beef extract were increased indicate that thismodification is a more efficient plating medium than the standard TGE agar and the first modified agar. ' The ratio of the counts on the double modified agar to the counts on standard TGE agar was l.‘2 and the ratio of the counts on the double modified agar to the counts on agar where Just the concentration of tryptone was increased was l.l#. The addition of an extra 0.} per cent of beef ex- tract probably introduces small ascunts of growth stimulat~ ing substances which.would account for the higher colony counts on the modified my. W | a ._ The third and final modification consisted of adding buffer salts to TOE. The concentration of salts The Comparative Plate Counts Obtained by Plating 23 Milk Samples on T.G.E. Agar and Two Modified T.G.E. Agar. T.G.E. Agar T.G.E. Agar T.G.E. Agar P1“. p1“. e5% trip- .5% trypteme tone plus .3% beef extract J 23 109.“ 8.6% 136.003 11.000 155.000 12.700 :32;g_1 The Comparative Plate Counts Obtained by Plating 18 Milk Samples on T.G.E. Agar'and Buffered T.G.E. Agar ‘ T.G.E. Agar 1 Buffered T.G.E. Apr E:; . Number of Arithmetic Geometric Arithmetie Geometric samples mean mean mean mean 18 617.000 152.000 402.000 119.000 - 25 - .added was as follows: 0,‘ per cent KaliPO‘ This medium was used to plate out 18 milk samples using standard TGE agar as a control. The results are shown in Table I. r ’ All of the 18 samples plated on the buffered TGE agar produced lower plate counts than when the samples were plated on the standard agar. The efficiency of the medium was lowered when the buffer salts were added. s possible explanation of this may be made on the basis that the organisms normally found in silk.are favored by a pH on the acidic side and the buffer salts would to some extent keep the hydrogen ion concentration near its initial value. The relationship of buffer salts to plating media efficiency should be checked further before any definite conclusion can be drawn. The use of the Frost ”little plate“ was fairly successful in the evaluation of the modified agars in the first part of this work. The use of the ”little plates“ to evaluate the modifications made on TGE agar met with‘ no success. Repeated attempts failed to produce results that were comparable to those obtained by the plating . method and even failed to produce results that were consist- ent in themselves. ’ H w -25.. was: A l per cent concentration of peptone substituted for the 0.5 per cent concentration of peptone in standard agar for water analysis produces a plating medium which is superior to any of the other modifications tried. The l per cent peptone agar exhibits greatest efficiency when the colony count on standard agar from river water samples is between 0 and 299. ' In the experiment where the effect of time of incubation was studied, 1 per cent peptone gave higher counts at 18. 2e, and A8 hours. f Standard agar gave the highest counts at the end of 7 hours. A The l per cent peptone agar gave higher colony counts from samplings in all growthlphases of m. Measuring'the colony sise of W by the Frost "little plates” further showed the superiority of a l per cent concentration of peptone. . In the modifications on TGE agar a superior ' plating medium was achieved when the concentration of tryptone in the formula was increased to l per cent. Increasing the concentration of beef extract to 0.6 per cent again improved the mgdiul. ' a The addition of buffer saltsto the formula of TGF: agar is a detriment to its efficiency. . The plating method of evaluating nutrient solid if ' - 27 - media is a laborious process which only gives good results when a very large number of samples are tested. It is also quite important to utilise samples which contain varied flora. .t more correct evaluation of the medium is obtained when the samples used most closely resemble the type of flora for which the medium is to be used. If by increasing the nutrients in a plating medium higher colony counts are obtained it may be assumed that either more of the same organisms are developing in the medium or that different organisms are growing where they would not grow before. Either of these developments is important since the ultimate aim in a plating medium which is used for quantitative work is the ability to grow all the viable organisms in the sample. 'REEi Abele. C. A. "Results of Bacterial Plate Counts of Milk on Three Media at Two Temperatures of Incubation.” we ug. Public fie gign g2. 821-46. (1939) - Cook, R. 0. "Quantitative Media for the Estimation of - Bacteria in Soils.” ggg:._§agt..l. 101, (1916) Darby, C. H. and Mallmann, w. L. "studies on Media for Coliform Organisms.” 193:. gm . gate 2 works Ass. 11, 689-706. (1939) De M. Gage, S. and Adams, G. 0. ”Studies of Media for the Quantitative Estimation of Bacteria in Water and Sewage.“ JQQr. Infect. Qig.ll, 358, (1902) Felts. V. 0. and Martin. w. H. "Comparison of Tryptone- Glucose-Skimmilk Agar and Standard Nutient Agar as Media for Determining the Bacterial Count in Ice 0ream."19ur. Dairy 591- 2;. 289-9#. (1938) Frost, u. D. "Rapid Method ofCounting Bacteria in Milk.“ We figs 255'255s (3-915) ~ Frost. w. D. "A Rapid Method of Countng Living Bacteria in Milk and Other Richly Seeded Materials." £231. We fie 889-890, (1916) Hook, A. E. and Fabian, F3 w. "Chemical and Bacteriological Studies on Peptones. Tech. Bull. 185, Michigan Agricultural EXperiment Station. (1943) Leifson. E. ”Preparation and Preperties of Bacteriological Peptones." Bu 1;. go ohns Hopkins Hogp..12. 179-99. (194)) Mallmann. w. L. and Breed, R. B. "A Comparative study of stuflard Agars for Determining Bacterial Counts in hater." WW )1. N?.4 .(1941) (J 3m- 5m, stgngggg Math o e minatio of Del Produc 8th Ed. Am. Public Health Ass. (l9g1) S n rd Method; for thew Examine 10 of w n ew th Ed. Am Public Health Ass. E1933). -29.. 'l b- H'J a L “5~i-' MIG 151949 NOV231953 .'_ 199255 ."1,'”‘ ‘ Olitzky THESIS I. OLITZKY M.S STUDY OF SOLID MEDIA WITH partiCULAR REFERENCE TO TECHNIQUES OF EVALUATING CONSTITUENTS 1947 MPH DATE , ! ROOM ’ DUE I BORROWER 5 NAME NUMBER —_ FT HT VT J __ r "5 guano HEALTH MICHIGAN STATE UNIVERSITY Ll BR