night blue and victoria blue as indicators in lipolysis media

NIGHT BLUE AND VICTORIA BLUE AS INDICATORS IN LIPOLYSIS MEDIA

BY AUDREY JONES AND T. RICHARDS

Department of Microbiology, The University, Reading

SUMMARY: Optimum conditions for the use of night blue and victoria blue es lipolysis indicators in fat emulsion agar medium required a dye strength of 1 : 15,000 in a medium of p H 8.0 containing 5% fat dispersed by hand shaking. Pour plates should contain 6 ml. and streak plates 10 ml. of the medium in a standard Petri dish. Incubation should be for 5 days a t 30”. The pinkish-mauve medium with clear blue-zoned lipolytic colonies gave the same results as butter fat agar without dye but treated with CuSO,, when tested with 963 pure cultures. The inhibitory powers of the dye were msessed and although strongly toxic in the aqueous phase to Gram-positive bacteria, victoria blue appears to have none to slight inhibitory power in the fat agar medium: night blue suppressed growth to about the same extent as tributyrin. The lipolytic flora of butter and to some extent milk shows a remarkable dominance of micrococci. Organisms lipolytic on fat agar media are able to produce appreciable acid in a fat emulsion in a liquid medium.

AN ideal medium for the detection of lipolytic micro-organisms should clearly differentiate between lipolytic and non-lipolytic types. When the hydrolysis products are water-soluble, as in the case of tributyrin, lipolysis is readily detected by the formation of a clear zone around the colonies in the turbid fat-emulsion agar. Unfortunately the ability of an organism to hydrolyse tributyrin does not imply that the organism can break down other and more complex natural fats. Colony counts of tributyrinolytic organisms may give no real indication of the rancidifying flora of natural fats, such as butter and cottonseed and olive oils.

Many of the fatty acids of the natural fats are insoluble in water, and although changes in the fat emulsion may be noted around lipolytic colonies, usually as an increased granularity and opacity, they are not very marked and can escape detection unless examination under lox to 50x magnificat,ion is used. In such cases an indicator is needed to reveal the changes to the naked eye. The formation of soaps by treating the incubated plates with an aqueous saturated solution of copper sulphate, or with strong dye solutions, greatly aids differentiation, but the colonies are thus rendered unfit for further cultural study by mutual contamination during flooding and washing and by the toxicity of such solutions to many micro- organisms.

Knaysi (1941) stated that effective indication of fatty acid liberation may be obtained by dissolving the bases of aniline dyes in the fat before it is incorporated in the agar medium. The acids combine with the relatively weak-coloured bases to give strongly coloured salts. We find this method has a very limited application, owing to the toxicity of many dyes, and even where there is no obvious inhibition

Dyes as indicators in lipolysis media 83

the colour differentiation is often less marked than in the cases where the dye is used in aqueous form.

The most successful kind of indicator is that which, like nile blue, is able to impart to the fat phase a colour which contrasts with the colour of the dye salts. In the case of nile blue a basic component dissolves in the fat phase at a slightly alkaline pH and imparts to the fat emulsion agar an orange-red colour against which the strong blue zones around lipolytic colonies show up very clearly. It is therefore unfortunate that this dye is highly toxic to many Gram-positive bacteria, including the significant staphylococci. Methods described by Knaysi to rid the medium of its inhibitory effect have proved unreliable in our experience, but further investigations are proceeding on this matter.

During the course of an investigation into the use of bases of the triphenylmethane series of dyes as lipolysis indicators, Richards (1946) discovered that both night blue and victoria blue exhibited a ' double staining ' property similar to that of nile blue. Aqueous solutions of these dyes are deep blue, but when neutral or slightly alkaline aqueous solutions were shaken with xylene, petroleum ether, or molten fat, what appeared to be the dark red base of the dye was extracted by the non-aqueous phase, giving i t a deep red colour and leaving the aqueous phase colourless or a t the most very pale. In the same way, when 10 ml. of nutrient agar containing 1 : 10,000 of the dye were shaken with 0.5 ml. of butterfat or olive oil, the medium changed to a pink-mauve shade. It was a t once apparent that the dyes might have possibilities as lipolysis indicators if they were free from the inhibitory characters of nile blue, and the investigations described below were made to secure data on this point.

EXPERIMENTS AND RESULTS

Preliminary experimental work

Dye solutions. Victoria blue (B.D.H.) dissolved readily in 1 : 15,000 concentration in distilled water, but night blue (B.D.H.) required the addition of a few drops of concentrated HC1 and warming before solutions of this strength could be obtained. Stock solutions of the dye containing 1 g. ofhowder in 250 ml. of distilled water were made, with HC1 addition where necessary, and after filtering the volume was made up to 1,500 ml. with distilled water. This particular strength of stock solution was made because preliminary experiments had shown that a final concentration of dye of 1 : 15,000 in the medium afforded the best compromise between good colour differentiation and possible inhibition, and the stock solution would thus be added in the convenient proportion of 1 : 10. For small-scale work, the solutions were sterilized by autoclaving, but when used for bulk media the unsterilized solutions were added to the medium before dispensing and sterilizing.

Pat substrate,. Fresh hand-churned butter made from ripened cream and slightly salted was used throughout the work. The butter was melted in an incubator a t 55" and filtered through paper. The resultant curd-free oil was sterilized in an autoclave a t 15 lb. for 20 min. Small amounts of fat were prepared a t frequent intervals

84 Audrey Jones and T . Richards

so that it was never more than 4 weeks old when used. Preliminary experiments established that when the fat was dispersed by hand shaking, a concentration of 5% (v/v) of fat in the medium gave optimum results.

The p H of the medium. With increase in pH above neutrality there was increasing depth of reddish colour in the medium after shaking, and the agar had to be a t least pH 7.8 before the fat phase would effectively take up the bases of the dyes, As several of the laboratory cultures tested were markedly inhibited above pH 8, that value gave the best compromise between colour and growth. Experiment showed that to obtain a final pH of 8 a t the incubation temperature of 30°, the medium had to be adjusted to pH 7-8 before sterilization.

The quantity of medium. Basal medium (see later) was tubed in quantities from 4 to 10 ml. and after sterilizing, appropriate quantities of dye and butterfat were added to give final dilutions of 1 : 15,000 and 5% respectively. The cooled, shaken media were poured into dishes containing a standard inoculum of a mixture of a non-lipolytic Gram-negative rod (Bact. coli) and a lipolytic Gram-positive coccus (Micrococcus sp.). After 5 days incubation a t 30°, it was clear that the Micrococcus sp. produced the best zones in the depth of medium given by 4 ml. in a Petri dish of 87 mm. internal diameter. The 5 and 6 ml. quantities gave results almost as distinctive, and had the advantage of not drying out during incubation as did the 4 ml. plates. It was therefore decided to use 6 ml. of medium for future work with pour plates. For surface streak plates of pure cultures, such thin layers gave no advantage and the usual 10 ml. quantities were dispensed.

It was thought that the enhanced lipolytic effect in shallow layers may have been due to the influence of oxygen tension. Indeed, in the anaerobic jar a lipolytic micrococcus grew but failed to give any indication of lipolysis, either as colour change or as physical change in the fat globules around the colonies. Oxygen supply thus might be an important factor in lipolysis and the point is being investigated further.

Preparation and use of media

Preparation of cowLplete media. The basal medium was made of peptone, 1%; yeast extract, 0.3%; sodium chloride, 0-5%;fagar, 2%, and was adjusted to pH 7.8. Before sterilization, 100 ml. of stock dye solution was added to each litre of basal medium. When the butterfat was added to the bulk medium, it was found impossible to keep it sufficiently dispersed to ensure a correct aliquot in each tube of medium, since the fat formed a top layer as dispensing proceeded. The medium, less fat, was therefore tubed in 6 or 10 ml. quantities and 0.3 or 0.5 ml. of molten butterfat added. The complete medium was then sterilized by autoclaving.

The 10 ml. amounts in 6 .< 8 in. tubes were frequently found to soil the cotton wool plugs when heated at 15 lb. pressure and so 10 Ib. for 20 min. was adopted as a routine measure.

Experiments were made on media in which the fat was emulsified by passing through a hand emulsifier of the domestic sort. In this type of medium, in which the globules have an average size many times smaller than in the hand shaken media,


the zones of lipolysis were smaller and paler. This effect of lipase on the smaller and more highly dispersed fat globules was unexpected and more work is to be done on this point.

Dispersal of fat before pouring. In tubes of sterile complete media the butterfat existed mainly as a surface layer and the fat had to be dispersed before pouring the plates. The tubes of media were melted, cooled to about 48" and fitted with sterile rubber stoppers. After fairly vigorous shaking the tubes were allowed to stand for a few minutes a t 45-48' to allow the froth to subside before their contents were poured. The amount of shaking naturally affected the degree of dispersion, and in experiments where the tubes were shaken with about one foot excursion from as many as 50 times to as few as 10 in 10 sec., 20-30 times produced the optimum effect, the zones being larger and more intensely coloured than in media shaken more or less often in that time.

Poured plates took on a pinkish-mauve tint, although it was sometimes necessary to incubate them for a little while a t 30" before the bluish coloration disappeared. This was probably due in part to the reaction becoming more alkaline on warming.

Incubation. Plates were incubated for 5 days before lipolysis was recorded, though it should be remarked that with most of the powerfully lipolytic organisms, such as staphylococci, a clear result was discernible in as little as 18 hr. at 30". Lipolysis was recorded as positive when a distinct blue zone was seen below or around the colony.

The inJluence of storage. With complete media that had been stored for some time less intense results were obtained and it became necessary to find the useful storage life. Media made with fresh and old dye stock solutions and used a t once gave identical results and no difference was seen after two and three months storage between media stored at about 4", a t about 15" and a t 30", but there was clearly a deterioration in these stored media after two months as compared with media freshly prepared. No deterioration was apparent after one month and the procedure was therefore adopted of keeping comple$e media in storage a t room temperature for not more than one month.

The effect of light. The poured medium was found to be markedly light sensitive and its pinkish-mauve colour faded in a day or so when exposed to daylight. Lipolytic zones remained blue much longer but the colour contrast in the medium was much reduced. Poured plates kept in the dark retain their original colour for much longer than a week and it is thus advisable to incubate and store them away from light.

Other media for comparative tests. For comparative tests tributyrin agar (Specification, 1940), butterfat agar for copper soap formation (Berry, 1933) and, in one experiment, nile blue agar were used. In each case the basal medium was the same. For tributyrin agar, 1 % of tributyrin was emulsified in a domestic hand emulsifier. Butterfat agar for the Berry method was prepared just like the night blue and victoria blue agars save that the dye was omitted and in the nile blue agar the final dye concentration was 1 : 10,000. The media were used as described above and lipolysis was judged by a clear zone around the colonies in tributyrin agar and as permanent colour changes in or around the colonies in the other media.


The inhibitory eflect of night blue and victoria blue

Tests on agar media. Before night blue or victoria blue agar could be adopted for routine surveys of lipolytic floras it was important to show that they possessed none of the inhibitory properties that mar nile blue media. In a preliminary experiment bacterial suspensions were diluted in Ringer's solution so that 1 ml. gave a countable plate on the basal medium. One ml. portions of the suspension were plated in triplicate on the basal medium, on butterfat agar, on basal medium with 1 : 15,000 night blue and on butterfat agar with 1 : 15,000 night blue. The experiment was repeated with victoria blue instead of night blue and the colonies were counted after 5 days a t 30". The results, which are given in Table 1, show that a t 1 : 15,000, either dye in the absence of butterfat was completely inhibitory to the micrococci but had no effect upon the Gram-negative bacteria. In the presence of the fat, the inhibitory effects of the dyes were almost eliminated, two of the micrococci, (3.5 and C.22, giving counts on the dye-butterfat media slightly less than on butterfat medium alone. The lower counts on the butterfat agar compared with basal agar may have been due partly to the difficulty of seeing minute colonies in the opaque and granular medium.

Table 1. The effect of butterfat, night blue and victoria blue on the numbers of colonies developing from standard inocula of five bacteria

Composition Mean no. of colonies/plate with culture* of medium I A >

c.5 c.11 c.22 B.2 B.18 Basal agar 127 83 178 50 101

126 80 107 44 109 Basal agar with 5 yo butterfat Basal agar with 596 butterfat and 103 74 120 41 103 1 : 15,000 night blue

0 0 0 54 98 Basal agar with 1 : 15,000 night blue

Basal agar 109 35 17 1 242 104

90 21 51 225 102 Basal agar with 5% butterfat Basal agar with

1 : 15,000 victoria blue 5% butterfat and 73 15 56 246 101

0 0 0 252 108 Basal agar with 1 : 15,000 victoria blue

*Cultures :-C.5, yellow micrococcus; C.ll, white micrococcus; C.22, orange micrococcus; B.2, Bacterium coli; B. 18, Achromobacter species.

l'ests in liquid media. An attempt to gain more quantitative information depended on establishing a stable emulsion in a liquid medium containing 5% butterfat and 1 : 15,000 dye, so that the rate of growth from a standard inoculum could be compared with that in a dye-free emulsion. This is probably more stringent, as an

Dyes as indicators i n lipolysis media 87

indication of inhibitory effect than whether a given cell or group of cells will form a visible colony on a solid medium.

Neither agar (0.1%) nor gelatin (0-5 to 5%) yielded a stable emulsion even after passage through a hand emulsifier, but 0*570 of gum tragacanth gave an emulsion which remained visibly stable for about 2 hr. and after separation it re-emulsified on shaking. It was first established that gum tragacanth itself did not inhibit growth a t 0.5y0. Then standard inocula of about 3,000 organisms were added to 5 ml. of basal broth in which was emulsified 5% butterfat with 0.5% gum tragacanth and in broth containing the same emulsion plus 1 : 15,000 night blue or victoria blue. One ml. of the inoculated emulsion was plated a t once to find out if an adequate inoculum had been introduced. The emulsions were incubated for 10 hr. a t 30°, being shaken every 2 hr. to re-establish the emulsion, and then the cultures were plated on basal agar. The results, given in Table 2, show that the Gram- negative rods grew equally well in all three emulsions. However, while one Micrococcus species and one Corynebacterium grew as well in the presence as in the absence of the dyes, the other three micrococci failed to increase ,and, indeed, the colony count was smaller than a t the start. The other Corynebacterium failed to grow even in the absence of the dyes. It seems that the dyes were more inhibitory to the growth of some Gram-positive bacteria in the liquid phase than to colony formation on agar.

Table 2 . The influence of two indicator dyes ( 1 : 15,000) on the development of bacteria in liquid gum tragacanth-butterfat emulsion media*

Culture r

Micrococcus aureus I Micrococcus aureus I1 Micrococcus conglomeratus Micrococcus flavus Corynebacterium species I Corynebacterium species I1 Achromobacter species I Achromobacter species I1 Bacterium coli

No. of organisms/ml.t after 10 hr. at 30" A

\ No dye Night blue Victoria blue

511):106 405 x lo6 454 x 10' 281 x lo6 44 36 205 x lo6 171 209 297 x lo6 252 - 400 x lo6 380 x lo6 - 111 0 0 350 Y 103 280 x 1 0 3 310 x lo3

190 x 106 2 1 9 i lo6 - 270 Y lo8 214 A lo8 254 x lo8

* Level of inoculation c. 600 organismslml. t Counted on basal agar.

The lipolytic flora of butter and milk. Further evidence about the inhibitory nature of the dye media under normal working conditions was sought by quantitative studies on the flora of 14 samples of butter and 8 of milk.

The standard technique was followed of plating three 1 ml. portions of each of a series of decimal dilutions in Ringer's solution. The media used were:-basal agar, butterfat agar with and without 1 : 15,000 night blue or victoria blue, and tributyrin agar. The plates were incubated at 30" for 5 days, when separate counts were made of all colonies, and of those that were lipolytic. The results are given in Table 3, from which it can be seen that within normal experimental limits the colony counts on the butterfat media with or without dye were not markedly different from those


on basal agar: they were, however, rather lower in some cases, especially in the case of night blue butterfat agar. Comparing plain butterfat agar with basal agar, in 22 samples 8 were slightly higher on butterfat and 14 lower. Comparing the dye- butterfat media with plain butterfat media, on night blue agar all 22 counts were lower than the plain butterfat but with victoria blue, 7 were higher and 15 lower. The results of tributyrin agar compared with basal agar show on the whole much lower counts, only 1 sample giving a slightly higher count. Broadly speaking, these tributyrin counts were lower to about the same extent as night blue agar. This inhibitory effect of tributyrin has been previously remarked on (Wolf, 1940). At the same time, the proportion of colonies which were lipolytic was clearly much higher than on the other media, a fact which has also been commented on in the past. The percentages of lipolytic colonies in the dye media were similar to those on butterfat agar treated with copper sulphate, which we have hitherto regarded as the most reliable medium.

Table 3. Colony counts and percentages of lipolytic colonies in 14 butter and 8 inilk samples

Sample

Butter 1 2 3 4 5 6 7 8 9

10 11 12 13 14

Milk 1 2 3 4 5 6 7 8

Colony count on*? w >

B. T.B. B.F. N.B. V.B. 63 38 85 37 58 97 71 126 68 76 85 41 112 55 39 66 55 58 51 63 56 54 50 48 58 78 60 77 73 74

188 138 157 146 170 177 143 166 76 169 197 162 175 142 177 5 1 52 39 6 19 84 79 87 29 55 43 27 32 21 36 89 78 82 76 79 67 52 54 15 44

Percentage of lipolytic colonies on

T.B. B.F. N.B. V.B. 7 A >

100 86 67 13 74 57 5

72 96 79 91 85 54 77

2 2 1 20 28 24 14 14 17 0 0 0 6 10 16 4 5 3 2 2 2 6 3 3 04 93 92 12 60 21 24 72 52

0 0 0 2 3 2 8 53 16

79 13 76 70

164 117 241 130 51 39

108 51 103 81 80 13

82 76 17 61 12 17 29 70 48 56 63 30 25 61

101 94 94 85 33 39 26 151 136 172 95 36 50 28 56 51 39 87 9 10 18

102 75 82 71 26 9 9 109 83 91 81 19 11 14 92 53 79 77 15 20 23

* Media: B., Basal agar; T.B., Tributyrin agar; B.F., Butterfat agar (CuSO, treatment);

t The figures for any one sample give the counts for plates of the same dilution. N.B., Night blue agar; V.B., Victoria blue agar.

Since micrococci had been inhibited in the tragacanth-fat emulsion experiment, and since they were believed to be significant members of the lipolytic flora, more than 25 colonies taken at random from each medium were tested for morphology and Gram-reaction. The proportions of these which proved to be Gram-positive cocci are given separately in Table 4 which reveals the dominance of Gram- positive cocci in the lipolytic flora of butter and milk. The majority of these colonies


were large and pigmented and were all catalase positive when tested with H,O,. Streptococci had previously been shown not to produce visible colonies on butterfat or tributyrin media, so there can be little doubt that the figures in Table 4 refer

Table 4.

Sample

Butter 1 2 3 4 5 6 7 8 9

10 11 12 13 14

Milk 1 2 3 4 5 6 7 8

The percentages of Gram-positive cocci in the lipolytic jiora of 14 butter and 8 milk samples on four media* Tributyrin

agar 60 58

100 0

84 58 90 80 98 50 82 0

25 59

90 12 21 16 98 90 88 70

Butterfat agar

96 96

0 96

100 96

. 80 97

100 100

0 100 100

64 12 48 32 94

100 70 90

-

Night blue agar 100 100 100

0 96

100 100 89

100 100 100

0 100 100

83 28 30 32 87 97 76 84

Victoria blue agar 98 96

100 0

96 92 96 87

100 100 100

0 100 100

100 12 55 24 92 97 79 90

* The results are based on 25 or more random colonies from each medium.

mainly to micrococci (including staphylococci). The large proportion of cases where all the 25 or more lipolytic colonies tested were micrococci is most striking, and in our experience species of Gram-negative rods such as Pseudomonas and Achromo- bacter, which have been reckoned of such importance previously, cannot be of much significance owing to the relative infrequency of their occurrence. However, the absolute dominance of micrococci was seen more often in butter than in milk; the butterfat media with or without dyes revealed absolute or almost absolute dominance of the micrococci in all the butter samples except two, where they were completely absent. Dominance of micrococci was less striking on tributyrin agar and this may be connected with the higher percentages of lipclytic colonies on this medium.

The lipolytic activities of surface streaks of pure cultures

Young broth cultures of many of the organisms isolated from butter and milk and some of the cultures from the laboratory collection were inoculated as streaks on the surface of the same lipolysis media as were used in the previous experiment, the object being t o establish the value of the media for use in a diagnostic test for lipolysis. The plates were incubated as before, and the results of tests on 962 cultures are given in Table 5, the organisms being grouped in broad categories since the


freshly isolated strains were not fully identified. There was very good agreement between the proportions of organisms showing lipolysis on the butterfat media, both with and without dye indicator, and again tributyrin gave much higher percentages of lipolytic organisms, this effect being most marked with the micrococci, the aerobic sporeformers and the yeasts. All the lipolysis media were unsuitable for the streptococci tested; this may have been due to the lack of fermentable carbohydrate.

Table 5. The incidence of lipolytic activity among pure cultures as revealed by four media

Type of culture

Micrococci Aerobic

Yeasts Pseudornonas

species Other Gram-

negative rods Streptococci

sporeformers

Number tested

730

46

51

31

94

10

Percentage of cultures showing lipolysis on*

T.B. B.F. N.B. V.B. 89 53 53 53

54 24 26 26

100 4 4 4

' 35 35 36 35

19 14 14 14

0 0 0 0

Total cultures 962 752 426 428 428

* Media: T.B., Tributyrin agar; B.F., Butterfat agar treated with copper sulphate; N.B., Night blue agar; V.B., Victoria blue agar.

Correlation between lipolytic action on agar media and production of acid rancidity in a fat emulsion

So far it has been assumed that lipolytic organisms produce a visible permanent change in the fat globules below and around the colonies growing on a fat emulsion agar. To check the truth of this assumption, 18 cultures representing Gram-positive and Gram-negative cocci and rods of both ' lipolytic ' and ' non-lipolytic ' kinds were cultivated on the usual lipolysis media (including in this case nile blue'agar) and also grown in a butterfat emulsion where their ability to produce acid W&B

determined. The emulsion was prepared from a mixture of fresh whole butter, 350 g.; KH,PO,,

1 g.; peptone, 1 g.; agar, 2.5 g.; and tap water (to 1,000 ml.) by heating to dissolve the agar and passing through a hand emulsifier. Quantities of 50 ml. were run into 6 oz. wide mouth bottles, plugged with cotton wool and sterilized in the autoclave. After cooling to 45", the emulsion was inoculated with 1 ml. of a young broth culture and incubated for 5 weeks a t 30". The titratable acidity was determined by extracting 25 ml. of emulsion and adding 50 ml. of ethanol (45% vjv aqueous) and 4-5 drops of phenolphthalein, and titrating with alcoholic 0.1N KOH.

Table 6 gives the results, the titratable acidity being expressed as ml. of 0.1N KOH/10 g. of butterfat. With few exceptions there was a good correlation between


the titratable acidity and the changes in the lipolysis media. The cultures fell into two broad groups, in which the acidity produced was greater or less than 10 ml. of 0-1N acid/l0 g. of butterfat. Of the 7 cultures in the first group, only 1, a micrococcus, was non-lipolytic on the 4 media on which i t grew, while of the 11 feeble acid producers, only 1 was positive on butterfat agar and 3 on tributyrin, with another 1 doubtful on the latter medium. The limitations of nile blue as a lipolysis indicator are clearly seen; none of the Gram-positive bacteria was able to grow.

Table 6. The association between acid producing pouter in butterfat emulsion and lipolytic activity in 18 pure cultures

Culture Acid? Growth and lipolytic activity on* (ml. 0.1N , h

KOH/10 g;. Tri- Butter-

Bacterium coli 1 Bacterium coli 2 Flavobaeterium Pseudomonas 1 Pseudomonas 2 Pseudomonas 3 Achrwmobacter Corynebacterium 1 Corynebaeterium 2 Bacillus subtilis Bacillus mycoides Micrococcus 1 Micrococcus 2 Micrococcus 3 Micrococcus 4 Micrococcus 5 Micrococcus 6 Micrococcus 7

butterfat7 butyrin fat

2.4 1.4 6 .O

10.0 25.0 92.0 28.5 4 .O 2.6 3 .2 3.5

16.0 3.5 5.5 4.0 7 .O

75.0 46 .O

7-7 G C

++ - ++ - t - ++ ++ ++ + ++ + ++ ++ + - + - ++ ++

++ - f - ++ + + - + + ++ ++ ++ ++

_ _

r-----h- G C

++ - ++ - + - ++ + ++ + ++ tS ++ + ++ - i - ++ - ++ - ++ - + - ++ + + - ++ -

++ +t _ _

Nile blue

7-L-

G C + - + - f - ++ ++ ++ ++ +t ++ ++ + _ _

Night blue

r A - - G C

++ - ++ - + - ++ + ++ + ++ + ++ A ++ - ++ - ++ * ++ - ++ - + - ++ 3z ++ - ++ - ++ ++ ++ ++

Victoria blue

G

++ ++

+

++

* G, growth; C, colour change. t after 5 weeks at 30’.

The effe$ of glucose in night blue agar

Stark & Scheib (1936), in their experiments with nile blue, used a medium with 0.5% of glucose. This addition would certainly enhance the growth of many micro- organisms on the fat emulsion media and would make possible the growth of lactic acid bacteria, but the inclusion of a fermentable carbohydrate might give rise to false positive reactions from the acid produced from the sugar.

Experiments were made on night blue agar with and without 0.5% of glucose which was added as a sterile aqueous solution. Tests were made using streaks of 10 non-lipolytic and 2 lipolytic cultures. Of the former, which included 4 streptococci and 1 lactobacillus, the 6 which produced acid in glucose peptone water also produced a blue zone on the glucose night blue agar but no colour on the same medium without glucose. The 4 non-lipolytic, non-glucose-fermenting organisms gave no colour change on either medium. The lipolytic species, only one of which


fermented glucose, gave similar colour changes on each medium. It is clear that the presence of fermentable carbohydrate in the medium, although giving enhanced growth of the lactic acid and many other bacteria, is most undesirable and may be responsible for an entirely false picture in lipolysis tests.

The use of dye bases

Knaysi (1041) claimed that inhibition by many indicator dyes could be avoided by incorporating the base of the dye in the fat before it was emulsified in the agar. In extensive unpublished work with a number of dyes we were unable to confirm this, and experiments were made to ascertain whether night blue and victoria blue could be usefully employed in this way.

The stained butterfat was prepared by mixing equal volumes of molten fat and O*lyo aqueous night blue and adding 2 ml. of saturated aqueous sodium carbonate to each 100 ml. of mixture. The mixture was well shaken in a separating funnel and when the fat had risen the aqueous solution was run off. The fat was well washed with hot water, filtered and sterilized. The product, which had a deep red colour, was incorporated in 5 ml. of basal medium in quantities from 1-6% and streaked with test cultures. The medium containing 5% of fat gave the best results but even so, some of the cultures grew less well than on the normal night blue medium and in these cases the colour changes were not so striking. As these stained-fat media are a little more troublesome to prepare than the normal medium there seemed little advantage and this method was discontinued.

DISCUSSION

From the foregoing results it seems clear that both night blue and victoria blue, particularly the latter, are satisfactory as indicators of lipolysis in butterfat agar plates and that they have advantages over other indicators which have been used in the past. Care must be taken to adjust the pH of the medium correctly; if this is done the blue-zoned lipolytic colonies stand out well in the pinkish medium and are easy to detect. The dye media proved equal to butterfat agar tested by copper soap formation for the detection of lipolysis, while not suffering from the latter’s defects of the mutual contamination of the colonies and often of the marked toxic effect upon the organisms of the strong copper solutions used. Although both dyes are inhibitory a t 1 : 15,000 in aqueous solution to Gram-positive bacteria, the results suggest that a t pH 8.0 and in the preserice of 5% butterfat emulsified in an agar medium there is no more than slight suppression of growth of these organisms.

Measurement of the ability of micro-organisms to produce acid rancidity in fats is a matter of some technical difficulty, but the titratable acidity formed by pure cultures in a liquid fat-emulsion medium did correlate well with ‘ lipolysis ’ on fat agar. While the results may have been influenced by the products of phosphate or peptone utilization it is unlikely that the broad picture was thereby affected. It is realized that the production of acid in such an emulsion does not mean that the same organisms would cause rancidity in butter, nor does it mean that high-acid


producing strains would lower the keeping quality of butter more than those forming little acid in the emulsion. The whole question of acid rancidity in fat is most complex and much work remains to be done.

The dominance of Gram-positive cocci in the lipolytic flora of butter and milk was a t first surprising. It may be that these organisms have been overlooked from time to time in the past, owing to the use of inhibitory nile blue media.. Of these cocci, it is clear that the most active fat-splitting organisms are the glucose-fermenting, acetoin-forming strains of micrococci, which Abd-el-Malek & Gibson (1948) group together as the ' Staphylococcus group ', although not all of the strains which belong to this group that we have examined are lipolytic. The character of lipolysis is not correlated with coagulase production, for out of 59 coagulase positive staphylococci examined 19 were feebly lipolytic or not a t all and all these were from the bovine udder. However, many of the pathogenic staphylococci produce very strong lipolytic action on agar containing emulsified fat, the effect being noticeable in some cases after overnight incubation.

Quite apart from their use in the quantitative examination of the lipolytic flora of food products, media such as those described should be used in preference to tributyrin agar in lipolysis tests for diagnostic work. The production of lipase seems to be a constant character in many groups of organisms and a great deal more use could be made of it in classification. The results with tributyrin are far too equivocal t o be reliable. Butterfat, being widely available and reasonably constant in composition, would be a suitable substrate. The present-day difficulties in the supply of butter might make the use of olive oil more attractive. Several experiments were done using sterile olive oil, B.P., and although extensive comparative tests have not been made excellent results were obtained using a 5% concentration. Even more desirable in many ways is the use of a chemically defined fat and, to the same extent as olive oil, triolein has been tested in a 5% concentration with very good results.

REFERENCES

ABD-EL-MALEK, T. & GIBSON, T. (1948). Studies in the bacteriology of milk. 11. The staphylo-

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(Received 22 April, 1952)

night blue and victoria blue as indicators in lipolysis media

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