DEMONSTRATION OF PHOSPHATASES AND LIPASE IN BACTERIAAND TRUE FUNGI BY STAINING METHODS AND THE EFFECT

OF PENICILLIN ON PHOSPHATASE ACTIVITY1

MILWARD BAYLISS, DAVID GLICK, AND ROBERT A. SIEMDepartment of Bacteriology and Imm'unology and the Department of Physiological

Chemistry, University of Minnesota, Minneapolis, MinnesotaReceived for publication November 7, 1947

A considerable amount of work is being done on the histochemical localizationof some enzymes in the tissues of higher animals and plants. However,al-though bacteria have been frequently studied for a few isolated substancessuch as glycogen, lipids, nucleic acids, and protein, no determined effort hasbeen made to analyze these lower forms of life for their various enzyme systemsby histochemical methods. The localization of enzymes and chemical con-stituents in bacteria offers possibilities of determining further details of theirinternal anatomy, and may help to shed light on the problem of the bacterialnucleus. In addition, such studies may aid in classifying microorganisms aswell as in giving a better understanding of their physiology.

This communication describes the results of applying the methods of Gomori(1946 a, b) for the localization of acid and alkaline phosphatases and lipase tobacteria and true fungi. The effect of penicillin, in concentrations sufficientto halt growth, on the acid and alkaline phosphatase stains of certain bacteriawas also studied, and the findings were checked with the quantitative effect ofthis antibiotic on the phosphatase activities in the culture suspensions of thesame organisms.Roche et al. (1946) reported that the exposure of dialyzed intestinal tissue

extracts to divalent cations such as those of magnesium and manganese in com-bination with alanine resulted in an activation of the alkaline phosphatasepresent. Accordingly, in this investigation magnesium and manganous ionsalong with alanine in each case were added separately to two lots of agar mediain order to determine the effect of these agents on the staining, not only of thealkaline phosphaitase, but also of the acid phosphatase and lipase in the bac-teria grown on these media.

EXPERIMENTAL PROCEDtJES

Bacterial smears were prepared in the usual manner by allowing a suspensionof the organisms in distilled water to dry on microscope slides. The slideswere not heated. After drying they were placed in a Coplin jar full of acetonefor 10 minutes, to be fixed. The acetone was rinsed from the slides with distilledwater, after which they were placed in a 0.1 M citrate buffer, pH 4.7, to remove

1 The work reported in this paper was made possible by grants from the Smith, Klineand French Laboratories, Philadelphia, Pennsylvania, and from the Medical Research Fundof the Graduate School of the University of Minnesota.

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naturally occurring inorganic phosphates and other substances that mightgive a positive test. Thus any staining that was obtained was due to enzymeactivity alone. The wash interval may vary, but 0.5 of an hour was found to besufficient. The slides were then removed, washed thoroughly, and placed inthe substrate solution to be incubated overnight. During the incubation periodthe enzyme, if present, hydrolyzes the substrate, releasing an anion that isimmediately precipitated, in situ, by cations in the substrate media. Theprecipitate is then converted to a compound that can be visualized in the micro-scope.The principle of the Gomori method is as follows: The alkaline phosphatase

activity is visualized by precipitation of the phosphate, which is liberated fromthe substrate, as the calcium salt; this compound is converted to cobalt phos-phate, and finally transformed to cobalt sulfide, which is black and can be easilyseen. In the case of acid phosphatase, lead is used in place of calcium becausecalcium phosphate is soluble at the lower pH values used. Lipase is visualizedby the formation of a precipitate of the calcium salt of the high molecular weightfatty acids that are set free by the enzyme action. This precipitate is thenconverted to lead sulfide in the same manner as for phosphatase.

Alkaline PhosphataseReagents:

(1) Citrate buffer. Add 2 volumes of 0.1 M hydrochloric acid to 8 volumes ofcitrate solution (21 g of citric acid plus 200 ml of 1 M sodium hydroxide made upto 1 liter with distilled water).

(2) Substrate. Combine 25 ml of 2 per cent sodium glycerophosphate,2 25ml of 2 per cent sodium barbital, 50 ml of distilled water, 5 ml of 2 per centcalcium chloride, and 2 ml of 2 per cent magnesium sulfate. Add a few drops ofchloroform and store in icebox. The stored solution will not deteriorate forseveral months.

(3) Ammonium sulfide solution. Add 2 or 3 drops of the ammonium sulfidesolution to a Coplin jar of distilled water.

(4) Cobalt nitrate solution. Two per cent aqueous solution.

Procedure:(1) Suspend organisms in distilled water on microscope slides and allow to

dry.(2) Fix in acetone for 10 minutes.(3) Rinse thoroughly with distilled water.(4) Immerse in citrate buffer for 30 minutes.(5) Rinse thoroughly with distilled water.(6) Place in the substrate medium overnight.(7) Rinse thoroughly with distilled water.(8) Immerse in the cobalt nitrate solution for 5 minutes.2 A product of the Eastman Kodak Company (52 per cent alpha- and 48 per cent beta-).

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19481 PHOSPHATASES AND LIPASES IN BACTERIA AND TRUE FUNGI

(9) Rinse thoroughly with distilled water.(10) Immerse in ammonium sulfide for 1 or 2 minutes.(11) Rinse thoroughly with distilled water.

Acid PhosphataseReagents:

(1) Citrate buffer. Same as for the alkaline phosphatase.(2) Substrate. Combine 6 ml of acetate buffer (30.3 ml of 1 M glacial acetic

acid plus 68 ml of 1 M sodium acetate made up to a liter), 5 ml. of 0.1 M leadnitrate, 37 ml of distilled water, and 2 ml of 3.2 per cent sodium glycerophos-phate.8 Make fresh each time.

(3) Ammonium sulfide. Same as for the alkaline phosphatase.

Procedure:

(1) Suspend organisms in distilled water on microscope slides and allow todry.

(2) Fix in acetone for 10 minutes.(3) Rinse thoroughly with distilled water.(4) Immerse in citrate buffer for 30 minutes.(5) Rinse thoroughly with distilled water.(6) Place in the suibstrate medium overnight.(7) Rinse with distilled water 5 or 6 times at 5-minute intervals.(8) Immerse in ammonium sulfide for 1 or 2 minutes.(9) Rinse thoroughly with distilled water.

LipaseReagents:

(1) Citrate buffer. Same as for the alkaline phosphatase.(2) Substrate. Stock solution I-Combine 150 ml of 30 per cent glycerol, 50

ml of 10 per cent calcium chloride, 50 ml of barbiturate buffer (29 ml of 0.1 Msodium barbital and 21 ml of 0.1 M hydrochloric acid), and make up to a literwith distilled water.

Stock solution II-five per cent aqueous solution of "tween 40" or "60,"4or "product 81."5The lipase substrate was prepared for use by adding 2 ml of stock solution

II to 50 ml of stock solution I.(3) Ammonium sulfide. Same as for the alkaline phosphatase.(4) Lead nitrate solution. Two per cent aqueous solution.

' See footnote 2.4Palmitate and stearate, respectively, of a synthetic ester made by the Atlas Powder

Company, Wilmington, Delaware.6 Synthetic stearic acid ester made by the Onyx Oil and Chemical Company, Jersey City,

New Jersey.

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Procedure:(1) Suspend organisms in distilled water on microscope slides and allow to dry.(2) Fix in acetone for 10 minutes.(3) Rinse thoroughly with distilled water.(4) Immerse in citrate buffer for 30 minutes.(5) Rinse thoroughly with distilled water.(6) Place in the substrate medium overnight.(7) Rinse thoroughly with distilled water.(8) Immerse in the lead nitrate solution for 10 minutes.(9) Rinse thoroughly with distilled water.(10) Immerse in ammonium sulfide for 1 or 2 minutes.(11) Rinse thoroughly with distilled water.By means of the King-Armstrong (1934) procedure, quantitative acid and

alkaline phosphatase activities were determined on 10 peptoiie culture suspen-sions of bacteria for correlation with the phosphatase stains on these organisms.

Penicillin was used in a final concentration of 100 units per ml in the sub-strate solutions employed for the staining reactions. The antibiotic was addedat the start of the digestion period. Separate trials showed that the same con-centration of penicillin in the culture media stopped the growth of all the organ-isms tested with the one exception of Alcaligenes faecalis. In the quantitativemeasurements of the phosphatase activity, the effect of penicillin was observedafter the culture suspensions were brought to a final concentration of 100 unitsper ml just before the enzyme measurement was begun.

RESULTS AND DISCUSSION

The intensity of the phosphatase or lipase stains that were given by the organ-isms was found to vary with the age of the culture, the older cultures givingweaker stains. Accordingly, 24-hour cultures were employed for all the workincluded in this report. The effect of magnesium or manganous ions along withalanine in the agar on the intensity of the enzyme staining is illustrated in ta-ble 1. It may be seen that certain bacteria that gave negative reactions couldbe activated to the point of yielding positive stains in some instances.The organisms used were selected from the museum stock of the Department

of Bacteriology, University of Minnesota. It was observed that in many casesvarious strains of the same organism varied considerably in their enzyme po-tencies. In some cases one strain of an organism was completely negative in thestaining reaction whereas another strain was strongly positive. With the bac-teria, both phosphatases did not occur together in the same organism as indicatedby the staining. This is not true of certain of the fungi (table 2). It is note-worthy that none of the fungi tested gave a lipase stain, whereas most of thebacteria did.The lipase stain very rarely revealed definite intracellular morphology. It

usually appeared that the enzymatic activity was diffuse, as evidenced by entireareas of golden-brown stain. Very fine strands were thought to be observed onseveral of the organisms such as Bacillus indicus and Bacillus megatherium, and a

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1948] PHOSPHATASES AND LIPASES IN BACTERIA AND TRUE FUNGI 311

check-on the motility indicated the possibility that the flagella were being stained.Along with the apparent strands noted in these lipase stains another unusualextracellular element was observed. It had the appearance of a spiral, and inmost cases appeared to be unattached to a cell. The significance of the strandsand the spirals and of their relationship, if any, to the organisms themselvesis not understood.

TABLE 1The effect of activators in the medium on the enzyme staining of certain bacteria grown

on agar

ORGANISM

Klebsiella pneumoniae (strain I)....Aerobacter cloacae..................Aerobacter aerogenes................

Bacillus mycoides...................Bacillus indicus.....................Alcaligenes faecalis..................Klebsiella pneumoniae (strain II)....Bacillus megatherium...............Bacillus subtilis.....................

CONTROL AGAR

Alka- Acid s0line i

po- phos- Cd

phatase phatase ;-

-+ _

-+

-

- -+

_ - ±

Mg-ALANINE AGAR*

Alka- Acid e,

r phatase .phatase pt 4

_- ++-

_-

+- + ++ - ++ +

Alka-linephos-

phatase

+

* Final concentration of magnesium sulfate, 0.1 molar; alanine, 0.017 molar.t Final concentration of manganous sulfate, 0.01 molar; alanine, 0.017 molar.

TABLE 2Enzyme staining of certain fungi

ORGANISM ALKALINE ACID LIPASEPHOSPHATASE PHOSPHATASE LPS

Saccharomyces cerevisiae ...................... + +Saccharomyces fragilis............................ +Candida albicans ................................. + +Cryptococcus sphaerica............................ +"Torula cremoris"....................... ........... + +Cryptococcus species ............................ +Geotrichum candidum............................. +

Figures 1 to 5 illustrate the appearance of the stained organisms in a fewinstances. It should be borne in mind that all organisms were treated withcitrate buffer of pH 4.7 before the staining reaction was carried out in order toremove extraneous substances that might give a positive test. When the organ-

isms were so treated and then subjected to the staining reagents (but not sub-strate), no stain was apparent. Thus any stain that these organisms developedlater represents enzyme and only enzyme. As another check against false-

Mn-ALANINE7AGARt

Acidphos-pha-tase

++

+

+Cd

*zA

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1948] PHOSPHATASES AND LIPASES IN BACTERIA AND TRUE FUNGI 313

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M. BAYLISS, D. GLICK, AND R. A. SIEM

positive reactions, both heat and fluoride were used to inactivate the enzymes.Boiling the fixed preparations for a short time or exposing the slides to a fluoridesolution resulted in negative staining in all cases.From the appearance of electronmicrographs of Bacillus mycoides, Knaysi and

Baker (1947) reported the presence of nuclei in these organisms. Their photo-graphs showed internal structures very similar to those observed in this inves-tigation under the light microscope after staining for alkaline phosphatase. In

*;

FIG. 5. BACILLUS SUBTILIS STAINED FOR LIPASE. X 1,267

both plant and animal tissues it has been shown that phosphatases are presentin nuclei. The possibility exists that the stained areas that were observed inthe present work may indicate nuclei, but whether or not this is so remains to beproved.A word of caution should be injected concerning the interpretation of the

localized areas of stain. These should not be taken too literally. Ionic andmolecular forces are operative that may cause certain deviations in the precipitatepattern and thus lead to a distribution not necessarily identical with the enzymetopography as it existed prior to the staining treatments.

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19481 PHOSPHATASES AND LIPASES IN BACTERIA AND TRUE FUNGI

The effect of penicillin on the phosphatase activities of 10 species of bacteriamay be seen in table 3. It is obvious that penicillin has little if any effect, eventhough the concentration used arrested growth in all cases except Alcaligenesfaecalis. When added to the substrate solutions employed for the enzyme-stain-

TABLE 3 &

The effect of penicillin on the phosphata8e activities of certain bacterial peptone-brothsuspensions

ORoGAMSM ALKALINE PHOSPHATASE ACID PHOSPEIATASE(UNTS*/100 ML SUSPENSION) (UNITst/100 ML SUSPENSION)

Klebsiella pneumoniae (strain I)Control ...........................Penicillin...........................

Aerobacter aerogenesControl ............................Penicillin ..........................

Bacillus mycoidesControl.............................Penicillin...........................

Bacillus indicusControl. .. .........

Penicillin...........................Alcaligenes faecalisControl. .. ............

Penicillin...........................Klebsiella pneumoniae (strain II)

Control.............................Penicillin...........................

Bacillus megatheriumControl.............................Penicillin...........................

Bacillis subtilisControl.............................Penicillin...........................

Bacillus terminalisControl.............................Penicillin...........................

0.00.0

0.00.0

18. 21. 46.35. 20. 46.

11.11.

0.0 2.0 6.017. 0.0 2.0

26. 13. 70.28. 13. 60.

1.01.0

59. 32. 37.61. 29. 36.

0.00.0

7.8 6.49.2 6.0

9.8 7.2 6.09.0 6.8 5.8

0.80.2

0.4 1.28.6 1.2

2.02.0

0.0 0.8 2.02.0 0.8 0.8

2.00.0

1.01.2

0.6 0.60.8 0.8

* Amount of enzyme acting on excess disodium phenyl phosphate at pH 9.0 and 37.5C that will liberate 1 mg of phenol in 3 hours.

t Amount of enzyme acting on excess disodium phenyl phosphate at pH 4.9 and 37.5C that will liberate 1 mg of phenol in 3 hours.

ing reactions, penicillin had no effect on the stains, as would be expected from thequantitative data.

If one compares the data in tables 1 and 3, it is clear that a negative stain doesnot necessarily indicate the complete absence of enzyme, but merely that theactivity is less than some low value. Thus the agid phosphatase stain wasnegative for Klebsiella pneumoniae even though a small degree of activity waspresent.

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M. BAYLISS, D. GLICK, AND R. A. SIEM

SUMMARY

The Gomori methods for acid and alkaline phosphatases and lipase wereemployed for the staining of -a variety of bacteria and fungi. Localized areaswithin the organisms gave positive stains in certain instances, suggestive of aninternal structure or inhomogeneity.Manganous and magiresium ions along with alanine in the culture agar were

demonstrated to activate the bacterial enzymes and markedly increase theirstaining intensity.

Penicillin, in concentrations sufficient to arrest completely the growth ofcertain bacteria, was found to have no significant effect on the phosphataseactivities of these bacteria, as demonstrated separately by staining ability andquantitative measurement.

REFERENCESGoMoRI, G. 1946a The study of enzymes in tissue sections. Am. J. Clin. Path., 16,

347-352.GoMoRI, G. 1946b Distribution of lipase in the tissues under normal and under pathologic

conditions. Arch. Path., 41, 121-129.KING, E. J., AND ARMSTRONG, A. R. 1934 A convenient method for determining serum

and bile phosphatase activity. Canadian Med. Assn. J., 31, 376-381.KNAYSI, G. 1947 Demonstration, with the electron microscope, of a nucleus in BaciUus

mycoides grown in a nitrogen-free medium. J. Bact., 53, 539-553.RocHE, J., VAN THOAI, N., AND ROGER, M. 1946 Inactivation et reactivation totales de

la phosphomonesterase alcaline (intestin) et interchangeabilit6 Ides m6taux actifs.Arch. intern. physiol., 54, 209-213.

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