CHARACTERIZATION OF THE ACTION OF LYSOZYME ONSTAPHYLOCOCCUS AUREUS AND ON MICROCOCCUS

LYSODEIKTICUS

RAYMOND A. KERN, MARY JANE KINGKADE, STANLEY F. KERN,AND OTTO K. BEHRENS

Lilly Ruesearch Labioratories, Eli Lilly and Company, Indianapolis 6, Indiana

Received for publication November 6, 1950

A recent report from this laboratory (Kingkade, Huff, and Behrens, 1948) (Ie-scribes experiments in which lysozyme was used to prepare bacterial cell lysatesfrom Staphylococcus aureus K1 for use as a possible medium for bacteriophagemultiplication. It was hoped that the cell wall would be ruptured allowing dis-persal of the intracellular constituents into the medium without appreciabledenaturation or destruction.

Since in many of the experiments filtration had been used to remove any in-tact cells from the cell lysates, experiments were undertaken to determinewhether the intracellular constituents had dispersed sufficiently to be filterable.'For this purpose electron micrographs were made of cells treated for varioustimes with lysozyme. At the same time turbidimetric readings and determina-tions of cell count, both direct and viable, were made to be correlated with theresults of electron micrography. Observations w^ere also made on the filtratesfrom the staphylococcal lysates.The action of lysozyme on Staphylococcus aureus K1 is accompanied by a grad-

ual decrease in density at the periphery of the cell. No evidence was found of rup-ture of a cell membrane with a spilling of the cellular contents. The cellular massesremaining in the lysates did not pass through bacterial filters. In contrast, lyso-zyme causes rupture of the cell wall of Micrococcus lysodcikliclus and a dispersalof the cell contents.

EXPERIMENTAL METHODS

The Staphylococcus aureus strain K1 and Mficrococcus lysodeikticus MB79(7R-2150)2 were grown at 37 C on nutrient agar (1 per cent Difco nutrient broth,0.85 per cent NaCl, 2.5 per cent agar) in toxin bottles. After incubating for 24hours, the cells were washed from the agar with saline. They were sedimentedby centrifugation, washed once with saline, and suspended in sufficient salineto give a concentration of 1 to 3 X 109 cells per ml.

Duplicate tubes containing 7.2 ml of cell suspension and 0.8 ml of lysozyme3in saline were incubated at 35 to 37 C with aeration. Control tubes containing

1 Dr. S. E. Luria had suggested that lysis might be incomplete.2 The S&aphylococcus aurents K, was obtained from Dr. A. P. Krueger and the Micro-

coccus lysodeikticus from Dr. H. H. Anderson.3 A crystalline preparation of lysozyme prepared from egg white by Armour Laboratories

was used.171

KERN, KINGKADE, KERN, AND BEHRENS

7.2 ml of cell suspension and 0.8 ml of saline were treated similarly. One controltube and one tube containing lysozyme were used for turbidimetric determina-tions with the Klett-Summerson photoelectric colorimeter making use of filterno. 54. Aliquots from the other two tubes were removed at suitable intervalsfor direct cell counts, viable cell determinations, and electron microscopy. Directcounts were made using a Levy-Hausser counting chamber. Viable- cells weredetermined by colony counts on nutrient agar (1 per cent Difco nutrient broth,0.85 per cent NaCl, 1 per cent agar) with suitable dilutions of the cell suspen-

TURBIDITY DIRECT COUNT VIABLE COUNT

0 20 40 60 80 loo 0 20 40 0 20 40 60 80 IODTIME IN MINUTES

Figure 1. Effect of varying concentrations of lysozyme on M. lysodeikticus. X-X =

0.006 mg per ml; 0-0 = 0.Q012 mg per ml; *- = 0.00015 mg per ml.

sions. Apparently this dilution largely prevents the continuing action of thelysozyme.

In the experiments with Staphylococcus aureus, sufficient lysozyme was addedto give a concentration of 3 mg per ml. Under these conditions a loss of 80 percent or more of the cells was observed in 30 minutes as determined by directcount. With Micrococcus lysodeikticus a lysozyme concentration of 0.0012 mgper ml produced lysis at a rate suitable for these experiments. The concentra-tion was found to be critical within a limited range (figure 1).The experiments to determine the effect of filtration on the removal of mate-

rial from the bacterial lysates were performed in the following manner: a sus-pension of Staphylococcus aureus K1 containing 1 to 3 X 109 cells per ml was pre-pared as described above. After the addition of 1 per cent of egg white as the

172 [VOL. 61

ACTION OF LYSOZYME

source of lysozyme, lysis was allowed to proceed for 12 minutes. The lysates werethen filtered according to the method of Krueger, Scribner, and Brown (1946).

Samples were prepared for electron microscopic observation by placing a dropof cell suspension on a "formvar" film over a supporting grid. After 1 minute thedrop was removed as completely as possible with a capillary tube, leaving a thinfilm of suspension on the specimen screen. Immediately after the removal of thesuspension, the sample was washed by adding a drop of distilled water, allowingit to stand 1 minute, and removing it with a capillary tube. The washing was re-peated one more time. After drying, the sample was shadow-cast at a 10° angle

TURBIDITY DIRECT COUNT VIABLE COUNT

0 2 3 4 0 1 2 3 0 1 2 3 4TIME IN HOURS

Figure 2. Effect of lysozyme on S. aureus and on M. lysodeikiicus: X-X = S. aureuswith 3.0 mg lysozyme per ml; 0-0 = M. lysodeikticus with 0.0012 mg lysozyme per ml.

with a platinum-palladium mixture and was observed at a magnification of about2,500 X in an RCA model B electron microscope.

RESULTS

The results of typical experiments with lysozyme and Staphylococcus aureusK1 and with Mlicrococcus lysodeikticus are shown in figure 2. With both of theseorganisms the viable count fell at a more rapid rate than did the turbidity anddirect count.A distinct difference is noted between the observations on the two organisms

and may be correlated writh the electron micrographic observations as notedbelow. With S. aurets a rapid initial drop in direct count to approximately one-

1951] 173

KERN, KINGKADE, KERN, AND BEHRENS

tenth of the original value is observed. This count is then maintained throughoutthe remainder of the period of observation. The viable count demonstrates thatonly a small percentage of the cells that are visible are still capable of multiplica-tion. It appears probable that these visible cells correspond to the slowly dis-integrating cellular masses that are observed with the electron microscope (figure

c-_. g-IN,INI -Figure S. S. aureus. Magnification 4,650 X. (A) Without lysozyme. (B) With 3 mg per

ml of lysozyme, sample taken immediately. (C) Like B after 1 hour at 37 C. (D) Like B after6 hours at 37 C.

3). The continuing disintegration is reflected to some extent in the continuingdecrease in turbidity.

Figure 3, A, depicts a field of typical unlysed S. aureus. The efficiency of thewashing procedure in removing dissolved materials is indicated by the absenceof salt crystals in the field. Figure 3, B, illustrates the immediate action of lyso-zyme on this organism. The time interval between the addition of lysozyme tothe cells and the beginning of the washing procedure was less than 1 minute.An effect of lysozyme is already apparent in the appearance of a less dense por-

174 [VOL. 61

ACTION OF LYSOZYME

tion around a generally irregular mass of greater density. In higher magnificationpictures the less dense portion is observed to be thinner than the dense portion.Conclusions concerning the specific absorption of the two regions are not war-ranted. However, it must be concluded that lysozyme must have acted on theless dense portion to the extent that it would flatten out on drying. The observed

Figure 4. S. aureus. Magnification 4,650 X>. (A) Sample taken immediately after additionof lysozyme. Washing of slide omitted. (B) Sample taken 2 hours after addition of lysozyme.Washing of slide omitted. (C) Like B. Sample dried without washing and thenl washed andredried. (D) Like B but washed on specimen screen before drying.

increase in diameter of the cell is consistent with this interpretation. Longer ac-

tion of the lysozyme on the cells, as illustrated in figure 3, C, D, brings aboutgradual dissolution of the outer material and a decrease in diameter of the re-

maining portion. These pictures were taken, respectively, after 1 and 6 hoursof lysozyme action. In spite of the long-continued action ef lysozyme, the cellsmaintain their individuality. Apparently lysozyme has little or no action on theinterior portion of the cell.

19511 175

KERN, KINGKADE, KERN, AND BEHRENS

The effect of the omission of the washing procedure is illustrated in figure 4,A, B, representing samples of S. aureus treated with lysozyme for less than 1minute and for 2 hours before drying on the "formvar" film. The presence ofdissolved and finely dispersed materials in the suspension is evidenced by thegeneral coating of the cells. The increase in the amount of this material with in-creased length of lysozyme contact substantiates the conclusion that this enzyme

Figure 5. M. lysodeikticus. Magnification 4,650 X. (A) Without lysozyme. (B) With 0.0012mg per ml of lysozyme; sample taken immediately. (C) Like B after 10 minutes at 37 C.(D) Like B after 60 minutes at 37 C.

gradually causes dissolution of some of the cellular material. These pictures alsosuggest that a process of drying without washing allows more extensive lyso-zyme action than when the supernatant fluid is washed away prior to drying.This result may be due to the increased concentration of the lysozyme duringthe drying procedure. Figure 4, C, represents a field of the S. aureus that wasdried without washing and was then washed and redried. The presence of thelarge amount of filamentous material in the background and covering the cells

176 [VOL. 61

ACTION OF LYSOZYME

indicates that this material of cellular origin is not readily dissolved or dispersedonce it has been dried. Figure 4, D, represents a field from the same suspensionthat had been washed on the specimen screen before drying.The more rapid and more complete dissolution of M. lysodeikticus by lysozyme

is reflected by all of the methods of observation. In contrast, with the Staphylo-coccus, the direct count falls rapidly to values that preclude accurate determina-tion. Sections A, B, C, and D of Figure 5 represent electron micrographs of fieldsof the Micrococcus prepared from cultures before lysozyme treatment and 1,10, and 60 minutes, respectively, following the addition of lysozyme. After 1

,. iS. "'-v.

Figure 6. M. lysodeikticus. Magnification 23,500 X. (A) With 0.0012 mg lysozyme after1 hour at 37 C. (B) Like A after 2 hours at 37 C.

minute of treatment with lysozyme the only change observed was a noticeableenlarging or swelling of the cells (figure 5, B). The apparent differentiation of cellconstituents observed with the Staphylococcus was not seen. With longer periodsof lysozyme action, the decrease in thickness of the cell made possible the ob-servation of cell bodies that remained unaffected even after 60 and 120 minutes.This is plainly shown in figure 6, A, B, representing higher magnification picturesof cells treated with lysozyme for 60 and 120 minutes, respectively.The pictures taken at 10 minutes indicate that many of the cells were already

ruptured, the break occurring between the two cell bodies. Each cell appearsto crack open, allowing the cell contents to disperse and leaving behind only thecell membrane and the two cell bodies. With longer action of the lysozyme the

1951] 177

KERN, KINGKADE, KERN, AND BEHRENS

cell membrane is also destroyed, so that as an end result the cell is lysed exceptfor the cell bodies.The authors are grateful to Jane Osborn for valuable assistance.

SUMMARY

The action of lysozyme on Micrococcus lysodeikticus differs from that onStaphylococcus aureus K1. With the Micrococcus, electron microscopic observa-tion showed that lysozyme first causes an enlarging or swelling of the cell. Thisis followed by a rupture of the cell wall with a dispersal of the cell contents.With the exception of two cells bodies (nuclei?), the cell contents dissolve in themedium. Finally the cell membrane is also destroyed leaving only the cell bodies.Turbidity, viable count, and direct count all reflect the rapid dissolution of thecells observed with the electron microscope.The Staphylococcus is not so sensitive to the action of lysozyme, and in addi-

tion a different type of cell destruction is observed. At no time is there any evi-dence of the splitting of a cell wall. Rather, a slow dissolution of any outer viscousportion of the cell takes place over a period of several hours, leaving an innermore dense portion. The viable count continues to decrease, reaching very lowlevels. In contrast, the direct count remains quite high, probably because ofvisualization of the cell masses.

Attention is directed to precautions that must be exercised in preparing mountsfor electron micrographs of materials undergoing enzymatic changes.

REFERENCESKINGKADE, M. J., HUFF, D. E., AND BEHRENS, 0. K. 1948 The use of lysozyme in studies

with a bacterial virus. Proc. Indiana Acad. Sci., 58, 63-68.KRUEGER, A. P., SCRIBNER, E. J., AND BROWN, B. B. 1946 Further observations on the

mechanism of phage action. J. Gen. Physiol., 30, 25-39.

[V-OL. 61178