DETECTION, ELIMINATION, AND PREVENTION OF CONTAMINATION OFCELL CULTURES WITH PLEUROPNEUMONIALIKE ORGANISMS

HENRY J. HEARN, JR., JULIUS E. OFFICER, VIRGINIA ELSNER, AND ARTHUR BROWN

U. S. Army Chemical Corps, Fort Detrick, Frederick, Maryland

Received for publication April 20, 1959

Recent reports indicate that a number ofestablished cell lines in various laboratoriesthroughout the world are contaminated withpleuropneumonialike organisms (PPLO) (Robin-son et al., 1956; Collier, 1957; Rothblat andMorton, 1958). The contamination is insidiousand is difficult to detect for the following reasons:(a) the microorganisms may fail to affect overlythe cultures, or they may induce cytopathogenicchanges irregularly, (b) the PPLO may reacha very light or no visible turbidity in liquidmedia, (c) the contaminants are often missed onagar plates because of their minute colony size,and (d) many PPLO are resistant to penicillin andstreptomycin, the antibiotics most commonlyused in tissue culture laboratories.

This report concerns the procedures whichhave been developed in our laboratories to detect,eliminate, and prevent contamination of cellcultures by PPLO or related microorganisms.

MATERIALS AND METHODS

Cell culture media. The following media wereused for maintaining the cultures: (a) yeastextract-proteose peptone no. 3 (Difco) mediumcontaining 10 or 20 per cent horse serum (YE-PPno. 390HoSj0) (Mayyasi and Schuurmans, 1956),(b) a 0.5 per cent lactalbumin hydrolyzate me-dium in Hanks' balanced salt solution with 10 percent calf serum added (lacto.5BSS89.5CaSjo), (c)Eagle's medium (1955) containing 10 per centhorse serum (EaglegoHoS,o), (d) medium 199(Morgan et al., 1950) containing 20 per centhorse serum (1998oHoS20). The horse serum wasobtained from horses quartered at Fort Detrick.The calf serum was obtained commercially orfrom a local slaughter house. All sera were rou-

tinely filtered through Selas filters of 03 porositybefore procedures described below were developedfor control of PPLO contamination.Media for isolation of microorganisms. Heart-

infusion broth (Difco) and blood agar base(Difco) were used for the isolation of PPLO.Both media were sterilized by autoclaving, then

cooled, after which filtered horse serum wasadded to each to a final concentration of 20 percent. No adjustment of pH was made. Petriplates (50 mm, disposable, plastic) containingserum agar were streaked with supernatantmedium and supernatant plus cells of all thecultures tested. At the same time, 0.2 ml ofcultures was added to 2 ml of serum-broth inrubber-stoppered Wasserman tubes. The inocu-lated plates and tubes were incubated at 37 C andexamined daily. Negative broth cultures weresubcultured 3 times at 3-day intervals in a 1 :10dilution for enrichment purposes. At each trans-fer, samples were streaked on agar plates.

Cell cultures. The cell cultures tested includedall those routinely maintained in our laboratoryas well as several obtained from different labora-tories in the United States. The cell cultures andthe media used for their maintenance are listed intable 1.

IlIicroscopic observations. Wet mounts fromserum broth or cell culture media were examinedby ordinary light microscopy and by phasecontrast microscopy for the presence of PPLO.In addition, some of the cell cultures were culti-vated in a simple perfusion chamber (Officer,1958), and were observed continuously over aperiod of 48 to 120 hr for the appearance ofPPLO.

Material for staining was prepared from liquidmedia, broth cultures, and agar colonies pressedon cover slips. Cells adhering to 11 by 22 mmcover slips were also examined. Fixation was bydry heat or absolute methanol. All preparationswere stained for 10 min in Giemsa diluted 4oin distilled water.

RESULTS

Isolation. PPLO were successfully isolatedand grown in both liquid and solid media asdescribed in Materials and Methods. Coloniesappearing for the first time on agar plates weretransferred at least 3 times in serum-broth andretested for growth each time on agar plates.

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TABLE 1Cell cultures tested for contamination with pleuropneumonialike organisms

Cell Culture Culture Medium Reference and Comment

L YE-PP no. 39oHoSio Sanford et al. (1948); 4 of 6 cultures tested from19980HoS20 6 different laboratories were positive

Chang's human liver cells Lact0.5BSS9oCaS,o Chang (1954); 2 of 2 cultures from 1 labora-tory were positive

Maben YE-PP no. 39oHoSjo Frisch et al. (1955); 2 of 2 cultures from 2 lab-oratories were positive

HeLa YE-PP no. 39oHoSio Scherer et al. (1953); 2 of 2 cultures from 2 lab-oratories were positive

Embryonic human lung YE-PP no. 39oHoSio Henle and Deinhardt (1957); 2 of 2 culturesEagles9oCaSjo from 2 laboratories were negative

Embryonic rabbit kidney YE-PP no. 39oHoS1o Westwood et al. (1957); 1 of 1 culture was neg-ative

Adult guinea pig lung Ye-PP no. 39oHoS1o Isolated in our laboratories; 2 of 2 culturesfrom 2 laboratories were positive

Adult mouse lung YE-PP no. 39oHoSio Isolated in our laboratories; 2 of 2 culturesfrom 2 laboratories were positive

Human amnion 19080HoS20 Fogh and Lund (1957); 1 of 1 culture was posi-tive

Controls, consisting of medium constituents, ora known PPLO-free culture, were also helpful ineliminating artifacts resembling PPLO colonies.

Identification. Minute colonies about 0.05 to0.2 mm in diameter could be seen by 48 hrmicroscopically and between 3 to 4 days macro-scopically if observed with oblique light. Growthof colonies usually ceased by the 6th day (figures1 and 2). The size of full grown colonies variedfrom about 0.2 to 2 mm depending on the type.Colonies often appeared homogeneously granularwith or without a raised center and a spreadingthin periphery typical of a so-called "fried egg"(figure 3). They may also appear vacuolated(figure 4).In table 1 are listed the numbers and kinds of

cultures tested for PPLO contamination and theresults obtained. Typical colonies isolated fromL, Hela, and Chang's liver cells may be seen infigures 5, 6, and 7, respectively. All cell lineswith the exception of HeLa and Chang's liverline showed the "fried egg" type of colony.

Broth cultures inoculated with PPLO, and thesupernatant fluid from cell cultures were exam-ined under phase-contrast. For all types it wasdifficult to distinguish the irregularly shapedgranules presumed to be microorganisms fromthe artifacts in uninoculated serum broth. Thesame was true of stained films. The PPLO type

from Chang's human liver cells when examined inthe culture flask was clearly distinguishable fromartifacts. Figure 8 shows the microscopic appear-ance of a Giemsa-stained preparation of thePPLO against a background of liver cells whichare unavoidably out of focus.

Artifacts. Nonliving artifacts have been aproblem for investigators attempting to identifysuspected microorganisms as PPLO (Laidlaw,1925; Brown et al., 1940). Two types of experi-ments were carried out with all the PPLO isolatedfrom the cultures in the early stages of this workto confirm the fact that the colonies were PPLOand not nonliving artifacts. Samples of brothcultures were irradiated with ultraviolet light(GE lamp, 15 w, 95 per cent light = 2537 A)and heated at 70 C for 10 min. Samples takenbefore and after treatment were streaked immedi-ately on serum agar plates, or streaked on agarplates after 3 serial transfers at 3-day intervalsin broth. Controls consisted of a known PPLOculture and sterile broth. The results of theseexperiments showed that colonies of PPLO ap-peared before but not after the treatments.

Treatment. An experiment was carried out withbroth cultures for the purpose of testing theorganisms for sensitivity to several antibiotics.Broth cultures and controls were spread on agarplates. Discs of penicillin, streptomycin, aureo-

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Figure 1 (top left). Pleuropneumonialike organism colonies from L cell cultures 48 hr after inoculation(X100).Figure 2 (top right). Same field as figure 1, 96 hr (X100).FigureS (center left). "Fried egg" type of pleuropneumonialike organisms isolated from L cells (X450).Figure 4 (center right). Pleuropneumonialike organism colony from L cells, vacuolated, 15 days (X200).Figure 6 (bottom left). Clump of L cells surrounded by pleuropneumonialike organism colonies (X 100) .Figure 6 (bottom right). Pleuropneumonialike organism colony adjacent to HeLa cell debris (X 100).

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Figure 7. Pleuropneumonialike organism colonyfrom Chang's human liver cells (X100).

mycin, terramycin, and chloroamphenicol andfuracin were placed on such plates. All the PPLOtested were found to be about equally susceptibleto aureomycin and terramycin, less susceptibleto chloroamphenicol and furacin and resistant topenicillin and streptomycin.Aureomycin was chosen as the antibiotic for

eliminating PPLO from the cultures. An attemptwas made to cure (permanent elimination ofPPLO) -the cultures by using doses of antibioticwhich were toxic for the cells. Toxic doses were

chosen because several investigators have usedsubtoxic doses of drug only to find that the PPLOcontamination returned soon after the drug was

removed (e. g., Rothblat and Morton, 1958;Doherty, 1958). The toxicity level of aureomycinfor the various cell lines ranged from about 25,ug/ml to 150 ,tg/ml. A number of experimentswere carried out using both nontoxic and toxicconcentrations of antibiotic. The most consistentand earliest cures were obtained when toxicdoses were used which ranged from 100lg/mlto 200 ,ug/ml, although near toxic doses of anti-biotic were able to cure some cell cultures. Theantibiotic at any selected concentration was

renewed every time a medium change was made,or the cells transferred, which meant a drugaddition was made every 2 to 3 days. It was

common for cultures showing toxic effects due tothe antibiotic to recover after removal of theantibiotic or after lowering its concentration to 5to 10 j,g/ml. While toxicity for the cells was

encouraged, care was observed in preventingextreme necrotic effects. The reason that toxic

doses were always found to be effective whereasnontoxic doses were erratic in effecting curesmay be because the organisms may grow intra-cellularly. Figures 9, 10, and 11 show a controland some PPLO infected cultures of human livercells. The infected cultures showed inclusionswhich were not found in control or treated cul-tures; the inclusions were interpreted as re-sembling PPLO or as being PPLO induced.Attempts were made to isolate PPLO through-

out the period of treatment. In addition, isola-tions were attempted at least once through eachof 4 cell transfers and medium changes after theaureomycin was removed. When tests were nega-tive for PPLO on cells after such treatment, thecells were tested again after being disrupted by 4cycles of freezing, thawing, and shaking withglass beads. Negative tests for PPLO that re-sulted after all these steps were used were ac-cepted as proof that the cell lines were cured.Each cell line has been tested occasionally sincebeing treated for periods ranging thus far from 9to 15 months. None have shown PPLO.

Prevention. The effective prevention of PPLOcontamination depends in part on the source ofthe contamination The sources of contaminationthus far suggested are (a) the serum,' (b) othermedium constituents, (e) the technician ascarrier, (d) bacterial contaminants that becomeconverted to L forms by the antibiotics oftenused in cell culture media (Holmgren,2 1959;New York Academy of Sciences, 1959), and (e)the source of tissue from which the original cellisolation was made. To eliminate all of the sourcesof contamination mentioned above except thelast, the following procedures were carried out:(a) all media were filtered through ST-1 or ST-3Seitz filters3 and tested before use and (b) all

I Several batches of sera were tested for pleuro-pneumonialike organisms and were negative, evenafter ultracentrifugation at 50,000 X G was carriedout for concentration purposes.

2 In a personal communication, Dr. NeldaHolmgren, Fort Detrick, states that she has con-verted a bacterial contaminant in her cell culturesto the L form with antibiotics, and reconvertedthe L form to the bacterial contaminant.

3 These pads filter out particles greater than 3ioor 1100 A, respectively, and are treated so thatmedia passed through such filter pads are nontoxicfor cell cultures. They were brought to our atten-tion by Dr. William Daniels of Fort Detrick and

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Figure 8a (top) and b (bottom). Microscopic appearance of pleuropneumonialike organism on coverslips taken from cultures of Chang's human liver cells. Cells in background are out of focus (X2000).

cultures were carried free of antibiotics on analternating schedule using stringent aseptictechnique. The latter was, in part, designed toprevent the not so easily recognized L form con-tamination as discussed above. PPLO-free celllines in our laboratories have been stored at-55 C to insure that they are not permanentlylost due to contamination or for other reasons.

The cells were stored according to a modifiedprocedure described by Scherer (Hanks et al.,1955). The main modifications are (a) the cellsare frozen (slowly) in the medium used forgrowth containing 5 or 10 per cent glyceroldepending on the cell types, and (b) very smallfluctuations in temperature are avoided.

DISCUSSION

It has been the main purpose of this paper topresent a reasonably simple procedure for thedetection, elimination, and possible prevention

were tested successfully in a filter press (by Dr.Daniels) under low pressure and in Seitz filters inour laboratory under vacuum with stock pleuro-pneumonialike organism types.

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Figure 9. Phase contrast photomicrograph of control, pleuropneumonialike organism-free, Chang'shuman liver cells (X2000).

Figure 10. Phase contrast photomicrograph of Chang's human liver cells contaminated with pleuro-pneumonialike organisms (X2000). Note inclusions.

of PPLO contamination in cell cultures. Indeveloping the procedures, various ideas concern-ing the source of PPLO contamination have beenconsidered. One possible source of contaminationdeserves special comment; it has been suggestedthat the contaminants referred to as PPLO may,in reality, be L forms that have arisen from con-taminating bacteria as the result of conversion

by the antibiotics often used in cell culture media(Smith et al., 1957; Holmgren, 1959 personalcommunication; New York Academy of Sciences,1959). It is worthy of note that one cured cultureof Chang's human liver line was maintained by anew worker in our group in the presence ofpenicillin and streptomycin. This line becamerecontaminated with PPLO. The same line

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Figure 11. Same as figure 10 with another kind of inclusion (X2000)

carried by several others at the same time usingthe same batch of medium without antibioticsremained free of PPLO. This is compatible withthe suggestion that the apparent PPLO contami-nation may be in reality the conversion of ordinarybacterial contaminants into bacterial L forms bythe antibiotics; other explanations are possible,however. It is difficult to distinguish betweenPPLO and L forms of bacteria (Dienes and Wein-berger, 1951; New York Academy of Sciences,1959). The possible relationships between bac-teria, PPLO, L forms, and protoplasts have beendiscussed elsewhere (Dienes and Weinberger,1951; Smith et al., 1957; Landman et al., 1958;New York Academy of Sciences, 1959).The methods for the detection of PPLO de-

scribed here have permitted a high percentage ofsuccessful isolations. The identification of thecontaminants as PPLO has been made mainly onthe basis of the colonial and microscopic appear-ance of the organisms (Breed et al., 1957; Dienesand Weinberger, 1951). These are the primarycriteria used by others, although serologicalmethods are sometimes used as further aids indiagnosis, especially by those attempting toidentify the host origin of the parasitic PPLO(e. g., Jungherr et al., 1955).

Several investigators have reported or informedus that they have failed to cure their con-taminated cultures, e. g., Rothblat and Morton(1958), Doherty (1958). Their failures may be

due to the insufficient antibiotic concentrationused or the return of cured cultures to mediacontaining penicillin or streptomycin. As ex-plained earlier, the antibiotic concentrationshould be high enough to be toxic to the cells, incases where the cultures are resistant to cure.Because cells may show signs of toxicity and stillremain viable for several days, at least onereplicate culture should be kept in the presenceof toxic doses of antibiotic through 2 or 3 mediumchanges over a period of 1 week before the anti-biotic concentration is decreased or removed. Thereturn of cured cell cultures to media containingantibiotics may be unwise because PPLO areresistant to several antibiotics or because thecontamination may, in reality, be due to the con-version of bacterial contaminants into L formsas discussed previously. It is recommended thatstock cell cultures be carried in media free ofantibiotics.

ACKNOWLEDGMENT

The authors wish to thank Dr. John B. Nelsonof the Rockefeller Institute for his helpful dis-cussion of the manuscript.

SUMMARY

Procedures have been given for the detection,elimination, and possible prevention of cell cul-ture contamination by pleuropneumonialikeorganisms (PPLO) or closely related micro-

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organisms. PPLO were isolated on serum agardirectly and after enrichment by rapid serialtransfers in serum broth. Identification wasmade on the basis of colony morphology andmicroscopic appearance of the organisms.Permanent elimination of the contaminants wasachieved by treating cultures with doses ofaureomycin which were toxic for the cells. Theprocedures recommended to prevent PPLO(re-)contamination include the use of filter padsof unusually small porosity, and the maintenanceof cell cultures in antibiotic-free media.

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COLLIER, L. H. 1957 Contamination of stocklines of human carcinoma cells by pleuropneu-monia-like organisms. Nature, 180, 757-758.

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MORGAN, J. F., MORTON, H. J., AND PARKER, R. L.1950 Nutrition of animal cells in tissue cul-ture. I. Initial studies on a synthetic me-dium. Proc. Soc. Exptl. Biol. Med., 73, 1-8.

New York Academy of Sciences 1959 Confer-ence on the biology of the pleuropneumonia-like organisms. To be published in the Ann.N. Y. Acad. Sci.

OFFICER, J. E. 1958 The growth and morpho-logical development of two psittacosis strainsin tissuie culture. Ph.D. Dissertation, GeorgeWashington University School of Medicine,Washington, D. C.

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