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TECHNICAL MANUSCRIPT 236

DIRECT FLUORESCENT TAGGINGOF MICROORGANISMS:

POSSIBLE LIFE DETECTION TECHNIQUE

Abe Pital

; : Sheldon L. JanowltzCharles E. Hudak

Evelyn IE. Lewis

AUGUST 1965

~J~~T ~E S A RM Y~ * eIR ATQ!RuES,

Best Aa

Best Available Copy

US. ARMY BIOLOGICAL LABORATORIESFort Detrick, Frederick, Maryland

TECHNICAL MANUSCRIPT 236

DIRECT FLUORESCENT TAGGIWI OF MICROORGANISMS:A POSSIBLE LIFE DETECTION TECH!NIQUE

Abe Pital

Sheldon L. JanowitzCharles E. Hudak

Evelyn E. Levis

Physical Defense DivisionDIRECTORATE OF MEDICAL RZ,•EARCH

Project IC62240LA071 August 1965

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The authors express their appreciation to Mr. Frank W. Wagner for fur-nishing atmospheric background samples, and to Mr. Franklin T. Vvtzel forcover slip preparations of hamster kidney cells. Thanks are also extendedto Dr. John H. Ladino for micio-Kjeldahl determinations and to Dr. Sidney IYaverbaum for helpful suggestions during the course of this study. Thetechnical assistance of Lt. Joseph N. Freund and Sp-4 R•hbet J. gammer, Jr.is gratefully acknowledged.

Microorganism and selected proteinaceous substances were directlytagged with fluorescein isothiocyanate. This approach suggested apossible application for detection of extraterrestrial life. A stableand apparently specific linkage was formed with protein, although non-protein substances were readily dertained. Substances such as soil andatmospheric debris did not exhibit any $ignifLcant affinity for the dye.

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OR.IGINAL* DOCUTMENT

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DIRECT FLUORESCENT TAGGING OF MICROORGANISMS:A POSSIBLE LIFE DETECTION TECHNIQUE

During experimental investigations on methods for tagging bacterialcells with fluorescent labels, it was found that microbial protein couldbe rapidly conjugated with fluorescein isothiocyanate (FITC). Thesimplicity and apparent specificity of the technique suggested a possible

A ~ ~ ~ ~ ie jU,-~Te 'In ources ofbiological material are often presumed to be soil and atmospheric debris.Some of the methods proposed for detecting extraterrestrial life are basedon the assumption that microorganisms may be a predominant form of lifeand consequently systems have been developed for detecting bacteria ortheir metabolic products. These systems depend on integrated metabolicreactions and growth of microorganisms. The use of simplified approachesmay be more realistic when applied to unknwn extraterrestrial environ-ments. However, all approaches are, of necessity, based on certainassumptions, and ultimately, a combination of different methoda may berequired for a definitive answer.

This preliminary report describes a ; .mpie staining procedure that isbased on direct conjugation of FITC with protein particulates. Basically,the method involves the conjugation of small concentrations of proteinwith FITC and approximates the ponditioni for labeling protein in thefluorescent antibody technique. A stable linkage with protein appearsto be formed; nonproteinaceous substances are readily destained. In corn-parison with fluorescent antibody, the direct conjugation of nonantibodyprotein has receiv.:d limited application in experimental studies. How-ever, a variety of froteins have been conjugated and these include solublebacterial antigens, virus particulates,* bovine albumin,, hormoneuenzymes, and amino acids. The present study was concerned with eo.ab-lishing an experimental basis for utilizing protein conjugation with FITCas a rapid aL] nonspecific method of biological detection.

Three classes of representative substanc¢s were utilized to evaluatestaining reactions with FITC. These consisted of (i) bacteria andselected protein-type substances; (ii) nonprotein biochemical substances;and (iii) predominantly inorganic materials that included shelf compounds,atmospheric debris, and soil samples.

All test substances were examined as smear rreparations on standardmicroscopic slides. Smears of individual and combined test materialswere initially prepared in distilled water droplets. Bacterial suspen-sions were adjusted to contain approximately leP cells per ml. Concen-trated atmospheric debris was collected by impaction on glass slidesand examined with ani without added organisms. Cover slip preparationsof hamster kidney cells were stained directly and mounted.

* Dr. James E. Smith, Syracuse University, permonal communication.

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A standard staining solution of FITC* was prepared with the followingreagents: 1.3 ml of 0.5 M carbonate-bicarbonate buffer (pH 9.6); 6.0 mlof 0.01 M phosphate-buffered saline (pH 7.2);* 5.7 ml of 0,85' -h,;iologicalsaline; and 5.3 mg of crystallinp FITC- added as a dry pwder. After mix-ing on a Vortex minfz; Lor 30 minutes at arbient room temperature, the solu-tivn *as centrifuged at 3000 rpm for 20 minutes to remove any insolubleparticulates. Preparations were either used immediately or stored in thedark at 4 C for no longer than 6 hours.

Duplicate air-dried and gently heat-fixed smears were covered with 0.1ml of FITC solution and stained in a moist chamber. Bacteria were treatedfor one minute at 37 C; other oubstances were stained for 20 minutes atroom temperature. Bacterial smears were warmed to 37 C prior to stsiiningat this temperature. Following staining, slides were rinsed and washedfor 10 minutes in 0.5 M carbonate-bicarbonate buffer at pH 9.6 and mountedin glycerol adjusted to pH 9.6 with 2% carbonate-bicarbona - buffer.Unstained controls were treated in the same manner. Slides were examinedby fluorescence microscopy as described in a previous publication. I

Experimental evidence for firm binding of FITC to bacterial proteinwas obtained by passage of conjugated cells through a Sephadex colum &Andby acetone treatment of stained cells. Two comparable cell suspensionswere prepared in the following manner. Six ml of a phosphate-buffered

saline suspension of Serratia marcescens (10 cells per ml) were combinedwith the standard conjugation reagents and mixed for 30 minutes at roomtemperature. The staining intensity of each preparation was examinedafter this treatment,

Conjugated cells from one preparation were passed through a G-25Sephadex column (coarse, nonbeaded, 28 cm :K 2.5 cm) equilibrated andeluted with 0.5 M carbonate-bicarbonate buf.-r (pH 9.6). Five fractionswere collected separately in conical centrifuge tubes. After centrifu-gation for 20 minutes at 3000 rpm, smears were prepared from the sedimentof each tube, allowed to dry, and then mounted in buffered glycerol.

* All fractions were examined for fluorescence intem.ity of stained cells.

After conjugation, the second suspension was centrifuged at 3000 rpmfor 20 minutes. The pell*t was washed with cold phosphate-buffered saline(pH 7.2) and finally suspended in one ml of buffered saline. Eight ml ofcold acetone were added and the contents mixed for several minutes. Aftermixing, the suspension was centrifuged and washed with buffered saline.Acetone extraction was repeated for a second time and the bacterial pellet

* Reagents and volumes used for the standard FITC solution approximate

ti.- conditions for conjugating 130 mg of antibody protein in thefluorescent antibody method. The antibody protein to be conjugatedis usually dissolved in phosphate-buffered saline.

** Obtained from Naltimore Biologic.1l Laboratories.

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was finally suspended in one ml of 0.5 M4 carbonate-bicarbonate buffer(j-,- 9.6). A drop from the suspension was placed on a slide, air-dried,and mounted in buffered glycerol.

The effect of pH on the FITC staining reaction was evaluated at twodifferent pH levels. Staining at pH 7.2 was performed with an FITC solu-tion containing 7.3 ml of buffered saline (pH 7.2), 5.7 ml of physiologicalsaline, and 5.3 m of FITC Staining at pH 9.6 was conducted with thestandard FITC solution, Smears of §. marcescens were prepared on slidesand prewarmed to 37 C. Staining solutions were applied for one minute at37 C. Atter &L..t-ag, biid- 1-4• _ qnd waxbed In acetone for 15minutes. Acetone-treated slides wei. again washed in distilled water for5 minuLes aud given a final wash in 0.5 M carbonate-bicarbonate buffer(pH 9.6). Smears were mounted in buffered glycerol.

The specificity of the FITC reaction was further evaluated by stain-ing prenarations of Bacillus authracis soil, and atmospheric debris withFITC solution and normal rabbit globulin conjugated with FITC. Additionalspecificity studies included (i) use of a staining solution cont~iningsodium fluorescein instead of FITC, and (ii) complete blockiir w .ITCstaining by pretreatment of bacterial cells with 2,4,dinitrofluorobenzenefor 24 hours.

The possible existence of protein impurities in sodium glycerophosphatewas determined by a modified Biuret reaction and absorbance at 280 aI%.

A micro-Kjeldahl procedure was used for determining the protein contentof glycogen.

Experimental data have shown that selected proteins were stained,nonprotein biochemical substances as well as inorganic compounds weredestained (Tables 1 and 2). However, such substances iz glycogen, impureRNA., and sodium glycerophosphate exhibited sow stairing to a greater o0lesser degree (Table 2). The latter results .may be explained on the basisof protein contamination, which in turn wo-ld depend on. origin of startingmaterials (e.g., anima" glycogen or fat) and degree of purification. Forexample, sodium glycerophosphate and glycogen were found to contain asignificant amount of protein-type impurity (Table 2).

The critical differentiation between atmospheric background and specificstaining of bacterial protein was evaluated with standard FITC solution.The results have shown that background was readily destained, but thatparticles of possible biological origin were brightly stained. This effectwas strikingly more apparent when bacterial cells were added to atmosphericdebris and soil (Table 3). Figure 1 Jilusttates staining of j. anthracisin atmospheric debris.

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TABLE I. STAINING OF SELECTED PROTEINS 4ND BACTERIAWITH STANDARD FITC SOLUTION!'

Staining / UnstainedTest Substance Intensity;-- Control

Protein-Polypept ide-Amino Acid

Egg Albumin 4+ -DL-CysteineHCl 2+ -Glvcyl-L-Tyrosine 3+ -Hamster &ioney Cells 4+ -Mouse Liver Powder 4+ -Wheat Germ 4+ -

Bacteria

Bacillus anthracis CD-3S 4+ -Bacillus anthracis (spores) 4+ -Bacillus subtils var. 4+ -Rrucella abortus 6NIH-1 3+ -Listeria monocyto enes ATCC7644 + -Salmonella typhosa ATCC9992-V 4+ -Serratia marcescens 8UK 4+ -

a. Protein-polypeptide-amino acid group stained for 20 minutesat room temperature; bacteria stained for one minute at 37 C.

b. Estl'iated brightness based on miaimal to maximal intensity(1+ to 4+); - - no staining.

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TABLE 2. STAINING OF NONPROTEIN BIOCHEMICAL SUBSTANCESAND INORGANIC COMPOUNDS

WITH STANDARD FITC SOLUTION!'

Stainini, UnstainedTest Substance Intensity-/ Control

Nonprotein Biochemical Substances

Glycogen +-_rv. fimlure) -

RNA (impure) 2+DNA (pure)+-Sodium Glycerophosphate 2

Inorganic Substances

Zinc Powder - -Stannous Chloride - -Potassium Permanganate - -

Mercuric Chloride - -Molybdenum Trioxide - -

Test substances stained for 20 minutes at roomtemperature.

b. Estimated brightness based on minimal to maximalintensity (1+ to 4+); ± - sever-l fluorescentparticles observed in each microscopic field;- -no staining.

c. Glycogen and sodium glycerophosphate containedC.6 and k n4% by weight of protein impurityrespectively; the c. 'entration of proteinimpurity in DNA and ,uA was not determined.

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TABLE 3. STAINING OF ATMOSPHERIC AND SOIL BACKGROUNDAS WELL AS ADDED TEST ORGANI S S

WITH STANDAID viITC SOLUTION!'

Staining UngtainedTest Substance Intensity-/ Control

soi,- +

B. anthracis in Soil 4

Atmospheric Debris-'

B. anthraci~s in Air Debris 4+

B. aubtil~is var. nizer (sporea) in 3.+-

Air Debris

B. abortus in Air Debris 3+

a. Test substances stained for 20 minutes at room temperature.b. Estimated brightness based on minimal to maximal. intensity

(14- to 4+): an average of one and si.x brightly stainedparticles r--'4din 30 microscopic fields for atocaphericand soil background respectively (come stained particles insoil resemble rod-like bacteria); - - no staining.

c. A total of 6 soil samples and 12 air samples were examined.

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Staining reactions illustrating the specificity of FITC solution ascompared with FITC-conjugated normal rabbit globulin are presented inTable 4. Experiment-al results have shown that FITC conjugated withprotein was not available for coupling with unlabeled protein (bacterialprotein). Specificity was dependent on the presence of proper proteincombining groups and available FITC. When sodium fluorercein was sub-stituted for FITC no specific fluorescence was observed. This could beexpected, since an isothiocyanate linkage wes not present for couplingwith protein. Pretreatment of bacterial cells with 2,4,dinitrofluorobenzenecompletely inhibitad staining with FITC and indicated effective blockingof combining sites on the bacterial protein.

TABLE 4 STAINING SPECIFICITY OF FITC SOLUTIONCOMPAEED WITH FLUORESCEIN-L BELED

NORMAL RABBIT GLOBULINA'

JstainIn ntensl.' ?-

Labeled NormalTest Substance FITC Rabbit Globulin

Atmospheric Debris *

B. anth-acia 4+

Soil ±

B. anthracis in Soil 4+

a. Test substances stained for 20 minutes atroom temperature.

b. Estimated brightness based on minimal tomaximal intensity (1+ to 4+); - - no staining;± - occasional fluorescent particles ofpcosible biological origin.

!*

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The effect of acetone extraction or passage through a Sephadex columnon the linkage of FITC to protein is hown in Table 5. The intensity offluorescence following such treatment did not appear to be affected, endindicated firm binding of FITC to bacterial protein. The staining reac-tions of a. arcescens mars treated vith solutions buffered at pH 7.2&a4A pF 9.6 are also presented in Table 5. They show that the fluorescenceintensik.-, of preparations stained at pH 7.2 is markedly reduced and impli-cates rapid conjugation at a higher pH (9.6) as the factor responsible forbrighter staining.' is

TABLE 5. DETERMINATION OF BIDING O- nTC TO ACMTERIAL PROTEIN

Method for Determining StainingFirtness of FITC Intensity of

Experiment Method of Staining;/ Binding Treated Cellek

1 Cells staineO in Passage through 4+solution at pH 9.6 Sephadex column

2 Cells stained in Acetone extraction 4+solution at pR 9.6

3 Cells stained on a "Acetone extraction 4+slide at pH 9.6 1

4 Cells stained on a Acetone extraction 2+slide at pH 7.2

a. Cells were stained in standard conjugation solution for 30 minutes atroom temperature; a one-minute staining period at 37 C was used forslide preparations

b. Estimated brightnt.,. was based on minimal to maximal intensity (1+ to4+); staining intensity remains unaffected following Sephadex andacetone treatment.

i ............

14

The staining technique described in this report was simple and did notrequire any unusual manipulations. Specific staining appeared to depend onthe availability of proper combining groups as well as the usual conditionsfor conjugating protein. A wider latitude in conjugation methodology ispossible with bacteri.l and other nonantibody proteins, because conditionsthat may affect antibody combining activity are of little :oncern in thepresent study.

It is impossible, of course, to predict the reactions of FITC in extra-terrestrial environments. Two major sources of error might be encountered:(i) reaction of FIN with nonprotein substances of unusual chemical con-figuration; and (ii) emission of green autofluorescence by nonproteinmaterials.

For satellite probes, the FITC method could be used 4n the followingmanner. Soil or dust particles are drawn into a previously filtered FITCsolution and reacted for several minutes. The reaction mixture is passedthrough a filter-type membrane and stained particles are deposited on thesurface. The membrane surface is given a buffer wash and particles arethen scanned with a sensitive phototube device.

Solutions of FITC buffered at pH 9.6 tend to become unstable afterseveral hours. Therefore, for extended missions, FMTC powder could bemixed with liquid reagent at the time of sampling. Instrumentatirn ofthe FITC reaction should not be any more complex than that proposed forpresent systems.

The FITC method is based on the assumption that proteinaceous sub-stances (particularly microorganisms) may exist in extraterrestrialenvironments and possess structural characteristics that are reminiscentof Earth protein.

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i1. Quimby, F.M. 1964. Concepts for detection of extraterrestrial life.

HASA, SP-56.

2. Coonm, A.H.; Creech, H.J.; Jones, R.N.; Berliner, E. 1942. Thedemonstrntion of pneumococcal antigen in tissues by the use tffluorescent antibody. J. Immunol. 45:159-170.

3. KaplanP, N.H.; Coon., A.H.; Deanna, .W. 1950. Localization of anti-gen in tissue cellsa IrT. Cellular distributiot of ineumococealpolysaccharides types II and III in the mouse. J. ep. Med. 91:15-30.

4. Marshall, J.D.; Eveland, W.C.; Smith, C.W. 1958. SupaLortty offluorescein isothiocyanate (Riggs) for fluorescent-antibody technicwith a modificetion of its applicatior, Proc. Sac. Exp. Bio4o. Med.98:898-900.

5. Eveland, W.C.; Griies, J.V. 1964. Use of fluoresatii-labeled sonic-disrupted bacterial antigen to demonstrate antibody-producing cellsBacteriol. Proc. p. 64.

6. Schiller, A.A.; Schayer, R.W. 1951. Fluorescein-conji.-'-ed plasmaprotein as an indicator of vascular permeability. Federation Proc.10: 120.

7. Mmcini, E.E.; Vilar, 0.; Dellacha, J.M. ; Gimano, A.; Castro, A.

"1959. Histological lu6alizatfon of fl,,orescent thyroid stimulatinghormone in rat tissues. Nature 184:1733-1734.

8. Cormack, D.H.; Easty, G.C.; Ambrose, E.J. 1961. Interaction ofenzymes vth normal and tumour cells. Nature 190:1207-1208.

9. laser, L.F.; Creech, H.J. 1939. The conjugation of amino acids withisocyanates of the anthracene and 1,2, benzanthracena series. J. An.Chaw. Sac. 61:3502-3506.

10. Pital, A.; Janowitz, S.L. 1963. Enhancemmnt of staining intensityin the fluorescent-antibody reaction.. J. Bacteriol. 86:888-889.

11. Gornall, A.C.; Bardwmill, C.J.; David, N.M. 1949. Determination ofserum proteins by mans of the biuret reaction. J. 1iol. Chai.177:751-766.

12. Nairn, i.C. 1962. Fluoresco-mt protein tracing, Chaptar 3. 1. & S.Liv!agstu.i Ltd., Edinburgh & Lodon.

13. Singer, S.L.; Schick, A.F. 1961. The preparation of specific utatnsfor electron micretropy prepared by the conjugation of antibodymolecules with ferritin. J. liophys. tiochem. Cytol. 9:519-537.

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I qEPORT TITLE

DIRECT FL1JORESCEWr TAGGINO OF M' 4.OORGANISMS: A POSSIBLE LTFE DETECTiONTECHNIQ11E

4 5scR#PTIV;E -do7,ý77. .1 ý-po,r ed nih,..- de.00)

I5 -AUTNON(S) ZooL.a.Vn,. 1r.- '"0, -701e)

Pital, Abe NKI Hudak, Charles E. _

Janowitz, Sh..ld~cn L. Lewis, Evclyn E.

III REPORT OATL TOTOTAL .0 OF PAGW.F 76. No, OF R(Prs

August 1465 ____ _____18 1 3

6 PRJECTNC 162241A07 ~ ::Technical Manuscript 236

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38 A-IRACT

Microorganisms and selected proteinaceous substances were directlytagged with fluorescein isothiocyanate. This approach suggested a poesibleapplication for detection of extraterrestrial life. A stable anid apparentlyspecific linkage was formed with protein, although nonprotain substanceswere readily destained. Substances such as 4oll and atmospheric debrisdiJ not exhiLit any significant affinity for the dye.

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