Bacterial Aerosol Samplers

III. Comparison of Biological and Physical Effects in Liquid Impinger Samplers

M. E. TYLER,' E. L. SHIPE,2 AN D R. B. PAINTER3

Fort Detrick, Frederick, Maryland

Received for publication May 15, 1959

The relationships between particle size discrimina-tion and biological recovery in bacterial aerosol sam-

plers were explored between 1948 and 1952 and are

reported here. Little experimental evidence concerningthese points has been published. Particularly deficienthas been the measurement of the possible destructive-ness of samplers for vegetative forms of bacteria. Ten-tatively quantitative data are presented indicating thatsuch killing may be a material factor in evaluating aer-

osols.Several excellent studies of the theoretical behavior

of aerosol particles have been published (May, 1945;Wells, 1955; Magill et al., 1956) and some of these con-

tain experimental data derived from nonliving mate-rials. Meager biological data under restricted conditionswere given by Phelps and Buchbinder (1941), duBuyet al. (1945), and Bourdillon (1948). Since the develop-ment of the all-glass impinger (AGI)4 as a useful sam-

pler, problems of particle discrimination in the inlettubes of samplers became obvious. As reported, it ap-

peared that larger bacterial droplets might be segregatedin the inlet tube of the AGI (Tyler and Shipe, 1959), an

effect which could be diminished or eliminated as in theShipe sampler (Shipe et al., 1959). The theoretical con-

siderations formed a basis for predicting that the curvedinlet tubes of the AGI and capillary impinger, and thevarious constrictions and protuberances in most liquidimpingers, would serve as impaction surfaces for par-

ticles above a few microns in equivalent diameter.However, such theories did not provide for the very

probable variance in density and configuration of bac-terial aerosol droplets. It was believed, therefore, thatonly by use of comparative experiments with bacteriaand physical tracers could a quantitative evaluation ofthese factors be attempted.The potential destruction of bacteria during the sam-

pling process has received little attention, despite sug-

gestions of their lability in disseminators (Rosebury,

l Present address: Department of Bacteriology, College ofAgriculture, University of Florida, Gainesville, Florida.

2Present address: Knoxville Branch Laboratory, Knoxville,Tennessee.

3Present address: Medical Department, Brookhaven Na-tional Laboratories, Upton, Long Island, New York.

4Later designated "AGI-4" indicating 4 mm clearance be-tween capillary jet and bottom of bottle.

1947) and aerosols (Wells, 1955). Indirect but strongevidence of such killing of Serratia marcescens and Esche-richia coli was found when comparing the AGI to lessforceful samplers (Tyler and Shipe, 1959; Shipe et al.,1959). Attempts to explore this aspect have met withsome success. Quantitative data are presented to show,tentatively, that the AGI, although quite efficient, dis-criminates against larger air-borne particles and causessignificantly great killing of vegetative bacteria, variablewith species, relative to other samplers.

MATERIALS AND METHODS

The bacterial suspensions, aerosol chambers, aerosolsamplers, disseminators, and bacterial assay techniqueswere described previously (Tyler and Shipe, 1959).Crystal violet solutions, used for physical studies, weredevised empirically by adding varying proportions of asaturated alcoholic solution to gelatin phosphate diluentuntil appropriate concentrations were attained for aero-sol measurement. 'No strong attempt was made to keepcrystal violet concentrations identical in all tests, sinceall data were relative and depended particularly onparticle size. Samples taken from dye aerosols wereassayed by washing the samplers thoroughly with 95per cent ethanol in measured volume, and evaluatingthe concentration of dye in a Klett-Summerson5 colori-meter with use of a standard curve made with knowncrystal violet concentrations.

Particle-size measurements were made with cascadeimpactors designed by May (1945). The impactors werecalibrated with ethylene glycol aerosols, and the massmedian diameters (MMD) of aerosol particles werecalculated. "Effective drop sizes" were estimated incrystal violet samples by washing off the slide of eachstage of the impactor with alcohol, measuring the dyecontent in a colorimeter, and calculating by May'smethod.

S. marcescens cells were tagged with radioactive phos-phorus (P32) by culturing single colony isolates in amineral base glucose broth to which Na2HP3204 wasadded before adjusting to pH 6.8. The flasks were incu-bated on a reciprocal shaker at 30 to 32 C. Empiricalvariations in the amount of p32 added and in the incu-bation period permitted selecting cultures in which the

Klett Manufacturing Company, New York, New York.

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p32 was sufficiently concentrated to permit use in aerosolstudies. A similar empirical approach was used in "tag-ging" S. marcescens cells with S35, using Na2S3504 as theadditive to the mineral glucose broth. To prepare sus-

pensions for dissemination, cultures were harvested andwashed twice in distilled water by centrifugation; thecells were resuspended in distilled water to approxi-mately 10 X 108 per ml immediately prior to dissemina-tion. Control samples were removed for plate count andtracer count. Tracer counting was accomplished by add-ing appropriate volumes (0.1 to 1.0 ml) of suspensionor sampling fluid to a stainless steel planchet, dryingunder an infrared lamp, and counting in a thin-windowGeiger6 counter. Appropriate corrections were made forbackground count and self-absorption; the latter was

essentially negligible for suspensions in distilled water.Another method also permitted direct correlation

between total and viable recovery. A stained cell7 tech-nique was adapted in which an aliquot of bacterial sus-

pension (or aerosol sample) was mixed with saturatedaqueous crystal violet (approximately 1:1). A smallquantity of the mixture was added to fill the chamberof a Spencer-Neubauer8 hemocytometer, and was

allowed to stand for 45 min to permit settling of cellsonto the ruled surface. All stained bodies were countedunder the oil immersion objective of a microscope, andthe number per ml of original sample was computedand recorded as "total cells." It was found during theseand other studies that various, apparently nonbacterial,particles occasionally interfered; it was only in "clean"aerosols consisting exclusively of droplets from a care-

fully prepared bacterial suspension that reliable resultscould be obtained. Distilled water for both suspensionand sampling was filtered through bacteriological filters,and glassware was rinsed carefully with filtered water.

EXPERIMENTAL RESULTS

Discrimination in sampler inlet tubes. Early attemptsto evaluate the numbers of bacterial droplets trappedin the inlet tube of the AGI were performed with mixedaerosols generated by a DeVilbiss9 nebulizer. The sus-

pensions contained approximately 0.8 X 108 Bacillussubtilis spores and 1.4 X 108 S. marcescens per ml andgave aerosols in the glass tube aerosol unit (Tyler andShipe, 1959) of <3.0j, MMD. In each of five trials,

six duplicate samplers were used, the samplers from eachtrial being refrigerated until all were available for assay,

from 1 to 3 hr. The refrigerated samplers were thenassayed by removing duplicate aliquots from the sam-

pling fluid. After the residual sampling fluid from the

6 Tracerlab, Inc., Waltham, Massachusetts.7 Detailed technique developed by Mr. Milton Shon, Fort

Detrick, Frederick, Maryland.8 American Optical Co., Buffalo, New York.9 The DeVilbiss Company, Toledo, Ohio.

capillary was removed with a sterile cotton pledget anddiscarded, the inlet tubes were rinsed twice with 5-mlaliquots of distilled water. The two washings from eachinlet tube were pooled, and they and the sampler ali-quots were diluted appropriately and plated by thesurface spreading method. Colonies of each bacteriumwere counted after the plates had incubated at 30 to32 C for 24 to 36 hr. The results (table 1) revealed thatrelatively few spores were trapped in the inlet tube whensampling small droplets. Recoveries of S. marcescensfrom the inlet were even less in proportion to those inthe sampling fluid. It was learned later (see below) thatthis could be explained in part by the death of cellstrapped in the inlet, during sampling, and during re-frigerated storage.A series of tests with larger aerosol particles was per-

formed in the test chamber (Tyler and Shipe, 1959) withexplosive dissemination of S. marcescens. Although notmeasured in this series, previous data indicated thatthe aerosol was of heterogeneous droplet size, averagingabout 5 A MMD at the first sampling time. The assayof samplers was performed as above, except that allsamples were plated within 15 min after completion ofsampling. The mean percentage of S. marcescens re-

covered from the inlet tube as compared to the totalfrom sampler plus inlet tube at the various samplingperiods was:

4 Min

Per cent total in inlet 22.5Standard deviation of

values .............. a12.2No. samplers 11

9 Min

13.418 Min Avg

14.7 16.7

4-6.3 --13.4 4±11.412 12 35

These values suggested that the larger particles, andperhaps earlier assay, gave much higher retention values

TABLE 1Apparent retention in inlet tube of all-glass impinger (AGI) of

Bacillus subtilis spores and Serratia marcescens collectedfrom aerosols of small droplets

B. subtilis Spores S. marcescens*

ia l-

No. No. from No. from No. per No. from No. from No. perinlet/ inlet/sampler inlet tota/ sampler inlet total no.

x 1O0 x 103 % X 103 X 1O3 %1 400t 1.930 0.49 540 0.980 0.182 392 0.689 0.18 597 0.865 0.143 353 0.469 0.13 432 0.446 0.104 320 0.464 0.14 336 0.348 0.105 330 0.396 0.12 519 0.369 0.07

Mean (60 samplers). 0.21 0.12S.D. (mean)............. -+-O. 15 4:0. 04

* The refrigerated samplers were assayed several hr afterthe samples were collected.

t Aerosol MMD (mass mean diameter) < 3.0,. Each valuerepresents the mean of 12 samplers.

S.D. = standard deviation.

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BACTERIAL AEROSOL SAMPLERS. III

in the inlet tube. It was noted that the percentage re-tained decreased between the first and second samplingperiod, possibly ascribable to a decrease in number oflarger droplets remaining air-borne. This was confirmedin another series, similarly performed, using morewidely spaced sampling intervals:

Per cent total in inlet...........Standard deviation of values....No. samplers....................

2 Min 29 Min

33 22416 :1:59 9

59 Min

7.3:11.89

To evaluate the relative retention of various parts ofthe inlet tube of the AGI, samplers were modified bycutting off various portions of the exterior tubulation:all of the curved portion only; and all external tubing,with the resulting edge of the joint ground smoothly.Sets of these samplers were tested in parallel with un-modified AGI in explosively disseminated aerosols in thetest tank (Tyler and Shipe, 1959). The summarized re-sults (table 2) illustrate two important findings: (a)that the modified samplers recovered, in both inlet andfluid, more than twice as many S. marcescens as theunmodified samplers; (b) that, since the apparent inletretention in the modified samplers was severalfoldgreater than in the unmodified, there must have beenappreciable destruction of S. marcescens impacted inthe full inlet tube during the sampling process. Thecontribution of particle fall-out to the differencebetween modified and unmodified samplers was dis-counted by consideration of the short sampling time(1 min) and the differences still apparent at later sampl-ing periods (not shown in data).The tests reported above demonstrated the need for

a more reproducible procedure than that afforded byspore aerosols, and for one which would not be com-plicated by considerations of bacterial delicacy for quan-titative estimation of droplet discrimination. Crystalviolet dye was chosen for its ease in handling, sensitivityin detection, and precision of measurement in a colori-

TABLE 2Apparent retention in modified inlet tubes of all-glass impinger

(AGI) of Serratia marcescens collected from static aerosols

Calculated No Inlet MeanColonies Recovered Re-

Sampler Modification No. covered/Samples TotalSampler + Sampler Samplerinlet wash fluid only

X 106 X 106 %

Unmodified ............... 26 11.8* 9.0 23.14t2.8 4--2.3 -:114.0

Curve removed.26 26.8 11.6 58.4:18.5 :--6.7 -:-11.5

Exterior tube removed.... 26 27.9 13.3 53.2-:18.5 j--7.3 -:-15.0

* Each value is average of samples taken at 2, 9, and 17min after dissemination in the test tank, 9 trials, and standarddeviation of the values.

meter. Solutions of crystal violet in distilled water orgelatin-phosphate diluent were prepared and dissemi-nated by a DeVilbiss nebulizer, Chicago'0 atomizer, orvariously adjusted Binks" nozzles into a laminar-flowwind tunnel. The concentration of dye, composition ofdiluent, and type of disseminator were empiricallytested to give a wide range of droplet sizes in the aero-sols. Particle size was determined by the effective dropsize technique (May, 1945) in cascade impactors oper-ated parallel to AGI. The AGI were operated withoutsampling fluid; tests with cotton collectors in seriesdownstream demonstrated that less than 1 per cent ofthe dye escaped in the exhaust. The amounts of dyecollected on slides at each cascade stage and theamounts collected in AGI inlet and bottle separatelywere assayed by washing carefully in 50 per centethanol, adjusting the volume to a known value, anddetermining quantity by transmission in a Klett-Sum-merson colorimeter and comparison with a standardcurve. The results are illustrated in figures 1 and 2.Despite some variability, the percentage of dye retainedin the inlet tube increased nearly linearly as the calcu-lated MMD of the droplets increased; at 2 ,u MMDthe retention was less than 10 per cent, whereas at 20A MMD retention averaged near 65 per cent. A morecritical analysis was permitted by calculating the per-centage of the total mass of droplets falling below vari-ous diameters and comparing with retention in the inlettube. Analysis showed that the logarithm of percentageretention varied inversely with mass percentage below5 ,u to a high level of correlation (figure 2). It was foundby this analysis that the retention was well below 5 per

10 See Rosebury (1947)."Binks Manufacturing Co., Chicago, Iflinois.

20

18

16

'4

; 12

°0 lo0

Sa080

a6

2

A

a

gz~~~r= 0.89

0 10 20 30 40 50 60 70 80Per cent retained in inlet

Figure 1. Effect of particle size on retention of crystal violetaerosols in the inlet tube of the all-glass impinger. MMD =

mass mean diameter.

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M. E. TYLER, E. L. SHIPE, AND R. B. PAINTER

cent when nearly 90 per cent of aerosol particle masswas below 5 ,u. Retention exceeded 60 per cent when lessthan 20 per cent of the particle mass fell below 5 ,u.These data confirmed earlier tentative conclusions frombiological tests that, in aerosols of very small particles,the AGI samples with little or no discrimination.

Tests performed with modified inlet tubes (table 2)had shown appreciable retention of larger particles evenwhen the exterior tubulation was removed. A few trialswere performed later with dried S. marcescens in whichvisual appearance only was used to estimate the loca-tions of principal retention in unmodified and modifiedinlet tubes of the AGI. These observations suggestedthat the curved portion was most important, althoughsome impaction occurred near the entrance even whenthe curve was removed. Furthermore, the quantity ofparticles impacted where the inlet tube constricted tocapillary bore increased visibly as the length of exteriortubulation was decreased. Quantitation of the amountof material collected at the constriction as related to theinlet tube as a whole was undertaken by the crystalviolet aerosol technique as described. The dye collectedin the constriction was removed by dipping the capillaryand lower 14 in. of tubing into solvent; the dye collectedin the upper portion of the inlet was then washed outas was that in the sampler bottle. Each portion wasanalyzed by the colorimetric method. It was found(table 3) that, although the proportion of dye in theconstriction was quite variable with aerosols of differentparticle sizes, there was a trend toward higher quanti-ties with smaller particles. Nevertheless, the total quan-tity collected in the constriction relative to collection inthe whole sampler was quite small, and the upper por-tion of the inlet tube was implicated as the major sourceof particle size discrimination in the AGI.

90

"'803:.2700.00600

o50._21-040

30

w20

1I

Killing of vegetative bacteria by liquid impingement.The possibility of killing some proportion of livingbacteria taken in by such samplers as the AGI was sug-gested by previous results as well as by theoretical esti-mates. Attempts to quantitate such an effect, if it oc-curred, were complicated by inability to separate clearlybetween loss by discrimination (impaction) and by kill-ing, and by the difficulty of estimating the number ofliving bacteria expected to be available in the aerosolfor sampling. Previous experience with B. subtilis sporesas tracers of assumed indestructibility led us to believethat the variability associated with germinating andclumping irregularities made them too insensitive forthis use; experimental attempts convinced us that thiswas true. Attempts to achieve unicellular particles inspore aerosols were unsuccessful.The attractive possibilities of tagging bacteria with

radioactive tracers, whose sensitivity to detection wassufficiently refined, led to several experiments. S. marces-cens, cultured with Na2HP3204, was found to take upsufficient tracer to permit measurement of it in aerosolsamples. Such suspensions were disseminated from dis-tilled water by a DeVilbiss nebulizer into the plasticsphere unit, giving an aerosol of <3.0 ,u MMD. Shipesamplers and AGI containing distilled water were usedin pairs to sample the aerosol. Immediately after sam-pling, each was assayed by bacterial plate count and byisotope count. The measured recoveries of viable cellsand of isotope were adjusted to a common basis by com-puting the percentage of each recovered in the sampleron the basis of the amount (0.15 ml) of suspension dis-seminated. Obviously, this did not compensate for lossesduring dissemination and passage through the appara-tus; it did give a comparable basis for comparing thesamplers. The results of five trials, with six pairs of sam-

TABLE 3Relation between particle size of crystal violet aerosols and

retention in the all-glass impinger (AGI) inlet tube*

Aerosol MMDt

r=-0.98 (log.)

oj.0 to0 0 0000 0m 1 5° ° - 2 %

0 0.5 l.0 l.5 2-.0Log. per cent retention

Figure 2. Effect of decreasing percentage of particles of crys-tal violet below 5 ,u on retention in the inlet tube of the all-glass impinger.

2.23.54.04.14.54.66.37.07.211.011.012.013.018.0

Dye in Constriction/Total Dye in Inlet

35.025.721.015.18.07.79.4

20.94.111.14.76.54.45.2

Dye in Constriction/Total Dye in Sampler

1.511.723.101.901.421.412.946.821.961.784.492.661.822.97

* Tests performed in a laminar flow wind tunnel.t MMD = mass mean diameter.

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plers in each (table 4) demonstrated that, by this tech-nique, the AGI collected slightly but not significantlymore tracer than the Shipe sampler. The Shipe sam-

plers, on the other hand, contained 23 per cent more

viable cells than the AGI, a highly significant difference.The ratio of viable cells to tracer count in each sampleralso, of course, indicated as 23 per cent higher viable:total recovery in the Shipe samplers.A similar series of tests was performed comparing the

Midget impinger12 and AGI. In four trials with sixpaired samplers per trial (table 4), it was found thatthe Midget impingers contained significantly less iso-tope and fewer viable cells, giving viable:total ratiosof the same magnitude. Controls indicated the pres-

ence of appreciable extracellular p32 in the aerosol sam-

ples, however, making interpretation questionable.The difficulty of obtaining firmly tagged cells with

p32 led to investigation of S35 as a tracer. Preliminarystudies showed that high isotope counts could be ob-tained with S. marcescens cultured with Na2S3504, andthat extracellular isotope was negligible in such cul-tures after washing with distilled water. Trials similarto those with p32 were performed in which the Shipesampler, Midget impinger, and AGI were tested in par-

allel in aerosols of tagged S. marcescens, <3.0 MMD,in the plastic sphere unit. A summary of six paralleltests (table 5) showed that the AGI and Shipe samplerscontained equal amounts of tracer, the Midget im-pingers having significantly less. The viable cell counts,

12 Mine Safety Appliance Company, Pittsburgh, Pennsyl-vania.

TABLE 4Relative viable and total recoveries in three samplers of Serratia

on the other hand, indicated higher recoveries in theShipe samplers and Midget impingers than in the AGI.An analysis of the ratios of viable:tracer recoveries inthe three samplers demonstrated superiority of theShipe sampler and Midget impinger over the AGI byabout 29 per cent; in the Midget impinger, of course,

this reflected lower total as well as viable recoveries.Concurrently with isotope tracer studies, another

technique for estimating total cell recovery was at-tempted. It involved staining cells of S. marcescens incontrol suspensions and aerosol samples with crystalviolet, and making stained cell counts in a hemocytom-eter. The assumption was made that all cells, livingor dead, were stained and countable. Trials comparingthe Shipe sampler and AGI with small aerosol drop-lets were performed by the usual method in the plasticsphere unit. Thirty-one comparisons were made, usinga suspension which gave a ratio of viable to stainedcells of about 0.5, the plate count approximating 10 X108 per ml. The results, calculated to numbers recov-

ered per L of aerosol sampled, were:

Plate Count Stained CellsX 106/L X 106/L

AGI .................... 7.1 16.8

±0.36 ±0.57Shipe samplers ......... 11.6 19.7

±0.54 40.63

Viable Count:Total Count

0.48±0.020.58

±0.02

Thus, it was apparent that, with these small particles,the Shipe samplers collected significantly more viablecells and more total cells than the AGI. More impor-tantly, the Shipe samplers contained a very signifi-

marcescens aerosols tagged with p32

SSuspension Counts per ml Viable Recovery* Tracer Recovery* Viable/TracerSampler Type Trial No.

Plate Tracer Test AGI Test AGI Test AGI

X 10 CpS X 104X % % % % %

Shipe samplert 1 7.2 4.4 10.7 8.1 25.7 21.5 43.3 32.52 13.0 18.8 11.0 9.4 22.2 23.6 50.5 41.73 13.0 18.8 10.6 9.3 22.8 24.7 45.2 35.04 7.1 9.7 8.2 7.2 26.5 29.2 30.5 24.35 7.1 9.7 9.9 8.7 26.2 32.7 38.5 26.8

Mean ................ ....... 10.1 8.2 24.8 26.4 41.5 32.0S.D. (mean) .1.8 =4=1.8 45.1 4:6.4 :4:9.3 47.9

Midget im- 1 0.83 32.9 10.5 14.1 15.1 21.4 71.3 67.8pingert 2 0.83 32.9 10.0 17.4 13.8 22.8 69.9 76.2

3 10.5 5.6 13.1 21.9 18.8 21.6 68.7 74.34 10.5 5.6 12.9 16.8 20.2 27.8 62.6 59.7

Mean ............ ................. 11.6 17.6 17.0 24.9 68.2 69.8S.D. (mean) .2.9 ±4.0 +4.3 ±6.0 ±12.1 ±12.2

* Percentage of total numbers of bacteria, or total tracer, sprayed from 0.15 ml that were collected in sampler fluid.t Six paired samplers per trial, mean represents 29 sampler pairs (one lost).t Six paired samplers per trial, mean represents 24 sampler pairs.AGI = all-glass impinger; S.D. = standard deviation.

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cantly higher proportion of viable: total cells. The ra-tio of viable: total cells in the Shipe samplers comparedfavorably with the ratio in the control suspension, butinsufficient data were collected from the latter to per-mit analysis of the correlation. On the assumption thatthe stained cell counts in the Shipe samplers repre-sented 100 per cent of recoverable aerosol, the Shipesamplers averaged 23 per cent greater viable cell re-recovery than the AGI.

Since technical difficulties were encountered withboth isotope tracer and stained cell techniques, testswere performed in which both methods were employed

TABLE 5Relative viable and total recoveries in three samplers of Serratia

marcescens aerosols tagged with S35

Sampler Type Mean Viable* Mean Tracer* Viable/TracerRecovery Recovery

All-glass impinger.... 9.0 (d1.7) 36.7 (f5.5) 24.4 (i3-7)Midget impinger ...... 10. 1 (a2.0) 29.2 (-5.1) 34.7 (-4.2)Shipe sampler ......... 12.3 (-i 2.3) 36.2 (di5.5) 34.4 (-:-5.5)

* Means of 6 samplers each, run in parallel. Control counts:14.1 X 108 per ml, plate count; 13.03 X 104 cps per ml, tracer.

TABLE 6Relative viable and total recoveries in the all-glass impinger

(AGI) and Shipe sampler of Serratia marcescens, with p32or stained cells as a physical measure

Mean Ratio Viable/Total, Per Cent Recovery*

Day Replicate Trial No. pt2 tracer Stained cell

AGI Shipe AGI Shipe

1 A 1 0.918 1.074 0.813 0.9772 0.971 1.026 0.705 0.944

B 1 0.723 0.755 0.653 0.8402 0.906 0.847 0.944 0.910

2 A 1 0.620 0.819 0.521 0.6922 0.594 0.791 0.512 0.705

B 1 0.851 0.881 0.766 0.7982 0.826 0.940 0.675 0.867

A 1 0.695 0.910 0.618 0.8632 0.871 0.935 0.466 0.738

B 1 0.728 1.227 0.634 0.9592 1.000 1.367 0.900 1.172

4 A 1 0.962 1.297 0.889 1.0992 0.977 1.309 0.834 1.047

B 1 0.995 1.021 0.826 1.0232 0.904 1.045 0.789 0.849

Mean .0.0.839 0.995 0.706 0.895

Ratio: AGI/Shipe. 84% 79%

* Per cent recovery as measured by plate count (viable), orradioactive or stained cell count (total). Each value represerts

on the same suspension of P32-tagged S. marcescens.

Duplicate comparisons of the Shipe sampler and AGIwere made in each of two replicate trials on each offour days in the plastic sphere unit (aerosol MMD<3.0 IA). Each aerosol sample was assayed for isotope(p32), stained cells, and viable cells by plate count. Theresults were adjusted to a common basis by computingpercentage recoveries based on control counts and vol-ume disseminated. Ratios of percentage of viable:totalrecovery (table 6) demonstrated the same relative re-

lationships between the Shipe samplers and AGI: theformer gave higher viable recovery in relation to totalrecovery. The difference in ratios between data ob-tained with isotope and stained cells was significant,although relatively small. The relative killing effect ofthe AGI compared to the Shipe was 16 per cent, esti-mated with p32 tracer; and 21 per cent, estimated withstained cells.

Killing effect of AGI with other bacterial species. Thedestructive effects of impingement shown with S. mar-

cescens suggested that there might be marked killingwith other species of bacteria. Two experiments tendedto confirm this, but data were inadequate to ascribehigh certainty to the quantitative values obtained.Tests with Brucella suis gave comparative recoverieswith the Shipe sampler 2- to 6-fold higher than withthe AGI, with precaution taken to equalize particlediscrimination. Comparative tests with small-particleaerosols of Pasteurella tularensis were reported'3 inwhich the Shipe sampler recovered 2- to 5-fold more

viable cells than the AGI.

DISCUSSION

The data presented here add quantitative evidenceregarding two parameters of aerosol sampling for bac-teria with the better models of liquid impingers: par-ticle discrimination by mass, and killing of vegetativebacteria. Combined with earlier reports of efficiency ofcollection (Tyler and Shipe, 1959), a fairly reasonableestimate of over-all usefulness of the AGI and relatedsamplers may be made. The properties of the AGI may

be summarized as follows: (a) it is highly efficientin removing bacteria from air, with less than 1 percent loss in exhaust air; (b) the standard inlet tube dis-criminates against air-borne particles of larger sizes, re-

moving by impaction most of those whose equivalentdiameters exceed 18 to 20 ,u, and collecting in the sam-

pling fluid more than 95 per cent of those below 5 ,;(c) the bacteria impacted in the inlet tube, if they are

as sensitive as S. marcescens, die rapidly in situ, mak-ing quantitative recovery by washing the inlet tubequestionable; and (d) the direct, normal impingementfrom a jet 4 mm above the bottom of the bottle, or

some other unknown force, causes material destruc-

13 Personal communication from Dr. Henry Eigelsbach, MBthe mean of 4 samplers.

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tion of living bacteria such as S. marcescens, B. suis, orP. tularensis.

Of the other types of liquid samplers examined, onlythe Shipe sampler appears to offer limited advantages.The liquid bubbler samplers, exemplified by the Midgetimpinger, are quite inefficient in collection althoughthey may at the same time be less destructive; certainlytheir inability to entrap smaller air-borne bacterial par-ticles is a serious defect. The Shipe sampler, in its pres-ent design, is slightly less efficient than the AGI forsmall particles; precise estimates were not made of thelow limit of particle sizes entrapped. It has the qualityof being relatively nondiscriminatory to larger air-borne particles; this is advantageous where knowledgeof total air-borne content is desired. Its greatest meritis its appreciably lesser killing effect for delicate bac-teria, presumptively resulting from the tangential modeof impingement into liquid.

Unfortunately, direct comparison of the liquid im-pingement type of sampler to other types is not pos-sible with published data. The assumption that im-pingers measure individual bacteria whereas agar im-pingement devices measure bacterial particles has notprevented subjective comparisons in some publications.The various studies made of such samplers as the Wells'centrifuge and the several agar impingement deviceshave not included direct comparison with samplers likethe AGI. More importantly, critical examination hasbeen made of only one or two of the significant param-eters. The studies of duBuy et al. (1945) and of Phelpsand Buchbinder (1941) lead one to believe that the rel-atively low collection efficiency may be a material de-fect in both centrifuge and agar impingement devices.Whether these and such newly designed samplers as theAnderson impactor (Anderson, 1958; Spendlove, 1957)kill significant numbers of vegetative bacteria seems tobe unknown. An important need for comparative evalu-ation is here made evident.

It is important to note that, despite relatively ex-tensive testing under highly standardized conditions,the replication of quantitative values reported here wasnot as precise as might be desired. This was due, in nosmall part, to the complex of variables involved in cre-ating, controlling, and sampling bacterial aerosols.Many variables are very difficult to control, standard-ize, or measure. In many instances, attempts to reducecomplexity by analysis and simplification of experi-mental procedure merely raised serious doubts of thevalidity of resulting data; interactions have been show-nto be of considerable influence. The logical conclusion,then, is that studies with aerosols must be carefullyplanned and analyzed for statistical validity, and inter-preted within objectively determined limits.No attempt was made herein to evaluate the rela-

tively subjective factors influencing choice of samplers.It is reasonable that different types of samplers may be

chosen to serve different objectives. It is obvious, how-ever, that the choice should be made with full knowl-edge of the quantitative values of the various param-eters affecting behavior of chosen samplers, or withrealization that lack of such information, objectivelymeasured, reflects on the validity of collected data.

ACK-NOWLEDGMENTS

Grateful acknowledgment is made of the skilled tech-nical assistance of Miss D. M. Chapman, Pfc. RufusFranklin, and Mr. Floyd Martin. Personnel of severaltechnical groups at Fort Detrick are thanked for coop-eration in permitting use of facilities in the study.

SUMMARY

The particle size selectivity of the inlet tube of theall-glass impinger (AGI) was evaluated by various bio-logical and physical techniques of aerosol study. It wasfound that, in general, the AGI collected in samplingfluid nearly all particles of bacteria or crystal violetdye below 5 ,u equivalent diameter. An increasing pro-portion of such particles were trapped by impaction inthe inlet tube as equivalent diameter increased; nearlyall particles of 20 u were so trapped. Serratia marcescensparticles impacted in the inlet tube lost viability rap-idly.

Comparisons of the AGI, Shipe, and Midget im-pinger samplers with isotope-tagged S. marcescens, andwith a stained cell technique of measuring total cell re-covery from aerosols, indicated that considerable de-struction of viable cells occurred in the AGI. This ap-proximated 20 to 25 per cent relative to the Shipesampler. Meager evidence suggested that the Shipesampler killed few if any S. marcescens. Tentative datafrom a very few tests with Brucella suis and Pasteu-rella tularensis indicated that the relative killing effectof the AGI may be of greater magnitude.The relative advantages and disadvantages of the

Shipe sampler, AGI, and other types are discussed.

REFERENCES

ANDERSON, A. 1958. New sampler for the collection, sizingand enumeration of viable airborne particles. J. Bac-teriol., 76, 471-484.

BOURDILLON, R. B. 1948 Studies in air hygiene. Med. Re-search Council (Brit.), Spec. Rept. Ser. No. 262.

DUBuY, H. G., HOLLAENDER, A., AND LACKEY, M. D. 1945A comparative study of sampling devices for air-bornemicroorganisms. Public Health Repts. (U. S.), Suppl.No. 184.

MAGILL, P. L., HOLDEN, F. R., AND ACKLEY, C. 1956 Airpollution handbook. McGraw-Hill Book Co., Inc., NewYork, New York.

MIAY, K. R. 1945 The cascade impactor, an instrument forsampling coarse aerosols. J. Sci. Instr., 22, 187-195.

PHELPS, E. N. AND BUCHBINDER, L. 1941 Studies on micro-organisms in simulated room environments. I. A studyof the performance of the Wells air centrifuge and of the

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settling rates of bacteria through the air. J. Bacteriol.,42, 321-344.

ROSEBURY, T. 1947 Experimental airborne infection. InMicrobiological Monographs. The Williams & Wilkins Co.,Baltimore, Maryland.

SHIPE, E. L., TYLER, M. E., AND CHAPMAN, D. M. 1959 Bac-terial aerosol samplers. II. Development and evaluationof the Shipe sampler. Appl. Microbiol., 7, 349-354.

SPENDLOVE, J. C. 1957 Production of bacterial aerosols in arendering plant process. Public Health Repts. (U. S.),72, 176-180.

TYLER, M. E. AND SHIPE, E. L. 1959 Bacterial aerosol sam-plers. I. Development and evaluation of the all-glassimpinger. Appl. Microbiol., 7, 337-349.

WELLS, W. F. 1955 Airborne contagion and air hygiene. Har-vard University Press, Cambridge, Massachusetts.

Chemistry and Microbiology of Forage-Crop Silage2 3A. G. KEMPTON4 AND C. L. SAN CLEMENTE

Department of Microbiology and Public Health, Michigan State University, East Lansing, Michigan

Received for publication May 25, 1959

The quality of hay-crop silage has often been relatedto the chemical composition of the finished product,but detailed investigations on the step-wise chemicalchanges occurring during the fermentation have sel-dom been reported. Conversely, the relationship be-tween silage quality and the changing bacterial spec-trum is not clearly understood. Although the bacterialchanges during the storage period have been plottedby several workers, the significance of their results hasbeen difficult to determine because simultaneous chem-ical studies were not included. Furthermore, most ex-periments have been conducted on miniature or experi-mental sized silos, and it has never been proved thatthese results are applicable to the fermentations occur-ring in farm sized silos.

Consequently, the main object of the present inves-tigation was to interrelate silage quality with both thechemical and bacterial changes which occur when grassesand legumes are ensiled in full-sized silos on typicalfarms. Since this study was completed, Langston et al.(1958) have published the results of similar investiga-tions with experimental sized silos.The chemistry of forage-crop silage has been compre-

hensively reviewed by Barnett (1954). It has been re-peatedly confirmed that when well preserved silagesare compared to spoiled silages the former have lowpH values, large amounts of lactic acid, and smallamounts of butyric acid and volatile base.The lactic acid bacteria are the most important1 Journal article no. 2446 from the Michigan Agricultural

Experiment Station.2 Part of a thesis submitted by the senior author, A. G.

Kempton, to Michigan State University in September, 1958, inpartial fulfillment of the requirements for the Ph.D. degree.

3 Presented in part at the Society for Industrial MicrobiologyAmerican Institute of Biological Sciences, Bloomington, In-diana, August 1958.

4 Present address: Soil Research Laboratory, Department ofAgriculture, Swift Current, Saskatchewan, Canada.

agents of silage preservation, although they are not theonly bacteria present in normal fermentations (Burkeyet al., 1953). Kroulik et al. (1955a, b) found very fewlactic acid bacteria on fresh crops, but normally therewere as many as 109 to 1010 per g within a day or twoafter ensiling. Although homofermentative rods carriedout the main lactic fermentation, pediococci and strep-tococci were observed in the early stages of the fermen-tation, and heterofermentive rods predominated laterin the storage period. The population of lactic acidbacteria can be modified by wilting (Kroulik et al.,1955b), mincing (Stirling, 1951), and other factors(Barnett, 1954).Beynum and Pette (1936) were the first to attribute

the butyric acid associated with spoiled silage to thelactate-fermenting activity of Clostridium tyrobutyri-cum. The characteristics of this organism have beendetailed recently by Bryant et al. (1952). Unfortu-nately, all reports of clostridia have been based on sporecounts. Rosenberger (1951) cautioned against the useof spore counts to measure lactate-fermenting activity,since these organisms do not form spores during thestage of their most rapid development, but only afteractive growth ceases. In our study, lactate-fermentinganaerobes were detected in the vegetative state.The course of the fermentation is undoubtedly influ-

enced by factors such as the plant species, the moisturecontent, and the maturity of the crop; but the relativeimportance of each factor has not been clearly defined.The silages studied for this report were prepared undera wide range of field conditions. Weather data werecollected and details of the harvesting procedure wererecorded in an attempt to relate silage qualky to en-vironmental conditions at the time of ensiling.

EXPERIMENTAL METHODS

The silages were prepared by individual farmers onrandomly selected farms in Michigan's Eaton and Ing-

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