JOURNAL OF CLINICAL MICROBIOLOGY, Sept. 1976, p. 296-305Copyright ©3 1976 American Society for Microbiology

Vol. 4, No. 3Printed in U.S.A.

Use of Droplet Plating Method and Cystine-LactoseElectrolyte-Deficient Medium in Routine Quantitative Urine

Culturing ProcedureTHOMAS R. NEBLETT

Henry Ford Hospital, Detroit, Michigan 48202

Received for publication 29 March 1976

Droplet plating of 0.01 ml of 10-2 dilutions of mixed sonically treated urinesonto cystine-lactose electrolyte-deficient agar permits formation of discrete,easily counted colonies within a small circumscribed area without interferenceby Proteus overswarm. Each colony is considered as arising from a single viablecell. The single dilution permits precise reproducible quantitation of urinebacteria population within the range 104 to 106 cells/ml of sample. Droplet-platedcounts were found to be consistently (approximately) double those determinedby standard pour plate quantitation. The method requires only inexpensivereadily available materials and has been performed routinely in a large-volumeclinical laboratory for several years.

Procedures to detect bacteriuria are amongthe most important functions of a clinical mi-crobiology laboratory and are quite often themost frequently requested. A properly per-formed urine culture enables the physician toknow numbers of organisms present in a sam-ple, the identities of probable pathogens, andtheir antimicrobial susceptibility patterns. De-finitive urine cultures also enable the clinicianto monitor the status of patients with indwell-ing catheters and to learn if a subsequent uri-nary tract infection was nosocomial. A labora-tory method should provide a convenient andreliable means to determine bacteriuria at asignificant level and enable technologists todistinguish between gram-positive and gram-negative varieties and have well-isolated colo-nies free from overswarm.A flood plate technique was found by Vejls-

gaard (5, 6) to have precision and reproducibil-ity to quantitate urine bacteria. Whole or di-luted urine was flooded onto the agar surfaceby pipette, and resulting colonies were counted.Mallmann and Broitman used the droplet plat-ing method to determine milk bacterial countsin which they deposited 0.1 or 0.2 ml of theappropriate dilution (2), and Reed and Reed (3)have described and evaluated the method forgeneral microbiological quantitation.

Cystine-lactose electrolyte-deficient (CLED)medium was designed to halt spreadingProteuswithout inhibiting growth. Its peptone contentenables other urinary tract organisms to grow,whereas its dye content permits detection oflactose fermenters by their yellow color (4).

This report describes application of the drop-let plating method and CLED agar to deter-mine numbers of urine bacteria. It has beenused in the author's laboratory for severalyears.

MATERIALS AND METHODSCulture routine. Urines are received in glass

screw-capped tubes (20 by 125 mm) which are placedonto a Labquake rocking platform mixer (Labin-dustries, Inc.) for 5 min to provide gross mixing.Tubes are placed 10 at a time into a low-power sonicoscillation bath (Esterline-Angus sonic cleaner,model EA 12, with a frequency of 40 kHz) for 90 sto break apart bacterial clumps. Greater exposureis unnecessary. However, kill will not occur whenurine samples are exposed for up to 900 s, shouldtubes inadvertently be left in longer.

Potential lethality of exposure by sonic treatmentto commonly isolated urine bacteria was investi-gated by exposing 18- to 24-h Trypticase soy brothcultures in glass screw-capped tubes to the oscilla-tion continuously for 15 min. Base line numbers ofviable organisms were determined by droplet plat-ing prior to sonic treatment. Samples were with-drawn at 3-min intervals during treatment anddroplet plated onto Trypticase soy agar. Counts pertime were determined for Escherichia coli, Staphylo-coccus aureus, Streptococcus faecalis, Pseudomonas,and Mycobacterium tuberculosis.Mixed sonically treated urine is diluted 1:100 by

adding 0.1 ml to a 9.9-ml saline blank autoclavedwith rubber stopper clamped in to prevent fluid loss.The dilution is mixed thoroughly on a Vortex appa-ratus, and CLED (3) agar plates are "droplet inocu-lated" on a level surface using 4 drops (0.01 ml) froma Corning no. 7099 disposable glass pipette (CorningGlass Works, Corning, N.Y.).

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DROPLET PLATE QUANTITATIVE URINE CULTURE 297

By allowing 0.01 ml of diluted urine to flow fromthe vertical 0.1-ml pipette, a pendant droplet willform but not fall (Fig. 1 and 2). The expulsion ofslightly more fluid will cause the droplet to fall, butthis should not be allowed to happen. Surface ten-sion will cause the droplet to become spherical, andit will likely roll or bounce prior to breaking on theagar surface. Breaking on contact can create anaerosol hazard plus result in an aggregation of colo-nies difficult to count. The suspended droplet is ap-plied to the agar medium by allowing the fluid, butnot the pipette tip, to touch the surface (Fig. 3 and4). The fluid spot is allowed to spread naturally onthe agar surface, which should be supported on alevel working area (Fig. 5 and 6). Its liquid phasewill enter the gel matrix by imbibition, depositingbacterial cells on the agar surface (Fig. 7). No glass

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FIG. 1. Diagram of the formation of a pendantdroplet. A 0.1 ml disposable glass pipette marked in0.01-mi graduations with 0.01 ml of urine diluted1/100 as a pendant fluid mass containing uniformlydispersed bacteria is shown

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FIG. 2. Photograph ofpendant droplet of dilutedurine containing 0.01 ml of fluid.

or wire spreader is used to distribute the deposit,and plates are not moved for 30 min or until all fluidhas disappeared. Use of a spreading instrument maypick up organisms from the deposit, thereby disturb-ing a critical small number. Four deposits are madeon each plate. At the end of 18 h discrete roundclusters of microcolonies will result, as shown inFig. 8. The count per milliliter of urine is an averageof the total count from four spots (Fig. 9) multipliedby 104. The number of colony types is recorded atread out, which takes place under low-power stereo-microscopy by both reflected and transmitted light.

Occasionally, large mucoid colonies will merge ina semiconfluent mass, but if the colony count is notexcessive (>106) and distinct arcs of colony periph-ery are discernible, they are counted (Fig. 10).

Comparison between pour and droplet platecounts. Simultaneous quantitative comparisonswere performed between a pour plate (1) method anddroplet plating at 10-5, 10-6, and 10-7 dilutions on156 bacteriuric urines. Standard pour plates weremade into tryptic soy agar from sonically treatedspecimens, and colony counts were performed on astandard back-lighted colony counter.

Reproducibility. Reproducibility of the method

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298 NEBLETT J. CLIN. MICROBIOL.

colonies than the standard pour plating tech-nique.

Variability indices between spots on platesare represented in Fig. 12. The range of valuesfor 121 samples was 0.049 to 2.0, with a mean of0.447. A larger number means a greater varia-tion between numbers of colonies per plate spot.Mean counts determined by droplet plating

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FIG. 3. Diagram ofa droplet touching the surface.Droplet should not be allowed to fall onto agar. Maybounce and roll or splatter, causing aerosols.

was determined by performing 100 sequential platesfrom the same dilution blank prepared from a bac-teriuric urine. The same urine quantitation was

performed 50 times using a freshly made dilutionblank each time.

Reproducibility between spots on a single platewas expressed as a variability index, determined asthe difference between high and low counts on theplate divided by the mean of the four replicates.Perfect reproducibility would equal zero.

RESULTSIn no instances did populations from soni-

cally treated broth culture tubes decline duringthe 15-min exposure period. Numbers of S. fae-calis, Pseudomonas, and S. aureus increasedslightly over base line numbers, suggestingbreakup of clumps.Comparison between pour plates and droplet

plates resulted in no growth by either methodsix times, and 54 did not permit direct compari-son, either by one or the other being uncounta-ble or by virtue of an extreme count ratio,exceeding either lOx or 0.1x. Mean dropletplate count was compared with mean pour platecount on 96 specimens. A scatter diagram ofratios is shown in Fig. 11. A value above theline (1.0) favors the droplet plating method, andone below favors the pour plate count. Meandroplet plate count was found to exceed mean

pour plate count by 1.87x. This would indicatethat the droplet plating quantitative methodresults in formation ofapproximately 50% more

j,.,

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FIG. 4. Diluted urine droplet being touched offonto agar surface. Pipette does not touch surface.

.... .

FIG. 5. Diagram of droplet that is allowed tospread freely on agar and dry 30 min It is not spreadmechanically.

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DROPLET PLATE QUANTITATIVE URINE CULTURE 299

FIG. 6. Touched-off droplet spreading freely on agar surface.

onto 100 sequential plates from the same di-luted bacteriuric urine on 2 successive daysresulted in mean counts remaining fairly con-stant until replicate 45 (day 1) (Fig. 13) andreplicate 75 (day 2) (Fig. 14), respectively. Forreplicates 1 to 40 on day 1 the average countwas 305,500, with a standard deviation of 30,328and a regression slope of -0.087, and for repli-cates 1 to 75 on day 2 the average count was341,400, with a standard deviation of 31,556 anda regression slope of -0.063. Decline in countsattributed to death of cells suspended in thedilution blank indicated that the amount oftime the population may be expected to remainconstant within a diluted urine is limited. Theslightly higher values obtained from the sameurine on a second day are attributable to asmall psychrophilic population increase duringrefrigeration.

Quantitation of urine colony counts using 50sequential different dilution blanks from thesame specimen (Fig. 15) resulted in an average

.............-_- -

FIG. 7. Fluid passes agar by imbibition, andseparated cells are deposited on the surface as colony-forming units. Average number of colony-formingunits multiplied by a factor ofl 04 gives the count.

count of 283,500, with a standard deviation of45,300 and a regression slope of + 0.162. Thecolony count was not found to decline as fromthe single blank experiment, which indicatedthe time that organisms were suspended indilution to be an important factor. These datasuggest that the method yields consistent andreliable counts, and that if a urine culture is tobe repeated it should be performed on a freshdilution made from the original specimen. Dilu-tion blanks should not be saved.

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300 NEBLETT

FIG. 8. Colonies formed within droplet-inoculated area. Diameter of spot is approximately 7 to 9 mm.

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DROPLET PLATE QUANTITATIVE URINE CULTURE 301

FIG. 9. Droplet-inoculated CLED plate after 18 h, showing four discrete colony clusters.

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302 NEBLETT

FIG. 10. Crowded, yet discernible, mucoid colonies.

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DROPLET PLATE QUANTITATIVE URINE CULTURE 303

lo - 0

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RATIOS

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FIG. 11. Scatter diagram ofratios ofmean dropletplate count and mean pour plate count of 96 speci-mens.

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DISTRIBUTION OF DROP PLATEVARIABILITY INDICES

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FIG. 12. Variability indices between spots on

plates.

DISCUSSION

The droplet inoculation method permits theformation of discrete, well-distributed colonies

within the inoculated area. If dispersal of cellswithin the urine is complete and its dilution isproperly performed, resulting colonies may beconsidered as arising from single viable cells.Failure to achieve complete dispersal will re-sult in a reduced number of colony-formingunits per spot, and a falsely low count mayresult (Fig. 16). The findings from this investi-gation that droplet-plated urines produce con-sistent counts approximately double those ob-tained by pour plating seems to cast reasonablequestion upon the viewpoint of Bailey and Scott(1) that the pour plate technique "remains themost accurate procedure yet devised for meas-uring the degree of bacteriuria." The compari-son indicates that organisms detectable by thedroplet plate method are not revealed by thepour plate technique. This may have far-reach-ing significance, since many of the rapid

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FIG. 13. Determination ofmean counts by dropletplating onto 100 sequential plates from the samediluted bacteriuric urine on 2 successive days, result-ing in fairly constant mean counts until replicate 45(day 1).

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FIG. 14. Determination ofmean counts by dropletplating onto 100 sequential plates from the same

diluted bacteriuric urine on 2 successive days, result-ing in fairly constant mean counts until replicate 75(day 2).

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FIG. 15. Quantitation of urine colony counts us-ing 50 sequential different dilution blanks from thesame specimen.

screening methods, such as dip-slides, etc., arecalibrated to the pour plate count as a standard.However, the difference between attainablecounts by the two methods may not assumepractical significance until the variation ex-ceeds one order of magnitude.The 10-4 dilution permits formation of count-

able colonies from urines having populationsbetween 10-4 and 10-6 organisms/ml. Only onedilution is needed to cover the range extendingone order of magnitude on either side of numer-ically significant bacteriuria. In routine prac-tice no more than 50 colonies would be countedfrom a single spot. Such urines would be re-ported as "colony count exceeds 500,000 colony-forming units/ml."The method has distinct advantage when

coupled with use of CLED medium. However,minor disadvantages are seen. Certain strepto-cocci and lactobacilli appear at 18 h as tinytransparent colonies. These enlarge upon pro-longed incubation but may be missed withoutlow-power stereomicroscopy to read plates ini-tially.The urine culture procedure in our labora-

tory also includes streaking 0.1 ml of undilutedurine on MacConkey agar, blood, and colistin-nalidixic acid blood agar plates. These latterplates are not inoculated by the droplet methodbecause in heavily populated urines isolatedcolonies may not result. Colistin-nalidixic acidinhibits gram-negative organisms and allowsgram positives to grow. However, certainstrains of S. aureus produce stunted coloniesand sometimes occur in reduced numbers oncolistin-nalidixic acid. Pseudomonas strainswill often grow on colistin-nalidixic acid.

In conclusion, the droplet plate method toestimate bacteriuria is reliable, reproducible,and able to detect microbial populations be-

FIG. 16. Uneven dispersion of cells by failure tobreak up clumps can result in falsely low numbers ofcolony-forming units, hence, low apparent count.

tween 104 and 106 organisms with a single dilu-tion step. Precision of the method is dependentupon thorough dispersal of cells within the dilu-tion fluid and a careful pipetting technique. Itssuccessful application to urine culturing is en-hanced by the use of CLED agar that inhibitsswarming of Proteus without toxicity or detri-ment to other species. The method yields countsgreater than are attainable by pour plating andhas the additional advantage that an averageof four counts per specimen is possible insteadof a single one. Numbers obtained representviable colony-forming units. Equipment andmaterials to perform the technique are readilyavailable and inexpensive.

ACKNOWLEDGMENTSI am indebted to Barbara Ross (retired) and Jonathon

Geiger (Harper Hospital, Detroit) for technological effortsduring standardization and evaluation of the method.

LITERATURE CITED1. Bailey, R. W., and E. G. Scott (ed.). 1974. Diagnostic

microbiology, 4th ed., p. 72-73. C. V. Mosby Co., St.Louis.

2. Mallmann, W. L., and S. A. Broitman. 1956. A surfaceplating technic for determining bacterial populationof milk. Am. J. Public Health 46:1018-1020.

3. Reed, R. W., and G. B. Reed. 1948. "Drop plate" methodof counting viable bacteria. Can. J. Res. Sect. E26:317-326.

4. Sandys, G. H. 1960. A new method of preventing

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DROPLET PLATE QUANTITATIVE URINE CULTURE

swarming of Proteus sp. with a description of a new

medium suitable for use in routine laboratory prac-tice. J. Med. Lab. Technol. 17:224-233.

5. Vejlsgaard, R. 1965. Quantitative bacterial culture ofurine (limit between contamination and significantbacteriuria), p. 468-477. In E. H. Kass (ed.), Prog-

ress in pyelonephritis. F. A. Davis Co., New York.6. Vejlsgaard, R., and T. Justesen. 1973. Quantitative

bacterial culture of urine. II. Evaluation of 10 differ-ent screening methods for the diagnosis ofbacteriuriacompared with results obtained by the dilution tech-nique. Acta Med. Scand. 193:147-159.

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