Comparison of Streaking Patterns, Plating Consistency, and Isolation of the WASP and InoqulA on Blood and MacConkey Agar.

Numerous publication have demonstrated that automatic plating creates moreconsistent streaking patterns and isolates more bacteria than manual plating;however, it is unclear what streaking patterns, systems, and plates are optimal forculture. In this study, we compared the WASP and InoqulA benchtop instrumentfor their ability to isolate pathogens from both urine and sputum specimens.Steaking methods were evaluated by comparing reporting results, need forisolation to complete ID and AST (≥5 isolated colonies), and technician preference.

Introduction

v

M. L. Faron1, Hasan Samra1, B. W. Buchan1,2, and Nathan. A. Ledeboer1,2

1 Medical College of Wisconsin, Milwaukee, WI, USA; 2 Wisconsin Diagnostic Laboratories, Milwaukee, WI, USA;

Method

Conclusions

➢ Precision plating of multiple pathogens demonstrate that both systems are highly accurate andreproducible. In most cases automatic plating did not require a purity plate, with the exceptionof bacterial burdens of 108 CFU or greater. This drop was most notable in bi-plate testing.

➢ There was little differences between culture reporting independent of plating methods.Differences observed were often linked to likely dilution differences (1uL vs 10uL plated) and inmost cases only less prevalent organisms were either detected or missing between platforms.

➢ There were a few limitations to this study. The inoqulA used was a benchtop model where thetechnologist is needed to inoculate the plates with a calibrated pipette. The current systems arefull automation and do not require human intervention for plating. In addition the automatedsystem uses it’s own liquid transfer system instead of a calibrated pipette, which may affectprecision of amount plated. Use of a calibrated pipette also allowed for a visual check to confirmadequate specimen is loaded as pipetting treated sputum specimens can be difficult. Finally, thenumber of zigzags for urine plating did not match, which could effect colony isolation.

Urine stored in boric acid was plated on blood and MacConkey agar. Specimenswere plated using the WASP 1uL and 10uL loop and 10uL of specimens wasinoculated to each plate with a calibrated pipette for the InoqulA instrument.Urine streaking patterns were used with the WASP at 27 zigzags and the inoqulA20 zigzags across the plate. A technologist reviewed each plate recordingpreliminary results and determining if subculture of any significant growth wasnecessary (≤5 isolated colonies). Significant growth was based on clean-catchurine culture guidelines. Furthermore, two technologists were blinded to thestreaking methods and ranked quality of streaking patterns from 1-3 to evaluatetechnologist preference. Sputum cultures enrolled in the study was treated withSLsolution following package insert prior to plating. Treated sputum specimenswere plated onto blood agar or MacConkey agar with either 1 or 10uL using theWASP or 10uL using the InoqulA. All streaking methods for sputum used a 4quadrant streak patter. Culture analysis was performed following guidelines below.

Figure 2. Evaluation of streaking patterns for testing of urine

Figure 4. Evaluation of Streaking patterns for sputum cultures

Figure 5. Technologist preference for automated streaking

Table 2. Site 3 Breakout of discordant sputum specimens

Figure 1. Precision of automated plating using spiked saline.

50

60

70

80

90

100

10^4 10^5 10^6 10^7 10^8

Percent of plate that had 5+ colonies isolated

WASP 1uL

WASP 10uL

InoqulA

WASP bi-plate

InoqulA bi-plate

Figure 1. (A) 0.5 McFarland standards of each isolate were plated in dilutions in triplicate. Bacterial colonycounts were compared to plating of 100uL at 103 cfu/mL to determine plating precision.

Table 1. Breakout of discordant urine specimen reportDiscrepant plate(s) Consensus Reportable

resultsDifference Possible explanation

InoqulA bi-plate >100K GNR, 20K GPC Missing 20k GPC Overgrowth of GNR

W1, InoqulA, W1-bi >100K GNR, 20K GNR #2 Missing 2nd GNR Overgrowth of GNR

W1, W1-bi >100K GNR, >100K GPCCH Additional 20K GNR Overgrowth of GNR

W1, InoqulA >100K GPC Multiple organisms (no workup)

Unknown

W-bi, InoqulA bi >100K GNR, 10K GNR #2 <10K GNR #2 Overgrowth of GNR

InoqulA, InoqulA-bi 10K GPCCL <10K (no workup) Plating differences near limit

InoqulA 10K GPCCL <10K (no workup) Plating differences near limit

W10, InoqulA-bi >100K GNR, >100K GPC Missing GPC Overgrowth of GNR

W1, W-bi <10K GNR and GPCCH (no workup)

>10K GNR and GPCCH Plating differences near limit

Table 1. Breakout of the specimens that were discordant between plating methods.The consensus row indicates the most common result shared between allspecimens. W1 indicates WASP 1uL, W10 indicates WASP 10uL, and bi indicates bi-plates used for plating. WASP bi-plates were struck using a 1uL loop. Cutoff of urinespecimen for significant growth is 10K.Figure 2. Comparison of the streaking methods for 150 urines were evaluated head to head for

overall concordance of specimen reporting and if enough colonies were available to performboth ID and sensitivity testing for significant organisms without purity plates.

Figure 3. Image examples of sputum plating for all methods.

Discrepant plate(s) Consensus Reportable results Difference Possible explanation

WASP 1uL Normal oral Flora (NOF) Pure culture yeast Fold dilution difference

WASP 1uL NOF Pure culture yeast Fold dilution difference

WASP 10uL 4+ GPCCH Additional 4+ GPCCH Overgrowth of GPCCL

WASP 1uL 4+ GNR, 4+ NOF, 2 colonies Beta Missing Beta Fold dilution difference

WASP 1uL 4+ GPCCL, 4+ NOF GPCCL part of NOF (no workup) Fold dilution difference

InoqulA NOF (1+ W1 and 3+ W10) 4+ GPCCL, 4+ NOF Sampling precision

WASP 1uL 4+ Yeast, 4+ NOF Yeast part of NOF (no workup) Fold dilution difference

WASP 10uL Yeast (1+ W1, 4+ InoqulA) Yeast part of NOF (no workup) Sampling precision

InoqulA 4+ GNR, 4+ GNR #2, 3+ NOF 4+ GPCCL, 4+ NOF Sampling precision

91.76 91.76 92.94

WASP 1 VS WASP 10

WASP1 VS INOQULA

WASP 10 VS INOQULA

% of plates that were concordant for reporting

95.29 94.12 91.76

WASP 1 VS WASP 10

WASP1 VS INOQULA

WASP 10 VS INOQULA

% of plates with ≥5 well isolated colonies

Figure 4. Comparison of the streaking methods for sputum were evaluated head to head foroverall concordance of specimen reporting and if enough colonies were available to performboth ID and sensitivity testing for significant organisms without purity plates. Plating methodsused quadrant streak pattern following Wisconsin Diagnostic Laboratories guidelines forsignificant organisms.

2.272.10

1.68

WASP 1 UL BAP WASP 10UL BAP INOCULA BAP

Average streak quality score for sputum specimens

Figure 5. Two technologists were blinded to the streak method and given each plate to gradefrom 1-3, with 3 being the great isolation. Scores were individual for each specimen, meaningthat both WASP and Inoqula A could be given a 3 on the same specimen. Averages were takenfor the two techs with standard deviation error bars.

Sputum Potential pathogen guidelines

Pathogens only

No NOF present

Pathogen: 2+

NOF: 2+

Pathogen: NOF

NOF: 2+

Pathogen: 2+

NOF: 2+

Pathogen predominant on Gram stain

Pathogen: 1+

NOF: 2+

Pathogen: 1

NOF: 1+

Pathogen not predominant on Gram stain

GNR: less than 3 types present

ID & SENS ID & SENS ID & SENS ID & SENSCALL NOF CALL NOF

GNR: 3 or more types present.

MULTIGNR MULTIGNR MULTIGNR MULTIGNR CALL NOF CALL NOF

S. aureus/ S. lugdunensis

ID & SENS ID & SENS ID & SENS ID & SENS CALL NOF CALL NOF

H. influenzae ID ONLY ID ONLY ID ONLY ID ONLY CALL NOF CALL NOF

N. meningitidis ID ONLY ID ONLY ID ONLY Discuss at roundsDiscuss at

roundsDiscuss at

rounds

M. catarrhalis ID (-LACT +) ID (-LACT +) ID (-LACT +) ID (-LACT +) CALL NOF CALL NOF

S. pneumoniae ID & SENS ID & SENS ID & SENS ID & SENS CALL NOF CALL NOF

Strep group A ID ONLY ID ONLY ID ONLY ID ONLY ID ONLY ID ONLY

Beta Strep not A ID ONLY (type) ID ONLY (type) ID ONLY (type) CALL NOF CALL NOF CALL NOF

Enterococcus ID ONLY ID ONLY ID ONLY CALL NOF CALL NOF CALL NOF

Yeast

ID to species if in pure culture

Rule out C. neoformans if

not in pure culture

Rule out C. neoformans

Rule out C. neoformans

CALL NOF CALL NOF CALL NOF

Urine isolate guidelines CFU/mL EXTENT OF WORKUP

1 Pathogen

Other nonpathogens

Other nonpathogens

>10,000

>10,000

<10,000

Full identification & susceptibility

None

Do not report

2 Pathogens

Other nonpathogens

Other nonpathogens

> 10,000

>10,000

<10,000

Full identification & susceptibility

None

Do not report

1 Pathogen

1 PathogenOther nonpathogens

Other nonpathogens

> 10,000

< 10,000>10,000

<10,000

Full identification & susceptibility

Minimal identificationNone

Do not report

3 or more Pathogens

Other nonpathogens

10,000 with 1 or 2 pathogens predominating

10,000

Full identification and susceptibility of the 1 or 2

predominating pathogens. Minimal

identification of remaining pathogens

None

3 or more Pathogens w/ or w/o other nonpathogens

Approximately equal amounts

of at least 3 pathogens

None

98.00 97.33 98.00

WASP 1 VS WASP 10

WASP1 VS INOQULA

WASP 10 VS INOQULA

% of plate that were concordant for reporting

96.00 96.67 95.33

WASP 1 VS WASP 10

WASP1 VS INOQULA

WASP 10 VS INOQULA

% plates with ≥5 well isolated colonies

2.44

2.081.87

WASP 1 UL WASP 10 UL INOQULA

Average streak quality score for urine specimens