Evaluation of Sample Disruption Techniques for High Throughput
Extraction of Live Escherichia Coli and Recombinant DNA from Spinach
Shari Garrett1, James Atwood1, Brandon Easparro1, Eric Clouser1

Introduction

Methods

The detection and enumeration of pathogens in food is important to ensure the safetyof foods throughout the food supply chain. Government agencies and food companies
use microbiological analysis to monitor contamination and risks within their food supply.
Traditional microbiological methods for detecting microorganisms in food are based on
culturing followed by standard biochemical identification. These traditional methods
are typically simple and inexpensive but can be time consuming and can require up to a
week for preliminary and confirmation testing. PCR rapid methods with high sensitivity
and specificity have been developed for pathogen detection. Samples are prepared byhomogenizing in a stomacher and incubated in a selective enrichment media. Since
the advent of bead mill technology, the time and efficiency of homogenizing a varietyof sample matrices has drastically improved. This is achieved through the high speed
shaking of a sample against small beads inside a sample vial. The high speed impact of thebeads against the sample produces efficient grinding and can process a variety of sample
matrices in less than half the time of traditional methods. Bead beating in conjunction
with PCR has the potential to speed the sample testing process by processing the samples
faster and eliminating the need for sample incubation.

Results
Level of Detection: The pGLO plasmid was extracted and detected via end-point PCR
after processing. Expected size of the pGLO plasmid was shown to be 714 bp. The CFUlimit of detection was analyzed post amplification via gel electrophoresis. A decrease inspot intensity is shown with a decrease in number of CFUs.
Rotor Stator
DH5α E.coli

pGLO




Manual Homogenization
Fisher 150 Homogenizer

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

Bead Beating
2.8 mm ceramic beads



Figure 8: Lane 1: 2.5 kb Marker, 2-7:
Genomic DNA, Lane 9: 100bp Marker, Lane
10: 10-2, Lane 11: 10-4, Lane 12: 10-5, Lane 13:
5x10-6, Lane 14: 2.5x10-6, Lane 15: 1x10-6

Figure 9: Lane 1: 2.5 kb Marker, 2-7:
Genomic DNA, Lane 9: 100bp Marker, Lane
10: 10-2, Lane 11: 10-4, Lane 12: 10-5, Lane 13:
5x10-6, Lane 14: 2.5x10-6, Lane 15: 1x10-6

Spot Volume Intensities: Spot volume intensities were analyzed via the Gel Doc EZ software(BioRad). The sensitivity was adjusted so that every band could be detected and analyzed.

Extraction of pGLO plasmidFigure 10: Gel Spot Intensities

Dilutions and Processing: After incubation, each culture was serial diluted by thefollowing dilution factors 1x10-2, 1x10-4,1x10-5, 5x10-6, 2.5x10-6 and 1x10-6 to produce twelve
total control colonies. ~5g of store bought spinach was added to each dilution (total12 inoculums). Prior to processing, 100 µl of control dilutions 1 x10-5 and 1 x10-6 wereplated on LB/Amp/Ara plates to confirm colony count. Six spinach inoculums (1x10-2 to1x10-6) were disrupted on the Fisher 150 Homogenizer rotor-stator homogenizer with
Hard Tissue tips for 1 minute at medium speed and the other six spinach inoculums(1x10-2 to 1x10-6) were disrupted on the Bead Mill 24 bead mill homogenizer at 4 m/s for
30 s in a 30 ml tube containing 6.3 g of 2.8 mm ceramic beads. After processing, 100 µl ofeach spinach homogenate was plated on LB/AMP/Ara plates. All plates were grown overnight at 37°C and counted to determine the number of viable recombinant colonies.

Dilution

PCR Amplification/Gel Electrophoresis

Rotor Stator SpotVolume (Int)

Bead Mill SpotVolume (Int)1,246,320
273,300
125,880
39,87051,96037,2001,554,420
155,220
58,23052,08041,73023,8201 x 10-2
1 x 10-4
1 x 10-5
5 x 10-6
2.5 x 10-6
1 x 10-6

Spot Volume to CFU Analysis: The number of CFUs were analyzed by taking the log2 of thevalues and plotting against the gel spot volumes for each dilution sample. A best fit linear
curve was applied. It is shown that the values are linear such that there is a correlation between
the number of CFUs to the abundance of detected amplicon.

Results
Live Cell Recovery and Viability: Bead mill and rotor stator homogenizers are powerful mechanical homogenizers and are capable of vigorously homogenizing avariety of sample types. Here, an inoculated foodstuff was processed to complete homogenization while still maintaining microbial load and viability. After diluting,inoculation and processing, the homogenate was plated for standard plate count. The cell viability and recovery was compared to an unprocessed control. Recombinant
GFP colonies fluoresce green under UV light. Both the rotor stator and bead mill can fully process spinach samples while also maintaining viable cells. Each processing
technique recovered a similar amount of recombinant colonies as in the controls.

Rotor Stator Sample ProcessingBead Mill Sample Processing

235‘-ATGGCTAGCAAAGGAGAAGAA - 3’
5’ - GTAGAGCTCATCCATGCCATGTG - 3’

2321

Figure 4: 1x10-5 Dilution

Figure 5: 1x10-6 Dilution

21y = 2.394x - 26.41
R² = 0.952

191715137

90

Hot Start/Denaturation
30 Cycles
Final Extension

Instrument

Sequence

9595597272120303060
5 min

50000

100000150000Gel Spot Volume

200000250000300000Figure 11: Rotor Stator CFU/Spot Volume Analysis

Unprocessed Control

Homogenate

Unprocessed Control

Homogenate

Bead Mill Live Cell Recovery
Figure 6: 1x10-5 Dilution

Figure 7: 1x10-6 Dilution

Homogenate

Unprocessed Control020000

40000

60000

80000

100000120000140000160000180000200000Gel Spot Volume

Figure 12: Bead Mill CFU/ Spot Volume Analysis

Conclusions• Both the Bead Mill 24 and the 150 are capable of fully homogenizing spinach without
disrupting surface bacteria• Both methods delivered a 76% cell recovery and a PCR detection limit of 1x10-6 CFUs/ml• A linear relationship was demonstrated between the number of CFUs to quantity ofDNA amplified via PCR, which is especially shown at the lower CFU limit• The vigorous processing of the Bead Mill 24 and 150 potentially has the ability toeliminate the pre-enrichment step and allowing pathogen detection to be completedin half the time.

PCR products were analyzed on a 2% agarose gel and stained using ethidium bromide. Spotvolume intensities were analyzed via Bio-Rad’s Gel DOC EZ Software.
Unprocessed Control

15119Amplifications were performed using the T100 Thermal Cycler by Bio-Rad.

17

13

11Figure 2: PCR Programy = 7E-06x + 11.77
R² = 0.842

19Log 2 CFUs

GFP-F
GFP-R

Sequence

Rotor Stator Live Cell Recovery
Log 2 CFUs

Instrument

Automated Homogenization
Fisher Bead Mill 24
Bead Mill Homogenizer

Serial Dilute

Standard Plating/Count
800 bp700 bp

Process

Transformation: Competent DH5 alpha E. coli cells were transformed with the pGLO
plasmid containing the GFP gene. The cells were spread onto LB/Amp/Ara plates and later
observed under UV light for detection recombinant colonies. Two recombinant colonies
were inoculated into two separate LB/AMP broth cultures and incubated overnight at 37°C.

Figure 1: Primers

800 bp700 bpGrow up Recombinant E.coli O/NMethods

GFP Gene Detection: 5 ng of each of the twelve spinach homogenates were amplified
using the below primers. These primers are designed to amplify 99.2% of the GFP gene.

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15E.coli Transformation

Herein, spinach samples were inoculated with a serial dilution of known levels ofrecombinant Escherichia coli expressing green fluorescent protein (GFP) and processedusing both bead mill and rotor stator homogenizers. Live cell recovery was evaluated via
standard plate counts and limit of detection of both methods were analyzed through PCR
amplification.

pGLO Extraction: The pGLO plasmid was extracted from each of the twelve spinachhomogenates using OMEGA BioTek’s E.Z.N.A Bacterial DNA Kit. The DNA concentrationsof each dilution was analyzed using a NanoDrop Spectrophotometer.

Bead Mill

Homogenate