Uncontrolled When Printed TABLE OF CONTENTS

Department of Microbiology

Quality Manual

Policy # MI_PFGE

Page 1 of 57

Version: 1.4 CURRENT

Section: Bacteriology Procedures Subject Title: Infection Control Pulsed-field Gel

Electrophoresis Prepared by QA Committee

Issued by: Laboratory Manager Revision Date: 2/2/2022

Approved by Laboratory Director:

Microbiologist-in-Chief

Next Review Date: 2/2/2024

Uncontrolled When Printed

UNIVERSITY HEALTH NETWORK/MOUNT SINAI HOSPITAL, DEPARTMENT OF MICROBIOLOGY

NOTE: This document is Uncontrolled When Printed.

Any documents appearing in paper form that do not state "CONTROLLED COPY " in red print are not controlled and should be checked

against the document (titled as above) on the server prior to use. Management System\UHN_Mount Sinai Hospital Microbiology\Standard Operating Procedures\Bacteriology Procedures\

TABLE OF CONTENTS

I. Introduction ............................................................................................................... 3

Isolates for Referred-Out PFGE.............................................................................................. 3

II. Procedure ................................................................................................................... 4

Pulsed-field Bench Workflow .................................................................................................. 4

Testing schedule ........................................................................................................................ 5

Test ordering, broth labeling and inoculation in preparation for typing ............................ 5

Cell Extraction Preparation ..................................................................................................... 7

For MRSA, VRE, GNB, GAS, GBS, ..................................................................................... 7 For Serratia marcescens ....................................................................................................... 12

Settings for CHEF-DR II/III instruments ............................................................................ 16

CHEF-DR II/III Module ....................................................................................................... 17

Ethidium bromide for staining .............................................................................................. 18

Weekly Ethidium Bromide Disposal ..................................................................................... 18

Weekly Ethidium Bromide Preparation ............................................................................... 19

Staining Protocol ..................................................................................................................... 19

How To Use The Gel DOC XR+ Camera ............................................................................. 19

BioNumerics Gel Analysis ...................................................................................................... 22

BioNumerics Naming CMRSA .............................................................................................. 24

III. Reporting .............................................................................................................. 25

PFGE Reporting Phrases ....................................................................................................... 25

Verifying Bionumerics Results .............................................................................................. 26

IV. Processing a Comparison Request (In-house and for PHL) ............................ 28

Documenting Request ............................................................................................................. 28

Comparing and Interpreting a Cluster in Bionumerics ...................................................... 29

Interpretation Example .................................................................................................. 30


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Sending a Report by E-mail ................................................................................................... 31

Appendix I – PFGE Record Sheet ................................................................................. 32

Appendix II - Documenting Comparison Requests in LIS ......................................... 34

Appendix III - Maintenance and Quality Control ....................................................... 35

Maintenance and QC CHEF PFGE Machines ..................................................................... 35

Before each run ..................................................................................................................... 35

Weekly .................................................................................................................................. 35 Monthly ................................................................................................................................. 35

Water flush and temperature check....................................................................................... 36 Maintenance of Gel Run Record........................................................................................... 36

Yearly .................................................................................................................................... 37

Appendix IV - Preparing PFGE Plugs of the Salmonella ser Branderup H9812 Standard

Strain ................................................................................................................................ 38

Appendix V - Preparing PFGE plugs for Serratia marcescens ................................... 39

Preparing the plugs ................................................................................................................. 40

Appendix VI - Reagents Preparation ............................................................................ 42

PFGE Reagents for Gram Positives ...................................................................................... 42

PFGE Reagents for Gram Negative Bacilli .......................................................................... 43

PFGE Reagents for Serratia marcescens ............................................................................... 44

Stock Reagents ........................................................................................................................ 45

TRIS Buffer Chart .................................................................................................................. 45

ENZYMES ............................................................................................................................... 46

Appendix VII - Calculations for Restriction Enzymes, MBI Fermentas Fast Digest enzyme/

buffer Chart ..................................................................................................................... 46

Appendix VIII - To Send Isolates To NML For: Spa typing ..................................... 52

Appendix IX – Reference Material .............................................................................. 53


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I. Introduction

Pulsed field gel electrophoresis (PFGE) typing or macro-restriction of micro-organisms is

performed for epidemiological purposes to determine the relatedness between the chromosomal

DNA of two or more isolates.

PFGE assists the Infection Control department by:

1. Providing an understanding of the transmission of isolates between patients.

2. Providing evidence of transmission of isolates from the environment to patients.

3. Determining whether isolates involved in recurrent infections are the same clone or

are newly acquired.

II. Specimen Collection

Isolates for PFGE (Routinely Done) In-House

ISOLATES FOR PFGE IN-HOUSE UHN MSH

New MRSA No Yes1

New VRE No Yes2

New Group A Streptococcus No Yes3

Any organism as part of a cluster or upon ICP's request Yes Yes

1. MSH New = 1st time isolated and every 3months thereafter, if no other positive in

between isolates.

2. 1st time isolated and once a year there after.

3. In-patient only or if identified by infection control to be a nosocomial GAS.

Isolates for Referred-Out PFGE

ISOLATES FOR PFGE PHL Baycrest

New MRSA Yes1

Any organism as part of a cluster upon ICP's request Yes

1. 1st time isolated and every 3months thereafter if no other positive in between isolates.

Note: VRE not routinely sent out.

III. Reagents / Materials / Media

See Analytical Process – Bacteriology Reagents_Materials_Media List QPCMI10001

https://eportal.mountsinai.ca/MSHPresentations/public/paradigm/D0024697.pdf


Quality Manual

Policy # MI_PFGE

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Electrophoresis Prepared by QA Committee

Issued by: Laboratory Manager Revision Date: 2/2/2022

Approved by Laboratory Director:

Microbiologist-in-Chief

Next Review Date: 2/2/2024

Uncontrolled When Printed



Any documents appearing in paper form that do not state "CONTROLLED COPY " in red print are not controlled and should be checked


II. Procedure

Pulsed-field Bench Workflow

DAY 1 DAY 2 DAY 3 DAY 4

PFGE 1 1. Retrieve incubated PF BHIB isolates

from incubator.

2. Check Bionumerics database to see

if done previously. Refer to chart

above for testing frequency

3. Process isolates up until “wash

stage"( can be left washing

overnight)

4. Enter isolates in Bionumerics, LIS

(plug date and PF done in isolate

window)

5. Create gel legends for each type and

log into “Gel Run Record” (in T

drive)

6. Document incubator temperatures,

lot number and expiry dates for

enzymes on gel legends

7. Check for new queries Respond

ETA for results

8. Answer queries.

9. Retrieve any query isolates as

necessary.

1. Prepare isolates for digest

2. Prepare machines (drain,

rinse, clean, change buffer,

inspect for broken electrodes)

3. Make agarose for each gel,

load plugs onto combs, affix

with agarose, chill, pour gel,

chill and load

4. Document machine

maintenance

5. Wash glass wash containers

and prepare for autoclaving.

6. Send green caps for washing.

7. Send glassware for washing

8. Check for new queries

Respond ETA for results

9. Answer queries.

10. Retrieve any query isolates as

necessary.

1. Make fresh stain

2. Stain/ Destain and

photograph gels

3. File and export data

from images

photographed into

Bionumerics.

4. Normalize gels

5. Name MRSA’s.

6. Report in LIS.

7. Prepare green caps for

autoclaving

8. Answer queries.

9. Subculture send out

organisms as required

10. Check for new queries

Respond ETA for results

11. Answer queries.

12. Retrieve any query

isolates as necessary. 13. Verifiy results

1. Make reagents

2. Prepare aliquots of

enzymes

3. Check inventory

4. Order enzymes,

supplies

5. Restock supplies

6. Prepare H9812 plugs

as required

7. Discard plugs > 3

months old

8. Update Bionumerics

database (Cleaning)

9. Retrieve any query

isolates as necessary.

10. Monthly-

temperature check;

Flow rate check and

adjustment


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Testing schedule

PFGE is run weekly in-house according to schedule, refrigerate pre-inoculated broths.

Incubate broths in 37ºC incubator @ 4:00p.m on day before for PFGE processing.

The run from incubated the broth to finish will take 3-4 days.

Test ordering, broth labeling and inoculation in preparation for typing

Isolates for typing come from different benches, but are handled in the same manner.

Routine Benches

When an isolate qualifies for PFGE:-

1. Order ^BHIB and ^PF in LIS.

2. From PF, select isolate type from keypad (i.e. MRSA, VRE, GNB etc).

3. Attach one large label to 10ml BHI broth for gram positive organisms. Alternately, a

Mueller Hinton plate can be used for gram negative organisms. Write organism name (i.e.

MRSA, VRE, etc) and any other relevant information e.g. (ESBL Class A-LF) on the

label.

4. Touch 1-3 colonies (dependent on colony size) of pure growth with a loop and inoculate

the broth/plate.

5. Put inoculated broth/plate into rack in PFGE fridge for batch testing.

6. The MRSA bench technologist is responsible for placing the BHIB rack into the 37oC

incubating shaker on the date before PFGE setting up. The only exceptions to this

protocol are:

a. Streptococcus pneumoniae - must be inoculated just prior to incubation onto 2 BA

plates (CO2).

PFGE Bench

1. Retrieve isolates for PFGE from shaker and plate rack

2. Put Brain heart infusion tubes / plates in order (alphabetical) within groups

3. Print required labels.

4. Put labels in the same order within groups.

5. Match labels to isolates

6. Place cultures @4oC until ready for processing.

7. Using labels check appropriate Bionumerics database MRSA, VRE, and GNB MSH for

repeats (right click on Last Name column and choose arrange by field for alphabetical

order).


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If an unwarranted repeat, document in LIS (Prev see LIS#........) at back of workcard.

8. Assign processing numbers to isolates. Write processing numbers on labels (1 large label

3 small labels) and on matching BHI tubes. Affix a small barcode label on the PFGE

record sheet (see Appendix I).

IN LIS

1. Document plug date under PF

(If isolate not on work list order PF)

2. Go to individual isolates and add isolate letter and pfdone in isolate window.

3. Ensure that the appropriate organism has been ordered in the PF window in LIS.

i.e.`MRSA`

IN BIONUMERICS

1. Add new entries

N.B:

a. Copy and paste demographics from LIS to avoid transcription errors.

b. Choose Organism name and comment (ESBL class A…etc.) from drop down menu for

consistency.


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Cell Extraction Preparation

For MRSA, VRE, GNB, GAS, GBS,

Step 1 Preparation:

1. Turn on 60ºC, 37 ºC water-bath and adjust the water levels as appropriate.

2. Ensure incubator/shaker on at 55 ºC.

3. Calculate how much of each enzyme is necessary for lysis (see Table 1).

TABLE 1: LYSIS ENZYME TABLE

Lysozyme L10

(10mg/mL)

Lysostaphin

1mg/mL (Sigma)

Lysozyme L100

(100mg/mL)

Mutanolysin

5U/µL (Sigma)

Proteinase K

20mg/mL (Roche)

MRSA 50µL per tube 10µL per tube 40µL per tube

VRE 40µL per tube 15 µL per tube 40µL per tube

GNB 40µL per tube

GAS / GBS 50µL per tube 50µL per tube 20µL per tube

4. Remove enzymes from freezer and allow them to thaw. Keep @4 ºC until ready for use.

5. Alphabetize labels for VRE, MRSA, etc and check Bionumerics for previous. Discard

repeats. See chart above to determine testing frequency.

6. Put labels in numerical order and BHIB to match.

7. Label BHIB lids and labels 1, 2 etc.

8. Label 2 sets of 1.5ml microfuge tube with the assigned number for each sample to be

sub-typed. (i.e. 1, 2,3 etc) (blue for MRSA, black for VRE, red for GNB).

9. Label 1 Vk. Tube for each (1, 2 etc.) (colour coded as above).

10. Aliquot 1.5 mls PIV Buffer (GP) into each tube Vitek tube. (SE buffer for GNB)

11. Label white-capped tubes for each isolate. (colour coded as above) (1, 2 etc.)

12. Label disposable plug molds 2 spots per isolate. (colour coded as above)

NB: If unable to proceed with making plugs this is a good place to break off.

13. Prepare 1% Seakem Gold Agarose(SKG) in Sterile de-ionized water(SDH2O) for making

all plugs.(0.1gm SKG in 10 mL SDH2O or .2gm SKG in 20mL SDH2O). Use 50mL tube.

14. Place tube in beaker with water and dissolve agarose in microwave for 30 secs to 1 min

(check at 30 sections). Keep in 58 ºC water-bath until ready for use.


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Step 2 Standardization:

1. Transfer broth from BHIB’s using transfer pipette into labeled microfuge tubes,

centrifuge @ 14,000 rpm for 1 minute to pellet.

2. Pipette off supernatant. Change pipette.

3. Calibrate Vitek colorimeter.

4. Re-suspend pellet with small amount of PIV buffer from the pre-labelled Vk. tube and

make 20% transmittance suspension for each isolate (SE buffer for Gram negatives)

Vortex to make a smooth suspension.

5. Add 200 uls (using 1000 ls pipette) of bacterial cell suspension to each labelled

microfuge tube.

6. Add 200 uls (using 200 ls pipette) 1% SeaKem Gold agarose. Mix and fill two plug

mold spots. (Make sure that there are no bubbles and that the molds have not been

overfilled).

7. Do this for each isolate. Solidify at 4C 5-10 minutes.

Step 3 Lysis & Preparation:

MRSA Lysis Enzymes

Make a master mix for lysis in 50 ml conical tube

2 mL EC Lysis Buffer per test plus 1

50 uls L10 (lysozyme) per test plus 1

10 uL Lysostaphin 1 mg/mL per test plus 1

Document lot # on gel legend sheet.

Aliquot 2mls EC Lysis Buffer/L10 mixture to labeled white-capped tubes for each

isolate

VRE Lysis Enzymes

Make a master mix for lysis in 50 ml conical tube

2 mL EC Lysis Buffer per test plus 1

40 uls L100 (lysozyme) per test plus 1

15 uL Mutanolysin(50000 U/mL) per test plus 1

Document lot # on gel legend sheet.

Aliquot 2mls EC Lysis Buffer/L10 mixture to labeled white-capped tubes for each

isolate

Immerse the plugs into the lysis buffer

1. Remove caps from tubes containing lysis buffer.

2. Break off plungers from disposable plug molds. Clean with alcohol and dry with Kim

wipe. Remove tape from bottom of wells and dispense plugs into the appropriate tubes by


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inserting plunger into well and tapping the top to remove plugs. Ensure plugs are

immersed in buffer!

3. Incubate 3 hrs at 37C on shaker.

TABLE 2:

ORGANISM LYSIS BUFFER PK BUFFER

Incubation

Temperature

Incubation

Time

Incubation

Temperature Incubation Time

MRSA 37ºC for 3hrs

minimum 55ºC 1hr

VRE and GAS 37ºC for 3hrs

minimum 55ºC 1hr

Enterobacteriacae and

Other gram negative bacilli N/A N/A 55ºC 3hrs minimum

4. Make gel legends. (7 tests for 10 well gel and 11 for 15 well) Use small label with

barcode for legend

5. Assign GEL ID # T:\Microbiology\BioNumerics\Gel run record

6. Make sure that there is 1 ladder every 5-6 wells. Record date made and date digested.

7. Write organism names on the legends.

8. Label 6-well storage plate for each isolate.

9. Dispense adequate amount (2.5mls per isolate) of Gram +ve Wash Buffer (TE buffer)

and Gram –ve Wash buffer into two 50 ml. conical tubes.

10. Pipette 2.5mls Gram +ve Wash Buffer (TE buffer) or 2.5mls Gram –ve Wash buffer

into each well of 6-well storage plate.

11. Pre-warm bottle of SDH2O @ 55C.

12. Pre-warm adequate Wash Buffers (Gram +ve and Gram negative) in 55C incubator.

13. Assemble the required amount of numbered green screen caps for wash step (put in

ascending order, lowest numbers on bottom).

14. Label microfuge tubes according to gel legend positions for restriction digest.Include

tubes for standards (H9812) also.

15. Aliquot 750 uL SDH2O into prelabeled microtubes. 16. Enter plug date and PF in isolate window in LIS and enter into Bionumerics.

* NOTE: NO LYSIS STEP FOR GRAM NEGATIVES! (Proceed to step 4.)


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Step 4 Removal Of Interfering Proteins

1. Calculate PK Buffer needed for each tube (2.0ml PKB and 40ul PK 50) and dispense

into pre-labelled white-capped tubes.

2. Document Lot # for PK.

For Gram negatives:

3. Break off plungers from disposable plug molds. Clean with alcohol and dry with Kim

wipe. Remove tape from bottom of wells and dispense plugs into the appropriate tubes by

inserting plunger into well and tapping the top to remove plugs. Ensure plugs are

immersed in PK solution! Incubate on shaker 55C 3 hours.

For Gram positives:

4. Decant lysis buffer into petri dish, using spatula.

5. Pipette 2 mls PK buffer mixture into each tube. Ensure plugs are immersed in PK

solution! Incubate on shaker 55C 30 min.

Step 5 Plug Washing:

1. Retrieve tubes from incubator

2. Put green appropriately labeled screw-cap on a 50 ml centrifuge tube labeled “Discard”

and decant P K buffer solution for each isolate, stacking one on top of the other as you

go. (Don’t exceed 6 caps, before starting a new stack)

3. Place green cap on top of each stack.

4. Remove stack from discard centrifuge tube.

5. Discard decanted PK buffer.

6. Return stack to the centrifuge tube.

7. Pour 50.0ml pre-warmed SDH2O in a sterile centrifuge tube labeled “SDH2O” and pour

through the assembled screw caps to rinse.

8. Place blue cap of a clean 50ml tube on the last cap sealing the top of the last green cap on

top of the stack.

9. Invert sideways to rinse through. (3 times).

10. Decant. Repeat steps 7-9, 2 more times.

11. Load assembled screw caps into glass wash dishes.

12. Pour enough TE buffer (or Gram –ve Wash buffer for Gram negatives) to fully

submerge green caps. Seal dishes tightly and wash for a minimum 60 minutes @ 55C

on shaker and then decant. (You can wash overnight at this point.)


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After wash is complete:

1. Pour pre-warmed 50.0ml SDH2O in a sterile centrifuge tube and pour through the

assembled screw caps to rinse.

2. Discard wash buffer in sink

3. Tap green caps on desk to shake plugs into bottom of cap.

4. Transfer plugs from green caps into corresponding 6 well storage plate.

N.B. OPTIONAL: Store plugs overnight or proceed to next step

Step 6 Restriction Enzyme Digestion of Plugs:

1. Calculate the amount of SDH2O, digest buffer and digest enzyme needed for the digest

mixture (see chart) (SeeT:\Microbiology\BioNumerics\Protocols\Calculation for Enzyme

and buffer chart.doc

2. Put plugs into to pre-labeled 6-well plate (plugs can be stored at 4C at this point).

3. Retrieve 1/2 plug with spatula and place it into the appropriate pre-labeled microfuge

tube with 250ul of SDH2O. Rotate for 15 minutes @ RT (25C) to equilibrate plug.

4. Pipette off all of the SDH2O from each tube.

TABLE 3.

ENZYME Fast digest

Sma 1 MBI

Fast digest

Xba 1

Fast digest Bcu 1

for Pseudomonas /

Stenotrophomonas

Fast digest Sfi1

Acinetobacter

QUANTITY per

sample

10µL 5 µL 5 µL 10 µL

INCUBATION

TIME

3hrs-O/N 3hrs- 3hrs-O/N 3hrs-O/N

INCUBATION

TEMPERATURE

37ºC 37ºC 37ºC 50 ºC

Buffer FD buffer FD buffer FD buffer FD buffer

5. Aliquot 200ls of the Enzyme/10x buffer mixture into each tube.

6. Digest plugs for a minimum of 3 hrs See Table 3.

7. Document Lot #s on Gel Legend sheet.

Restriction Enzyme Digestion of Standards (S.branderup)

8. Calculate how many standards needed for gels. There are 10 in each tube and they should

be cut in half for use (6 per tube).


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9. Proceed as in STEP 6.

10. Document date standard was made and date it was digested on Gel Legend sheet.

11. NOTE: IF USING PREDIGESTED PLUGS MAKE SURE YOU WASH THEM IN

WATER FIRST! (15 minutes in SDH2O @ RT (25C) to equilibrate plug.

Refrigerate digested plugs until ready for use

For Serratia marcescens

Step 1 Preparation:

1. Turn on 60ºC, 37 ºC water-bath and adjust the water levels as appropriate.

2. Turn on Incubator and Shaker oven to 55 ºC

3. Calculate how much of each enzyme is necessary for Lysis (see Table 1)

TABLE 1: LYSIS ENZYME TABLE

Proteinase K 20mg/mL(Roche)

Serratia marcescens 10 µL per plug

4. Remove PK from freezer and allow them to thaw. Keep @4 ºC until ready for use.

5. Alphabetize labels for Serratia and check Bionumerics for previous. Discard repeats. See

chart above to determine testing frequency.

6. Put labels in numerical order and BHIB to match.

7. Label BHIB lids and labels 1, 2 etc.

8. Label 2 sets of 1.5ml microfuge tube with the assigned number for each sample to be sub-

typed. (i.e. 1, 2,3 etc).

9. Add 10 µL PK to one set of the microfuge tubes.

10. Label 1 Vk. Tube for each (1, 2 etc.).

11. Aliquot 1.5 ml Serratia Cell Suspension Buffer (SCSB) into each tube Vitek tube.

12. Label white-capped tubes for each isolate. (1, 2 etc.).

13. Label disposable plug molds 2 spots per isolate. (Keep at 4 ºC until ready to use).

14. Prepare 1% Seakem Gold Agarose (SKG) in SCSB for making all plugs.(0.1gm SKG in

10 mL SCSB ). Use 50mL tube.


Keep in 58 ºC water-bath until ready for use.


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Step 2 Standardization & Plug Making

1. Transfer broth from BHIB’s using transfer pipette into labeled microfuge tubes (without the

PK), centrifuge @ 14,000 rpm for 1 minute to pellet.

2. Pipette off supernatant. Change pipette.


4. Re-suspend pellet with small amount of SCSB from the pre-labelled Vk. tube and make 20%

transmittance suspension for each isolate .Vortex to make a smooth suspension.

5. Add 200 uls (using 1000 ls pipette) of bacterial cell suspension to each labelled microfuge

tube (with pre-aliqoted 10 ls PK).

6. Add 200 uls (using 200 ls pipette) 1% SeaKem Gold agarose. Mix and fill two plug mold

spots. (Make sure that there are no bubbles and that the molds have not been overfilled).

7. Do this for each isolate. Solidify at 4C 5-10 minutes.

Step 3 Removal Of Interfering Proteins

1. Calculate PK Buffer needed for each tube (2.0ml Serratia Lysis/PK buffer (SLPK) and

40ul PK 50) and dispense into pre-labelled white-capped tubes.

2. Document Lot # for PK.

3. Break off plungers from disposable plug molds. Clean with alcohol and dry with Kim-wipe.

Remove tape from bottom of wells and dispense plugs into the appropriate tubes by inserting

plunger into well and tapping the top to remove plugs. Ensure plugs are immersed in PK

solution! Incubate on shaker 55C 3 hours.

Step 4 Preparation

1. Turn on incubator/shaker and bring to 55C.

2. Make gel legends. (7 tests for 10 well gel and 11 for 15 well) Use small label with barcode

for legend.

3. Assign GEL ID # T:\Microbiology\BioNumerics\Gel run record

4. Make sure that there is 1 ladder every 5-6 wells. Record date made and date digested.

5. Write organism names on the legends.

6. Label 6-well storage plate for each isolate.

7. Dispense adequate amount (2.5mls per isolate) of Gram –ve Wash buffer into two 50 ml.

conical tubes.

8. Pipette 2.5mls Gram –ve Wash buffer into each well of 6-well storage plate.

9. Pre-warm bottle of SDH2O @ 50C.

10. Pre-warm adequate Wash Buffer in 55C incubator.


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11. Assemble the required amount of numbered green screen caps for wash step (put in

ascending order, lowest numbers on bottom).

12. Label microfuge tubes according to gel legend positions for restriction digest.Include tubes

for standards (H9812) also.

13. Aliquot 750 uL SDH2O into prelabeled microtubes. 14. Enter plug date and PF in isolate window in LIS and enter into Bionumerics.

Step 5 Plug Washing:

1. Retrieve tubes from incubator.

2. Put green appropriately labeled screw-cap on a 50 ml centrifuge tube labeled “Discard” and

decant P K buffer solution for each isolate, stacking one on top of the other as you go. (Don’t

exceed 6 caps, before starting a new stack).

3. Place green cap on top of each stack.

4. Remove stack from discard centrifuge tube.

5. Discard decanted PK buffer.

6. Return stack to the centrifuge tube.

7. Pour 50.0ml pre-warmed SDH2O in a sterile centrifuge tube labeled “SDH2O” and pour

through the assembled screw caps to rinse.

8. Place blue cap of a clean 50ml tube on the last cap sealing the top of the last green cap on top

of the stack.

9. Invert sideways to rinse through. (3 times).

10. Decant.

11. Load assembled screw caps into glass wash dishes.

12. Pour enough Gram –ve Wash buffer for Gram negatives to fully submerge green caps.

Seal dishes tightly and wash for a minimum 60 minutes @ 55C on shaker and then decant.

(You can wash overnight at this point.)


1. Pour pre-warmed 50.0ml SDH2O in a sterile centrifuge tube and pour through the assembled

screw caps to rinse.

2. Discard wash buffer in sink.


4. Transfer plugs from green caps into corresponding 6 well storage plate.

N.B. OPTIONAL: Store plugs overnight or proceed to next step


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Step 6 Restriction Enzyme Digestion of Plugs:

1. Calculate the amount of digest buffer needed for the digest (see chart)

(SeeT:\Microbiology\BioNumerics\Protocols\Calculation for Enzyme and buffer chart.doc

2. Put plugs into to 6-well plate (or store plugs at 4C).

3. Retrieve 1/2 plug with spatula and place it into the appropriate tube with 750ul of SDH2O.

Rotate for 15 minutes @ RT (25C) to equilibrate plug.

4. Pipette off all of the SDH2O from each tube.

5. Aliquot 200ls of the Enzyme/10x buffer mixture into each tube.

6. Digest plugs for a minimum of 3 hrs @ 37oC with Xba 1.

7. Document Lot #s on Gel Legend sheet.

Restriction Enzyme Digestion of Standards (S.branderup).

8. Calculate how many standards needed for gels. There are 3 in each tube and they should be

cut in half for use (6 per tube).

9. Proceed as in STEP 6.

10. Document date standard was made and date it was digested on Gel Legend sheet.

11. NOTE: IF USING PREDIGESTED PLUGS MAKE SURE YOU WASH THEM IN

WATER FIRST!

Refrigerate digested plugs until ready for use

Step 7 Gel Preparation and Loading:

1. Turn on the 60C waterbath

2. Prepare Electrophoresis machines (clean and add new buffer)

Drain existing buffer NOTE: Buffer used only once.

Rinse gel box with de-ionized water

Check drain with syringe by placing syringe in each hole and moving the plunger up

and down blowing in air to make drain is clear.

Check electrodes and change if broken.(please document on QC sheet that electrodes

have been changed)

Clean lid and insides of gel box and check that gel boxes are level

Make fresh 0.5xTBE for running and casting gels. (600mls 10x TBE +11400mls

de-ionized water in designated carboy). Instructions for making 0.5xTBE also on

front of carboy.

Fill gel boxes (2000mls 0.5xTBE per box).

Turn on machine and watch to make sure that there are no bubbles in the line.

Document QC in Maintenance Log for each machine being used.


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3. Clean and assemble the appropriate gel casting form and comb. Place a 10 or 15-well

0.75 mm comb into the tray, making sure to adjust comb height using a glass slide.

Note:Make sure screws are tight. 4. Place comb holder on it’s back with comb on the underside (furthest away from you).

5. Place sticker on each casting stand to identify gel.

6. Prepare 130ml of 1% Seakem Gold agarose (1.3g) using 0.5X TBE (130 mls) and

microwave to melt the agarose. Boil 1% SKG mixture (1 min 30 secs –swirl, return to

microwave.) Boil ~ 1 minute more until bubbling rapidly and completely dissolved.

7. For 15 well gels use 190ml 0.5 TBE andand 1.9 gms Seakem gold. Boil as above.

8. Allow agarose to cool to 60C in water bath.

9. Retrieve restricted plugs and standards and position at the bottom edge of comb

according to gel legend Note: Be sure to remove excess digest fluid from plug with

Kim-wipe. 10. Take out 0.5ml agarose with transfer pipette and dispense 1 drop on each plug to fix to

comb.

11. Allow to solidify 10 minutes.

12. Gently place comb upright in stand and pull as far forward as possible.

13. Pour agarose into casting stand (avoiding bubbles) and allow it to solidify10 minutes.

14. Refrigerate at 4C for 10 minutes.

15. Remove comb.

16. Load machines making sure to identify gels and document machine number, initial

current in mA, run date, and person loading for each machine on Gel Legend sheet.

Settings for CHEF-DR II/III instruments

Please note: there are 2 different power supply control modules.

1. Turn on power. Set temperature of cooling module to 12C. Ensure that no large bubbles

are caught in tubing. Pump setting should be between 80-100.

2. Press gel firmly in place and ensure the level of 0.5 x TBE buffer covers the top of the

gel. Ensure the orientation of gel corresponds to direction indicated on top of lid.

3. Close lid.


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CHEF-DR II/III Module

` Staph Entero GAS Gm –ve S. marces

To set:

Initial time = press Block + Volts 5.3 sec 5.0 sec 5.0 sec 5.0 sec

Final time = press Volts + Run 34.9 sec 25 sec 35.0 sec 35.0 sec

Volts 6 6 6 6

Run Time 20 hrs 22 hrs 20 hrs 20 hrs

Press Start.

Press Run Time to display run time countdown.


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Run Parameters Bionumerics\Protocols\Run parameters for PFGE.doc

Ethidium bromide for staining

***Use extreme caution when dealing with Ethidium bromide solutions.

This solution is very toxic.

Always handle the gels with double gloves when placing into the stain.

Goggles should also be worn.

Weekly Ethidium Bromide Disposal

1. Add one (1) Amresco de-staining tea bag per 1000ml of used stain and leave overnight.

2. On the following day, remove tea-bag with forceps and discard in yellow bag for

incineration.

3. Discard decontaminated solution down sink.

Organism Enzyme Initial

Switch

Time

Final

Switch

Time

Run

Time

Voltage PµLe

Angle

Enterobacteriace Xba 1 5.0sec 35.0 sec 20

hours

200/6volts 120°

Ps. aeruginosa

Ps. Maltophilia

B. cepacia

Bcu 1 5.0sec 35.0 sec 20

hours

200/6volts 120°

Serratia marcescens Xba 1 2.0sec 25.0 sec 20

hours

200/6volts 120°

Acinetobacter

baumanii complex Sfi 1 5.0sec 35.0 sec 20

hours

200/6volts 120°

Staphylococcus

MRSA

Sma 1 5.3 sec 34.9 sec 20

hours

200/6volts 120°

VRE Sma 1 5.0 sec 25.0 sec 22

hours

200/6volts 120°

Streptococcus

Group A

Sma 1 5.0 sec 35.0 sec 20

hours

200/6volts 120°

Listeria

monocytogenes

4.0 sec 40.0 sec 22

hours

200/6volts 120°

Clostridium

difficile

Sma 1 1.0 sec 40.0 sec 22

hours

200/6volts 120°


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Weekly Ethidium Bromide Preparation

1. Fill a 1000ml glass cylinder with de-ionized water.

2. Pour into ethidium bromide green top stain container (STAINING SOLUTION).

3. Add 50µl of ethidium bromide (ETBR) 10 mg/ml for a final concentration of 0.5µg/ml.

Staining Protocol

N.B. Remove excess buffer from gels before putting them into staining solution so that stain is

not further diluted.

1. Freshly made stain: Stain for 30 minutes and de-stain in de-ionized water for 30 minute

washes x 2.

2. Previously used stain: Stain for 45 minutes and de-stain in de-ionized water for 30 minute

washes x 2.

How To Use The Gel DOC XR+ Camera

DO NOT USE DIRTY GLOVES WHEN TOUCHING THE COMPUTER.

1. Load the gel into the camera chamber.

2. Double click on Image lab icon.

3. Under Protocol click “New”

4. Click Select under application.

5. Go to nucleic acids and select ethidium bromide.

6. Load the gel into the camera chamber.

7. Click on “Position gel”.

8. Click OK to “Filter 1”.

9. Go to imaging area and click on the “Enter image area” button to adjust the Camera Zoom

for gel size (for a 10well gel enter 15.7in the first box; 15well gel enter 20.1) and adjust as

needed.

10. Center the gel with lanes at very top so that the bottom of the gel is also visible.

11. Go to Image exposure and select intense bands for first picture. If bands are too difficult to

see also take a second picture (-1) using the faint band setting.

12. Go to “Display options” and de-select “Highlight saturated pixels”.

13. Click on “Position gel” to do final adjustments of gel position.

14. Click OK to “Filter 1”.


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15. Click on Run Protocol.

16. Wait for second picture.

17. Adjust the contrast of the picture by using the contrast icon.

18. Go to File and choose Save as in V:\drive\Bionumerics\Gel pictures\Image lab files.

19. Name file by choosing previous file and changing number and date. Gel 20XX-month-day

MRSA/MRSA/GNB-gel number i.e. 2021-04-06 MRSA-22.

20. Click on Save.

21. Go to File and Click on Export….Export for Pulsenet Save in V:\drive/Bionumerics/Gel

pictures/20XX gel pictures in appropriate folder. (File is now ready for import into

Bionumerics database).

22. Go to File and choose print.

23. In the Image Preview toolbar click on print. Make sure that Mitsubishi P93D printer is

highlighted.

24. Settings for printing are set as defaults. (Landscape; paper size 1280x1920; Option- enlarge

Image to fit; high density paper).

25. Click on print.

26. Close Image Lab window.

27. Clean the camera chamber with Deionized water and Kim-wipes

http://www.777icons.com/libs/design/contrast-icon.gif


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Assessing Gel Quality

1. After taking picture go to Gel –Run record

2. Fill out No. of lanes that have failed if any

3. Choose reason for failure and corrective action

Gel Status Corrective action

Lanes skewed Possibly broken electrode

Ladders did not work degraded Re-digest new ladder and run QC gel

Ladders did not work – no DNA Re-digest new ladder with right enzyme

All DNA lanes too short Re-digest and re-run, power interruption

Bottom lanes of ladder missing Possible chiller failure. Check temperature

Gel broke Re-make gel with right agarose concentration

Lane Status Corrective action

Only one top band of DNA Failed enzyme digest, re-digest and re-run

No DNA Subculture for purity. Reprocess with longer lysis

Failed repeat lysis Send to NML? Livestock strain

Faint (ghost) bands Rule out mixed culture

Broken band Re-digest plug and re-run

Plug fallen off comb Re-process re-run


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BioNumerics Gel Analysis

1. Double click BioNumerics icon.

2. Choose the correct database for analyzing/saving gel results:

Database Organism Experiment Type

UHN-MSH MRSA MRSA, SA, CNST PFGE Sma1

UHN-MSH VRE Enterococcus species

(including VRE)

PFGE Sma1 5:25-22 hrs

ESBL-PSEUDO-SERR-

ACINETO

Enterobacteriaciae,

Pseudomonas

Acinetobacter

Serratia

PFGE 5:35 (20) in use

Serratia 2:25 in use

UHN-MSH OTHERS

(NOT Others_conn)

GBS, GAS,

Haemophilus,

Leptotrichia

Listeria,

Strep pneumo

PFGE Sma1

3. Click Experiment types and highlight the correct experiment type.

4. Check for File name in the Fingerprint files field. If the File name is not there, then click

Create new fingerprint file . Browse: V: Bionumerics: Gel pictures: Gel pictures: 20XX

Gel pictures: MRSA or VRE or GNB….

5. Double click the fingerprint file to open.

6. Choose OK.

7. File should appear as new (N) in the Fingerprint files.

8. Highlight and check mark the new file in the Fingerprint files.

9. Click Open the fingerprint data symbol to load the image

10. Highlight the correct Experiment Type

11. Click OK

12. Enlarge the picture.

13. Strips should be highlighted

14. Click Lanes.

15. Choose Auto search lanes.

16. Enter the number of the lanes, click OK.

17. Adjust the width of strip by using the make strip larger or smaller symbols if necessary.

18. Click Edit.

19. Choose Edit tone curve.


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20. Choose Linear and Enhance weak bands, click OK.

21. Left click on middle green node at the top of the box, and drag to just above the ladders. Left

click on middle green node at the bottom of the box, and drag to just below the ladders.

22. Click Curves at the bottom left on toolbar.

23. Click Edit settings symbol.

24. Remove check mark from background subtraction and least square filtering, click OK.

25. Click Curves.

26. Choose Spectral analysis.

27. Click Edit.

28. Choose Edit settings.

29. Click Apply from background subtraction field, and enter the number from background scale.

Click Apply least square filtering from Filtering field, and enter the number from cutoff

scale. Click OK.

30. Close Spectral analysis window.

31. Click Normalization on bottom toolbar.

32. Click H9812, attach Use selected lane as reference lane symbol to H9812. (two or three)

33. Click auto assign reference position symbol.

34. Choose Using bands, click OK.

35. Check appropriate assignment of bands on H9812 only.

36. Add new band by pressing ctrl + left click on specific position, or to manually move cursor

by pressing Tab key + left click and press Tab key + right click to choose assign reference

position.

37. Click Show normalized view symbol.

38. Click Bands on bottom toolbar

39. Click Bands on top.

40. Choose Auto search bands.

41. Choose Search all lanes.

42. Remove lanes from H9812 by pressing shift + left click and drag from top to bottom of the

bands, press Delete key.

43. Check the rest bands and make sure the bands are assigned correctly with original gel picture.

44. Click show normalized view symbol.

45. Save the fingerprint file.

46. Close the window, Click No.

47. Under Database entries, select the patient either by last name or Lab No and check mark the

selected patient.

48. Bring selected entries to the TOP: select Database -> Entries -> Move selection to top

49. Right click the File name under Fingerprint files field.

50. Choose Open highlighted fingerprint file.


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51. Attach the Fingerprint file to patient’s information by highlighting position, left clicking the

arrow, and dragging over to patient’s information. A green dot will appear on the right

column beside the patient’s information.

52. Close the window. This is final for VRE and ESBL. Give to another technologist for

verification and then file gel legend in appropriate binder.

53. For MRSA, see BioNumerics naming CMRSA. (under V\BioNumerics/Protocol)

BioNumerics Naming CMRSA

1. Double click BioNumerics icon.

2. Double click UHN-MSH MRSA.

3. Double click File name on Fingerprint files window (i.e. 2021-03-01 MRSA-23) to open

Fingerprint file entries.

4. Select all entries by pressing Click on highlighted entry.

5. Close experiment file.

6. In Bionumerics main window Click Copy Selection icon to copy selected entries.

7. In Comparisons window, double click 1 NML+MSH (IN USE)17.11.

8. Click Paste selection icon to paste selected entries into comparison.

9. Click the Show image icon to bring up picture.

10. Click Cluster analysis (similarity matrix) icon .

11. Choose Dice on Band based on page 1 similarity coefficient (Optimization: 0.81%,

Tolerance: 1.06%), click Next.

12. Choose UPGMA on Method on page 2 cluster analysis, click Finish.

13. Click Bring selected entries to top icon .

14. Click on the isolate of interest to highlight

15. Click Arrange entries by similarity icon .

16. Choose Yes when asked if you want to use the existing similarity matrix

17. Scroll over to the epidemic type for the strains that have a Similarity Index of >80% with the

unknown isolate and record the epidemic type on your Gel Legend for reporting.

18. Repeat this process until all the strains have been named.

19. If no isolates in database with a similarity Index of >80%, check isolate for purity/ID

20. Send for spa typing if requested only 21. Troubleshoot all lanes that have failed


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Naming CMRSA -10:

On Gel Run record, document:

a. Gel, ladder or lane failures

b. Proposed corrective action

c. Fill in outcome

III. Reporting

PFGE Reporting Phrases

A. Standard MRSA PFGE Report

“SmaI Pulsed field pattern:” CMRSA-2

“Based on the closest pattern match using the National Microbiology Laboratory (NML) MRSA

Database and a similarity index of at least 80%.”

H9812

CM

RS

A 2

*

CM

RS

A 1

0

H9812

CM

RS

A 2

Key Bands are highlighted in Pink

For CMRSA 2, that band is closer to 216.9, while the

CMRSA 10 band is between 244.4 and 216.9

* This was a 92% match with CMRSA 10 in bionumerics but it

did not have the key band when that band is missing, send the

organism to Sunnybrook for sccmec and PVL if needed.

CMRSA-10 is PVL positive.

OR

Send organism to NML for testing.


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Confirmation to follow from NML. (to add this comment when isolate possibly more than one

epidemic type).

If not requested to send to NML report as “Other than..”

When typing report is available then an updated report will be issued which will replace the

PFGE results above with the following:

“Updated Report::

“SmaI Pulsed field pattern:” CMRSA-2………ETC

“As reported by the National Microbiology Laboratory,

1015 Arlington St., Winnipeg, MB, Canada, R3E 3R2

NML Specimen No. Nxx-xxxxx”

B. Standard Organisms-other-than-MRSA PFGE Report

Pulsed-field gel electrophoresis (PFGE) has been completed on this isolate. For results, please

contact the microbiology laboratory

Verifying Bionumerics Results

1. Open appropriate database.

2. Go to File name window.

3. Check mark and highlight the Gel ID.

4. Right click on Gel ID.

5. Click the Open the fingerprint data symbol on top the Fingerprint files bar.

6. Go to normalization window and check band assignments and alignment of ladders (top band

assignment =668.9. Must have 5 bands below the double bands173.4/167.1 the lowest

molecular wt=33.3). N.B. For VRE top band sometimes differentiates into 2 bands.

7. Move to Bands and make sure all the bands are correct.

8. Close data file.

9. Right click to Open highlighted fingerprint file.

10. Check gel legend with linked entries.

11. FOR VRE/GNB Initial and return to PFGE tech for filing.

12. For CMRSA

13. Open fingerprint file.

14. Select all entries.

15. Close window

16. In Bionumerics main window


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17. Copy selection and paste into 1 NML+MSH (IN USE)17.11

18. Click the Show image icon to bring up the picture.

19. Click the Cluster analysis icon, chose Dice and UPGMA, click Finish.

20. Click Bring selected entries to top icon .

21. Highlight the top entry.

22. Click Arrange entries by similarity icon to do Cluster analysis to confirm the epidemic

group, chose Yes

23. Similarity index has to be >= 80%.

24. Initial Gel legend if you agree with name given.

25. If you do not agree, consult with PFGE tech. to come to a consensus.

26. If unable to agree, the isolate must be re-run to check reproducibility of differences.

27. Arrange entries by last name and check for previous isolate to see if the result matches.

28. Go to LIS and check resulting for each isolate on front and back of card. If any reportable

discrepancies a corrected report must be sent.

29. Return Gel legend to PFGE tech for filing.


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IV. Processing a Comparison Request (In-house and for PHL)

Documenting Request

Comparison requests can be received via e-mail with FORM.

a. E-mail requests should come with a completed PFGE comparison request form

1. Give request form a file name(Date /Organism/requestor)

2. Print both e-mail and request form

3. Check status of isolates that need to be compared i.e. previously done/ in progress/ not

yet processed/ not yet received. (If inadequate picture in database please re-run)

4. Check that comparison requests are for the same organism and phenotypes and not mixed

organisms/phenotypes without a good explanation.

5. Check urgency of request i.e. ASAP or next week’s run

6. Respond to e-mail with projected completion date of comparison. (If urgency not stated,

ask if next week’s run will be O.K.)

7. Document request in LIS for each isolate if PFGE not yet done. See

T:\Microbiology\BioNumerics\Protocols\Documenting Comparison requests in LIS.doc

Appendix 1

8. Fill in information in the Query Log and document as requested for units.

See.\..\..\BioNumerics\PFGE FORMS\Query Log.xls

b. Telephone requests

1. Fill out PFGE comparison request form PFGE COMPARISON REQUEST FORM and

proceed as above for an e-mail request

c. Faxed requests from Baycrest

1. Faxed requests may come with a completed PFGE comparison request form. Some

requests may be for in-house PFGE or for send out to PHL.

2. For in-house PFGE proceed as above (Steps 1-8). Copy of completed comparison report

must be submitted for billing.

3. For PHL send-out

Check Soft to determine if isolates were previously sent to PHL or if it is

necessary to pull from freezer .Print labels/note freezer # and PHL PF#s

Enter code for PFGE at PHL (phlpf) in isolate window for VRE (should already

be there for MRSA if previously sent)

Subculture necessary isolates and print PHL forms for each isolate that is being

sent.


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On the PHL form for the reference strain put a small label for each of the other

strains in the comparison and fill out the information showing PF #s for those

already sent for PFGE.

Attach a copy of the hospital request form to the PHL request forms to go with the

isolates.

Send a faxed response to requestor of the comparison with the date isolates were

sent to PHL.

Give request form a file name(Date of request /Organism/Hospital) e.g.

2011.11.17 VRE CHC

Fill in information in the Query Log Including date sent to PHL and document as

requested for units. See T:\Microbiology\BioNumerics\PFGE FORMS\Query

Log.xls

File request in Binder on PFGE shelf “ÓTHER HOSPITAL QUERIES. PHL”

PHL will send comparison report directly to the hospital requesting it.

Comparing and Interpreting a Cluster in Bionumerics

1. Select all entries requested and create a new comparison in comparison window.

2. Save comparison giving an appropriate name (Date Org Requester)

Example: “2015.06.03 MRSA Wong”

3. When Gels have been processed Open comparison

4. Show images by choosing the Show image icon for the appropriate experiment.

5. Choose Cluster analysis icon using DICE and UPGMA

Note: make sure the Optimization is 0.81 and Tolerance is 1.06, click Next and click Finish

6. Click the Dendrogram display setting icon

7. Check mark Show node information, click OK, % similarity indices will appear.

8. Compare two entries by choosing Analysis (on top of the tool bar), pick compare two entries

9. PFGE pattern comparison is based on Tenover’s criteria using the reference strain (s) as

indicated (J.Clin. Microbiol. 1995 33:2233-2239). Interpretation is as follows:

Indistinguishable: same number and size of fragments as reference strain

Closely related: differs from reference strain by 1-3 fragments

Possible related: differs from reference strain by 4-6 fragments

Unrelated: differs from reference strain by >6 fragments

DNA relatedness based on the DICE co-efficient using Bionumerics software is also

shown. In general, a Dice co-efficient of correlation that is <75% correlates with

Tenover’s criteria for relatedness.


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Note: all results from PFGE analysis must be analyzed in the light of epidemiologic

data. PFGE types have been assigned for use in this comparison only.

Indistinguishable patterns have been given the same type name. Related patterns

share the same letter in their type name.

10. If given a reference, i.e. an isolate against which all other strains should be compared, then

call that isolate PFGE Query type “A” and place it at the top of the dendogram. If given more

than one reference, then label the next “B” etc. Using Tenover criteria assign types to other

entries as follows:

Indistinguishable strains should have the same designation.

Closely or possibly related patterns should be labeled with the same letter as the

reference but would be given a number to suggest they are related but are not

identical i.e. A1. A2, A3 etc.

Unrelated patterns should be labelled with different letters following alphamerical

order.

Patterns with indistinguishable patterns should be given the same letter/number

11. If no organism is designated as reference, then choose an arbitrary reference or if there is a

cluster(s), call the organisms in the largest cluster “A”, then the next cluster “B” etc. and

different strains C; D; E; etc.

12. Fill out the Interpretation field as appropriate according to Tenover’s criteria. Remember that

all isolates should be compared to the reference strain(s) if present (see example below).

13. Save comparison.

Interpretation Example

In this comparison:

3 different reference strains were given. None of the isolates in question clustered

with the reference strains. In the D cluster the isolate D1 could be interpreted as

closely related to D however D is not the reference strain so the correct interpretation

is as shown.

Dice (Opt:1.00%) (Tol 1.0%-1.0%) (H>0.0% S>0.0%) [0.0%-100.0%]

PFGE - Sma 1 5:25-22hrs

100

80

60

100

100

96.4

89.7

62.1

100

63.9

58.2

53.9

45.6

PFGE - Sma 1 5:25-22hrs

Key

K0052813

K0220690

K0020800

K0042696

K0172793

J7172552

K0032180

K0061859

K0232800

I6173584

G9293228

Spec. Date

2010-08-05

2010-08-22

2010-08-02

2010-08-04

2010-08-17

2010-05-17

2010-08-03

2010-08-06

2010-08-23

2009-06-17

2008-01-29

Last Name

MACPHERSON

ALLADIN

GAUCI

PAQUETTE

SCOTT

SIMAAN

GROVES

MILLER

BARKOPOULOS

LABIB

HUYNH .

MRN

3735047

3870535

805433037

3724442

3778117

2635075

3886761

2880613

3259712

2286685

2523196

UNIT

GIP

10CMS

14S

GIP

10CMS

7BMOT

7BMOT

7BMOT

7AMOT

7NCSB

7NCSB

Comment

Phenotypic vanA

Phenotypic vanA

Phenotypic vanA

Phenotypic vanA

Phenotypic vanA

Phenotypic vanA

Phenotypic vanB

Phenotypic vanB

Phenotypic vanA

Phenotypic vanA

Phenotypic vanA

TYPE

D

D

D

D

D1

A

E

E

F

C

B

Interpretation

Unrelated to reference strains





Reference




Reference

Reference


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Both E. faecium phenotypic vanA and vanB strains were requested in the same

comparison so it was necessary to include a comment field

Sending a Report by E-mail

1. Open Comparison

2. Arrange information fields as per reporting form. See...\PFGE FORMS\UHN-MSH

Report Template.doc

3. From the comparison window click on the printer icon for a print preview then go to File:

Printer set -up to change orientation of page to Landscape.

4. Maximize window and arrange display on one page by dragging the yellow bars to the

left or right as necessary.

5. Go to “Layout” show field names click once.

6. When satisfied with the display go to File: copy page to clipboard. Under preferred

clipboard format choose “ENHANCED METAFILE”

7. Go to START on bottom toolbar choose programs/Microsoft Office/Microsoft Word

double click for a blank new document

8. Right click, choose paste to add file.

9. Left click on the comparison, a border will appear

10. Click Picture Tools on top bar, click the crop icon and tidy up the picture.

11. Right click and choose copy.

12. Go to T:\Microbiology\BioNumerics\PFGE FORMS\UHN-MSH Report Template.doc

13. Delete both highlighted instruction and existing picture.

14. Paste new picture into form by right click, choose paste.

15. Fill out the top right hand corner of the form using drop down boxes where applicable

which include Date Ordered, Date of Report, and Status.

16. Choose the right organism and enzyme from the drop down boxes.

17. Click Review on top the bar, choose Restrict editing.

18. Click Stop Protection on bottom right corner, Restrict Permission appear. Edit the fields

of Ordering physician, Requested by, and Technologist’s Initials

19. Please make a final check of report before saving see

T:\Microbiology\BioNumerics\PFGE FORMS\UHN-MSH Report Form check list.doc

20. Save the report in V/Bionumerics/PFGE 20XX in appropriate folder; giving the file a

name as follows “Date/Organism/Requester” ex. 2015.06.03VREWong

21. Print a copy of the report for filing with the original e-mail.

22. Go to File: Send to: Mail recipient (as attachment) Add e-mail address


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o MSH: requesting ICP cc. Infection Control Team and Lab-PFGE team

o UHN requesting ICP cc. UHN IC and Dr. Hota

23. Document date report sent in LIS under Call.

24. Document date report sent in Query Log.

25. File report with original e-mail in Completed Queries book.

Appendix I – PFGE Record Sheet

See Next Page


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UHN/MSH MICROBIOLOGY PFGE RECORD _____________GEL ID: 2016-_________ Lane Patient ID Ward Organism Comment Lane Patient ID Ward Organism Comment

1 H9812 S. branderup

Made: Digested

11

2 12

3 13

4 14

5 15

6

7

8

9

10 H9812 S. branderup

Made:

Digested

ENZYMES:

Lysostaphin/Mutanolysin

Manufacturer: Sigma

Lot#:_____________

Lysozyme

Manufacturer: Sigma

Lot#:_____________

Proteinase K 20mg/ml

Manufacturer: Roche

Lot#:_____________

RESTRICTION

Enzyme:____________

Manufacturer: Thermo

Lot#________________

Exp. Date: __________

Buffer _FDB Thermo

Lot#________________

MACHINE

Number______

Current______ma

Run Date: ________

Loaded by: ________

Normalized by: _______

Reported by: _______

Verified by: _______

LIS report checked by: ___

Enter in Gel Run Record:___

TEMPERATURES: MIWM2 Water Bath Temp (58

0C + 2.0):_____

MIACM12-10 Incubator (55oC + 2.0):______

MIACM12-11 Incubator (37C+ 2.0):______

MIWM10 Water Bath/Shaker(37C+ 2.0):____ Plugs Made by: _______ on 2016.______


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Appendix II - Documenting Comparison Requests in LIS

Find Patient in LIS

PFGE already begun:

Go to back of work-card under PF

Add PFQ from keypad

Choose “F” for IC query and “E” for Call

Choose 3 IN:`RQUST`for results add date of e-mail

When comparison is complete and results sent

Go to back of work-card

Add Call

Choose 2 OUT:Manual`FAX`results and change Fax to E-mail.

Add date of report.

Work-card should look as below:

5. BHIB PF

6. PF PFQ

7. PFQ IC query CALL

8. CALL IN:`RQUST`for results E-mail

9. CALL OUT:Manual`E-mail`results

If necessary to retrieve isolate from Freezer

Add Pull and SUBBA


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Appendix III - Maintenance and Quality Control

Maintenance and QC CHEF PFGE Machines

Before each run:

Agarose removal

1. With the gel-box full of buffer remove the flat rectangular plastic trap at front of gel box

2. Attach a syringe to a front port of the gel-box

3. Draw back the syringe so that it fills with buffer.

4. Quickly depress the syringe so that the buffer rapidly flows back into the gel-box

pushing any small pieces of agarose out of the drainage holes

5. Remove agarose with a swab

6. Drain old buffer by removing clamps and allowing buffer to run into 2L cylinder provided.

7. Turn on pump for 30 secs to flush the tubing and drain.

8. Pour approximately 500ml of de-ionized water into gel box to rinse.

9. Wipe sides and cover of gel box with a wet and then dry kim-wipe.

10. Check that gel- box is level.

11. Fill gel-box with fresh buffer

12. Fill out QC sheets.

Weekly

1. Check electrodes with gloves on, gently wriggle each electrode to see if it is loose.

(The electrode will usually break if it is loose or thin)

2. If the electrode breaks or is thinner at the ends replace electrodes and document change on QC

sheet

Monthly

Flow rate (Goal 1L per/min)

With gel-box full of buffer, disconnect the tubing at the back of box and put the end into a 1L

graduated cylinder (top at desk height).

1. Set timer for 1 min

2. Turn on the pump and simultaneously start timer

3. Shut off pump when the minute is up.

The amount of buffer in the cylinder is the flow rate per min.

4. Adjust the dial on the pump and retest the flow rate until you get 1L per min or as close to it

as you can.

5. Document flow rate for each machine making sure that they are all within the same 50ml

radius.

6. Fill out QC sheets


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N.B.: If an adjustment is made make a note of setting change on pump.

Water flush and temperature check.

Temperature goal 12°C

1. Fill the gel-box with 2L of de-ionized water

2. Turn on the pump and chiller.

3. Set chiller temperature to 12°C and press actual reading button

4. When the actual temperature is achieved, place a thermometer in the centre of the gel-box

and close lid.

5. Check the thermometer reading.

6. Adjust temperature of chiller to get desired reading if necessary.

7. Empty the gel-box and the tubing of water and re-fill with buffer.

8. Fill out QC sheets.

N.B.: If an adjustment is made make a note of setting to be used to achieve the desired

temperature on chiller.

Please make note of any unscheduled maintenance on the back of QC sheets

Maintenance of Gel Run Record

This record is for

1. Documenting all incidents that might occur during gel processing

2. Gathering statistics as a quality indicator for PFGE processing.

At the end of each run Fill in:

No. of pictures

No. of test lanes

Failed lanes

No. of control lanes

No. of failed control lanes

At the end of each month

Under Gels fill in totals for:

a. No. of Gels

b. No. of pictures

c. No. of test lanes

d. Failed lanes

Under Molecular Marker fill in totals for:-

a. No. of control lanes

b. Failed lanes


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Yearly

Clean procedure of PFGE gel box with CLR solution

1. Make up 1% CLR dilute solution: add 20mL of CLR into 1980mL of dH2O.

2. Empty the PFGE gel box first and then fill the gel box with 2000mL of 1% CLR dilute

solution and run for 4 hours with the temperature setting at 25C.

3. Discard the 1% CLR dilute solution, replace with 2000mL of dH2O and flush 5 times

with new dH2O each time until bubble disappear.

4. Refill the PFGE gel box with 2000mL of another new dH2O and run overnight.

5. On next day, replace with 2000mL of freshly made TSB buffer solution, flush 3 times,

and run overnight with new TSB buffer solution.

6. Repeat this procedure yearly or as needed if the gel picture background is blurry.


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Appendix IV - Preparing PFGE Plugs of the Salmonella ser Branderup H9812 Standard

Strain

This protocol used is same for Gram negative bacilli.

1. Labels for H9812 S. branderup can be found in the PFGE QC record

2. Go to Lis order entry and edit date made for each new batch.

3. Print labels one large label per tube, each tube containing 3 plugs

4. Make as many plugs as are feasible on a Wednesday run

Process according to the protocol for gram negative bacilli.

5. Store plugs in glass tubes with 2.5-5mls of Gram negative wash buffer

6. QC new plugs by running on a gel along with old plugs for comparison

7. Plugs can be cut into 3 pieces, pre-digested with Xba1 and stored in 50mM EDTA @

4°C until needed (2 weeks).


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Appendix V - Preparing PFGE plugs for Serratia marcescens

Subcultures from overnight growth on MH plates or 10ml BHI broth

1. Keep subcultures @4ºC until ready for use

2. Prepare all that is needed for processing before beginning

3. Work through until plugs are in lysis buffer.

Preparation

1. Turn on 58ºC water-bath

2. Turn on incubator/shaker oven to 55 ºC

3. Calculate how much Proteinase K (PK) is necessary for each plug and Serratia Lysis/PK

buffer (10ul PK per plug and 40ul PK per 2ml aliquot Serratia lysis/PK buffer).

4. Remove Proteinase K from freezer and allow it to thaw. Keep @4 ºC until ready for use

5. Aliquot 10uL PK to eppendorf tubes for making plugs

6. Make up SL/PK buffer with PK and dispense 2 mLs for each isolate. Keep @4 ºC

7. Keep plug molds @4 ºC

8. Prepare 1% Seakem Gold Agarose (SKG) in Sterile Serratia cell suspension buffer

(SCSB) for making all plugs.(0.1gm SKG in 10 mL SCSB or .2gm SKG in 20mL SCSB).

Use 50mL tube.


Keep in 58 ºC water-bath until ready for use

Standardization


2. From broth spin 1.5mL @ 14,000 rpm and discard supernatant using a transfer pipette.

3. Re-suspend cell button with 250uL of suspension buffer (Serratia Cell suspension buffer-

SCSB)

4. Add to pre-labelled tube (1.5mL of SCSB)

5. Vortex to make a smooth suspension

6. Add organism until equivalent to 20% transmittance.

7. From plate, add organism to pre-labelled tube (1.5mL of SCSB) until equivalent to 20%

transmittance.

N.B. Keep cell suspensions on ice while making plugs.


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Preparing the plugs

Plug Making

Working with one specimen at a time.

1. Aliquot 200µls of bacterial cell suspension into a micro-fuge tube containing 10ul PK

and mix gently

2. Add 200µls of 1% Seakem gold agarose in SCSB. Mix gently.

3. Immediately fill two plug molds making sure that there are no bubbles and that the molds

have not been overfilled.

4. Allow plugs to solidify @4ºC for 5-10 minutes

5. Remove caps from tubes containing SL/PK buffer

6. Clean plungers for plugs with alcohol and dry with Kim wipe.

7. Remove tape from bottom of wells and dispense plugs into the appropriate tubes by

inserting plunger into well and tapping the top to remove plugs.

Make sure that plugs are immersed in the buffer. 8. Incubate Serratia plugs @ 55°C for 3 hrs. to overnight

Plug Washing

1. Pre-warm sterile de-ionized water, Gram positive wash buffer and Gram negative wash

buffer @ 55ºC

2. Arrange green caps in order according to processing numbers with a cap at end of row

3. Using a 50ml tube as a discard container, place each green cap over 50ml tube and decant

plugs from SL/PK buffer tube into appropriate cap.

4. Mount caps one on the other with the lowest number at the bottom.

5. Screw the blue cover of a clean 50ml tube on the last cap sealing the top of the last green

cap.

6. Add 50ml of pre-warmed sterile distilled water to a clean 50ml tube

7. Screw bottom cap of row onto tube and up-end, allowing water to run through caps and

wash all plugs

8. Discard water and repeat step 8.

9. Remove blue cap and replace with green cap as new cover.

10. Put row of caps into the glass container. Add 500ml of Gram positive/negative wash

buffer.

11. Incubate @ 55ºC for 1 hr. with a spin speed of 66 in incubator/shaker.(Wash can be left

overnight)


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12. Discard wash buffer


14. Transfer plugs from green caps into corresponding wells

N.B. OPTIONAL: Store plugs overnight or proceed to restriction digest with Xba1.


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Appendix VI - Reagents Preparation

PFGE Reagents for Gram Positives

Gram Positive PK Buffer (500 mL)

450 mL 0.5M EDTA pH 9.0-9.5

50 mL 10% Sarkosyl (SLS) *

Mix together. Do NOT Autoclave

TE Buffer (1L)

(Gram Positive Wash Buffer) 2 mL 0.5M EDTA pH 9.0-9.5

10 mL 1 M Tris HCl pH 7.6

Make up to 1L with DdH20 and autoclave.

Gram Positive PIV Buffer (500 mL)

5 mL 1M Tris HCl pH 7.6

100 mL 5M NaCl

395 mL DdH2O

Mix together and autoclave.

EC Lysis Buffer (500 mL)

3 mL 1M Tris HCl pH 7.6

100 mL 5M NaCl

100 mL 0.5M EDTA pH 9.0

2.5 mL 10% NP40 *

10 mL 10% Deoxycholate *

25 mL 10% Sarkosyl *

Make up to 500 mL using sterilized DdH2O.

*Do NOT autoclave reagents containing Sarkosyl, Deoxycholate or NP40.


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PFGE Reagents for Gram Negative Bacilli

SE Buffer (1L)

50 mL 0.5M EDTA (pH 7.4)

15 mL 5M NaCl

935 mL DdH20


Gram Negative Wash Buffer (1L)

20 mL 1M Tris HCl (pH 8.0)

100 mL 0.5M EDTA (pH 8.0)

Make up to 1L with DdH2O and autoclave.

GNB Lysis/Proteinase K Buffer (1L)

200 mL 0.5M EDTA (pH 8.0)

20 mL 10% Deoxycholate

50 mL 20% Sarkosyl

730 mL DdH2O

Do NOT autoclave.

Buffer for storing digested H9812 S.

branderup plugs (50mM EDTA)

10 mL 0.5M EDTA ph 8.0

90 mL SDH2O


* Do NOT autoclave reagents containing Sarkosyl, Deoxycholate or NP40. Filter sterilize.


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PFGE Reagents for Serratia marcescens

Cell Suspension Buffer (SCSB):

10ml of 1M Tris pH 8.0

20ml of 0.5M EDTA pH 8.0

Dilute to 100 ml using ddH2O

10% Deoxycholic acid

10g Deoxychoclic acid poswer

100ml DH20

Cell Lysis Buffer (SCLB):

0.5M EDTA 93.05gms

1% Sarkosyl, Sodium salt

(N-Laurylsarcosine) 5.0gms

NaOH pellets 20.0gms

Add 400mls of DH20 to dissolve

Adjust ph to 9-9.5

Bring volume to 500ml

Store @4ºC


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Stock Reagents

5M NaCl

292.2 g NaCl

Dissolve in 1 Litre of DdH2O and autoclave.

0.5M EDTA

20 g NaOH pellets

Dissolve in 800 mL DdH2O

(put flask on stirrer and can add small amount of

heat to dissolve)

Add 186.1 g EDTA

When dissolved, put in cylinder and top up to

1L with DdH2O

Test pH and add more NaOH pellets until desired

pH of 8 or 9 is reached.

Divide into 2 x 500 mL bottles and autoclave.

Sarkosyl (L. Laural sarcosine)

10% solution = 10 grams/ 100mL

20% solution = 20 grams/ 100mL

TRIS Buffer Chart

pH at Temperature g/L for 1.0 M Solution

5 °C 25 °C 37 °C Trizma HCL Trizma Base

7.76 7.20 6.91 140.4 13.4

7.97 7.40 7.12 132.2 19.4

8.07 7.50 7.22 127.0 23.6

8.18 7.60 7.30 121.2 27.8

8.58 8.00 7.71 88.8 53.0


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ENZYMES

Lysostaphin

5mg powder rehydrate in 5mls of

SDH2O

Final conc: 1mg/ml

Aliquot: 100uls

Lysozyme 10mg/ml

50mg powder in 5ml SDH2O

Aliquot: 1 ml

Lysozyme 100mg/ml

500mg in 5 ml

Aliquot: 500uls

Mutanolysin

Rehydrate in SDH2O

Final conc: 5U/ul

10,000U(powder) in 2mls SDH2O

Aliquot: 200uls

ProteinaseK

Rehydrate in SDH2O

Final conc: 20 mg/ml

Add 5 mls SDH2O to 1 vial of

powder

Aliquot: 1 ml.


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Appendix VII - Calculations for Restriction Enzymes, MBI Fermentas Fast Digest enzyme/

buffer Chart

N.B Use 1/3 of plug for Ladder and ½ for VRE, MRSA and GNB

No. of

samples

Pre-restriction

Wash all plugs

( patients and ladder) in

750uls of SDH2O

@RT for 15-20min

Gram Positive

-Restriction

@ 37ºC 2hrs to

overnight

Gram Negative

-Restriction

@ 37ºC 2hrs to

overnight

1 Total mix=200uls ddH2O 170 uL

FD buffer 20 uL

FD Sma1 10 uL

ddH2O 175 uL

FD buffer 20 uL

FD Xba1 5 uL

2 Total mix=400uls ddH2O 340 uL

FD Buffer 40 uL

Sma1 20 uL

ddH2O 350 uL

FD Buffer 40 uL

Xba1 10 uL

3 Total mix=600uls ddH2O 510

FD Buffer 60

Sma1 30

ddH2O 525

FD Buffer 60

Xba1 15


FD Buffer 80

Sma1 40

ddH2O 700

FD Buffer 80

Xba1 20


FD Buffer 100

Sma1 50

ddH2O 875

FD buffer 100

Xba1 25

6 Total mix =1200uls ddH2O 1020

FD buffer 120

Sma1 60

ddH2O 1050

FD buffer 120

Xba1 30


FD buffer 140

Sma1 70

ddH2O 1225

FD buffer 140

Xba1 35


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No. of

samples

Pre-restriction

Wash all plugs


750uls of SDH2O

@RT for 15-20min

Gram Positive

-Restriction

@ 37ºC 2hrs to

overnight

Gram Negative

-Restriction

@ 37ºC 2hrs to

overnight


FD buffer 160

FD Sma1 80

ddH2O 1400

FD buffer 160

FD Xba1 40

9 Total mix =1800uls ddH2O 1530 uL

FD buffer 180 uL

FD Sma1 90 uL

ddH2O 1575 uL

FD buffer 180 uL

FD Xba1 45 uL


FD buffer 200 uL

FD Sma1 100 uL

ddH2O 1750 uL

FD buffer 200 uL

FD Xba1 50 uL


FD buffer 220 uL

FD Sma1 110 uL

ddH2O 1925 uL

FD buffer 220 uL

FD Xba1 55 uL


FD buffer 240 uL

FD Sma1 120 uL

ddH2O 2100 uL

FD buffer 240 uL

FD Xba1 60 uL


FD buffer 260 uL

FD Sma1 130 uL

ddH2O 2275 uL

FD buffer 260 uL

FD Xba1 65 uL


FD buffer 280 uL

FD Sma1 140 uL

ddH2O 2450 uL

FD buffer 280 uL

FD Xba1 70 uL


FD buffer 300 uL

FD Sma1 150 uL

ddH2O 2625 uL

FD buffer 300 uL

FD Xba1 75 uL


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No. of

samples

Pre-restriction

Wash all plugs


750uls of SDH2O

@RT for 15-20min

Gram Positive

-Restriction

@ 37ºC 2hrs to

overnight

Gram Negative

-Restriction

@ 37ºC 2hrs to

overnight


FD buffer 320 uL

FD Sma1 160 uL

ddH2O 2800 uL

FD buffer 320 uL

FD Xba1 80 uL

17 Total mix =3400 ddH2O 2890 uL

FD buffer 340 uL

FD Sma1 170 uL

ddH2O 2975 uL

FD buffer 340 uL

FD Xba1 85 uL


FD buffer 360 uL

FD Sma1 180 uL

ddH2O 3150 uL

FD buffer 360 uL

FD Xba1 90 uL


FD buffer 380 uL

FD Sma1 190 uL

ddH2O 3325 uL

FD buffer 380 uL

FD Xba1 95 uL


FD buffer 400 uL

FD Sma1 200 uL

ddH2O 3500 uL

FD buffer 400 uL

FD Xba1 100 uL


FD buffer 420 uL

FD Sma1 210 uL

ddH2O 3675 uL

FD buffer 420 uL

FD Xba1 105 uL


FD buffer 440 uL

FD Sma1 220 uL

ddH2O 3850 uL

FD buffer 440 uL

FD Xba1 110 uL


FD buffer 460 uL

FD Sma1 230 uL

ddH2O 4025 uL

FD buffer 460 uL

FD Xba1 115 uL


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No. of

samples

Pre-restriction

Wash all plugs


750uls of SDH2O

@RT for 15-20min

Gram Positive

-Restriction

@ 37ºC 2hrs to

overnight

Gram Negative

-Restriction

@ 37ºC 2hrs to

overnight


FD buffer 480 uL

FD Sma1 240 uL

ddH2O 4200 uL

FD buffer 480 uL

FD Xba1 120 uL


FD buffer 500 uL

FD Sma1 250 uL

ddH2O 4375 uL

FD buffer 500 uL

FD Xba1 125 uL


FD buffer 520 uL

FD Sma1 260 uL

ddH2O 4550 uL

FD buffer 520 uL

FD Xba1 130 uL


FD buffer 540 uL

FD Sma1 270 uL

ddH2O 4725 uL

FD buffer 540 uL

FD Xba1 135 uL


FD buffer 560 uL

FD Sma1 280 uL

ddH2O 4900 uL

FD buffer 560 uL

FD Xba1 140 uL

29 Total mix =5800 uls ddH2O 4930 uL

FD buffer 580 uL

FD Sma1 290 uL

ddH2O 5075 uL

FD buffer 580 uL

FD Xba1 145 uL


FD buffer 600 uL

FD Sma1 300 uL

ddH2O 5250 uL

FD buffer 600 uL

FD Xba1 150 uL


FD buffer 620 uL

FD Sma1 310 uL

ddH2O 5425 uL

FD buffer 620 uL

FD Xba1 155 uL


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No. of

samples

Pre-restriction

Wash all plugs


750uls of SDH2O

@RT for 15-20min

Gram Positive

-Restriction

@ 37ºC 2hrs to

overnight

Gram Negative

-Restriction

@ 37ºC 2hrs to

overnight


FD buffer 640 uL

FD Sma 1 320 uL

ddH2O 5600 uL

FD buffer 640 uL

FD Xba1 160 uL


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Appendix VIII - To Send Isolates To NML For: Spa typing

1. Refer to Sendout Manual FEDEX section for instructions on preparing specimens for

shipping.

2. Ensure to send To NML attn: Dr. Michael Mulvey

3. Please use the MicrobiologyNML Requisition.





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Appendix IX – Reference Material

Reference Material:

PFGE Reference Material

Troubleshooting links:

PGFE Tips and Tricks to Success and Interpretation of Results

PFGE Troubleshooting Tips

PFGE comparison request form:

PFGE COMPARISON REQUEST FORM

https://eportal.mountsinai.ca/MSHPresentations/public/paradigm/D0023977.PDF




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Record of Edited Revisions

Manual Section Name: Infection Control Pulsed-field Gel Electrophoresis

Page Number / Item Date of Revision Signature of

Approval Annual Review May 30, 2007 Dr. T. Mazzulli

Annual Review September 20, 2008 Dr. T. Mazzulli

Annual Review April 12, 2009

Added Reporting section October 15, 2010 Dr. T. Mazzulli

Annual Review October 15, 2010 Dr. T. Mazzulli

Reviewed and Updated May 15, 2011 Dr. T. Mazzulli

Added comparison request for Rouge Valley and other

hospitals

December 15, 2011 Dr. T. Mazzulli

Annual Review December 15, 2011 Dr. T. Mazzulli

Changed UHN new MRSA and VRE to not routinely set

up

March 21, 2012 Dr. T. Mazzulli

Updated Comparing a cluster May 14, 2012 Dr. T. Mazzulli

Updated workflow August 14, 2012 Dr. T. Mazzulli

Annual Review August 14, 2012 Dr. T. Mazzulli

Added Serratia marcescens procedure December 18, 2012 Dr. T. Mazzulli

Annual Review August 27, 2013 Dr. T. Mazzulli

PFGE comparison request form May 16, 2014 Dr. T. Mazzulli

Add on organisms on PFGE Bionumerics Run Parameters

Annual Review

Updated headers/footers

November 06, 2014 Dr. T. Mazzulli

p.9 Lysis Enzyme table added: GAS / GBS row with 50ul

Lysozyme L100 100mg/mL, 50 ul Mytanolysin 5U/uL

(Sigma) and 20ul Proteinase K 20mg/mL (Roche)

February 03, 2015 Dr. T. Mazzulli

Appendix VIII – Trouble shooting web link February 08, 2015 Dr. T. Mazzulli

P.4 GAS from MSH note 3 added: if identified by

infection control to be a nosocomial GAS

May 16, 2015 Dr. T. Mazzulli

Appendix VIII – Reference Material: added folder of

reference material PDFs


Annual Review:

Removed New ESBL and p.aeruginosa from work.

Removed link for “PFGE set up preprocessing”

p.7 PFGE bench: #3 Changed to print labels, removed link

June 04, 2015 Dr. T. Mazzulli


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Approval from #7 “isolates for inhouse pfge”

Changed hybridization oven to incubator/shaker through

manual

p.11 PK BUFFER changed to 1hr for MRSA/VRE/GAS

p.13 Step 6 #3 added put 250ulSDH2O in mirofuge tube

p.17 Step 6 #3 added: add 750ul SDH2O to tube

p.17 Removed Note “plugs can be digested and stored in

50mM EDTA until ready for use”

Removed reference to PULSE WAVE 760 Module

p.21 removed Ethidium bromide stain removal bath section

Updated save file names

p.23 updated Bionumerics Gel analysis procedure

p.26 Removed standard reporting phrase when PFGE is in

progress section.

p.31 updated Sending a report by Email section added

MSh and UHN cc to (MSH)lab-PFGE team, (UHN) Dr.

Hota, Dr. Gardam, Dr. Lemieux

p.35 Weekly QC (removed Note marker H9812S)

Appendix IV, removed #4 Print QC for making molecular

marker/fill in information

Appendix V: Plug Washing, step 11, add 500ml gram

pos/meg wash buffer12: changed speed to 66

Appendix VI: added 10%deoxychoclic acid box

Made Calculations for Restriction Enzymes its own

appendix.

p.3 Isolates for Referred-Out PFGE: remove Rouge valley

p.5 procedure added #11 destain

Remove Appendix II - Thiourea Use for PFGE protocol

Appendix VIII refer to Send out Manual Fedex section.

Insert on Page 23 of 51, #2 - Choosing the Correct

Database for Analyzing/Saving Gel Results Table

October 30, 2015 Dr. T. Mazzulli

In section: Comparing and Interpreting a Cluster in

Bionumerics added to step 5:

Note: make sure the Optimization is 0.81 and Position

tolerance is 1.06

Click Position tolerances

Check the Optimization (0.81) and Position tolerance

April 08, 2016 Dr. T. Mazzulli


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Approval (1.06) setting

Click OK

Table 3 for Pseudomonas / Stenotrophomonas changed to

fast digest Bcu 1 from Spel and on run parameters (p.20)


Annual Review June 04, 2016 Dr. T. Mazzulli

In Naming CMRSA section, added paragraph “Naming

CMRSA-10)”

Added new appendix 1 (further appendices renumbered)

10 and 15 lane recording sheets.

Appendix VI: Gram positive PK buffer updated:

450 EDTA (from 475),

50ml 10% Sarkosyl from (25ml 1%)


In Naming CMRSA section, added paragraph “Naming

CMRSA-10)”

Added new appendix 1 (further appendices renumbered)

10 and 15 lane recording sheets.

Appendix VI: Gram positive PK buffer updated:

450 EDTA (from 475),

50ml 10% Sarkosyl from (25ml 1%)

October 08, 2016 Dr. T. Mazzulli

Annual Review

Added yearly PFGE to VRE from UHN


Annual Review May 30, 2018 Dr. T. Mazzulli

Minor format change September 14, 2018 Dr. T. Mazzulli



Full document review included in all updates. Bi-annual review conducted when no revision had

been made within 2 years.

Page Number / Item Date of Revision Edited by:

Minor formatting change April 11, 2021 Jessica Bourke

Updated procedure May 13, 2021 Wayne Chiu

Updated send report by email section Nov 26, 2021 Wayne Chiu

Added yearly maintenance section Feb 2, 2022 Wayne Chiu


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Page Number / Item Date of Revision Edited by:

Documents

Uncontrolled When Printed TABLE OF CONTENTS