Policy & Procedure Manual / Policy # MI\IC\PF\v17 / Page 1 of 51
Section: Infection Control Manual / Subject Title: Infection Control Bench Pulsed-field Gel Electrophoresis
Pulsed-field Gel Electrophoresis
Table of Contents
I.Introduction
Isolates for Referred-Out PFGE
IV.Procedure
Pulsed-field Bench Workflow
Testing schedule
Test ordering, broth labeling and inoculation in preparation for typing
Cell Extraction Preparation
For MRSA, VRE, GAS, GNB
For Serratia marcescens
Settings for CHEF-DR II/III instruments
CHEF-DR II/III Module
Ethidium bromide for staining
Weekly Ethidium Bromide Disposal
Weekly Ethidium Bromide Preparation
Staining Protocol
How To Use The Gel DOC XR+ Camera
BioNumerics Gel Analysis
BioNumerics Naming CMRSA
IV.Reporting
PFGE Reporting Phrases
Verifying Bionumerics Results
V.Processing a Comparison Request (In-house and for PHL)
Documenting Request
Comparing and Interpreting a Cluster in Bionumerics
Interpretation Example
Sending a Report by E-mail
Appendix I - Documenting Comparison Requests in LIS
Appendix II - Thiourea Use for PFGE protocol
Appendix III - Maintenance and Quality Control
Maintenance and QC CHEF PFGE Machines
Before each run
Weekly
Monthly
Water flush and temperature check.
Maintenance of Gel Run Record
Appendix IV - Preparing PFGE Plugs of the Salmonella ser Branderup H9812 Standard Strain
Appendix V - Preparing PFGE plugs for Serratia marcescens
Preparing the plugs
Appendix VI - Reagents Preparation
PFGE Reagents for Gram Positives
PFGE Reagents for Gram Negative Bacilli
PFGE Reagents for Serratia marcescens
Stock Reagents
TRIS Buffer Chart
Appendix VII - Calculations for Restriction Enzymes, MBI Fermentas Fast Digest enzyme/ buffer Chart
ENZYMES
Appendix VIII - To Send Isolates To NML For: Spa typing
Appendix VIII – Reference Material
Record of Edited Revisions
Appendix VIII – Reference Material: added folder of reference material PDFs
I.Introduction
Pulsed field gel electrophoresis (PFGE) typing or macro-restriction of micro-organisms is performedfor epidemiological purposes to determine the relatedness between the chromosomal DNA of two or more isolates.
PFGE assists the Infection Control department by:
- Providing an understanding of the transmission of isolates between patients.
- Providing evidence of transmission of isolates from the environment to patients
- Determining whether isolates involved in recurrent infections are the same clone or are newly acquired.
- Specimen Collection
Isolates for PFGE (Routinely Done) In-House
ISOLATES FOR PFGE IN-HOUSE / UHN / MSHNew MRSA / No / Yes2
New VRE / No / Yes1
New Group A Streptococcus / No / Yes3
Any organism as part of a cluster or upon ICP's request / Yes / Yes
- 1st time isolated and once a year there after.
- MSH New = 1st time isolated and every 3months thereafter, if no other positive in between isolates.
- In-patient onlyor if identified by infection control to be a nosocomial GAS.
Isolates for Referred-Out PFGE
ISOLATES FOR PFGE PHL / BaycrestNew 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
LABORATORY MANUAL
UNIVERSITY HEALTH NETWORK/MOUNT SINAI HOSPITAL, DEPARTMENT OF MICROBIOLOGY
NOTE: This is a CONTROLLED document for internal use only. Any documents appearing in paper form are not controlled and should be checked against the document (titled as above) on the server prior to use.
T:\Microbiology\New Manual\Live Manual\IC\Infection Control Pulsed-field Gel Electrophoresis.doc
Department of MicrobiologyPolicy & Procedure Manual / Policy # MI\IC\PF\v17 / Page 1 of 51
Section: Infection Control Manual / Subject Title: Infection Control Bench Pulsed-field Gel Electrophoresis
IV.Procedure
Pulsed-field Bench Workflow
MONDAY / TUESDAY / WEDNESDAY / THURSDAY / FRIDAYPFGE 1 /
- Retrieve incubated PF BHIB isolates from incubator.
- Check Bionumerics database to see if done previously. Refer to chart above for testing frequency
- Process isolates up until “wash stage"( can be left washing overnight)
- Enter isolates in Bionumerics, LIS (plug date and PF done in isolate window)
- Create gel legends for each type and log into “Gel Run Record” (in T drive)
- Document incubator temperatures, lot number and expiry dates for enzymes on gel legends
- Check for new queries Respond ETA for results
- Answer queries.
- Retrieve any query isolates as necessary.
- Prepare isolates for digest
- Prepare machines (drain, rinse, clean, change buffer, inspect for broken electrodes)
- Make agarose for each gel, load plugs onto combs, affix with agarose, chill, pour gel, chill and load
- Document machine maintenance
- Wash glass wash containers and prepare for autoclaving.
- Send green caps for washing.
- Send glassware for washing
- Check for new queries Respond ETA for results
- Answer queries.
- Retrieve any query isolates as necessary.
- Destain
- Make fresh stain
- Stain/ Destain and photograph gels set up on Tuesday.
- File and export data from images photographed into Bionumerics.
- Normalize gels from Tuesday's run.
- Name MRSA’s.
- Report in LIS.
- Prepare green caps for autoclaving
- Answer queries.
- Subculture send out organisms as required
- Check for new queries Respond ETA for results
- Answer queries.
- Retrieve any query isolates as necessary.
- Check for new queries Respond ETA for result
- Answer queries.
- Finish reporting queries as required
- Make reagents
- Prepare aliquots of enzymes
- Check inventory
- Order enzymes, supplies
- Restock supplies
- Prepare H9812 plugs as required
- Discard plugs > 3 months old
- Update Bionumerics database (Cleaning)
- Retrieve any query isolates as necessary.
- Check for new queries Respond ETA result
- Answer queries.
- Check Didi worklist in LIS for PF ordered but not given to bench
- Finish reporting queries as required
- Prepare for Mondays run (Monday’s steps 1, 2 & 4.) Labels can than be grouped and placed in box labeled “Labels for PFGE in Progress”
- Weekly-check electrodes Monthly- temperature check; Flow rate check and adjustment
- Retrieve any query isolates as necessary.
PFGE 1/2 / Verifiy results from Monday's run
LABORATORY MANUAL
UNIVERSITY HEALTH NETWORK/MOUNT SINAI HOSPITAL, DEPARTMENT OF MICROBIOLOGY
NOTE: This is a CONTROLLED document for internal use only. Any documents appearing in paper form are not controlled and should be checked against the document (titled as above) on the server prior to use.
T:\Microbiology\New Manual\Live Manual\IC\Infection Control Pulsed-field Gel Electrophoresis.doc
Department of MicrobiologyPolicy & Procedure Manual / Policy # MI\IC\PF\v17 / Page 1 of 51
Section: Infection Control Manual / Subject Title: Infection Control Bench Pulsed-field Gel Electrophoresis
Testing schedule
- PFGE is run weekly in-house, refrigerate pre-inoculated broths/plates from Monday until Sunday.
- Incubate broths/plates at 37ºC in walk in incubator on Sunday @ 4:00p.m. for PFGE processing on Monday.
- The run incubated on Sunday finishes on Wednesday
N.B
A failed or additional run can be done on Thursday (or when possible Wednesday to Friday) if required. It is fine to let it run over the weekend and attend to it on Monday.
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:-
- Order ^BHIB and ^PF in LIS.
- FromPF, select isolate type from keypad(i.e. MRSA, VRE, GNB etc).
- Go to the print label screen, and print three(3) labels.
- 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.
- Touch 1-3 colonies(dependent on colony size) of pure growth with a loop and inoculate the broth/plate.
- Put inoculated broth/plate into rack in PFGE fridge for batch testing.
- The MRSA bench technologist on the weekend is responsible for placing the BHIB rack into the 37oC walk-in incubator on Sunday @4:00p.m. and the PFGE The only exceptions to this protocol are:
- Streptococcucs pneumoniae- must be inoculated just prior to incubation onto 2 BA plates (CO2).
PFGE Bench
- Retrieve isolates for PFGE from shaker and plate rack
- Put Brain heart infusion tubes / plates in order (alphabetical) within groups
- Print required labels
- Put labels in the same order within groups
- Match labels to isolates
- Place cultures @4oC until ready for processing
- Using labels check appropriate Bionumerics database MRSA( UHN-MSH MRSA) GNB (UHN-MSH ESBL-MDRO) for repeats(right click on Last Name column and choose arrange by field for alphabetical order)
If an unwarranted repeat, document in LIS (Prev see LIS#...... ) at back of workcard.
- Put labels in numerical order with groups. Assign processing numbers to isolates Write processing numbers on labels (1 large label 3 small labels) and on matching BHI tubes
IN LIS
- Go to worklist :ZDIDI
- Mark all isolates document plug date under PF
(If isolate not on work list order PF)
- Go to individual isolates and add isolate letter and pfdone in isolate window
- Ensure that the appropriate organism has been ordered in the PF window in LIS. i.e.`MRSA`
- Check isolates within date range of order numbers that have not been marked.
- Investigate as to why broth is not received.
- Document in LIS- PF that broth was not received and request isolate from bench.
IN BIONUMERICS
- 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.
Cell Extraction Preparation
Routine Bench
- Subculture organism from pure growth to 10mL BHI broth
- Refrigerate broths Monday to Sunday. Incubate at 37ºC on Sunday @ 4:00p.m.for PFGE processing on Monday. (For long weekends that have holiday Mondays, incubate on Monday.)
PFGE Bench Monday
- Determine isolates for PFGE by checking Bionumerics and LIS databases to eliminate repeats and account for missed isolates.
For MRSA, VRE, GAS, GNB
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 or GAS / 40µL per tube / 15 µL per tube / 40µL per tube
GNB / 40µL 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 58ºC water-bath until ready for use.
Step 2 Standardization:
- Transfer broth from BHIB’s using transfer pipette into labeled microfuge tubes, centrifuge @ 14,000 rpm for 1 minute to pellet.
- Pipette off supernatant. Change pipette.
- Calibrate Vitek colorimeter.
- 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
- Add 200 uls (using 1000ls pipette) of bacterial cell suspension to each labelled microfuge tube.
- Add 200 uls (using 200ls 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)
- 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 mLEC 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 2mlsEC 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 mLEC 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 2mlsEC Lysis Buffer/L10 mixture to labeled white-capped tubes for each isolate
* NOTE: NO LYSIS STEP FOR GRAM NEGATIVES! (Proceed to step 4.)
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 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 BUFFERIncubation 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.
Step 4Removal 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:
- Retrieve tubes from incubator
- 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)
- Place green cap on top of each stack
- Remove stack from discard centrifuge tube
- Discard decanted PK buffer
- Return stack to the centrifuge tube
- Pour 50.0ml pre-warmed SDH2O in a sterile centrifuge tube labeled “SDH2O” and pour through the assembled screw caps to rinse
- 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
- Invert sideways to rinse through. (3 times)
- Decant. Repeat steps 7-9, 2 more times.
- Load assembled screw caps intoglass wash dishes.
- Pour enough TE buffer (or Gram –ve Wash buffer for Gram negatives) to fully submerge green caps. Seal dishes tightlyand wash for a minimum 60 minutes@ 55C on shaker and then decant. (You can wash overnight at this point.)
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 Spe1 for Pseudomonas / Stenotrophomonas / Fast digest Sfi1 AcinetobacterQUANTITY 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.