Testing the Efficiency of Aerosol Containment During Cell Sorting

Ingrid Schmid1, Lance E. Hultin1, John Ferbas1

1 UCLA School of Medicine, Los Angeles, California
Publication Name:  Current Protocols in Cytometry
Unit Number:  Unit 3.3
DOI:  10.1002/0471142956.cy0303s01
Online Posting Date:  May, 2001
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Abstract

Production of droplets and microdroplets (aerosols) is part of the normal operation of a cell sorter. These aerosols may contain toxic, carcinogenic, or teratogenic fluorophores or known or unknown pathogens from viable biological specimens. Most newer models of commercially available instruments incorporate features designed to reduce the production of aerosols and prevent their release into the room. This unit presents two protocols for assessment of aerosol containment on jet‐in‐air flow sorters. In both procedures, lytic T4 bacteriophage is run through the instrument at high concentrations to tag aerosol droplets. The instrument is tested in normal operating mode and in simulated failure mode. Aerosols are detected by plaque formation on susceptible E. coli lawns. With the continuing increase in the sorting of viable human cells, it is vital for cytometrists to be aware of the potential dangers.

     
 
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Table of Contents

  • Basic Protocol 1: Testing of Aerosol Containment in Flow‐Sorting Systems by Monitoring Gravitational Deposition of Droplets
  • Alternate Protocol 1: Testing of Aerosol Containment in Flow‐Sorting Systems by Use of an Air Sampler
  • Support Protocol 1: Preparation of T4 Bacteriophage Stock
  • Support Protocol 2: Preparation of E. Coli Lawns
  • Support Protocol 3: Titration of the T4 Bacteriophage Suspension to be Used in the Aerosol‐Containment Test
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Testing of Aerosol Containment in Flow‐Sorting Systems by Monitoring Gravitational Deposition of Droplets

  Materials
  • Dilution broth (see recipe)
  • T4 bacteriophage stock solution (see protocol 3), appropriately titered (see protocol 5)
  • 12 × 75–mm culture tubes (Falcon)
  • Electronic balance
  • Deflected‐droplet (jet‐in‐air) cell sorter to be tested (e.g., FACStar and FACSvantage from Becton Dickinson, MoFlo from Cytomation, EPICS series from Coulter, and Ortho Systems from Ortho Diagnostics; see unit 1.2)
  • Petri dishes containing confluent E. coli lawns (see protocol 4) and dish with bottom agar only as control
  • 35° to 37°C warm room or incubator

Alternate Protocol 1: Testing of Aerosol Containment in Flow‐Sorting Systems by Use of an Air Sampler

  • Andersen single‐stage air sampler (model #N6IACFM; Grayseby‐Andersen) with two‐port manifold

Support Protocol 1: Preparation of T4 Bacteriophage Stock

  Materials
  • T4‐susceptible E. coli (ATCC #11303)
  • Nutrient broth (see recipe)
  • T4 bacteriophage, lyophilized (ATCC #11303‐B4)
  • Chloroform
  • 25‐ and 250‐ml Erlenmeyer flasks
  • 10.2 × 10.2–cm sterile 12‐ply gauze pads (e.g., Johnson & Johnson Steri‐Pads)
  • 35° to 37°C warm room and orbital shaker or shaking incubator
  • 50‐ml conical polypropylene centrifuge tubes, sterile
  • Sorvall Omnispin R centrifuge with 42011‐type rotor (or equivalent)
NOTE: All reagents and equipment coming into contact with live cells must be sterile, and proper sterile technique should be followed accordingly.

Support Protocol 2: Preparation of E. Coli Lawns

  Materials
  • Bottom agar (see recipe)
  • Soft agar (see recipe)
  • 40° to 50°C water bath
  • 100‐mm petri dishes
  • Additional reagents and equipment for growing E. coli in nutrient broth (see protocol 3)
NOTE: All reagents and equipment coming into contact with live cells must be sterile, and proper sterile technique should be followed accordingly.

Support Protocol 3: Titration of the T4 Bacteriophage Suspension to be Used in the Aerosol‐Containment Test

  Materials
  • Dilution broth (see recipe)
  • T4 bacteriophage stock suspension (see protocol 3)
  • Petri dishes containing bottom agar (pre‐prepared; see protocol 4, step )
  • Hanks' balanced salt solution (HBSS; appendix 2A) or other sheath fluid for flow cytometry that will maintain viability of T4 bacteriophage
  • 12 × 75–mm culture tubes
  • 15‐ml culture tubes
  • Cell sorter to be tested (see protocol 1)
  • Additional reagents and equipment for preparing E. coli lawns (see protocol 4) and for determining and adjusting flow rate of cell sorter (see protocol 1, steps to )
NOTE: All reagents and equipment coming into contact with live cells must be sterile, and proper sterile technique should be followed accordingly.
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Figures

Videos

Literature Cited

Literature Cited
   Andersen, A.A. 1958. New sampler for the collection, sizing and enumeration of viable airborne particles. J. Bacteriol. 76:471‐484.
   Bakker, A.M. 1992. Evaluation of a biological containment system for a fluorescence‐activated cell sorter. M.S. thesis. University of San Francisco.
   Ferbas, J., Chadwick, K.R., Logar, A., Patterson, A.E., Gilpin, R.W., and Margolick, J.B. 1995. Assessment of aerosol containment on the ELITE flow cytometer. Cytometry 22:45‐47.
   Giorgi, J.V. 1994. Cell sorting of biohazardous specimens for assay of immune function. Methods Cell Biol. 42:359‐369.
   Merrill, J.T. 1981. Evaluation of selected aerosol‐control measures on flow sorters. Cytometry 1:342‐345.
   Sattar, S.A. and Ijaz, M.K. 1987. Spread of viral infections by aerosols. CRC Crit. Rev. Environ. Control 17:89‐131.
   Schmid, I., Nicholson, J.K.A., Giorgi, J.V., Janossy, G., Kunkl, A., Lopez, P.A., Perfetto, S., Seamer, C.L., and Dean, P.N. 1997. Biosafety guidelines for sorting of unfixed cells. Cytometry 28:99‐117.
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