Equipment Chapter 5

Laminar Flow Hood

Horizontal Laminar Flow Hood Suitable for preparing medium, sterile reagents and culturing nonprimate cells Suitable for dissecting nonprimate cells

Class II or Class III Cabinet Hazardous materials – any primate, human cell lines, virus-producing cultures, radiosiotopes or carcinogenic or toxic drugs Follow local and national biohazard regulations before installation

Measurements of a laminar hood Working surface are of 1200 mm (4 ft) wide x 600mm (2 ft) deep Quiet Cleaning both inside working area and below work surface to avoid spillage Comfortable to sit Flexibility with front screen (never remove)

Pipette Cylinders Made up of polypropylene One per workstation – freely distributed in the laboratory Allow 2 hours stand of pipettes in disinfectants

Aspiration Pump Suction pressure – remove medium or other reagents from culture flask Effluent – discharged into container (hypochlorite added) Switch pump before use

Vacuum pump Same principle Waste collected in container (emptied) Switch pump before use Avoid using contaminated vacuum lines

Service Carts Place all items on service carts Carry soiled materials and restock hoods

Inverted Microscope Look at cultures Good quality optics Long-working distance between condenser and objectives Provision of CCD camera and monitor Fluorescence microscopes

Centrifuges Increase concentration of cells Wash off reagents High speed – damage to cells and agglutination Large-scale suspension cultures – refrigerated centrifuge – 4 x 1L or 6 x 1L

Sterile Liquid Handling – Pipetting and Dispensing Removal of fluids Rubber bulb is cheap and simple use Pipetting aids – weight and feel of instrument Pipettors/Micropipette Pipettes and tips

Dispensing mechanisms Large-volume dispensing (> 100 ml) 100 ml pipette or graduated bottles or bags or > 500 mls (peristaltic pumps – different flows – also for Repetitive dispensing) Single fluid transfers (10- 10,000 L)

Multipoint Pipettor Fill many vials at one time (based on the number of tip holding capacity)

Plate Filler Automatic filling device Load multiple plates Sterile or nonsterile conditions

Cell Counter Hemocytometer – counts number of cells Cell counter – automatic counting of cells in solution

Incubator Culture cells Safety thermostat Resistant to corrosion – clean Shelving is perforated Routine maintenance of culture is important Uniform density exp- place in metal trays

Humid Co2 Incubator Humidifying tray – controls CO2 tension CO2 monitoring device – draws air from incubator into sample chamber, determines concentration of CO2 and injects pure CO2 Gold wires or Infrared detectors reset zero of CO2 detector Cannot culture flasks – use hot rooms gassed with CO2 supply Frequent cleaning is essential – dismantle shelves

Water-jacketed with circulating fan and Dry-walled with no circulating fan

Differences Water-jacketed Water jacket distributes heat evenly around cabinet Hold temperature longer in power failure Air circulation by fan – keeps CO2 level and temperature uniform Dry-walled Incubators No water jacket Cannot hold temperature longer in power failure No fan Less fungal contamination on walls

Magnetic Stirrer Used for suspension cultures Speed = 50 rpm Capable of stirring 10 L fluid

Preparation and Sterilization Wash up – Soaking baths or sinks - Pipette washer – Connected to Deionized water (final rinse) and regular tap cold water - Pipette drier – Dry pipettes (rack or drying oven)

Water Purifier

Rinsing glassware – Deionized or reverse osmosis water Powdered media and diluting concentrations – Ultra pure water Reverse osmosis – charcoal filtration- deionization-micropore distillation Purified water should not be stored. Must be recycled continuously to avoid growth of microorganisms

Autoclaves

Other equipment Balances, pH meter, Hot plate magnetic stirrer, automatic dispenser, conductivity meter (ionic concentration), refrigerator and freezer, Computer and networks, PCR cycler, syringes and needles, sterilization filters, paper towels and swabs, disinfectants (chlorine based disinfectants) etc

Glassware Washing Machine Water for washing should be heated to 80 degree C Last step – rinsing with deionized water degree C - Avoid cracking of dishes - Recycle water should be discarded - Drier is recommended

Specialized Equipment Microinjection facilities – can inject directly into cell – example nuclear transplantation or dye injection Colony Counter – Cell counting Centrifugal elutriator – Special centrifuge to separate cells of different sizes Flow Cytometer – Counts cell populations based on light scatter, absorbance and fluorescence

This project is funded by a grant awarded under the President’s Community Based Job Training Grant as implemented by the U.S. Department of Labor’s Employment and Training Administration (CB ). NCC is an equal opportunity employer and does not discriminate on the following basis: against any individual in the United States, on the basis of race, color, religion, sex, national origin, age disability, political affiliation or belief; and against any beneficiary of programs financially assisted under Title I of the Workforce Investment Act of 1998 (WIA), on the basis of the beneficiary’s citizenship/status as a lawfully admitted immigrant authorized to work in the United States, or his or her participation in any WIA Title I-financially assisted program or activity.

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