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 = controlled @ 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

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 - 50-60 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-15-162-06-60). 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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