basic requirement for laboratory design i. the laboratory design 1. design concepts and layout a. to...
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Basic Requirement For Laboratory Design
I. The Laboratory Design
1. Design Concepts and Layout a. To allow sterile handling of culture cells b. Minimum movement of people past or through the clean area c. Adequate separation of the clean area for sterile handling of cells from dirty operation
2. Facilities Essential to Cell Culture
a. Laminar flow cabinet
d. Iinstrument and equipment bench
e. Media fridge ( sterile working solution only)
f. Freezer for culture reagents requiring storage at - 20oC or below
g. Preparation area for media and other solution
h. General cold storage facility for chemicals and non- sterile reagents
b. CO2 Incubator
c. Storage space for sterile equipment and solution
RefrigeratorCO2 incubator
microscope
Laminar Flow
Washing area
Storage and preparation
Working area
Working area
Preparation area
soaking washing
Deionize water supply
Pipette washer
If possible In a separate room:
a. Liquid nitrogen freezers for cell stock b. General preparation area
c. Storage area for unopened plastic ware
d. Sterilization oven and autoclave
e. Drying oven
f. Water purification system
g. Washing-up area
Others:
1. Warm room at 37oC for large scale culture
2. Laminar flow for tissue dissection for primary culture
3. Electronic cell counter ( for large number in cell growth kinetic research)
4. Air conditioning
5. Cold room at 4oC for media storage or liquid nitrogen freezer
II. Services
1. Water supply to sink 2. Gas tap for Bunsen burner 3. CO2
4. Ultra pure water water may be used for:
a. Solvent for culture media
b. Solvent for supplement
c. For rinsing of glassware
tapwater water softener cartridge RO
ultrapurification system
ultrapure water
Direct-Q™ 5 Ultrapure Water Systems
A. Laminar flow hoods
III. Equipping the laboratory
class II cabinet offers:
Front screen which minimize the turbulence drown in front side
Exhausting air flow is mostly recycled
Equipped air flow monitor alarm system
Vertical Hood
Horizontal Hood
How to choose a laminar flow?
1 . Does the hood require an internal power point or gas tap?
2. Is the inside of the hood easy to clean?
3. Will it be necessary to open the front of the hood regularly to introduce large vessel?
4. Would it be useful for the height of the working surface of the hood to be adjustable
B. CO2 Incubator
1. Temperature
Mammalian cells : 37 oC
Avian cells : > 37 oC
Mammalian skin cells : <37 oC
Cold blooded animals: their normal expose range
CO2 Incubator
2. Gas phase CO2: maintain pH of culture medium
pH7.2---7.4 concentration: 5% 10% for serum free medium
NaOH+H2CO3 NaHCO3+H2O Na++HCO3-+H2O (similar effect to neutralize pH if add alkali)
Inclusion of pyruvate increase production of CO2
LeibovitzL15 has high sodium pyruvate, lack NaHCO3 does not require CO2
CO2+H2O H2CO3 H++ HCO3-
NaHCO3 Na+ + HCO3-
(add NaHCO3 additionally in the medium)
Buffer used for medium
Incorporate into medium to stabilize pH
Relationship Between NaHCO3, CO2 and HEPES
compound Eagle’s MEM Hank’s salt Low HCO3- buffer Eagle’s MEM
Earl’s salt
DMEM
NaHCO34mM 10mM 26mM 44mM
CO2 Atmospheric and evolved from culture
2% 5% 10%
HEPES 10mM 20mM 50mM ---
Oxygen
Rely mostly on glycolysis in cell culture
Dissolved O2 is toxic by producing free radical ----------
may be reduced by free radical scavager, i.e.
- mecaptoethanol or dithiothreitol
Higher O2 percentage for organ culture 95%
Keep depth of medium within 2-5 mm( 0.2-0.5% mL/cm2)
3. Humidity
change water frequently
avoid microbial contamination
Water plate
C. Microscope
Inverted microscope
for observation of :
cell morphology, degree of spreading,
membrane blebbing, proportion of
multinucleate, vaculation, microbial
contamination
Fluorescent microscope
Fluorescent microscope
D. Centrifuge bench top centrifuge
cell collection
E. Fridges and freezer( -20oC, -70oC, liquid
nitrogen)
media storage
F. Miscellaneous small items of equipment
Water bath 37oC
Submersible magnetic stirrer
Aspirator jar
Culture plastic ware and associate small consumable items
A. The most commonly used items of plastic ware:
1. For growing cells( electric treatment on surface of culture plate
flat bottomed flask
petri-dish
multi-well dish
conical flask roller bottle
Tubes
culture flask 25 cm2 75cm2 175 cm2
culture dish
petridish: 35mm, 60mm, 90mm,…. Multiwell dish 6 wells,12 wells,24 wells,…
Chamber Slides
2. For handling solutions and cell suspensions
Volumetric pipettes
Plastic Pasteur pipette
Micropipette tips
Centrifuge tube
15 ml centrifuge
50 ml centrifuge
3. For storage
Sample tube
Eppendorf tube
Cryotube
Large volume screw capped bottle
For large scale adherent cell culture:
anchorage dependent culture:
▪roller bottle
▪ micro carrier beads
▪ meshes
Suspension culture:
▪ spinner flask
▪ conical flask on a rotating platform
▪ static culture on nonadherent plastic ware
Spinner flaskconical flask on a rotating platform
B. Filters for sterilizing tissue culture solutions
pore size: 0.2um---0.45um
How to choose the filters
Low protein binding
Minimum dead space
Large volume
C. Pipettes micropipettes with disposable autoclaveable tips
volume: 1---20 ul
100---1000 ul
20---200 ul
5 ml
Disposable Pasteur pipettes
Glass volumetric pipette
volume 5---25 ml
D. Pipette aid or multichannel micropipette
E. Glassware for tissue culture use
F. Miscellaneous small items
IV. Washing re-usable tissue culture equipment
1. Soaking
residual medium must be removed
Inadequate wash may introduce toxic products into the new the new solution
dried on medium will be difficult to removed
after soaking, glass ware must be
a. rinse with cold tap waterb. remove all tap waterc. remove all markers
2. washing
Use detergent or acid
Machine or hand wash
Rinse with R.O. water after wash
3.washing pipette
Soak in the plastic cylinder
Wash with sonicator or pipette washer
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