Lecture 3

Question 1

Why is it important to have a dedicated culture lab?

Answer 1

contamination risk, people walking by frequently, other lab materials can be an issue (like bacteria used in the lab)

Question 2

describe air flow considerations in a cell culture lab

Answer 2

positive pressure is ideal. air pushes out of the room so no contamination comes in.
negative pressure is ideal for containment. air sucks in so hazards are kept inside the room

Question 3

requirements for lab areas

Answer 3

1. sterile handling areas: if no hood. no through traffic, only use tissues in the area
2. Laminar flow hoods/biosafety cabinets: greater sterility
3. service bench: store required equipment
4. quarantine and containment: for testing if new cultures are clean. negative air pressure

Question 4

Incubation requirements

Answer 4

clean air, low disturbance, specific temperature and gas phase composition for the cell type. can use a warm room or incubator (better)
warm room: for larger labs, convenient shelves, watch out for yeast/bacteria and avoid constant light (for media)
incubator: better for small labs, loses more heat, can control CO2

Question 5

preparation area requirements

Answer 5

media preparation: not very common or economical. has balances, pH meters, bottling, filters, etc
wash up area: minimal if using plastics, autoclaves and ovens, deep sinks
storage: sterile for liquids, glassware, disposables, and non sterile for gloves, etc. Liquid N2 and CO2 tanks (need adequate ventilation

Question 6

Types of laminar flow hoods and limitations

Answer 6

horizontal: air flows out of hood towards you, protects products and not you! good for preping medium (w/out antibiotics), nontoxic sterile reagents, culturing non human/primate cells, dissecting non human/primate cells
vertical: air flows down and circulates, protects products and you! use for potentially hazardous materials like human materials and cells, infections materials, radioisotopes, and toxic chemicals

Question 7

BSC class II vs class III

Answer 7

class II is the normal one and class III has the funny gloves in the glass and prevents ALL contact

Question 8

Things used for sterile liquid handling

Answer 8

pipettes (glass vs plastic), Pipette Controllers, micropipettors (not for routine culture), peristaltic pump (for large volumes), syringes (for filter sterilizing), automation (for large scale production)
determine choice by ease of use, cost vs efficiency, sterilization, safety, accuracy, reproducibility

Question 9

types of microscopes

Answer 9

Inverted microscope (essential!) for phase contrast, fluorescence. can have camera/monitor attached
dissecting microscope

Question 10

cell counting essentials

Answer 10

hemocytometer: simplest, cheapest, good viability

electronic counters: good for many cell lines, can be more accurate (sometimes)

Question 11

incubation essentials

Answer 11

Dry incubator: tight temp control, heated water jacket
Humid CO2 incubator: same as dry but can also control atmosphere humidity and CO2 concentration and O2 concentration, added water tray for humidity, must be cleaned often

Question 12

prep of media tools

Answer 12

water purifiers: water is simplest but most crucial reagent! purified water is needed for rinsing, dissolving, and diluting

Question 13

sterilization tools

Answer 13

sterilizing oven: dry heat sterilization
stem sterilization (autoclave): pressure cooker, weed and dry cycle
sterilization filters

Question 14

list the ESSENTIAL lab equipment

Answer 14

laminar flow hood
incubator
CO2 cylinders
balance 
autoclave
fridge and freezer
inverted microscope
washing sink
water purifier
water bath
centrifuge
liquid nitrogen freezer/storage
hemocytometer