Kuliah 7, Equipment and Biosafety in Cell Culture laboratory

8/7/2019 Kuliah 7, Equipment and Biosafety in Cell Culture laboratory

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Equipment and

Biosafety

in

Cell Culture Laboratory


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` Requirement

- Essential

- Beneficial- Useful


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` Laminar flow hood

Usually, one hood is suffient for two or three

people.

Horizontal flow hood

Cheaper and provide the best sterile protection

for cell culture


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Suitable only for preparing medium, other sterilereagents and culturing nonprimate cells

Potentially hazardous materials, a Class II or Class IIIbiohazard cabinet should be used

Important to familiarize yourself with local and nationalbiohazard regulations before installing equipment.


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` CO2 Incubator

Double chamber is preferable to one large

incubator it can accommodate more cultures with

better temperature control.

Many incubator have a heated water jacket to

distribute heat evenly around cabinet, thus

avoiding the formation of cold spot


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` More expensive, but their ease of use and

superiors control of temperature and CO2 tension.

` Determine the concentration of CO2, and injectspure CO2 into the incubator to make up any

deficiency.


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` Sterilizer

The simplest and cheapest sterilizer is a domestic

pressure cooker that generates 100 kpa above

ambient.

Bencth top autoclave give automatic programming

and safety locking.

Leave sufficient space around the sterilizer formaintenance and ventilation


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` Refrigerator and Freezers

Domestic refrigerator or freezer is quite efficient

and cheaper than special laboratory equipment.

Cold Room is easier to access more economical

in terms of space than are several separate

refrigerators


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` Inverted microscope

Large enough to accommodate culture dish

Invest in microscopes with a high quality optics,

long working distance phase contrast condenseand objective, with provisions for a camera.


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` Washing UpSoaking baths or sinks should be deep enough so thatall your glassware can be totally immersed indetergent during soaking.

` Water Purifications

Required for rinsing glassware, dissolving powderedmedia, and diluting concentrates.

Deionized or reverse water, ultrapure water.


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` Centrifuge

Periodiccaly, cell suspensions require

centrifugations to increase the concentration of

cells or to wash off reagent.

Small bench top centrifuge, preferably with

proporsionally controlled braking is suffient for

most purposes.


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` Cryostorage Container

- The choice of freezer is determined by

three factors:

a. Capacityb. Economy and static holding time

c. Convinience of access of contents

- Liquid Nitrogen Tank


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` Balance

` Hemocytometer

It is essential to have some means of countingcells.

Hemocytometer slide is the cheapest option and

has the added benefit of allowing cell viability tobe determined by dye exclusion


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` Cell counter

` Aspiration pump

` pH meter

` Conductivity Meter

` Osmometer

` Upright microscope

`

Dissecting microscope


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` Temperature Recording

` Magnetic Stirrer

` RollerRacks

` Fluid handling

- Removal of fluid

- Nonsterile dispensing

- Simple pippeting

- Pipettors


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` Low temperature Freezer

` Glassware washing Machine

` Video Camera and Monitor

` Colony Counter` Controlled Rate Freezer

` Centrifugal Elutriator

` Flow cytometer


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` Pippetes

` Culture Vessels

` Sterile Containers` Syringes and Needles

` Sterilization Filters


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` Contamination by microorganism remains a major

problem in tissue culture

bacteria, mycoplasma, yeast, and fungal spores

` Contaminations can be minor and confined to one

or two cultures and infect a whole experiment, can

be widespread and wipe out your entire stock


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` Mycoplasmal infection, invisible under regular

microscopy, presents one of the major threats

` Undetected, it can spread to other cultures aroundthe laboratory.

` Essential to back up visual checks with

mycoplasma test


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` Quite Area

` Work Surface

` Personal Hygiene` Reagents and media

` Culture


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` Swabbing

` Capping

` Flaming` Handling Bottles and Flasks

` Pippeting

` Pouring


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` Horizontal,

- the airflow blows from theside facing you, paralel to

the work surface, notrecirculated

- most stable airflow andbest sterile protection to the

culture and reagents


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` Vertical,

- air blows down from

the top of the hood onto

work surface and isdrawn through the work

surface and either

recirculated or vented

- more protection to the

operator


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` Class II vertical flow biohazard hood should be

used if potentially hazardous material is beinghandled

human or primate derived cultures,

virally infected cultures, etc

` HEPA filters above the work surface should be

monitored for airflow and holes

` UV lights are used to sterilize the air and expose

work surfaces in laminar-flow hoods between

uses.


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` BSC Class I is

designed to protect

the product

` BSC Class II (Types

A, B1, B2, and B3) is

designed to protect

personnel, productand enviroment.


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` Cell culture laboratory has a number of particular

risks associated with culture work.

` Risk Assesssment is an important principle that

has become incoporated into most modern safetylegislation.

` Standard Operating Procedures (SOP)

if the procedure is deemed to carry any significant risk

beyond the commonplace then a SOP should be defined


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` Safety regulation

- Occupational Health and Safety

Administration (OSHA)

- Biosafety in Microbiological andBiomedical Laboratories (BMBL), CDC.


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` General Safety Operator

It is responsibility of the institution to provide the

correct training.

Determine that the individual is already trained

Equipment

A general supervisor should be appointed to be in

charge of all equipment maintenance.


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Glassware and sharp items

The common form of injury in tissue culture resultsfrom accidental handling of broken glass and syringesneedles

Chemical toxicity

DMSO, powerful solvent and skin penetrant, carrymany substances through the skin.

Mutagens, carcinogens, cytotoxic drugs

Gases

CO2, O2, N2 are not harmful in small amounts


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Liquid Nitrogen

Three major risks are associated with liquid nitrogen:

frostbite, asphyxiation and explosion

Fire

Associated with tissue culture stem from the use bunsen

burners for flaming, together with alcohol for swabbing

and sterilization.


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` Radiation

- IngestionRadiolabeled compounds can be ingested by

being splashed on the hands or via aerosol

generated by pipetting or the use of syringe

- Labeled reagents

Irradiation from higher energy and

emmiters (32P, 125I, 131I, 51Cr)

- High energy sources

x rays macchines, UV sources


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` Biohazards

- Human biopsy material- Genetic Manipulation

- Disposal

` Sample should be handled with caution- Transport specimen in double wrap container

- Enter all specimens into logbook on receipt, and

place the specimens in a secure refrigerator

marked with biohazard label- Carry out dissection and subsequent culture work

in a designated Class II biohazard hood.


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` Enables the professional (biosafety officer,

responsible official) to:

- Become familiar with proposed work activities(procedures, equipment, personnel)

- Be knowledgeable and credible partner with the

investigator to develop a safe and secure

environment for the work


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` Review all activities associated with infectious

materials

- Proposed work activities

- Personnel- Storage

- Transfer and transport

- Destruction


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` Safety risk group (RG)RG 1 :No or low individual and community riskUnlike to cause human or animal diseases

RG 2 :Moderate individual risk, low community riskCan cause diseases but unlikely to be a serioushazard.Lab exposure may cause serious infection, but

effective treatment and preventive measure areavailable and risk of spread of infection is limited.


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` RG 3 :

High individual risk, low community risk,

usually causes serious human or animal diseasesbut does not ordinarily spread. Effective treatmentand preventive measures are available

RG 4 :

High individual and community risk, Usually causesserious human or animal diseases and can be

readily transmitted. Effective treatment andpreventive measure are not usually available

Kuliah 7, Equipment and Biosafety in Cell Culture laboratory

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