Archive for May, 2014

Laminar Air Flow and Plant Tissue Culture

May 17th, 2014

Tissue culture is a biological method of asexual propagation where a very small piece of tissue (shoot apex, leaf section, or even an individual cell) is excised (which is known as Explants) and placed in sterile /aseptic culture in a test tube/petri dish/tissue culture container containing a special culture media.

A tissue culture is used for rapid multiplication of an organism. A very specialized laboratory with sterile/aseptic conditions is required for tissue culture. All the procedure involved in tissue culture should be carried out in a special ventilated cabinet and these conditions are provided by Laminar Air Flow.

The culture medium is a proper mixture of the nutrients (organic and inorganic), vitamins, sugars, minerals and plant hormones especially Cytokinin and Auxin for shoot and root initiation.

Steps involved in Tissue Culture are

  • Explants ; excision of the plant parts.
  • Place the explants in a tissue culture container/test tubes/petri dishes.
  • Multiplication ; Tissue grows and produce small plants.
  • Rapid multiplication by transfer of fresh culture.
  • Transplanting the plant into some kind of acclimation container.

Laminar Air Flow provides a work area with aseptic/sterile conditions for the tissue culture. Laminar Air Flow has continuous displacement of air (it provides streamline flow of air) that passes through HEPA (High Efficiency Particulate Air) filter that removes the particulates from the air.

Laminar Air Flow are equipped with a UV lamp that should be turned on about 10-20 minutes before being used to sterilize the shell or cabinet or the surface of the Laminar Air Flow to avoid any kind of contaminations. Wipe down the surface with ethanol before and after each use.

Laminar Air Flow can be vertical and horizontal. In the Vertical Laminar Air Flow the air blows down from the top of the cabinet. Vertical Laminar Air Flow is best for working with hazardous specimens since the aerosols that are generated in the cabinet are filtered out before they are released into the surrounding environments.

Horizontal Laminar Air Flow are designed such that the air flows directly at the operator hence they are not useful for working with hazardous specimens but are best protection for Tissue/cell culture.

Laminar Air Flow provides aseptic/sterile conditions for the preparation of Culture Media. All the culture vessels, test tubes, pipette, tip boxes, stocks of sterile eppendorfs should be opened only in the Laminar Air Flow to avoid any kind of contaminations. Culture Media cannot be prepared outside the Laminar Air Flow because it can get contaminated and there would not be any result.

What is Laminar Hood/Laminar Air Flow?

May 17th, 2014

Laminar Hood sometimes also known as Laminar Air Flow is an enclosed bench designed to prevent contaminations like biological particles (SPM) or any particle sensitive device. This closed cabinet is usually made up of stainless steel without any gap or joints where spores might collect.

Air is drawn through a High Efficiency Particulate Air (HEPA) filter and blown in a very smooth flow or streamline flow (when a fluid flows in a parallel layer with no disruption between the layers)

Laminar Hoods are equipped with a shortwave ultraviolet germicidal lamp to sterilize the shell or cabinet or the surface of the Hood. The Hoods should be turned on about 10-20monutes before being used to kill the germs. It is important to switch off this light during use as it may cause mutations and may give any exposed skin sunburn and may cause cataracts too. It is required to wipe down the cabinet surface with ethanol before and after use and it’s also required to keep the hood free of clutter as possible because this will interfere with the laminar flow air pattern.

Components of the Laminar Hood are

  • A Blower
  • High Efficiency performance Air filter
  • A Plenum Chamber (Pressurized housing containing air at positive pressure)

Why you need Laminar Hood?

  • It provides clean air to the working area.
  • It provides a constant flow of air out of the work area to prevent room air from entering.
  • The air that flows out from the Hood, removes contaminants introduced into the work area by personnel.

The most important  part of a laminar flow hood  is a High Efficiency Particulate Air filter (HEPA). Room air is taken into the unit and passed through a pre-filter to remove gross contaminants (lint, dust etc). The air is then compressed and channeled up behind and through the HEPA filter in a laminar flow fashion. The HEPA filter removes nearly all of the bacteria from the air.

What Safety Precautions should be taken for the Laminar Hood?

  • Large objects should never be placed near the back of the hood. These objects contaminate everything downstream and disrupt the laminar flow pattern of air too, which normally suspends the contaminants and removes them from the area.
  • Waste and other items should never enter the hood. All calculations should be done before entering the hood. And clutter should be less.
  • Hands should be cleaned by ethanol.Do not touch your hair, face or clothing while working.
  • Excess dust should be removed from items before introducing them into the hood.

Items for Autoclaving

May 16th, 2014

Autoclave uses moist heat sterilization for sterilizing and decontaminating the equipments. Autoclave sterilizes the equipments by subjecting them to high pressure saturated steam at 121 degree C and 15 pounds of pressure per square inch depending for around 15-20 minutes on the size of the load and the contents.

As autoclave works by allowing steam to enter and maintaining extremely high pressure for at least 15 minutes therefore heat labile products cannot be sterilized using the autoclave such as plastics etc. Some materials present specific hazards when autoclaved such as production of toxic or noxious gases. There is a limit for the materials which can be autoclaved and which cannot. (As stated earlier heat labile articles cannot be autoclaved).

Articles that can be autoclaved are listed below;

  • Glass wares such as beakers, conical flasks glass rods and knifes.
  • Cultures and stocks of infectious material.
  • Culture dishes and related devices.
  • Culture media (Luria agar and Luria broth).
  • Discarded live and attenuated vaccines.
  • Contaminated solid items such as: Petri dishes, Eppendorf, pipette tips, pipettes, gloves, paper towel.

Articles that cannot be autoclaved are listed below;

  • Heat labile products
  • Sealed articles
  • Radioactive elements
  • Volatile articles
  • Chlorinated compounds
  • Corrosive chemicals
  • Articles contaminated with chemotherapeutic agents
  • Some plastics
  • Articles that can be denatured under the steam.