Preparation of laboratory ware

 

Preparatory methods include: cleaning using physical, chemical and biological methods

GLASSWARE PREPARATION

 

Steps of cleaning glassware: Courtesy of Labpedia

·         Decontaminate the glassware by presoaking in 5% bleach or by boiling.

·         Can use any detergent or cleaning powder.

Detergent should meet the following conditions:

Ø  It can soften the local water supply.

Ø  It should be able to remove organic material at a temperature of 60 °C.

Ø  It should have a neutral pH after rinsing with water.

Ø  Glassware should be free of the microbiological organism after the following rinsing.

 

·         Autoclave may be the alternate method.

·         If the glassware is soaked in water after use, that is ideal. A wire brush may be used to scrub off any dirt.

·         If leftover, then keeps in the detergent solution overnight.

·         Now rinse with tap water, followed by a rinse with deionized water.

 

Chemical methods of cleaning

The following chemicals are used in cleaning followed by rinsing with deionized water:

·         Water Soluble Solutions, e.g., sodium chloride or sucrose solutions.

·         Water Insoluble Solutions. e.g., solutions in hexane or chloroform.

·         Strong Acids. e.g., concentrated hydrochloric acid or sulfuric acid (Wash under the fume hood).

·         Strong Bases, e.g., 6M sodium hydroxide or concentrated ammonium hydroxide. (Wash under the fume hood).

·         Weak Acids, e.g., acetic acid solutions or dilutions of strong acids such as 0.1M or 1M hydrochloric acid or sulfuric acid.

·         Weak Bases, e.g., 0.1M and 1M sodium hydroxide

Common chemical disinfectants used are: Chlorine-releasing chemicals e.g. hypochlorite, aldehydes (formaldehyde and glutaraldehyde), ethanol or propanol is 70 to 80% V/V and phenols (hycolin, clearsol, stericol, and printol).

In the case of new glassware:

·         Boil the glassware in a detergent solution, which will cause the lysis of the organism.

·         Cool and again wash thoroughly in tap water and followed by the distilled water.

·         Dry in a hot air oven.

·         Sterilize by autoclave

 

Test to check the effectiveness of cleaning glassware:

·         Observe the glass surface as the final rinse water drains off.

·         The water should move with a sheeting action, leaving a thin film over the whole surface.

·         If the film breaks up into droplets or the surface is unevenly wet, it means the piece is not clean

 

Drying Glassware

It is done immediately after washing; it has three methods:

·         Rinse it 2-3 times with acetone. This will remove any water and will evaporate quickly.

·         Dry in hot air oven

·         Air drying

·         Glassware is rinsed with a water-miscible organic solvent and then exposed to a stream of air or nitrogen

 

 

 

PLASTICWARE PREPARATION

Procedure to clean the plastic ware:

·         There should be well cleaned and then rinse with deionized water before sterilization.

·         Polystyrene, polyvinyl chloride, styrene-acrylonitrile, and polyethylene are not auto-clavable, so these may be sterilized with gas (ethylene oxide).

·         Except for the Teflon, no other plastic ware should hot-air sterilized because of oxidative degradation.

·         Plastic ware may be dried in an oven at 110 °C.

**Plastics such as Polypropylene, Teflon, and polymethyl pentene may be repeatedly autoclaved under normal conditions. Polycarbonate should be autoclaved at 121 °C for 20 minutes.

 

STERILISATION

Sterilization refers the complete removal or elimination of microorganisms from an environment.

The heat, irradiation and filtration are the three physical agents and various chemicals are as chemical agents available for sterilization.

Terms

Cidal means to kill the microorganism

Static means inactivation or inhibition, but not killing. They prevent multiplication of the organism.

Sepsis refers break down of a living tissue by organisms and is accompanied by inflammation and pus formation.

1. Heat

At high temperature, all the macro molecules lose their structure and ability to function called as denaturation. Similarly, the heat coagulates the protein and oxidizes the elements lead for lethality.

·         Direct flaming: showing the objects or equipment directly to the flame leads the sterilization. This is also called as incineration. The inoculation needles, spread rods, forceps can be sterilized using this technique.

·         Boiling: The liquids boiling also possible. In this method, only the vegetative forms are killed in few minutes whereas the spores are not.

·         Dry heat: Causes oxidation of cells. Normally high temperature and longtime are required for complete sterilization using this method. 160-180°C for 2- 3 hours are required for complete sterilization. Example: use of hot air oven for sterilizing glass wares e.g. conical flasks, beakers, petri dishes, pipettes can be sterilized using this technique.

·         Moist heat: Causes denaturation and coagulation of protein. To kill vegetative structure of bacteria, yeast and molds - 80°C for 5-10 min; To kill mold spores - 80°C for 30 min; bacterial spores - 121°C, 15 lbs/sq.inch pressure for 15 min. are the optimum moist heat.

2. Filtration

Membrane filters such as Millipore filters. Filters with a pore of 0.22 micrometers are sufficient for the bacteria.

3. Radiation in the form of:

·         Ultraviolet light causes damage to bacteria.

·         Radiation in the form of beta and gamma X Rays is used for surgical pads.

·         Supersonic and ultrasonic waves, 9000 cycles per second or above, are used to rupture and disintegrate the cells

4. Chemical agents:

The following are different classes of chemicals involved in sterilization.

·         Phenol and Phenolics - 5% aqueous solutions kill vegetative forms of microorganisms. Some of their derivatives are lysol, dettol, cresol etc.

·         Alcohols - They are bactericidal and fungicidal but not sporicidal. They denature proteins and are solvents of lipids.

·         Halogens - Cause oxidation and direct halogenation of proteins thus inhibiting the activity of proteins e.g. hypochlorite, iodophores etc.

·         Surfactants - Surface active agents, good wetting and solubilizing agents e.g. Soap, detergent etc.

·         Alkylating agents - They substitute alkyl groups for hydrogen of reactive groups in nucleic acids and proteins, causing disruption of metabolic pathways e.g. Formaldehyde, glutaraldehyde, ethylene oxide (gaseous agent)

·         Heavy metals - React with sulfhydryl group (SH) of enzyme and make them inactive. Mercury, copper and silver are commonly used heavy metals.

·         Antibiotics - Antibiotics are low molecular weight molecules produced as secondary metabolites by microorganisms, which kill or inhibit the other microorganisms.