Laboratory:- Part 7 – Laboratory Glassware cleaning and Sterilization

1. In any laboratory, the proper cleaning of the glassware is the key to get the correct result.
2. The best test to see the cleanliness of the glassware is:
   1. Observe the glass surface as the final rinse water drains off.
   2. The water should move with a sheeting action, leaving a thin film over the whole surface.
   3. If the film breaks up into droplets or the surface is unevenly wet, it means the piece is not clean.
   4. This process can be made easy if:
      1. Empty all the glassware after the tests.
      2. Rinse with water.
      3. Soak in the solution of detergent.
         

         Glassware washing summary (Easy way to clean the glassware)

   5. For the sensitive tests, use the disposable test tubes.
      

      Laboratory glassware types

Glassware falls into the following categories:

The thermal properties of the glassware are changed by changing the ratio of boron oxide (B2O3). Glassware containing Boron oxide is called borosilicate glass. These are the most commonly used in the laboratory.

Advantages of the borosilicate glass:

1. These are free from Zinc group elements.
2. These are free from heavy metals like arsenic and antimony.
3. These are resistant to heat and thermal shock.
4. There are resistant to corrosion.
5. Their size changes very little on heating, so they are preferred when heating or sterilizing heat.

Another type is called soft glass:

1. These are boron free glassware and also called soft glass.
2. Advantages are:
   1. These have high resistance to alkali.
   2. These are used for a strongly alkaline solution.
   3. Their common use is in solutions or digestion involving the strong alkali.
3. Disadvantage:
   1. Its thermal resistance is much less as compared to borosilicate glass.

Types of laboratory glassware:

1. Pyrex consists of borosilicate.
   1. Borosilicate glass is free of zinc and heavy metals.
   2. These resist heat, corrosion, and thermal damage.
   3. These are very good for heating and sterilization.
2. Corex consists of aluminosilicate.
   1. These are made strong chemically rather than by heat.
   2. These are 6 times stronger than borosilicate glass.
   3. These are resistant to alkali and scratching.
   4. This glass can withstand high heat >250 °C.
   5. Examples are cylinders and centrifuge tubes.
3. Vycor is also called corning brand glass.
   1. This has high silica and is acid and alkali resistant.
   2. These can withstand high temperatures (heat shock).
   3. It can withstand extremes of acids and alkali.
   4. These can be heated to 900 °C.
   5. These can withstand heat shock from 900 C to 0 °C.
4. The low actinic glass is amber-colored.
   1. These are amber or red color glass due to the presence of some chemicals in them.
   2. Protects the serum from light.
   3. Used to handle bilirubin, carotene, and vitamin A. These are sensitive to the light when it is in the range of 300 to 500 nm.
5. Flint glass has a high index of refraction.
   1. These are soda-lime glass consisting of silicon, calcium, and sodium oxide.
   2. These are the cheapest of all.
   3. These are not resistant to high temperatures.
   4. It cannot tolerate heat shock.
   5. Resistant to the chemical is not strong.
   6. This glass is easy to melt and shape. These are used to make bottles and some disposable laboratory glassware.
   7. This glassware must be rinsed with water when used the first time.

• The most important step is to keep the glassware after use in a 3% Lysol(or any other disinfectant) solution for disinfection.
• For suspicion of Tuberculosis, use 1% sodium hypochlorite. This can also kill the HB virus.

Options to clean the glassware:

1. In most of the big labs, glassware is washed in three steps:
   1. Automatic washer.
      1. Followed by a special rinsing cycle.
      2. Then keep in the automatic dryer (below 100 °C).
      3. Or rinse the glassware with a water-miscible organic solvent and then exposing to a stream of air or nitrogen.
2. The most common use is the chemicals.
3. The most common method to clean glassware:
   1. Decontaminate the glassware by presoaking in 5% bleach or by boiling.
      1. Any detergent or cleaning powder can be used.
         1. Autoclave may be the alternate method.
      2. If the glassware is soaked in water after the use, that is ideal.
      3. If leftover, then keeps in the detergent solution overnight.
      4. Now rinse with tap water, followed by a rinse with deionized water.
   2. The most common disinfectant are:
      1. 1. Chlorine-releasing chemicals where chlorine is active against gram-positive and negative bacteria, including HIV and HB viruses.
            1. Examples are hypochlorite (bleach solution),  used in domestic and laundry.
         2. Aldehydes are formaldehyde and Glutaraldehyde.
         3. Alcohol used in ethanol or propanol 70 to 80% V/V.
         4. Phenols like hycolin, Clearsol, Stericol, and Printol.
   3. In the case of new glassware:
      1. Boil the glassware in a detergent solution, which will cause the lysis of the organism.
      2. Cool and again wash thoroughly in tap water and followed by the distle water.
      3. Dry in a hot air oven.
      4. Sterilize by autoclave at 15 lbs for 20 minutes.
   4. In the case of hand wash:
      1. The detergents must be nonionic, metal-free, and not highly alkaline.
      2. Also, ensure adequate rinsing.

Test to check the cleaning of glassware

1. Check the final rinse water, which should move with a  sheeting action, leaving a thin film over the surface.
2. If the film breaks up into droplets, or the surface is unevenly wet, then it indicates that the glassware is not clean.

We will discuss first the chemical options:

Cleaning Basics steps are:

1. It’s generally easier to clean glassware if you do it right away.
2. When detergent is used, it can use commercially available as Liquinox or Alconox.
   1. The detergent should meet the following criteria:
      1. It can soften the local water supply.
      2. It should be able to remove organic material at a temperature of 60 °C.
      3. It should have a neutral pH after rinsing with water.
      4. Glassware should be free of the microbiological organism after the following rinsing.
3. Much of the time, detergent and tap water is neither required nor desirable.
   1. You can rinse the glassware with the proper solvent.
   2. Then finish up with a couple of rinses with distilled water.
   3. A final rinse follows this with deionized water.

 Common Lab Chemicals used for Glassware are:

1. Water Soluble Solutions, e.g., sodium chloride or sucrose solutions.
   1. Rinse 3-4 times with deionized water.
2. Water Insoluble Solutions. e.g., solutions in hexane or chloroform. Rinse 2-3 times with ethanol or acetone.
   1. Rinse 3-4 times with deionized water,
3. Strong Acids. e.g., concentrated HCl or H2SO4. Wash Under the fume hood.
   1. Carefully rinse the glassware with copious volumes of tap water.
   2. Rinse 3-4 times with deionized water. Then let it dry.
4. Strong Bases, e.g., 6M NaOH or concentrated NH4OH. Wash Under the fume hood.
   1. Carefully rinse the glassware with copious volumes of tap water.
   2. Rinse 3-4 times with deionized water, then let it dry.
5. Weak Acids, e.g., acetic acid solutions or dilutions of strong acids such as 0.1M or 1M HCl or H2SO4.
   1. Rinse 3-4 times with deionized water and then let it dry.
6. Weak Bases, e.g., 0.1M and 1M NaOH and NH4OH. Rinse thoroughly with tap water to remove the base.
   1. Then rinse 3-4 times with deionized water and let it dry.

Washing Special Glassware used for Organic Chemistry:

1. 1. Rinse the glassware with the appropriate solvent.
   2. Use deionized water for water-soluble contents.
   3. Use ethanol for ethanol-soluble contents, followed by rinses in deionized water.
   4. Rinse with other solvents as needed, followed by ethanol, and finally deionized water.
   5. If the glassware requires scrubbing, scrub with a brush using hot soapy water, rinse thoroughly with tap water, followed by rinses with deionized water

Washing with soap and water:

1. Soak the glassware in soap solution for at least 10 to 15 minutes or leave overnight.
2. Scrub with brush or cloth or sponge if needed.
3. Rinse thoroughly with tap water.
4. Again rinse with distilled or deionized water.
5. If you need this glassware soon, then rinse with acetone or ethanol.

Burets (Cleaning)

1. Wash with hot soapy water, rinse thoroughly with tap water.
2. Then rinse 3-4 times with deionized water.
3. Burets need to be thoroughly clean to be used for quantitative lab work.

Pipettes and Volumetric Flasks (cleaning)

1. May need to soak the glassware overnight in soapy water.
2. Clean pipette and volumetric flasks using warm soapy water.
3. The glassware may require scrubbing with a brush.
4. Rinse with tap water followed by 3 – 4 rinses with deionized water.

Drying Glassware

1. If glassware is to be used immediately after washing and must be dry.
2. Rinse it 2-3 times with acetone. This will remove any water and will evaporate quickly.
3. This is not a great idea to blow air into glassware to dry it.
4. Acetone may be used for a final rinse of sensitive or urgently needed glassware. The solvent is miscible with water and helps dilute and wash away the remaining water from the glassware.

Wash labware as quickly as possible after use:

1. If a thorough cleaning is not possible immediately, put glassware to soak in water.
2. If labware is not cleaned immediately, it may become impossible to remove the residue.
3. Put into acid water (a 1% solution of hydrochloric or nitric acid) before washing. Can keep for several hours in this solution.
4. Brushes with wooden or plastic handles are recommended as they will not scratch or abrade the glass surface.

Chromic acid use:

1. If glassware becomes unduly clouded or dirty or contains coagulated organic matter, it must be cleaned with chromic acid.
2. When the chromic acid solution is used, the item may be rinsed with the cleaning solution or filled and allowed to stand.
3. Time depends on the amount of contamination on the glassware. Relatively clean glassware may require only a few minutes.
4. If more contaminated, then keep overnight.
5. Special types of precipitates may require removal with nitric acid or sulfuric acid. No doubt, these are very corrosive.

Hematological glassware needs special precautions:

1. Do not use detergents because if there is a minute concentration, that may lead to RBCs’ hemolysis.
2. So for general Tubes, pipettes, and slides, wash these thoroughly under tap water. Can use a brush to remove any leftover from the glassware.
3. Keep the hematology used material in a dichromate solution for 12 to 24 hours. Then again, wash thoroughly with tap water.
4. Allow draining.
5. Dry in the hot oven.

Blood pipettes need the following method to clean:

1. With the help of a suction pump or handheld suction pump, draw tap water through these pipettes.
2. Use distilled water for suction and washing.
   1. Last can use acetone for the same purpose.
3. Let them dry in the air.
4. If there are blood microclots, keep them in 10% potassium hydroxide for 12 to 24 hours.

Conclusion  and Important facts:

1. Glassware should be cleaned as soon as possible.
2. In the case of delay, put the glassware in water.
3. In the case of late cleaning, the residue may not be possible to remove.
4. New glassware that is slightly alkaline needs to be soaked in acid water (1% HCl or HNO3) for several hours before washing.

Plastic labware

1. Whenever possible, use plasticware instead of glassware.
   1. Advantage:
      1. Plasticware is unbreakable.
      2. It does not release ions like glassware.
   2. Disadvantage:
      1. It tends to bind various solutes and leads to the surface-bound material into the subsequent solution.
      2. Polyethylene is permeable to water vapor, even in tightly stoppered bottles. This will leads to the concentration of the reagents.
      3. Polyethylene is not inert and may bind or absorbs the proteins, dyes, iodine, stains, and picric acid.
2. Types of plasticware:
   1. Polyethylene.
      1. Disadvantages:
         1. It is permeable to water vapors. This will leads to the concentration of the reagents and calibrators.
         2. It is not completely inert, and it can absorb proteins, dyes, stains, iodine, and picric acid.
   2. Polypropylene.
      1. Advantage:
         1. It can withstand high temperatures.
         2. It can be sterilized.
      2. Disadvantage:
         1. It absorbs pigments and tends to become discolored.

Sterilization

• The following methods can do the sterilization:

1. Dry Heat sterilization has limited value. Prolonged exposure may cause damage.
2. Hot air oven where heat is transferred by convection, conduction, or radiation.
   1. The temperature of 100 °C for one hour can destroy the nonsporing organism. Fungal spores need 115 °C for one hour.
   2. While other bacteria at 160 °C temperature is needed for one hour.
3. Incineration where the flame is an effective way of sterilization. Flame heat is needed for the loop for culture.
4. Moist heat is the most reliable method of sterilization. This is the most lethal agent to kill microorganisms.
   1. Microbial death is due to coagulation and denaturation of the protein and enzyme.
5. Boiling is not effective in killing spore-bearing bacteria and for surgical instruments.
6. Steam sterilization or Tyndallization is exposure to steam at 100 °C for 90 minutes. This good means to sterile the media which contain sugar.
7. The autoclave is heating water under pressure, which boils at progressively higher temperatures. This method is good for rubber material and surgical instruments.
8. Membrane filters are Millipore filters. Filters with a pore of 0.22 micrometers are sufficient for the bacteria.
9.  Seitz filter is a disposable asbestos pad filter.
10. Flaming when the material is wetted by alcohol and then flamed. This method is rapid.
11. Ultraviolet light causes damage to bacteria.
12. Radiation in the form of beta and gamma X Rays used for surgical pads.
13. Supersonic and ultrasonic waves, 9000 cycles per second or above, are used to rupture and disintegrate the cells.

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