Interdisciplinary Nanotechnology Centre

Need of SOPs in laboratory work

Standard operating procedures in a laboratory are needed for following reasons

1. To improve and maintain the quality of laboratory service and identify problems associated with poor work performance

2. To provide laboratory staff with written instructions on how to perform test consistently to an "acceptable standard" in the laboratory

3. To provide written standardized techniques for use in the training of laboratory personnel

4. To facilitate the preparation of a list of essential reagents, chemicals and equipment’s

5. To promote safe laboratory practice

How to safely perform work involving hazardous materials or hazardous operationsin chemical and microbiology synthesisof Nanoparticles

SOP focus on

A. Process (e.g., Nanoparticles synthesis, distillation, Instruments handling)

B. Hazardous Chemicals and Solvents

C. Safety in Lab

D. Laboratory Equipment

E. Cleaning &Disinfection

F. Sterilization

G. Waste Management

A. Process

Before proceeding, go through description of the process, directions for reaction set-up, general procedures for eliminating or minimizing risks and emergency response for “what-if” cases.Find the process or type of process involving hazardous chemicals. For operating sophisticated instruments like XRD, UV-Vis-NIR etc, must follow the SOP of the particular instrument. For nanoparticle synthesis or Chemical process or Reaction Set-up consider the following

• A literature search

• Seek help from peers in your laboratory or from other groups in the department who arewell versed in the experimental set-up and procedures.

• Review the experiment set-up, use of proper glassware set-up or a pressure vessel,heating, and cooling system.

• List the circumstances under which a particular laboratory operation, procedure, activity requires prior approval from the Principal Investigator (PI), laboratory supervisor, or other personnel.

B.Hazardous Chemicals and Solvents

Before reaction, find out limitations on the quantities and types of materials to be used. List the hazardous chemicals (or class of chemicals) involved, including any hazardous products or by-products. Safety Data Sheets (SDSs) for all chemicals are readily accessible online. MSDSs for most chemicals are available through the chemical manufacturer.

C. Safety in Lab

Safety in the laboratory is the responsibility of all who work in it. Use latex gloves, lab coatand lab musk.

D. Laboratory equipment’s

Objectives

• To provide basic information regarding various parts of commonly used laboratory equipment’s.

• Basic information regarding use of equipment’s.

• Information regarding maintenance of laboratory equipment’s.

Method of teaching

• Lecture method

• OHP presentation

• Live demonstration

MICROSCOPE

The microscope is an essential instrument for the diagnosis of disease. Itrequires careful maintenance to prevent damage to mechanical and ocular parts and also to stop fungi from obscuring the lenses.

Centering of Condenser

1. Place a slide preparation without coverslip on the stage. Lower the condenser and open the iris diaphragm. Examine with low power objective x10. Look through the eyepiece and bring the slide into focus.

2. Close the diaphragm. A blurred circle of light surrounded by a dark ring appears in the field.

3. Raise the condenser slowly until the edges of the circle of light are in sharp focus.

4. Adjust the centering screws of the condenser so that the circle of light is in exact center of the field. Then check with other objectives.

Adjusting the diaphragm

Open the diaphragm completely. Remove the eyepiece and look down the tube,

The upper lens of the objective will be seen to be filled with a circle of light. Close the diaphragm slowly until the circle of light takes up only two-thirds of the surface. Do this for each objective as it is used.

Routine maintenance

Microscopes must be installed in well ventilated environment, away from chemicals.

Cleaning the optical surfaces- The optical surfaces (condenser, objectives, eyepieces) must be kept free of dust with a soft camel hair brush. If dust is found inside the eyepiece, unscrew the upper lens and clean the inside. Oil residue on the lenses should be removed with special lens tissue paper, absorbent paper or medical gradecotton wool. The optical surfaces may be finally cleaned with a special solution consisting of the following.

- 80% petroleum ether & 20% 2-propanol.

NOTE Do not use 95% ethanol, xylene or toluene for cleaning the lenses, since these substances dissolve the cement of lens.

Cleaning the instrument

Heavy contamination can be removed with mild soapy solutions. Grease and oil can be removed with the special cleaning solution as described above. The instrument should then be cleaned with a 1:1 mixture of distilled water and 95% ethanol. This mixture is not suitable for clearing the optical surfaces.

Precautions

• Never use ordinary paper to clean the lenses.

• Never touch the lenses with your fingers.

• Never clean the support or the stage with xylene or acetone.

• Never clean the inside lenses of the eye pieces and objectives with cloth or paper (this might remove the anti-reflective coating); use a soft camel-hair brush.

• Never keep the microscope in a closed wooden box in hot humid climate. .

• Never press the objective on to the slide, since both the slide and the objective may break.

• Keep the mechanical stage clean.

• Do not dismantle the optical components, as this may cause misalignment.

• Never carry the microscope by the limb with one hand; use both hands, one under the foot, the other holding the limb.

• When changing a bulb, avoid touching the glass with your fingers, as finger prints reduce the intensity of illumination.

• If the mains voltage fluctuates excessively, use a voltage stabilizer.

• Keep the microscope under an air tight plastic cover when not in use.

CENTRIFUGE

Principle - A centrifuge machine, by exerting a centrifugal force greater than that of gravity, is able to sediment particles suspended in a fluid. The particles are compacted at the bottom of the centrifuge tubes as a deposit.

To calculate the relative centrifugal force (RCF) for an individual centrifuge, measure the radius (r) of the rotor arm (in mm) and the number of revolutions per minute (rpm) and calculate:-

RCF= 1.12 x 10-6 x r x (rpm)².

Components of a centrifuge

A centrifuge consist of

• A central draft or spindle (A) that rotates at high speed.

• A head (e), fixed to the shaft, with buckets for holding the centrifuge tubes.

USE OF LABORATORY BALANCE

Analytical balance- This balance has two pans suspended from a crossbeam inside a glass case

Instructions for use

1. Place the balance on a firm level bench away from vibrations, draughts and direct sunlight.

2. Always ensure that the crossbeam is at rest while placing and removing the weights and the substance to be weighed on the pans.

3. Check that the pans are balanced (after closing the glass case) by unscrewing the beam release screw.

4. Always place the substance to be weighed on a piece of paper or in watch glass or porcelain dish.

5 Always use forceps to pick up weights

MICROPIPETTES

Micropipettes with disposable tips are frequently used to measure small volumes. They are available in a variety of volume, ranging from 20 μl to 200 μl and 100μl to 1000μl. The micropipettes may be of fixed volume or variable volume. Used tips are disposed of directly into disinfectant using an ejector mechanism.

E. Cleaning & Disinfection

Objectives

• To provide basic information regarding cleaning of new & reusable glassware and containers for sample collection.

• To provide information regarding commonly used disinfectants.

Method of teaching

• Lecture method

• OHP presentation

• Live demonstration

Glassware

New Glassware

Glassware that has never been used may be slightly alkaline. In order to neutralize it: use 1-2% solution of hydrochloric acid. (Add 60 ml concentrated HCL in 3 liters of water.) Immerse new glassware and leave for 24 hr. Rinse twice with ordinary water and once with demineralized water. Dry the glassware.

Dirty Glassware

• Preliminary rinsing - Rinse twice in cold or lukewarm water (Rinse glasswareused for fluids containing protein immediately before drying).

• Soak for 2-3 hrs. in a container of water mixed with washing powder or liquiddetergent. Brush the inside of glassware with a test tube brush.

• Remove articles one by one and rinse under running tap water, then soak themall in ordinary water for 30 minutes.

• Place containers (beakers, flasks, measuring cylinders) on a draining rack. Testtubes are placed upside down in a wire basket for draining.

• For drying, place the baskets either in hot air oven at 60°C or in a sunny spot andcover with a fine cloth.

• Plug the containers with non-absorbent cotton wool or cover the mouths withnewspaper.

• Place in a cupboard to protect it from dust.

Pipettes

• Once a pipette has been used, rinse it immediately in a stream of cold water toremove blood, urine, serum, reagents etc.

• Next place the pipettes in a large plastic cylinder full of water. If pipettes havebeen used for infectious material place them in disinfectant solution for 4 hrs.

• Then clean as routine glassware in detergent solution and water as above.

Blocked Pipette

• Place blocked pipettes in a cylinder filled with dichromate cleaning solution andleave for 24 hours.

• Next pour the dichromate solution into another cylinder (can be used 4 times) andrinse the pipette in the cylinder thoroughly under tap water.

• Remove the pipettes from cylinder one at a time, check thoroughly for anyobstruction and rinse again.

• Soak in ordinary water for 30 minutes, change water and soak again for 30minutes.Dry in hot air oven at 60°C or air dry.

Microorganism containing Flasks, Test tubes, Petri dishes

Autoclave method

• Place the container with specimens in the autoclave and sterilize at120° C for 30 minutes.

• After the containers have cooled, empty the contents into the sink or lavatory.

• Clean with detergent and water as in glassware.

F. Sterilization

Sterilization is the process of freeing an article from all living organisms, including bacteria and their spores.

Disinfection is the destruction of pathogenic bacteria but not necessarily of the resistant spores.

Sterilization can be done by

1)Physical methods (heat, radiation, filtration)

2)Chemical method (disinfectants)

Objectives

• To provide information regarding various equipment’s & methods used for sterilization.

• Basic information regarding preparation and procedure for sterilization.

• Precautions & quality control to be observed in sterilization procedure.

Method of teaching

• Lecture method

• Live demonstration

Autoclaving

• It provides moist heat at temperature above 100°C and at pressure greater thanatmospheric pressure.

• 
  As the steam condenses on the cooler load, it releases both thermal energy andmoisture, which together denature all microbial proteins.

• It is used for all materials that are water-containing, permeable or wettable andnot liable to be damaged by the process.

• Autoclaving is the most reliable method and most widely used method forsterilization of culture media and surgical supplies.

• It is also used for sterilization of infectious waste before discarding.

Preparation of material for autoclaving

Glassware

• 
  Specimen tubes, petri dishes etc should be wrapped in autoclavable polythenebags and tied with string.

• If autoclavable polythene bags are not available then use kraftpaper,cotton orgauze for wrapping the tubes/ petri dishes etc.

.

Sterilization procedure

• Fill the bottom of the autoclave with water but the water should not touch thebasket.

• Put the basket containing the material to be sterilized in the autoclave along withsterilization indicator papers.

• Close the lid, making sure the rubber washer is in its groove. Screw down the lidclamps evenly and firmly but not too tightly.

• Open the air outlet valve and begin heating the autoclave.

• When a jet of steam appears at the air outlet wait for 3-4 minutes till the jet ofsteam becomes uniform and continuous (shows that all air has been removed)close the air outlet valve, tighten the lid clamps and reduce the heat slightly.

• Watch the pressure gauge-when it reaches the 15 pounds mark- then regulatethe heat to maintain at this pressure.

• Start timing now when the pressure is stable at 15 pounds- this is the holdingtime.

Removing load from autoclave

• Turn off the heat after holding period is finished. When pressure gauge showsthat the autoclave is at atmospheric pressure, open the discharge tap very slowlyto allow air to enter as cooling of steam proceeds.

• Unscrew the lid clamps and take off the lid and remove the baskets of sterileequipment.

• During cooling process, sterilized items should never be placed on cold metalsurfaces as moisture will condense onto the items and contaminant them. Thesterile load should be kept in wire mesh racks or baskets till it gets cooled.

Precautions while using an autoclave

1. In the autoclave, all parts of the load to be sterilized must be permeated by steam.

2. Steam should be hot, saturated and dry (free from particles of liquid water).

3. All the air must be removed from the autoclave chamber and articles of the load, so that the load is exposed to pure steam during sterilization.

4. For sterilization, exposure of microbes to 121°C for 15 minutes at 15 pounds pressure is required.

5. Avoid autoclaving small and large volumes in the same load because with the same holding period (eg. 20 min) small amounts (eg. 10 ml) may be damaged by overheating and large amounts (eg 2000 ml) may not be sterilized.

6. Do not fill the bottle, tubes, flasks etc to more than 75-80% of their capacity as the contents overflow on expansion during heating.

7. Tubes, flasks and bottles should be closed with cotton wool stoppers, loose slip on metal caps or loosely applied screw caps.

8. There is 3-5% loss of water from media due to evaporation on cooling. So prepare media by adding 5% extra distilled water.

9. Never touch the drainage tap, outlet valve or safety valve while the autoclave is under pressure.

10. Never heat the autoclave too quickly to bring up the pressure once the outlet valve is closed.

11. Never leave the autoclave unattended while the pressure is rising.

12. Never open the lid before the pressure has dropped to normal.

13. During sterilization make sure that the lid is secured and no steam escapes.

14. Never leave the autoclave to cool for too long because if the discharge tap is not opened, vacuum is created and media gets dehydrated.

Waste management

Objectives

• To provide information regarding various categories of hospital waste.

• Basic information regarding collection, segregation & disposal of waste.

Method of teaching

• Lecture method

• OHP presentation

• Live demonstration

Collection and segregation:

Waste should be collected and segregated at the site of generation. Segregation (classification) of waste denotes separation of waste into various categories according to its nature. It aims to keep the harmful and infected waste separate from the harmless and non-infectious waste. For this purpose, specifically coloureddustbins and plastic bags are used