# An Instructional Material on Basic Concepts in Laboratory Techniques

Ajeet Kumar

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An Instructional Material

On

BASIC CONCEPTS IN LABORATORY TECHNIQUES

COURSE NO.: PGS-504

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Authored by

Dr. AJEET KUMAR

Assistant Professor-cum-Scientist (Sr. Scale)

Department of Soil Science

Sugarcane Research Institute

Dr. Rajendra Prasad Central Agricultural University
Pusa (Samastipur)-848125, Bihar, India

July-2024
# CONTENTS

**Objective of the course:** To acquaint the students about the basics of commonly used techniques in laboratory.

## Lecture outlines

<table><thead><tr><td>Sl. No.</td><td>Topic</td><td>Page No.</td></tr></thead><tbody><tr><td>1.</td><td>Laboratory Safety measures while working in Laboratory</td><td>4-7</td></tr><tr><td>2.</td><td>Handling of chemical substances and handling techniques of solutions</td><td>8-9</td></tr><tr><td>3.</td><td>Basic Laboratory Equipment- Identification and Use of burettes, pipettes, measuring cylinders, flasks, separatory funnel, condensers, micropipettes and vaccupets.</td><td>10-21</td></tr><tr><td>4.</td><td>Cleaning and drying of glassware, Methods of drying of solvents / chemicals.</td><td>22-24</td></tr><tr><td>5.</td><td>Preparation and dilution of solutions of various strengths; Preparation of different agro-chemical doses in field and pot applications</td><td>25-33</td></tr><tr><td>6.</td><td>Preparation of solutions of acids; Neutralization of acid and bases; Preparation of buffers of different strengths and pH values, Electric wiring and earthing</td><td>34-42</td></tr><tr><td>7.</td><td>Instruments Used In Soil Microbiology Laboratory - Use and handling of microscope, vacuum pumps, viscometer, thermometer, magnetic stirrer, micro-ovens, incubators, sand bath, water bath, oil bath;</td><td>43-54</td></tr><tr><td>8.</td><td>Working principle, use, different Parts their Functions and precautions of Hot Air oven and Auto clave used in soil microbiology Lab</td><td>55-61</td></tr><tr><td>9.</td><td>Preparation of Media, Types and Sterilization of Culture Media-Heat sterilization, Radiation, Chemical methods, Mechanical removal of organisms.</td><td>62-66</td></tr><tr><td>10.</td><td>Isolation, and Streaking methods Used in soil microbiology Laboratory, Compositions of different Media</td><td>67-73</td></tr><tr><td>11.</td><td>Sterilization Techniques for Glassware</td><td>74-75</td></tr></tbody></table>

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## Suggested Readings

Purr A K. 2000 CRC Hand Book of Laboratory Safety. CRC Press.

Gabb, M.H. & Latchem, W.E 1968. A handbook of Laboratory Solutions Chemical Pub. Co.
# Laboratory Safety Measures

As with any place of work, safety is an important consideration in soil-plant analysis laboratories, and one that is frequently overlooked. Safety is in the interest of the employees who work there and the organizations that operate the laboratories. All staff, irrespective of grade, technical skill or employment status should be briefed on all aspects of safety upon commencement of work. Periodic reminders of such regulations should be given to encourage familiarity with respect to regulations.

While rules pertaining to safety can be extensive, we have endeavored to concisely list the more important ones within different categories of concerns. These have been adapted from **laboratory safety guides developed by Kalra and Maynard (1991)** and Okalebo et al. (1993).

## A. General Attitude

1. Develop a positive attitude towards laboratory safety.

2. Observe normal laboratory safety practices.

3. Maintain a safe and clean work environment.

4. Avoid working alone

## B. Instrument Operation

1) Follow the safety precautions provided by the manufacturer when operating instruments.

2) Monitor instruments while they are operating.

3) Atomic Absorption Spectrophotometer must be vented to the atmosphere. Ensure that the drain trap is filled with water prior to igniting the burner.

4) Never open a centrifuge cover until machine has completely stopped

## C. Accidents

1. Learn what to do in case of emergencies (e.g., fire, chemical spill, etc.). Fire fighting equipment must be readily accessible in the event of fire. Periodic maintenance inspections must be conducted.

2. Learn **emergency First Aid**. First Aid supplies are a necessity and laboratorystaff should be well trained in their use. Replacement of expended supplies must take place in a timely fashion.
3. Seek **medical attention** immediately if affected by chemicals, and use FirstAid until medical aid is available.

4. Access to **eye-wash fountains** and safety showers must not be locked. Fountains and showers should be checked periodically for proper operation.

## D. Chemicals

1. Use **fume hoods** when handling concentrated acids, bases or other hazardous chemicals.

2. **Do not pipette by mouth;** always use a suction bulb.

3. When diluting, always **add acid to water**, not water to acid.

4. Many **metals/salts are extremely toxic** and may be fatal if swallowed. Wash hands thoroughly after handling such salts.

5. Chemical spills should be cleaned promptly.

6. All waste bins emptied regularly.

7. All reagent bottles should be clearly labeled and must include information on any particular hazard like..

* Poisonous,

* Corrosive,

* Explosive

* Inflammable substances.

## E. Muffle Furnaces, Ovens, Hot Plates

1. Use **forceps, ongs**, or **heat-resistant gloves** to remove containers from hot plates, ovens or muffle furnaces.

## F. Handling Gas

1. Cylinders of compressed gases should be secured at all times.

2. A central gasfacility is preferred.

## G. Maintenance

1. All electrical, plumbing, and instrument maintenance work should be done by qualified personnel.

2. Fume hoods should be checked routinely.

3. As most equipments operate on low wattage, an Uninterruptible Power Supply
(UPS) provides stable power and allows the completion of any batch
measurement in the event of power outage.

## H. Eating and Drinking

1. Do not eat, drink, or smoke in the laboratory. This is essential both for reasons of health and to reduce contamination.

2. Specific areas should be designated for staff breaks.

3. Do not use laboratory glassware for eating or drinking.

4. Do not store food in the laboratory.

## I. Protective Equipment

Use personal safety equipment as follows:

* **Body Protection:** Use laboratory coat and chemical-resistant apron.

* **Hand Protection:** Use gloves, particularly when handling concentrated acids, bases, and other hazardous chemicals.

* **Dust Mask:** Essentially needed when grinding soil, plant samples, etc.

* **Eye Protection:** Use safety glasses with side shields. Persons wearing contact lenses should always wear safety glasses in the laboratory. Make sure that your colleagues know that you wear contact lenses. Contact lenses should never be worn around corrosives.

* **Full-Face Shield:** Wear face shields over safety glasses in experiments involving corrosive chemicals.

* **Foot Protection:** Proper footwear should be used; sandals should not be worn in the laboratory

## J. Waste Disposal

1. Liquid wastes should be poured carefully down a sink with sufficient water to dilute and flush it away. Keep in mind that local ordinances often prohibit the disposal of specific substances through the public sewerage system.

2. Dispose off chipped or broken glassware in specially marked containers.

## K. Continuing Education

1. Display in a prominent place posters on "Laboratory Safety" which pictorially
describe various phases of laboratory activities.

2. Similarly, posters depicting First Aid after laboratories accidents should be prominently displayed. Such posters are not for ornamentation; they are for the protection of laboratory personnel, who should be thoroughly conversant with all procedures and eventualities.

3. If the laboratory is a part of a large institution, the laboratory staff should know the Safety Officer or person responsible for safety. If it is a small operation, one laboratory staff member should be responsible for safety.

## L. Contamination

The most insidious enemy in any laboratory is contamination and, therefore, its sources must be identified and eliminated. Some common sources of contamination are:

* External dusts blown from the surrounding environment;

* Internal dust resulting from cleaning operations;

* Cross-contamination derived from handling many samples at the same time (e.g., handling plant and soil samples together);

* Failure to store volatile reagents well away from the samples;

* Washing materials, particularly soap powder; and

* Smoking in the laboratory.
# Handling of chemical substances and handling techniques of solutions

## Chemical Handling & Disposal

### DO'S

* ✓ Always remember the three 'A's of acid handling - Always Add Acid to water or base. Slowly.

* ✓ Always treat any liquid in chemical area as acid because few acids like HF may look and feel like water.

* ✓ All spills and leakage should be cleaned up immediately.

* ✓ Safety equipments such as fire extinguishers, Eye wash basin and safety showers shall be kept clean and readily accessible.

### DON'TS

* ☒ Do not mix acids with solvents or flammables. A violent reaction may occur pour solvents down the sink.

* ☒ Do not drain.

* ☒ Do not dip your hand into a chemical even when wearing gloves.

* ☒ Do not inter-change the bottle caps. Put the same cap back on the bottle making sure that it is tight.

### DO'S

Do remember the following:

Acid handling requires the use of latex gloves

* ✓ Solvent and chemicals in miscellaneous category require the use of polythene gloves

* ✓ Check the gloves for any pin-holes prior to use

* ✓ Wash your hands before eating, even if gloves were used while handling the chemical

* ☒ Required safety equipments like plastic sleeves, aprons, rubbers, boots, safety glasses, fume hoods, fume shields etc. shall be used even while handling small quantities or hazardous chemicals.

* ☒ Demarcate separate areas for storing acids, base, solvents, wet chemicals, dry chemicals etc.

* ☒ Working alone shall be avoided when handling dangerous chemicals.

* ☒ Before draining any chemical in the central drain, ensure the compatibility of the chemical with the material of which the drain is made of and other chemicals normally flowing through that drain.
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Always rinse empty bottles of chemicals before disposing them of in the well-designated areas.

DON'TS

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Do not store chemicals near heat source.

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Do not store chemical containers (empty or full) at a height more than three feet above floor level.

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Do not store acids and solvents together.

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Do not wait to see whether the chemical you spill over you gives burn or not. Rinse the affected areas for 15-20 minutes and then report to a doctor. Also alert others so that they can help you in rinsing the chemical off you and in cleaning up the spill.

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Do not take hazardous chemicals if you are under the influence of Alcohol/drug etc. or are feeling sleepy.

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Don't come in direct contact with hazardous chemicals while in use/working.
Basic Laboratory Equipment

Chemistry is the science that deals with the composition and properties of substances and various elementary forms of matter. Chemistry, as are all sciences, is a discipline based on observation. ωIn lecture, you will learn the principles and theories that, to date, best explain the observations that have accumulated. The problem is that, if all you have is lecture, then it is all too easy to forget that these theories apply to the “real world”. The laboratory experience is, by design, your opportunity to see these principles and theories in practice.

1. Beaker

o A beaker is a simple container for stirring, mixing and heating liquids commonly used in many laboratories.

o Beakers are generally cylindrical in shape, with a flat bottom. Most also have a small spout (or "beak") to aid pouring.

o Beakers are available in a wide range of sizes, from 5 milliliter up to 2 liter.

o Beakers are commonly made of glass (usually borosilicate glass, pyrex®), but can also be in metal or certain plastics.

o Borosilicate glass mainly constitute from silica and boron oxide.

o Borosilicate glasses are known for having very low coefficients of thermal expansion making them resistant to thermal shock.

o Beakers are often graduated, that is, marked on the side with lines indicating the volume contained.

o These marks are not intended for obtaining a precise measurement of volume, but rather an estimation.

o Do not measure volume or prepare solutions at definite concentrations using a beaker

o The presence of a lip means that the beaker cannot have a lid.

o However, when in use, beakers may be covered by a watch glass to prevent contamination or loss of the contents, but allowing venting via the spout.

o Alternatively, a beaker may be covered with using a piece of parafilm.

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2. Erlenmeyer Flask

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An Erlenmeyer flask, also known as a conical flask, is a widely used type of laboratory flask which features a flat bottom, a conical body, and a cylindrical neck.

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It is named after the German chemist Emil Erlenmeyer, who created it in 1860.

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The Erlenmeyer is usually marked on the side (graduated) to indicate

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the approximate volume of contents.

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Do not measure volume or prepare solutions at definite concentrations using an Erlenmeyer flask

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The opening usually has a slight rounded lip so that the Erlenmeyer can be easily stoppered using a piece of cotton wool, rubber bung or similar.

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Erlenmeyer flasks are extremely useful in the lab setting for stirring the contents by hand by swirling the flask

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Erlenmeyer are used in chemistry labs for titration, as they can be held and the contents mixed single-handed leaving the other hand free to add reagent.

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Titration, also known as titrimetry, volumetry, is a common laboratory method of quantitative chemical analysis that is used to determine the unknown concentration of an identified analyte.

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A reagent, called the titrant is prepared as a standard solution. A known concentration and volume of titrant reacts with a solution of analyte in the presence of indicator to determine concentration.

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Erlenmeyer are also used in microbiology for the preparation of microbial cultures.

### 3. Volumetric flask

* A volumetric flask (measuring flask or graduated flask) is a piece of laboratory glassware, a type of laboratory flask, calibrated to contain a precise volume at a particular temperature.

* Volumetric flasks are used for precise dilutions and preparation of standard solutions.

* These flasks are usually pear-shaped, with a flat bottom, and made of glass or plastic.

* Solutions with definite concentrations only used in volumetric flasks.

* The neck of the volumetric flasks is elongated and narrow with an etched ring graduation marking. The marking indicates the volume of liquid contained when filled up to that point.

* Do not heat any volumetric equipment.

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## 4. Graduated measuring cylinder

* A graduated cylinder, measuring cylinder or mixing cylinder is a piece of laboratory equipment used to measure the volume of a liquid.

* Graduated cylinders are generally more accurate and precise than laboratory flasks and beakers. However, they are less accurate and precise than volumetric glassware, such as a volumetric flask or volumetric pipette.

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* For these reasons, graduated cylinders should not be used to perform volumetric analysis.

* A traditional graduated cylinder is usually narrow and high (so as to increase the accuracy and precision of volume measurement) and has a plastic or glass bottom and a "spout" for easy pouring from the measured liquid.

## 5. Graduated pipettes

* Graduated pipettes are a type of macro pipette consisting of a long tube with a series of graduations, as on a graduated cylinder or burette, to indicate different calibrated volumes.

* They also require a source of vacuum; in the early days of chemistry and biology, the mouth was used. Graduated pipettes commonly come in 1, 2, 5, 10 and 25 mL volumes and they are highly accurate.

* To avoid accidental ingestion of potentially harmful substances, a variety of pipetters have been developed, both entirely manual and electrically assisted

* Pipettes are made of borosilicate glass; disposable graduated pipettes are often made of polystyrene.

* Graduated pipettes are often graduated in one of two ways:

* Mohr, backward or drain-out pipettes have a 0 mL mark just above the end of the pipette.

* Serological, forward or blow-out pipettes have no 0ml mark as that corresponds to an empty pipette.

* Do not use graduated pipettes for measuring hot solutions or do not heat them!

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## 6. Volumetric pipettes

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Volumetric pipettes or bulb pipette allow the user to measure a volume of solution extremely accurately (accuracy of four significant figures).

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These pipettes have a large bulb with a long narrow portion above with a single graduation mark as it is calibrated for a single volume (like a volumetric flask).

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Typical volumes are 10, 25, and 50 mL.

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Volumetric pipettes are commonly used to make laboratory solutions from a base stock as well as prepare solutions for titration.

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## 7. Burette

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A burette (also buret) is a device used in analytical chemistry for the dispensing of variable, measured amounts of a chemical solution.

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A burette is distinguished from a pipette by the fact that the quantity delivered is variable. Thus in a titration, one solution is dispensed with a pipette, and another solution is added to it from a burette in aliquots of varying size.

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Burettes may be designated for use at a particular temperature. If used at another temperature they should be subject to calibration.

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Do not heat volumetric equipment!

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## 8. Retort Stand

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A retort stand, sometimes called a ring stand, is a piece of scientific equipment, to which clamps can be attached to hold test tubes and other equipment such as burettes which are most often used in titration experiments.

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It is also used in filtering, and extraction.

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The stands are usually made of a chemically impervious metal and may be covered with aluminium foil to further protect the base, on which may sit a hot plate, magnetic stirrer, heating mantle, or some other apparatus.

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## 9. Burette Clamp

* A burette clamp is chemical or biological laboratory equipment

* It is used for firmly holding test-tubes, burettes and other glassware in place when performing experiments such as titration.

* There are usually of two types, a single burette clamp and a double burette clamp.

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## 10. Iron ring and clamp

* An iron ring, sometimes called an iron support ring, is used in chemistry labs to stabilize flasks mounted to a ring stand.

* Some iron rings include a clamp, while others require a utility clamp to attach to a ring stand.

* A test tube clamp is used for holding a test tube.

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## 11. Test tubes and Test tube rack

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A test tube, also known as a culture tube or sample tube, is a common piece of laboratory glassware consisting of a finger-like length of glass or clear plastic tubing, open at the top, usually with a rounded U-shaped bottom.

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Test tubes are available in a multitude of lengths and widths, typically from 10 to 20 mm wide and 50 to 200 mm long.

<figure><img src="image_45.png" /></figure>

A test tube has either a flat bottom, a round bottom, or a conical bottom.

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Some test tubes are made to accept a ground glass stopper or a screw cap.

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Test tubes are widely used by chemists to hold, mix, or heat small quantities of solid or liquid chemicals, especially for qualitative experiments and assays.

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## 12. Mortar and Pestle

* The mortar is a bowl, typically made of hard wood, ceramic or stone.

* The pestle is a heavy club-shaped object, the end of which is used for crushing and grinding.

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➤ The substance to be ground is placed in the mortar and ground, crushed or mixed with the pestle.

## 13. Evaporating Dish

❖ An evaporating dish is a piece of laboratory glassware used for the evaporation of solutions and supernatant liquids, and sometimes to their melting point.

❖ Most are made of porcelain or borosilicate glass.

<figure><img src="image_50.png" /></figure>

## 14. Crucible

✓ A crucible is a container that can withstand very high temperatures and is used for metal, glass, and pigment production as well as a number of modern laboratory processes.

✓ While crucibles historically were usually made from clay, they can be made from any material that withstands temperatures high enough to melt or otherwise alter its contents.

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✓ Crucibles are used for calcination.

✓ The calcination process involves heating a substance in a crucible or over an open flame until it is reduced to ashes.

## 15. Bunsen burner

➤ A Bunsen burner, named after Robert Bunsen, is a common piece of laboratory equipment that produces a single open gas flame, which is used for heating, sterilization, and combustion.

➤ The device in use today safely burns a continuous stream of a flammable gas such as natural gas (which is principally methane) or a liquefied petroleum gas such as propane, butane, or a mixture of both.

➤ The amount of air mixed with the gas stream affects the completeness of the combustion reaction.

➤ Less air yields an incomplete and thus cooler reaction, while a gas stream well mixed with air provides oxygen in an equimolar amount and thus a complete and hotter reaction.

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## 16. Pipeclay triangle

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A pipeclay triangle (or ceramic triangle) is a piece of laboratory apparatus that is used to support items being heated by a Bunsen burner or other heat source.

<figure><img src="image_54.png" /></figure>

It is made of wires strung in an equilateral triangle on which are strung hollow ceramic tubes.

<figure><img src="image_55.png" /></figure>

The triangle is usually supported on a tripod or iron ring.

<figure><img src="image_56.png" /></figure>

Unlike wire gauze, which primarily supports glassware such as beakers, flasks, or evaporating dishes and provides indirect heat transfer to the glassware, the pipeclay triangle normally supports a crucible and allows the flame to heat the crucible directly.

<figure><img src="image_57.png" /></figure>

## 17. Wire Gauze

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There are two types of wire gauze used in chemistry: a normally woven wire gauze, and one with a white circle imprinted on it.

<figure><img src="image_59.png" /></figure>

The latter has a ceramic composite intended to aid in the dispersal in heat.

<figure><img src="image_60.png" /></figure>

The normal set-up is a ring stand which a support ring attached to it. The wire gauze is centered on the ring stand as to allow the open flame to be in contact with it to a certain extent, depending on the desired heat.

<figure><img src="image_61.png" /></figure>

<figure><img src="image_62.png" /></figure>

In the context of laboratory experimentation, the combination of the wire gauze plus the added ceramic composite greatly aids in the even dispersal of heat.

## 18. Laboratory Tripod

<figure><img src="image_63.png" /></figure>

Tripod is three-legged equipment, generally used as a platform of some sort.

<figure><img src="image_64.png" /></figure>

The word is derived from Greek word tripous, meaning "three feet".

<figure><img src="image_65.png" /></figure>

A tripod stand has three legs and is widely used in laboratories to conduct various experiments.

<figure><img src="image_66.png" /></figure>

This equipment used to support and hold various flasks, beakers and other glass ware during experiments.

<figure><img src="image_67.png" /></figure>

The stands specially constructed using light metals for light weight and ease of carrying.

<figure><img src="image_68.png" /></figure>
## 19. Watch Glass

o A watch glass is a circular, slightly convex-concave piece of glass used in chemistry as a surface to evaporate a liquid, to hold solids while being weighed, or as a cover for a beaker.

o The latter use is generally applied to prevent dust or other particles entering the beaker; the watch glass does not completely seal the beaker, and so gas exchanges still occur.

o When used as an evaporation surface, a watch glass allows closer observation of precipitates or crystallization, and can be placed on a surface of contrasting colour to improve the visibility overall.

<figure><img src="image_69.png" /></figure>

## 20. Tongs and Test Tube Holder

<figure><img src="image_70.png" /></figure>

Tongs are used for gripping and lifting tools, of which there are many forms adapted to their specific use.

<figure><img src="image_71.png" /></figure>

## 21. Ground glass joint

<figure><img src="image_72.png" /></figure>

❖ Ground glass joints are used in laboratories to quickly and easily fit leak-tight apparatus together from commonly available parts.

<figure><img src="image_73.png" /></figure>

## 22. Funnel

o A funnel is a pipe with a wide mouth, good for feeding, often conical mouth and a narrow stem.

o Laboratory funnels are funnels that have been made for use in the chemical laboratory.

o There are many different kinds of funnels that have been adapted for these specialized applications.

o Filter funnels, thistle funnels (shaped like thistle flowers), and have stopcocks which allow the fluids to be added to a flask slowly.

<figure><img src="image_74.png" /></figure>
## 23.Separatory Funnel

<figure><img src="image_75.png" /></figure>

A separatory funnel, also known as separation funnel, separating funnel, or colloquially sep funnel, is a piece of laboratory glassware used in liquid-liquid extractions to separate (partition) the components of a mixture into two immiscible solvent phases of different densities.

<figure><img src="image_76.png" /></figure>

<figure><img src="image_77.png" /></figure>

Typically, one of the phases will be aqueous, and the other a non-polar lipophilic organic solvent such as ether, MTBE, dichloromethane, chloroform, or ethyl acetate.

## 24.Spatula and Glass Rod

* ✓ A spatula is a small implement with a broad, flat, flexible blade used to mix, spread and lift materials.

* ✓ Spatula is used for weighing chemicals.

* ✓ Glass rods (or stirring rods) are used as stirrers in laboratory environments.

* ✓ Like most laboratory glass, stir rods are made of borosilicate (commonly known as pyrex).

<figure><img src="image_78.png" /></figure>

## 25.Forceps

<figure><img src="image_79.png" /></figure>

Forceps or forcipes are a handheld, hinged instrument used for grasping and holding objects.

<figure><img src="image_80.png" /></figure>

Forceps are used when fingers are too large to grasp small objects or when many objects need to be held at one time while the hands are used to perform a task.

<figure><img src="image_81.png" /></figure>

## 26.Wash Bottle

* o A wash bottle is a squeeze bottle with a nozzle, used to rinse various pieces of laboratory glassware, such as test tubes and round bottom flasks.

* o Wash bottles are sealed with a screw-top lid. When hand pressure is applied to the bottle, the liquid inside becomes pressurized and is forced out of the nozzle into a narrow stream of liquid.

* o Most wash bottles are made up of polyethylene, which is a flexible

<figure><img src="image_82.png" /></figure>
solvent-resistant petroleum-based plastic. Most bottles contain an internal dip tube
allowing upright use.

o Wash bottles may be filled with a range of common laboratory solvents and reagents, according to the work carried out in that lab.

o These include: deionized water, detergent solutions and rinse solvents such as acetone, isopropanol or ethanol.

**27. Condenser**

<figure><img src="image_83.png" /></figure>

A condenser is a piece of laboratory glassware used to cool hot vapors or liquids.

<figure><img src="image_84.png" /></figure>

A condenser usually consists of a large glass tube containing a smaller glass tube running its entire length, within which the hot fluids pass.

<figure><img src="image_85.png" /></figure>

The ends of the inner glass tube are usually fitted with ground glass joints which are easily fitted with other glassware. During reflux, the upper end is usually left open to the atmosphere or vented through a bubbler or a drying tube to prevent the ingress of water or oxygen.

<figure><img src="image_86.png" /><figcaption>Liebig
Condenser</figcaption></figure>

<figure><img src="image_87.png" /><figcaption>Alihn
Condenser</figcaption></figure>

<figure><img src="image_88.png" /><figcaption>Vigreux
condenser</figcaption></figure>

<figure><img src="image_89.png" /><figcaption>Graham
Condenser</figcaption></figure>

<figure><img src="image_90.png" /></figure>

The outer glass tube usually has two hose connections, and a coolant (usually tap water or chilled water/anti-freeze mixture) is passed through it. For maximum efficiency, and to maintain a smooth and correctly directed thermal gradient so as to minimize the risk of thermal shock to adjacent glassware, the coolant usually enters through the lower fitting, and exits through the higher fitting.

**28. Desiccator**

➤ Desiccators are sealable enclosures containing desiccants used for preserving moisture-sensitive items such as cobalt chloride paper for another use.

➤ A common use for desiccators is to protect chemicals which are hygroscopic or which react with water from humidity. Desiccators are sometimes used to remove traces of water from an almost-dry sample.

<figure><img src="image_91.png" /></figure>
## 29. Round and Flat bottom flask

<figure><img src="image_92.png" /></figure>

Round-bottom flasks are types of flasks having spherical bottoms used as laboratory glassware, mostly for chemical or biochemical work.

<figure><img src="image_93.png" /></figure>

They are usually made of heat-resistant borosilicate glass.

<figure><img src="image_94.png" /></figure>

There is at least one tubular section known as the neck with an opening at the tip. Two or three-necked flasks are common as well.

<figure><img src="image_95.png" /></figure>

Reagent flasks are usually a flat-bottomed flask, which can thus be conveniently placed on the table or in a cabinet.

<figure><img src="image_96.png" /></figure>

These flasks cannot withstand too much pressure or temperature differences, due to the stresses which arise in a flat bottom, these flasks are usually made of weaker glass than reaction flasks.

<figure><img src="image_97.png" /></figure>

<figure><img src="image_98.png" /></figure>

<figure><img src="image_99.png" /></figure>

## 30. Buchner funnel and flask

<figure><img src="image_100.png" /></figure>

A Buchner funnel is a piece of laboratory equipment used in filtration.

<figure><img src="image_101.png" /></figure>

It is traditionally made of porcelain, but glass and plastic funnels are also available.

<figure><img src="image_102.png" /></figure>

On top of the funnel-shaped part there is a cylinder with a perforated plate separating it from the funnel.

<figure><img src="image_103.png" /></figure>

## 31. Filter Paper

<figure><img src="image_104.png" /></figure>

Filter paper is a semi-permeable paper barrier placed perpendicular to a liquid or air flow.

<figure><img src="image_105.png" /></figure>

It is used to separate fine solids from liquids or air.

<figure><img src="image_106.png" /></figure>

Filter paper comes in various porosities and grades depending on the applications it is meant for. The important parameters are wet strength, porosity, particle retention, flow rate, compatibility, efficiency and capacity.

<figure><img src="image_107.png" /></figure>

<figure><img src="image_108.png" /></figure>

There are two mechanisms of filtration with paper; volume and surface. By volume filtration the particles are caught in the bulk of the filter paper. By surface filtration the particles are caught on the paper surface. Filter paper is mostly used because even a small piece of filter paper will absorb a significant volume of liquid.
## 32. Pasteur Pipette

✓ Pasteur pipettes, also known as droppers or eye droppers, are used to transfer small quantities of liquids.

✓ They are usually glass tubes tapered to a narrow point, and fitted with a rubber bulb at the top.

✓ The combination of the Pasteur pipette and rubber bulb has also been referred to as a teat pipette.

<figure><img src="image_109.png" /></figure>
Cleaning and Drying of Glassware, Drying of solvents/chemicals

Good lab practices require the use of clean glassware. Glass must be both physically and chemically clean; even sterile for many cases. Most of the glassware accidents take place during cleaning. Following are some important reminders to wash and dry glassware.
Cleaning of glassware involves the following steps

* In the first step the glassware to be washed is soaked in detergent overnight so that the contaminants may become loose, and any grease left can also be removed.

* The particles which cannot be removed by rinsing are scrubbed using brushes or scouring pads.

* Trace quantities of the contaminants are generally removed using aqua regia or other mild acids which cannot be removed by normal scrubbing. It is important for some sensitive experiments and this step eliminates the chances of any cross contamination.

* After they are washed with detergents and mild acids, they are thoroughly rinsed with water to remove the traces of acids and detergents and are then placed upside down on a rack and allowed to dry.

* Personal protective equipments, especially eye protection goggles and chemically resistant gloves should be worn during washing.

* After using, glassware should be washed as quickly as possible. As the glassware left unwashed longer, it takes long time to clean. If necessary, use harder cleaning apparatus and keep and leave glassware in soapy water.

* Do not overload sinks, dishwashers, or soaking bins.

* Keep glassware clear of the sides of the sink.

* Do not use worn out brushes as they may scratch the glass.

* In case if you need to use caustic for cleaning purposes, it is advised to ask help from an experienced lab user in the safe usage of caustic cleaning agents, especially prior to using aqua-regia, chromic acid or other reactive solutions.

* Leave glassware drying on towels, lined basket, or slip-resistant pads. Place glassware away from the edge of the bench. Large containers may be hung on pegs to dry.

* Pipettes and tips may be cleaned in a cylinder or tall jar of water with appropriate disinfectant (e.g. for biologically contaminated tips). To prevent tips from breakage, you may put a cotton pad or glass wool at the bottom.

* To remove residue or loose particles, new glassware should be washed prior to use.
# Drying of solvents/chemicals

These days many laboratories use a commercially available solvent purification system, others will distil solvents using more traditional techniques. Tetrahydrofuran, dichloromethane, dimethylformamide, chloroform, acetonitrile, methanol, diethyl ether and toluene are all commonly used solvents, and in many cases they are required in anhydrous form. In some cases there are multiple ways to dry a given solvent. Here are some suggestions:

## Specific solvents

1. **THF (Tetrahydrofuran):** distilled from sodium benzophenone ketyl. Add sodium wire and benzophenone to a volume of THF (pre-dried over calcium hydride or 4A molecular sieves), heat at reflux/under nitrogen for several hours until the solvent turns deep blue in colour. This indicates the solvent is dry, and you can distill off the volume you require. With time (weeks-maybe months if used correctly) the still will turn orange, this indicates that it is time to make a new one!

2. **DCM (Dichloromethane):** pre-dry over calcium hydride, then distil over calcium hydride... unfortunately no colorimetric indicator to tell you when the solvent is dry.

3. **MeCN (Acetonitrile):** pre-dry by shaking with type 4A molecular sieves, the distil over calcium hydride. Dried acetonitrile can be stored over 4A molecular sieves.

4. **Diethyl ether:** distilled from sodium benzophenone ketyl (see THF), will turn a deep purple/blue colour when dry.

5. **Methanol:** For most purposes, drying over 3A molecular sieves overnight followed by distillation is sufficient. Alternatively, the methanol can be dried from magnesium methoxide. Magnesium turnings (5 g) and iodine (0.5 g) are refluxed in a small (50-100 mL) quantity of dry methanol (from a previous batch) until all of the magnesium has reacted. The mixture is diluted (up to 1 L) with reagent grade methanol, refluxed for 2-3 hours then distilled under nitrogen.

6. **DMF (N,N-dimethylformamide):** Decomposes slowly at room temperature and more rapidly at reflux, releasing dimethylamine and carbon monoxide. This decomposition is catalysed by acidic and basic impurities, and standing DMF for several hours at room temperature with basic drying agents such as calcium hydride or sodium hydroxide leads to noticeable decomposition. Dry DMF can be prepared by drying overnight over barium oxide or 4A molecular sieves, followed by decantation of the drying agent and vacuum distillation (~20 mmHg is a sufficient vacuum to
lower the boiling point over DMF to a reasonable value). Dry DMF can be stored over 4A molecular sieves.

## General Precautions

As a general precaution ethers can produce explosive peroxides, making distillation of these solvents hazardous. If one is available, use a blast guard (really thick piece of pyrex? attached to a stand), and do not use stills once they have passed their use-by date. The process of distillation also removes the stabilisers that are added to the ethers, consequently distilled ethers should not be stored for long periods of time. For directions on handling sodium etc. consult 'reagant specific hazards'.

## Quenching of solvent

Stills that use metals (THF) should be quenched by pouring any excess solvent into a large container filled with isopropanol or tert-butanol. The reaction may become exothermic, so addition of the solvent to the alcohol must be done slowly. The remaining metal in the still can be quenched like normal.
# Preparation and dilution of Solutions of Various Strengths

Understanding how to prepare solutions and make dilutions is an essential skill which is necessary knowledge needed for doing any experiment.

**SOLUTIONS:** A simple solution is basically two substances that are evenly mixed together. One of them is called the solute and the other is the solvent. Solution can be composed from one or more solute dissolved in a solvent forming a homogenous mixture.

* ✓ Solute is the substance to be dissolved (sugar)

* ✓ Solvent is the one doing the dissolving (water)

<figure><img src="image_110.png" /></figure>

<figure><img src="image_111.png" /></figure>

## A. Preparation of Solutions

Solution concentration define as: quantity of a substance dissolved in per unit quantity of another substance (the relative amounts of solute and solvent in a solution).

* There are different ways to express concentration:

* 1. Molarity.

* 2. W/V %.

* 3. W/W %.
**1. Molarity:**

Molarity defines as: the number of moles of solute in one liter of a solution.

Molar = number of mole/volume in L

$$ \text{Molarity} = \frac{\text{moles of solute (mole)}}{\text{volume of solution in (L)}} $$

Remember that:

$$ \text{Mole} = \frac{\text{weight (g)}}{\text{molecular weight (g/mole)}} = \frac{\text{Wt}}{\text{M.W}} $$

$$ \text{Molarity} = \frac{\text{weight (g)}}{\text{volume (L)} \times \text{M.W}} $$

<figure><img src="image_112.png" /></figure>

1 Molar solution is a solution in which 1 mole of solute is dissolved in a total volume of 1 liter(1000ml).(0.5 Molar (M) solution: that mean there are 0.5 mole dissolved in 1L ..etc)

<figure><img src="image_113.png" /></figure>

Units of molarity are : M, molar or mole/L

Example:

**How to prepare 2M of NaCl in 100 ml?**

Concentration = 2M, Solution volume= 100 ml

So, **two ways to solve it**

(1)

2 mole of NaCl present in 1000 ml [ or 1Liter ] of solvent (dis.H₂O)

And we know that,

No of mole = weight (g) / molecular weight.

So,

[2 mole= weight (g) / 58.5]

$$ \text{weight (g)} = 2 \times 58.5 = 117 \text{ g.} $$

This weight needed if 1000 ml is required to be prepared. Since we need to prepare only 100 ml.

$$ 117 \text{ g} ====> 1000 \text{ ml.} $$

$$ ? \text{ g} ====> 100 \text{ ml.} $$

$$ [(100 \times 117)/1000] = 11.7 \text{ g} $$

11.7 g of NaCl dissolved in small volume of dis.H₂O, then complete the volume up to 100 ml
(2)

$$Molarity= 2M$$

$$Solution volume= 100 ml$$

$$Convert to L = 100/1000 = 0.1L$$

$$Molecular weight (M.W) = 58.5 g/mole$$

$$Weight = ?$$

So:

$$Weight = Molarity \times volume in L \times M.W$$

$$Weight = 2 \times 0.1 \times 58.5 = 11.7g$$

11.7 g of NaCl dissolved in small volume of dis.H2O, then complete the volume up to 100 ml. weight

Practically how to prepare 2M NaCl ?

1. Place a beaker in a balance and zero the balance.

2. Weight 11.7 grams of NaCl, in the beaker and dissolve it in a little water (less than 100 ml).

3. Once the solid is dissolved the volume is transferred to 100 ml volumetric flask.

4. Brought up to a final volume 100 ml by water

2. W/V % :

$$W/V\% = Weight/Volume \ Percentage \ Concentration.$$

W/V% define as : The number of grams of solute dissolved in 100 mL of solution (% = 100).

$$\frac{W}{V}\% = \frac{\text{weight of solute in (g)}}{\text{volume of solution in (ml)}} \times 100$$

For example: 3 W/V% NaOH

Mean 3 grams of NaOH is dissolved in 100 ml of the solution weight

Example:

How to prepare 50ml of 4 W/V % NaOH?

4% NaOH

Mean 4 grams of NaOH is dissolved in 100 ml of the solution

SO,

How many grams of NaOH I need to prepare 50ml of 4%NaOH solution?

4g ------> 100 ml

? -------> 50 ml

The Weight in grams of NaOH needed to prepare 4% NaOH is = (4 x 50)/100 = 2 g.
So, 2 grams of NaOH is dissolved in little water and the volume made up to 50 ml.

### 3. W/W %:

W/W%

Weight/Weight Percentage Concentration.

W/W% define as: the number of grams of solute dissolved in 100 gram of solution. (% = 100).

$$W/W\% = \frac{\text{weight of solute in (g)}}{\text{weight of solution in (g)}} \times 100$$

* The concentrations of many commercial acids are given in terms of w/w%.

* In order to calculate the volume of the stock solution required for a given preparation the density (specific gravity) of stock solution should be provided.

$$\text{Weight (wt)} = \text{volume (ml)} \times \text{SG} \times \text{w/w\% (as decimal)}$$

* To calculate w/w% as decimal = (w/w)/100,

* For example: w/w%= 13%

$$\frac{13}{100} = 0.13$$

### Example:

How to prepare 100 ml with 0.4 M HCL solutions starting with the concentrated HCL solution provided with: (W/W% = 36%, S.G = 1.15)?

How many ml of concentrated HCl we need to make 0.4M HCl solution?

Weight = volume (ml) x SG x w/w% (as decimal)

Important Note! : the volume in this formula is not the required volume in the question, it is the volume of the concentrated HCl that you must add to make the solution

First we must calculate the weight by the following:

From molarity formula

$$ 
\begin{align*}
\text{Mole} &= \text{Molarity} \times \text{volume in liter} \\
&= 0.4 \times 0.1 = 0.04 \text{ mole}
\end{align*}
$$

$$ 
\begin{align*}
\text{Weight} &= \text{mole} \times \text{MW (Note: The MW of HCl = 36.4)} \\
&= 0.04 \times 36.5 = 1.46 \text{ g}
\end{align*}
$$

Second: Weight (wt) = volume (ml) x SG x w/w% (as decimal)

$$1.46 = \text{volume} \times 1.15 \times 0.36$$

$$\text{Volume} = 3.53 \text{ ml}$$

So, 3.53 ml of stock (i.e. concentrated HCl) solution is needed and the volume made up to 100 ml by the addition of water.
## B. Dilution of Solution:

<figure><img src="image_114.png" /></figure>

Dilution of solution: means to add more solvent without the addition of more solute. To make it less concentrated.

<figure><img src="image_115.png" /></figure>

1. Volume to volume dilutions (ratio).

2. Preparing dilutions by using the V1XC1=V2XC2 formula.

3. Serial Dilutions.

1) Volume to Volume Dilution (ratio):

* ✓ This type of dilutions describes the ratio of the solute to the final volume of the dilute solution.

* ✓ For example: to make 1:10 dilution of 1M NaCl solution, one part of the 1M NaCl solution, should be mixed with nine parts of water, for a total of ten parts.

* ✓ Therefore 1:10 dilution means

* ✓ 1 part of 1M NaCl + 9 parts of water.

Thus, If 10 ml of the 1:10 dilution was needed, then 1 ml of 1M NaCl should be mixed with 9 ml of water

* ✓ If 100 ml of 1:10 dilution was needed, then 10 ml of the 1M NaCl should be mixed with 90 ml of water. [The final concentration of NaCl in both cases will be 0.1 M(1/10) = 0.1]

Example:

<figure><img src="image_116.png" /></figure>
Example:

How to prepare 2:10 dilution of a solution (A) with 7M, but the total volume is 20ml not 10 ml?

$$2 \text{ ml} \rightarrow 10 \text{ ml}$$

$$? \text{ ml} \rightarrow 20 \text{ ml}$$

$$= (2 \times 20) / 10 = 4 \text{ ml}$$

So,

4 ml from solution (A) of 7 M is needed and complete volume up to 20 ml (adding 16 ml water).

Note: [16 ml water = 20 ml - 4 ml].

How to know the concentration of solution A after dilution?

First we will find the DILUTION FACTOR by the following:

Dilution factor (D.F) = final volume / aliquot volume

$$=10/2 = 5$$

Then we will divide the stock concentration (before dilution) by the D.F:

$$7/5 = 1.4\text{M}$$

Note: To find out the stock concentration you will multiply the diluted concentration by the D.F

## 2) Preparing dilutions by using the V1XC1=V2XC2 formula.

<figure><img src="image_117.png" /></figure>

Sometimes it is necessary to use one solution to make a specific amount of a more dilute solution.

* To do this the following formula can be used:

$$V1XC1 = V2XC2$$

Where:

* V1= Volume of starting solution needed to make the new solution (volume of stock solution).

* C1= Concentration of starting solution (stock solution).

* V2= Final volume of new solution.

* C2= Final concentration of new solution.

How many ml of 1M solution we need to make 5 ml of 0.25M solution?

$$V1XC1 = V2XC2$$

Where:

$$V1 =? \quad C1= 1\text{M} \quad V2= 5\text{ml} \quad C2 = 0.25\text{M}$$

$$\text{So: } (V1) \times (1\text{M}) = (5\text{ml}) \times (0.25\text{M})$$
$$V1 = (5 \times 0.25)/1 = 1.25 \text{ ml}$$

So 1.25 ml of the 1M solution is needed (starting solution) then complete the volume up to 5 ml by diluent (generally water).

### 3) Serial Dilutions.

- It is a stepwise dilution of a solution, where the dilution factor is constant at each step.

- The source of dilution material for each step comes from the diluted material of the previous step.

$$\text{Dilution factor (D. F)} = \frac{\text{final volume}}{\text{aliquot volume}} = \frac{10}{1} = 10 \ (\text{for each step})$$

<figure><img src="image_118.png" /></figure>
**A. preparation of solution**

**Q.No.1:** You are provided with solid NaOH, Prepare 50ml with 0.08M NaOH solution.

Calculation:

<figure><img src="image_119.png" /></figure>

☐ To prepare the 0.08M NaOH solution ...g of solid NaOH should be dissolved in a little volume of water then the volume made up to ...ml, by the addition of water.

**Q.No.2:** You are provided with solid NaCl, Prepare 50ml with 1.5 w/v% solution of NaCl.

Calculation:

<figure><img src="image_120.png" /></figure>

☐ To prepare the 1.5 w/v% solution ...g of NaCl should be dissolved in little water and the volume made up to ...ml by the addition of water

**Q.No.3:** Prepare 100ml with 0.4 M HCl solutions starting with the concentrated HCl solution you are provided with: (w/w%= 36, S.Gr=1.15).

Calculation:

<figure><img src="image_121.png" /></figure>

✓ To prepare the 100ml of 0.4M HCl solution ...ml of stock (i.e. concentrated HCl) solution is needed and the volume made up to ...ml by the addition of water.

✓ Measure and record the pH value of the acid you prepared...

✓ Calculate the pH of the acid (pH= - log [H+])

✓ Determine your accuracy? ...

**B. Solution dilution**

1) Prepare 50ml with 1:20 dilution using the 0.08M NaOH solution you previously prepared.

Calculation:

<figure><img src="image_122.png" /></figure>

☐ To prepare the 1:20 dilution ...ml of the starting solution (0.08M NaOH) is needed and volumemade up to a final volume of ml
2) Prepare 100ml of 0.2M HCl from the previously 0.4M HCl solution you previously prepared.

Calculation:

<figure><img src="image_123.png" /></figure>

☐ To prepare the 0.2M HCl ...ml of the starting solution (0.4M HCl) is needed and volume made upto a total volume of ...ml by adding water

3) Starting with a 2.0 M stock solution of hydrochloric acid, prepare 8ml of four standard solutions(1 to 4) of the following Molarity respectively (dilution 2:8):

(1) ... M (2) ... M (3) ... M (4) ... M.

Calculation:

<figure><img src="image_124.png" /></figure>

❖ To prepare standard solution 1: ... ml of the stock 2.0M solution is needed and volume made up to ... ml with distilled water.

❖ To prepare standard solution 2-4: ... ml of the previously diluted solution (8.00×10⁻² M) is taken and volume is made up to a final volume of ... ml by the addition of distilled water

Home work

1. A student needed to prepare 1L of a 1M NaCl solution, which of the following methods is more accurate in preparing the solution? Why?

a) Weighing 58.5g of solid NaCl carefully, dissolving it in 300ml of water, then adding 700ml of water.

b) Weighing 58.5g of solid NaCl carefully, dissolving it in a small volume of water then making the final volume up to 1L by adding water.

2. List the most important points to be considered when preparing solutions.

3. A solution was prepared by taking 6ml of a 0.22M solution and then the volume was made up to a final volume of 30ml. What is the concentration of the final solution?

4. How would you prepare 80ml of a 1:25 dilution of a 2.1M KCl solution?

5. How would you prepare 50ml of a 6% NaCl solution?
## Preparation of solutions of acids

Common acid solutions can be prepared using the handy table below. The third column lists the amount of solute (acid) that is used to make 1 L of acid solution. Adjust the recipes accordingly to make larger or smaller volumes. For example, to make 500 mL of 6M HCl, use 250 mL of concentrated acid and slowly dilute to 500 mL with water.

### Tips for Preparing Acid Solutions

Always add acid to a large volume of water. The solution may then be diluted with additional water to make one liter. You'll get an incorrect concentration if you add 1 liter of water to the acid. It's best to use a volumetric flask when preparing stock solutions, but you can use an Erlenmeyer flask if you only need an approximate concentration. Because *mixing acid with water* is an exothermic reaction, be sure to use glassware capable of withstanding the temperature change (e.g., Pyrex or Kimax). *Sulfuric acid* is particularly reactive with water. Add the acid *slowly* to the water while stirring.

### Recipes for Acid Solutions

<table><thead><tr><th>Name / Formula / F.W.</th><th>Concentration</th><th>Amount/Liter</th></tr></thead><tbody><tr><td><strong>Acetic Acid</strong></td><td>6 M</td><td>345 mL</td></tr><tr><td>CH<sub>3</sub>CO<sub>2</sub>H</td><td>3 M</td><td>173</td></tr><tr><td>F.W. 60.05</td><td>1 M</td><td>58</td></tr><tr><td>99.7%, 17.4 M</td><td>0.5 M</td><td>29</td></tr><tr><td>sp. gr. 1.05</td><td>0.1 M</td><td>5.8</td></tr><tr><td><strong>Hydrochloric Acid</strong></td><td>6 M</td><td>500 mL</td></tr><tr><td>HCl</td><td>3 M</td><td>250</td></tr><tr><td>F.W. 36.4</td><td>1 M</td><td>83</td></tr><tr><td>37.2%, 12.1 M</td><td>0.5 M</td><td>41</td></tr><tr><td>sp. gr. 1.19</td><td>0.1 M</td><td>8.3</td></tr><tr><td><strong>Nitric Acid</strong></td><td>6 M</td><td>380 mL</td></tr><tr><td>HNO<sub>3</sub></td><td>3 M</td><td>190</td></tr><tr><td>F.W. 63.01</td><td>1 M</td><td>63</td></tr><tr><td>70.0%, 15.8 M</td><td>0.5 M</td><td>32</td></tr><tr><td>sp. gr. 1.42</td><td>0.1 M</td><td>6.3</td></tr></tbody></table>
<table><tr><td><strong>Phosphoric Acid</strong></td><td>6 M</td><td>405 mL</td></tr><tr><td>H<sub>3</sub>PO<sub>4</sub></td><td>3 M</td><td>203</td></tr><tr><td>F.W. 98.00</td><td>1 M</td><td>68</td></tr><tr><td>85.5%, 14.8 M</td><td>0.5 M</td><td>34</td></tr><tr><td>sp. gr. 1.70</td><td>0.1 M</td><td>6.8</td></tr></table>

<table><tr><td><strong>Sulfuric Acid</strong></td><td>9 M</td><td>500 mL</td></tr><tr><td>H<sub>2</sub>SO<sub>4</sub></td><td>6 M</td><td>333</td></tr><tr><td>F.W. 98.08</td><td>3 M</td><td>167</td></tr><tr><td>96.0%, 18.0 M</td><td>1 M</td><td>56</td></tr><tr><td>sp. gr. 1.84</td><td>0.5 M</td><td>28</td></tr><tr><td></td><td>0.1 M</td><td>5.6</td></tr></table>

## Acid Safety Information

You should always wear protective gear when mixing acid solutions. Be sure to wear safety goggles, gloves, and a lab coat as well. Tie back long hair and make sure your legs and feet are covered by long pants and shoes. It's a good idea to prepare acid solutions inside the ventilation hood because the fumes can be noxious, particularly if you are working with concentrated acids or if your glassware isn't absolutely clean. If you do spill acid, you can neutralize it with a weak base (safer than using a strong base) and dilute it with a large volume of water.
# Neutralisation of acid and bases

## Acid-Base Neutralization

A solution is neutral when it contains equal concentrations of hydronium and hydroxide ions. When we mix solutions of an acid and a base, an acid-base neutralization reaction occurs. However, even if we mix stoichiometrically equivalent quantities, we may find that the resulting solution is not neutral. It could contain either an excess of hydronium ions or an excess of hydroxide ions because the nature of the salt formed determines whether the solution is acidic, neutral, or basic. The following four situations illustrate how solutions with various pH values can arise following a neutralization reaction using stoichiometrically equivalent quantities:

1. A strong acid and a strong base, such as HCl(aq) and NaOH(aq) will react to form a neutral solution since the conjugate partners produced are of negligible strength:

$$HCl(aq) + NaOH(aq) \rightleftharpoons NaCl(aq) + H_2O(l)$$

2. A strong acid and a weak base yield a weakly acidic solution, not because of the strong acid involved, but because of the conjugate acid of the weak base.

3. A weak acid and a strong base yield a weakly basic solution. A solution of a weak acid reacts with a solution of a strong base to form the conjugate base of the weak acid and the conjugate acid of the strong base. The conjugate acid of the strong base is a weaker acid than water and has no effect on the acidity of the resulting solution. However, the conjugate base of the weak acid is a weak base and ionizes slightly in water. This increases the amount of hydroxide ion in the solution produced in the reaction and renders it slightly basic.

4. A weak acid plus a weak base can yield either an acidic, basic, or neutral solution. This is the most complex of the four types of reactions. When the conjugate acid and the conjugate base are of unequal strengths, the solution can be either acidic or basic, depending on the relative strengths of the two conjugates. Occasionally the weak acid and the weak base will have the same strength, so their respective conjugate base and acid will have the same strength, and the solution will be neutral. To predict whether a particular combination will be acidic, basic or neutral, tabulated K values of the conjugates must be compared.

## Neutralization Reactions

This is the general format for a neutralization reaction:

$$Acid + Base \rightarrow Salt + Water$$
It is important to note that neutralization reactions are just a specific type of double displacement redox reaction. Remember the rules for writing displacement reactions.

1. Figure out what the reactants and products will be.

* The cations will switch places in the products for double replacement reactions.

* The element will replace the cation in the reacting compound and result in a new product for single replacement reactions.

2. Make sure that the entire compound formulas are correctly written based on the oxidation state of the elements involved.

3. Balance the equation.

**Example:** Predicting the outcome of a neutralization reaction

Write the balanced chemical equation for the neutralization of HCl with Mg(OH)₂.

Hint: neutralization reactions are a specialized type of double replacement reaction.

$$2\text{HCl} + \text{Mg(OH)}_2 \rightarrow \text{Mg(Cl)}_2 + 2\text{H}_2\text{O}$$

Since HCl is a strong acid and Mg(OH)₂ is a strong base, the resulting solution would be neutral.

## Stomach Antacids

Our stomachs contain a solution of roughly 0.03 M HCl, which helps us digest the food we eat. The burning sensation associated with heartburn is a result of the acid of the stomach leaking through the muscular valve at the top of the stomach into the lower reaches of the esophagus. The lining of the esophagus is not protected from the corrosive effects of stomach acid the way the lining of the stomach is, and the results can be very painful. When we have heartburn, it feels better if we reduce the excess acid in the esophagus by taking an antacid. As you may have guessed, antacids are bases. One of the most common antacids is calcium carbonate, CaCO₃. The reaction, not only neutralizes stomach acid, it also produces CO₂(g), which may result in a satisfying belch.

$$\text{CaCO}_3\text{(s)} + 2\text{HCl}\text{(aq)} \rightleftharpoons \text{CaCl}_2\text{(aq)} + \text{H}_2\text{O(l)} + \text{CO}_2\text{(g)}$$

Milk of Magnesia is a suspension of the sparingly soluble base magnesium hydroxide, Mg(OH)₂. It works according to the reaction:

$$\text{Mg(OH)}_2\text{(s)} \rightleftharpoons \text{Mg}^{2+}\text{(aq)} + 2\text{OH}^{-}\text{(aq)}$$

The hydroxide ions generated in this equilibrium then go on to react with the hydronium ions from the stomach acid, so that:

$$\text{H}_3\text{O}^+ + \text{OH}^- \rightleftharpoons 2\text{H}_2\text{O(l)}$$
This reaction does not produce carbon dioxide, but magnesium-containing antacids can have a laxative effect. Several antacids have aluminum hydroxide, $Al(OH)_3$, as an active ingredient. The aluminum hydroxide tends to cause constipation, and some antacids use aluminum hydroxide in concert with magnesium hydroxide to balance the side effects of the two substances.

## Culinary Aspects of Chemistry

Cooking is essentially synthetic chemistry that happens to be safe to eat. There are a number of examples of acid-base chemistry in the culinary world. One example is the use of baking soda, or sodium bicarbonate in baking. $NaHCO_3$ is a base. When it reacts with an acid such as lemon juice, buttermilk, or sour cream in a batter, bubbles of carbon dioxide gas are formed from decomposition of the resulting carbonic acid, and the batter “rises.” Baking powder is a combination of sodium bicarbonate, and one or more acid salts that react when the two chemicals come in contact with water in the batter. Many people like to put lemon juice or vinegar, both of which are acids, on cooked fish. It turns out that fish have volatile amines (bases) in their systems, which are neutralized by the acids to yield involatile ammonium salts. This reduces the odor of the fish, and also adds a “sour” taste that we seem to enjoy.

<figure><img src="image_125.png" /><figcaption>Figure: A neutralization reaction takes place between citric acid in lemons or acetic acid in vinegar, and the bases in the flesh of fish.</figcaption></figure>
Pickling is a method used to preserve vegetables using a naturally produced acidic environment. The vegetable, such as a cucumber, is placed in a sealed jar submerged in a brine solution. The brine solution favors the growth of beneficial bacteria and suppresses the growth of harmful bacteria. The beneficial bacteria feed on starches in the cucumber and produce lactic acid as a waste product in a process called fermentation. The lactic acid eventually increases the acidity of the brine to a level that kills any harmful bacteria, which require a basic environment. Without the harmful bacteria consuming the cucumbers they are able to last much longer than if they were unprotected. A byproduct of the pickling process changes the flavor of the vegetables with the acid making them taste sour.

## Summary

The characteristic properties of aqueous solutions of Bronsted-Lowry acids are due to the presence of hydronium ions; those of aqueous solutions of Bronsted-Lowry bases are due to the presence of hydroxide ions. The neutralization that occurs when aqueous solutions of acids and bases are combined results from the reaction of the hydronium and hydroxide ions to form water. Some salts formed in neutralization reactions may make the product solutions slightly acidic or slightly basic.
Preparing Buffer Solutions

The pH of the mobile phase (eluent) is adjusted to improve component separation and to extend the column life. This pH adjustment should involve not simply dripping in an acid or alkali but using buffer solutions, as much as possible. Good separation reproducibility (stability) may not be achieved if buffer solutions are not used. A buffer solution is prepared as a combination of weak acids and their salts (sodium salts, etc.) or of weak alkalis and their salts. Common preparation methods include: 1) dripping an acid (or alkali) into an aqueous solution of a salt while measuring the pH with a pH meter and 2) making an aqueous solution of acid with the same concentration as the salt and mixing while measuring the pH with a pH meter. However, if the buffer solution is used as an HPLC mobile phase, even small errors in pH can lead to problems with separation reproducibility. Therefore, it is important to diligently inspect and calibrate any pH meter that is used. This page introduces a method that does not rely on a pH meter. The method involves weighing theoretically calculated fixed quantities of a salt and acid (or alkali). Consider the important points below.

Denoting Buffer Solutions

A buffer solution denoted, "100 mM phosphoric acid (sodium) buffer solution pH = 2.1," for example, contains phosphoric acid as the acid, sodium as the counterion, 100 mM total concentration of the phosphoric acid group, and a guaranteed buffer solution pH of 2.1.

Maximum Buffer Action Close to the Acid (or Alkali) pKa

When an acetic acid (sodium) buffer solution is prepared from 1:1 acetic acid and sodium acetate, for example, the buffer solution pH is approximately 4.7 (near the acetic acid pKa), and this is where the maximum buffer action can be obtained.

Buffer Capacity Increases as Concentration Increases

The buffer capacity of an acetic acid (sodium) buffer solution is larger at 100 mM concentration than at 10 mM, for example. However, precipitation occurs more readily at higher concentrations.

Beware of Salt Solubility and Precipitation

The salt solubility depends on the type of salt, such as potassium salt or sodium salt. Salts precipitate out more readily when an organic solvent is mixed in. In addition, avoid using buffer solutions based on organic acids (carboxylic acid) as much as possible for highly sensitive analysis at short UV wavelengths. Consider the various analytical conditions and use an appropriate buffer solution, such as an organic acid with a hydroxyl group at the α position to restrict the effects of metal impurity ions.
# Electric wiring and earthing

In the case of household wiring, the conductor itself is usually copper or aluminum (or copper-sheathed aluminum) and is either a solid metal conductor or stranded wire. Most wires in a home are insulated, meaning they are wrapped in a nonconductive plastic coating. Electricity moves through any metal conductor, such as a wire or the metal contacts and other components inside a switch or receptacle.

Electrical wiring is an electrical installation of cabling and associated devices such as switches, distribution boards, sockets, and light fittings in a structure. Wiring is subject to safety standards for design and installation. Allowable wire and cable types and sizes are specified according to the circuit operating voltage and electric current capability, with further restrictions on the environmental conditions, such as ambient temperature range, moisture levels, and exposure to sunlight and chemicals. Associated circuit protection, control, and distribution devices within a building's wiring system are subject to voltage, current, and functional specifications. Wiring safety codes vary by locality, country, or region. The International Electrotechnical Commission (IEC) is attempting to harmonise wiring standards among member countries, but significant variations in design and installation requirements still exist.

## Wiring codes

Wiring installation codes and regulations are intended to protect people and property from electrical shock and fire hazards. They are usually based on a model code (with or without local amendments) produced by a national or international standards organization, such as the IEC. The electrical current must move in a loop, or circuit. If the circuit is broken at any point, the flow of power stops. In general, copper and aluminium are used to make electrical wires and cables. However, you should only choose copper cables when choosing wires for your property. Additionally, select multi-stranded wires rather than single-stranded wires.

## Plug wiring colours

<table><thead><tr><td>Wire</td><td>Colour</td></tr></thead><tbody><tr><td>Live</td><td>Brown</td></tr><tr><td>Neutral</td><td>Blue</td></tr><tr><td>Earth</td><td>Yellow and Green</td></tr></tbody></table>

Wiring devices provide proper control and connections in your electrical devices and wiring. Wiring devices are needed for efficient, reliable, and safe electrical wire connections
in your home or business. They can protect circuits and prevent fire and shock hazards. A conduit wiring system is best for domestic and commercial installations. It provides proper protection to the installation against shock, fire hazards mechanical damage. Protected from external damage due to rodents, short circuit. Conduit is durable and strong, can last for a long time. Hot wire is used as the initial power feed to a circuit. It carries the current from the power source to the outlet. Acting as the first instance of a circuit, they are always carrying electricity, meaning it is dangerous to touch a hot wire while there is a power source feeding it.

## Wire Colour in India

Live wire is usually red in colour, the neutral wire is usually black in colour and the earth wire is usually green in colour. The electricity delivered to your home by the electric utility is at 120 and 240 volts. This is called "nominal voltage." This is the voltage as measured at the utility transformer outside your home. Nominal voltage will vary about plus or minus 5 percent under ideal conditions

### Three basic principles of wiring:

1. Keep the length of the wire to a minimum to avoid an unnecessary voltage drop on the conductor.

2. Connect wires solidly to panels and switchboards.

3. Size and select the type of wires to match the current-carrying requirements of the load.

## Building Blocks of the Circuit

* ✓ Rule 1 - Electricity will always want to flow from a higher voltage to a lower voltage.

* ✓ Rule 2 - Electricity always has work that needs to be done.

* ✓ Rule 3 - Electricity always needs a path to travel.

## Earthing

Earthing is used to protect you from an electric shock. It does this by providing a path (a protective conductor) for a fault current to flow to earth. It also causes the protective device (either a circuit-breaker or fuse) to switch off the electric current to the circuit that has the fault. An earthing system (UK and IEC) or grounding system (US) connects specific parts of an electric power system with the ground, typically the Earth's conductive surface, for safety and functional purposes. The choice of earthing system can affect the safety and electromagnetic compatibility of the installation.
# Instruments Used In Microbiology Lab with Principle and Uses

## List of Instruments used in Microbiology Lab

1. Analytical Balance

2. Autoclave

3. Bunsen burner

4. Centrifuge

5. Colony Counter

6. Deep Freezer

7. Homogenizer

8. Hot plate

9. Hot air oven

10. Incubator

11. Laminar Air Flow/ Laminar Hood

12. Magnetic Stirrer

13. Microscope

14. pH Meter

15. Spectrophotometer

16. Vortex Mixture/ Vortexer

17. Water Bath

18. Water Distiller

19. Culture tube

20. Test tube

21. Inoculation needle/rod/loop

22. Laboratory Heat Sealer

23. Vacuum pump

24. Microwave ovens

25. Lab incubators

26. Sand bath

27. Oil bath
# 1. Analytical Balance

An analytical balance is a type of balance that is commonly used for the measurement of mass in the sub-milligram range.

## Working Principle

<figure><img src="image_126.png" /></figure>

These types of balances are made with a measuring pan enclosed in a transparent covering that prevents small particles or air currents from getting collected on the pan.

<figure><img src="image_127.png" /></figure>

An electric analytical balance uses the force necessary to counteract the mass rather than measuring the mass itself.

<figure><img src="image_128.png" /></figure>

An electromagnet is used to create a force required to achieve a balance with the mass of the substance and the resulting force is displayed.

## Uses

* As they are highly precise and based on advanced technology, analytical balances are explicitly used in laboratories for the effective completion of tasks like weighing test materials and sampling amounts, formulation, density determination, purity analysis, quality control testing, and material and conformance testing

# 2. Autoclave

An autoclave is a pressurized chamber used for the process of sterilization and disinfection by combining three factors: time, pressure, and steam

## Working Principle

<figure><img src="image_129.png" /></figure>

Autoclaves use steam as their sterilization agent. The basic principle of an autoclave is that all the items within the autoclave come in direct contact with the steam for a particular period irrespective of the nature of the material- whether it is liquid, plastic ware, or glassware.

<figure><img src="image_130.png" /></figure>

The amount of time and the temperature depends on the type of material being sterilized and the increase in temperature of the cycle allows for shorter periods.

## Uses

* Autoclaves are mostly used for the sterilization of medical or laboratory equipment with the capacity of sterilizing a large number of materials at once.

* They are commonly used for the preparation of culture media during laboratory applications.

# 3. Bunsen burner

Bunsen burner is a standard tool used in laboratories, named after Robert Bunsen. It is a gas-fueled single open flame.
## Working Principle

<figure><img src="image_131.png" /></figure>

This burner is made with a metal tube on a flat base with a gas inlet at the bottom of the tube, which may have an adjustable valve. On the sides of the tube are openings that can be adjusted with a collar to control the amount of air that can enter.

<figure><img src="image_132.png" /></figure>

Once the burner is connected to a gas source, the gas is forced by the gas pressure so that the gas reaches the top where the flame is ignited with a match or a lighter.

## Uses

* It is commonly used for processes like sterilization, combustion, and heating. In medical or microbiology laboratories, it is commonly used for micro-loop sterilization.

## 4. Centrifuge

A centrifuge is a device that allows the rotation of an object about a single axis, where an outward force is applied perpendicularly to the axis. A laboratory centrifuge is motor-based and allows the rotation of a liquid sample resulting in the separation of the components of the mixture.

## Working Principle

<figure><img src="image_133.png" /></figure>

A centrifuge works on the principle of sedimentation, where the high speed of the rotation causes the denser particles to move away from the center while smaller, less dense particles are forced towards the center.

<figure><img src="image_134.png" /></figure>

Thus, the denser particles settle at the bottom while the lighter particles are collected at the top.

<figure><img src="image_135.png" /></figure>

In a laboratory tabletop centrifuge, the sample tubes are aligned at an angle so that the particles have to travel a shorter distance before they hit the bottom.

## Uses

* The primary application of a centrifuge is the separation of particles suspended in a suspension. It can be used for the separation of cell organelles, nucleic acid, blood components, and separation of isotopes.

## 5. Colony Counter

A colony counter is used to estimate the density of a liquid culture by counting the number of CFU (colony forming units) on an agar or culture plates. It is used for counting microorganism which is not visible by naked eye. The magnifying glass (convex lense) is fitted in this equipment.
## Working Principle

<figure><img src="image_136.png" /></figure>

This instrument can accommodate different sizes of plates which are scanned on top with UV, white light and/or fluorescent illumination.

<figure><img src="image_137.png" /></figure>

One can accomplish the counting either manually with the touch pressure or with a digital counter.

## Uses

* A colony counter is primarily used for counting the number of colonies present on a culture plate to estimate the concentration of microorganisms in liquid culture.

# 6. Deep Freezer

## Working Principle

<figure><img src="image_138.png" /></figure>

Deep freezers are based on the principle that under extremely low temperatures, there is **minimum microbial growth** which allows for the protection and preservation.

<figure><img src="image_139.png" /></figure>

Based on this principle, we can even preserve cultures over a long period of time without any change in the concentration of the microorganisms.

## Uses

* A deep freeze can be used for the preservation of different things used in the laboratories for a very long period of time. Deep freezers are used in laboratories to store and preserve isolated microbes for a more extended period of time.

# 7. Homogenizer

Homogenizer is a device used in laboratories for the mixing of various liquids and materials like tissue, plant, food, soil, and many others.

## Working Principle

<figure><img src="image_140.png" /></figure>

This instrument is based on the principle that when large globules in coarse emulsion are passed under high pressure through a narrow orifice, they break down into smaller particles giving a more uniform and stable mixture.

<figure><img src="image_141.png" /></figure>

A homogenizer has a metal rod with narrow parallel openings in the form of a comb at the end which acts as the orifice for the homogenization process.

## Uses

* A homogenizer is primarily used to disrupt cells to acquire cell organelles for different microbiological processes.

* It is used in the preparation step before the extraction and purification of different macromolecules like proteins, nucleic acids, and lipids.
## 8. Hot plate

A hot plate is a stand-alone appliance used in microbiology laboratories as a table-top heating system.

### Working Principle

- Unlike the traditional ways of producing heat through fire, a hot plate produces heat by the flow of electricity.

- On a hot plate, electricity runs through the coils which have a high level of electrical resistance. The resistance in the coils converts the electrical energy into heat energy which causes the coils to release heat.

### Uses

- In a laboratory, hot plates are used to heat glassware and its components.

- They are used over water baths as water baths might be hazardous in case of any spills or overheating.

## 9. Hot air oven

A hot air oven is an electrical device that is used for sterilization of equipment, dried glassware or samples using dry heat.

### Working Principle

- A hot air oven is a type of dry heat sterilization which is performed on dry materials and on substances that do not melt or catch fire under high temperatures.

- There are **two types of hot air ovens** based on the working principle

- **Forced air hot air oven:** In this type of hot air oven, the heated air inside the oven is distributed throughout the oven with a fan. This prevents the rising of hot air towards the top while keeping the cold air at the bottom. This allows for the adequate heating of materials inside the oven.

- **Static air hot air oven:** In this type of oven, the heat is produced by coils present at the bottom of the oven with no fan. The hot air rises and doesn't allow the effective sterilization of the materials.

- The equipment inside the oven acquires heat and passes the heat towards the center, one layer at a time which allows for effective dry heat sterilization.

### Uses

- A hot air oven can be used to sterilize materials like glassware, metal equipment, powders, etc.

- It allows for the destruction of microorganisms as well as bacterial spores.
## 10. Incubator

An incubator is a device used in laboratories for the growth and maintenance of microorganisms and cultures. Incubator provides an optimal temperature, pressure, moisture, among other things required for the growth of microorganisms.

### Working Principle

- The incubator is based on the principle of maintaining a proper atmosphere for the growth of microorganisms.

- Incubators have a heating system that allows for the temperature within the incubator to be adjusted according to the type of organism allowed to grow inside.

- Similarly, they are provided with adjustments for maintaining the concentration of CO₂ to balance the pH and humidity required for the growth of the organisms.

- Variation of the incubator like a **shaking incubator**, which allows for the continuous movement of the culture required for cell aeration and solubility studies.

### Uses

- Incubators have a wide range of applications including cell culture, pharmaceutical studies, hematological studies, and biochemical studies.

- Incubators can also be used in the steam cell research area.

## 11. Laminar Air Flow/ Laminar Hood

Laminar Hood is a closed device primarily for processes or instruments sensitive to microbial contamination.

### Working Principle

- A Laminar Hood is made up of stainless steel, avoiding joints and corners to prevent the accumulation of bacterial spores.

- This device creates a sterile environment with the flow of sterile air through a **High-Efficiency Particulate Air (HEPA) filter** and shortwave **ultraviolet germicidal lamp** that sterilizes the workstation.

- Laminar Air Flow has to **turn on 15 minutes before to ensure complete sterilization** and the workstation should be **cleaned with ethanol** before and after use.

### Uses

- Laminar Hood is used to conduct processes that are sensitive to contamination.
* It is used for experiments related to isolation of microorganism, plant tissue culture and for the experiments of genetic transformation.

## 12. Magnetic Stirrer/culture tube stirrer

Magnetic Stirrer is a device used in microbiology laboratories for the purpose of mixing the liquids/ or the material which is present in culture tube.

### Working Principle

<figure><img src="image_142.png" /></figure>

This device consists of a rotating magnetic or an electromagnet creating a rotating magnetic field that allows the stir bar (a piece of heavy metal) to move around in the vessel.

<figure><img src="image_143.png" /></figure>

It is coupled with a heating system to heat the liquid while it is mixed.

### Uses

* It is usually used for mixing various liquid components in a mixture in a chemical or microbiology laboratory.

* This device is used in place of other stirrers as it is noise-free and because the size of the stir bar is so tiny, there is less chance of contamination.

<figure><img src="image_144.png" /></figure>

## 13. Microscope

Microscopes are devices that allow the observer to have an exceedingly close view of minute particles.

### Working Principle

<figure><img src="image_145.png" /></figure>

There are different types of microscopes, each of which works on its respective principles. However, there is some commonality in them.

<figure><img src="image_146.png" /></figure>

The basic principle in a microscope is magnification. Based on the relative position of the object from the lens or electromagnets, different positions, nature, and magnification of the image can be achieved.
<figure><img src="image_147.png" /></figure>

Different types of microscopes are developed to cater to the specific needs of the observation. However, the common theme is magnification.

## Uses

* Based on the type of microscopes, different microscopes are used for different purposes.

* They are primarily used for the observation of minute particles which cannot be observed with naked eyes.

# 14. pH Meter

A pH meter is a device used in laboratories that measure the H-ion concentration in water-based solutions to determine the acidity or alkalinity of the solution. A **pH meter** is often termed a "potentiometric pH meter" as it **measures the difference** in electric potential between the **reference** and a **pH electrode**.

## Working Principle

<figure><img src="image_148.png" /></figure>

In a potentiometric pH meter, single or multiple glass electrodes, connected to a bulb selective to hydrogen ions, are attached to a metal rod.

<figure><img src="image_149.png" /></figure>

When the bulb with the electrodes is dipped into a solution, hydrogen ions in the solution exchange with positive charges on the electrode generating an electrochemical potential which is displayed in terms of pH units on display.

## Uses

* A pH meter is primarily used to measure the acidity of microbial cultures, soil, and water treatment plant.

* It can be used to measure the acidity level in wine and cheese during their production.

# 15. Spectrophotometer

The spectrophotometer is an optical instrument for measuring the intensity of light in relation to wavelength. Based on the amount of light absorbed by a coloured solution, a quantitative analysis of the solution can be done.

## Working Principle

<figure><img src="image_150.png" /></figure>

Spectrophotometry is based on the **Beer-Lambert Law**, which states the absorbance of light by a solution (of a particular wavelength) is directly proportional to the concentration of the substance.

<figure><img src="image_151.png" /></figure>

Different wavelengths of light are passed through a solution as different substances have better absorbance at different wavelengths. Based on the absorbance of a particular wavelength, the quantitative analysis of a solution can be done.
## Uses

* In a microbiology laboratory, a spectrophotometer is applied for the measurement of the substance concentration of protein, nucleic acids, bacterial growth, and enzymatic reactions.

# 16. Vortex Mixture/ Vortexer

A vortex mixture is one of the basic technologies used for the **mixing of samples in glass tubes or flasks** in laboratories.

## Working Principle

<figure><img src="image_152.png" /></figure>

It is based on the simple principle of causing reactions and homogenization by agitating the mixture.

<figure><img src="image_153.png" /></figure>

Motorized draft shafts present on the mixer oscillate and transfer the movement to the sample tubes causing the sample fluids to undergo turbulent flow.

## Uses

* Vortex mixer is mostly used for the mixing of various sample fluids in the sample tubes and also allows for the homogenization of cells and cell organelles.

# 17. Water Bath

Water Bath is a conventional device that is used for chemical reactions that required a controlled environment at a constant temperature.

## Working Principle

<figure><img src="image_154.png" /></figure>

A sensor in the device transfers water temperature to a reference value which is then amplified and a control system generates a signal for the heating system which heats the water to the desired temperature.

## Uses

* Water baths are primarily used for heating samples under a controlled temperature.

* These are suitable for heating chemicals that might be flammable under direct ignition.

<figure><img src="image_155.png" /></figure>

# 18. Water Distiller
A water distiller is a device that purifies water by the process of distillation. This is commonly used in, microbiology laboratories, organic chemistry laboratories and medical laboratories.

## Working Principle

<figure><img src="image_156.png" /></figure>

A water distiller is based on the principle of distillation.

<figure><img src="image_157.png" /></figure>

According to this process, water is first brought to a boil and then condensed into liquid form to obtain pure distilled water.

## Uses

* It is used to obtain distilled water required for lab tests as well as for the preparation of culture media.

## 19. Culture Tube

Culture tubes are often used in microbiology lab for handling and culturing all kinds of live organisms, such as molds, bacteria, seedlings, plant cuttings, etc. It is also used to store samples and other fluids. Mouth of tube is plain cut because rounded mouth causes problems in removing cotton plug of tube.

<figure><img src="image_158.png" /></figure>

## 20. Test Tube

Test tubes are widely used by chemists to handle chemicals, especially for qualitative experiments and assays. Their spherical bottom reduces mass loss when pouring, make them easier to wash out, and allow convenient monitoring of the contents.

<figure><img src="image_159.png" /></figure>

## 21. Inoculation needle/rod/loop

An inoculation needle is equipment used in the lab of microbiology. It is used in the transfer of microbial organisms from plate culture to needle by first sterilizing the needle to
prevent contaminations. Inoculation needle is made up of **Nichrome alloy** {Nickel, Chromium, Manganese & Fe} which is heat resistant.

An inoculation loop, also known as a **smear loop** is a simple tool, primarily used by microbiologists **to take and transfer inoculum of a microorganism culture.**

## 22. Laboratory Heat Sealer

This instrument is used to seal the **pouches, plastic bags, sachets** that are used in packaging of biofertilizer. The apparatus is used to ensure the intactness or band of the plastic bags and other packaging bags that are used to pack different consumable as well as non-consumable goods.

## 23. Vacuum pump

Vacuum pumps are mechanical devices that enable the removal of air and gas molecules from a sealed area to create an area devoid of air or gas, their purpose is to clean and seal. Vacuum pumps come in wet or dry variants depending on the media being pumped through them.

## 24. Microwave ovens

Microwave ovens are used for **heating and defrosting in laboratories**. Improper use of a microwave can pose a number of hazards including: Ignition of flammable vapors. Electrical shock from ungrounded or faulty units. Microwave ovens used in laboratories for the rapid heating of material-either to dry them completely or to subject a work piece to sudden thermal stress or electric field stress. **Determinations of the moisture content in soil or leaf tissue samples, for example, can be made within tens of seconds** rather than hours. It is often assumed that placing a load within a microwave oven will result in it being heated evenly as well as quickly.

## 25. BOD incubators

BOD (Biological Oxygen Demand) incubators are essential equipment in the laboratory that provides a temperature-controlled environment to support growth of microbiological cultures. **Here the materials kept in desired temperature for desired period of time.**

## 26. Sand bath

A sand bath is common laboratory equipment made from a container filled with heated sand. It is used **to provide even heating for another container during a chemical reaction.**
<figure><img src="image_160.png" /></figure>

## 27. Oil bath

An oil bath is a type of heated bath used in a laboratory, most commonly used to heat up chemical reactions. It's essentially a container of oil that is heated by a hot plate or (in rare cases) a Bunsen burner. It is used in laboratories for reactions that require heating temperatures up to 200 degrees Celsius. Oil baths provide uniform heat compared to other heaters. An oil bath is a heating device which uses boiling oil as the temperature regulator. Since different oils have different boiling points, it is possible to obtain a temperature near to the desired temperature by selecting oil with a boiling point as close as possible to the desired temperature.

<figure><img src="image_161.png" /></figure>
# Working principle, different Parts, their Functions and precautions of Hot Air oven and Auto clave

## 1. Hot air oven:

<figure><img src="image_162.png" /></figure>

A hot air oven is an instrument that is used to heat up the dried glassware at a uniform temperature.

<figure><img src="image_163.png" /></figure>

A hot air oven is used to sterilize the dried glassware at 180-200 °C temperature.

<figure><img src="image_164.png" /></figure>

The temperature range of a hot air oven is controlled by using a temperature regulator.

* Kept the material for a period of 3-4 hour.

* Hot air oven also known as **forced air circulating oven**.

* The forced air circulation provided by the oven ensures the temperature uniformity throughout the oven

* We can sterilize Glassware (like petri dishes, flasks, pipettes, test tubes and culture tube), Powder (like starch, zinc oxide, and sulfadiazine) and Materials that contain oils, Metal equipment (like scalpels, scissors, and blades) by using hot air oven.

* The process of dry heat sterilization using a hot air oven Originally developed by Louis Pasteur

<figure><img src="image_165.png" /></figure>

To destroy microorganisms and bacterial spores, a hot air oven provides extremely high temperatures over several hours.

<figure><img src="image_166.png" /></figure>

In a hot air oven first, the surface of the material is sterilized then the temperature slowly enters the centre of the item.

## Working Principle of Hot air oven

<figure><img src="image_167.png" /></figure>

Sterilization by dry heat is performed by conduction. The temperature is consumed by the surface of the objects, then moves towards the core of the object, coating by coating. The whole object will ultimately attain the temperature needed for sterilization to take place. Dry heat causes most of the injury by oxidizing particles. The primary cell components are damaged and the organism dies. The temperature is kept for about an hour to eliminate the most ambitious of the resistant spores.

## Dry Sterilisation

<figure><img src="image_168.png" /></figure>

It is originally used in a hot air oven. This method is proper for items like glassware, powders, oil containing materials, metal material. Hot air oven acts in a manner so that items stored inside the oven don't grab fire or meltdown. It primarily acts on the
principle of structure where heat moves on the outside of the substance and then to the core of the item. It is termed a dry sterilization process because the procedure is performed by employing the hot air.

<figure><img src="image_169.png" /></figure>

While the air shifts hot, it becomes thinner and transfers towards the ceiling of the chamber. If air hits the roof of the chamber it travels towards the ground of the chamber. It helps in the circulation of air within the chamber. This current flow assures the proper and uniform heating everywhere in the chamber. This is approaching a longer method than autoclaving or moist sterilization.

## Hot air oven Parts and Functions

1. **External cabinet:** The External cabinet is made of stainless sheets. It covers the inner chamber.

2. **Glass wool insulation:** The space between the inner chamber and external cabinet is filled with Glass wool. It provides insulation to the hot air oven.

3. **Inner chamber:** The inner chamber of the hot air oven is made of Stainless steel.

4. **Tubular air heaters:** They help to generate heat within the inner chamber. Two Tubular air heaters are located on both sides of the inner chamber.

5. **Motor-driven blower:** It helps in uniformly circulating the air within the chamber.

6. **Temperature sensor:** It measures the temperature within the hot air oven and displays it on the controller screen.

7. **Tray slots:** The inner wall of the chamber contains several try slots that hold the trays.

8. **PID temperature controller:** It maintains the accurate temperature during the entire cycle. It also controls the temperature and also displays the temperature values.

9. **Load indicator:** It indicates the hot air oven is overloaded.

10. **Mains on/off switch:** It helps to turn on/ turn off the hot air oven.

11. **Safety thermostat:** It is also known as an over-temperature protection device. It keeps your oven and specimen safe in case of controller malfunction.

<figure><img src="image_170.png" /></figure>
## 2. AUTO CLAVE

An autoclave is a machine that provides a **physical method of sterilization** by killing bacteria, viruses, and even spores present in the material put inside of the vessel using steam under pressure. Autoclave sterilizes the materials by heating them up to a particular temperature for a specific period of time. The autoclave is also called a steam sterilizer. The autoclave is considered a more effective method of sterilization as it is based on moist heat sterilization.

<figure><img src="image_171.png" /></figure>

Autoclave Parts/ Components

### a. Pressure Chamber

* The pressure chamber is the main component of a steam autoclave consisting of an inner chamber and an outer jacket.

* The inner chamber is made up of stainless steel or gunmetal, which is present inside the outer chamber made up of an iron case.

* The autoclaves used in soil microbiology laboratories have an outer jacket that is filled with steam to reduce the time taken to reach the sterilization temperature.
* The inner chamber is the case where the materials to be sterilized are put.

* The size of the pressure chamber ranges from 100 L to 3000 L.

## b. Lid/ Door

* The next important component of an autoclave is the lid or door of the autoclave.

* The purpose of the lid is to seal off the outside atmosphere and create a sterilized condition on heat inside of the autoclave.

* The lid is made airtight via the screw clamps and asbestos washer.

* The lid consists of various other components like:

### Pressure gauge

o A pressure gauge is present on the lid of the autoclave to indicate the pressure created in the autoclave during sterilization.

o The pressure gauge is essential as it assures the safety of the autoclave and the working condition of the operation.

### Pressure releasing unit/ Whistle

o A whistle is present on the lid of the autoclave is the same as that of the pressure cooker.

o The whistle controls the pressure inside the chamber by releasing a certain amount of vapor by lifting itself.

### Safety valve

o A safety valve is present on the lid of the autoclave, which is crucial in cases where the autoclave fails to perform its action or the pressure inside increases uncontrollably.

o The valve has a thin layer of rubber that bursts itself to release the pressure and to avoid the danger of explosion.

## c. Steam generator/ Electrical heater

* An electrical steam generator or boiler is present underneath the chamber that uses an electric heating system to heat the water and generate steam in the inner and the outer chamber.

* The level of water present in the inner chamber is vital as if the water is not sufficient; there are chances of the burning of the heating system.

* Similarly, if the water is more than necessary, it might interfere with the trays and other components present inside the chamber.
### d. Vacuum generator (if applicable)

* In some types of autoclaves, a separate vacuum generator is present which pulls out the air from the inside of the chamber to create a vacuum inside the chamber.

* The presence of some air pockets inside the chamber might support the growth of different microorganisms. This is why the vacuum chamber is an important component of an autoclave.

### e. Wastewater cooler

* Many autoclaves are provided with a system to cool the effluent before it enters the draining pipes.

* This system prevents any damage to the drainage pipe due to the boiling water being sent out of the autoclave.

* The autoclave works on the principle of moist heat sterilization where steam under pressure is used to sterilize the material present inside the chamber.

* The high pressure increases the boiling point of water and thus helps achieve a higher temperature for sterilization.

* Water usually boils at 100°C under normal atmospheric pressure (760 mm of Hg); however, the boiling point of water increases if the pressure is to be increased.

* Similarly, the high pressure also facilitates the rapid penetration of heat into deeper parts of the material, and moisture present in the steam causes the coagulation of proteins causing an irreversible loss of function and activity of microbes.

* This principle is employed in an autoclave where the water boils at 121°C at the pressure of 15 psi or 775 mm of Hg.

* When this steam comes in contact with the surface, it kills the microbes by giving off latent heat.

* The condensed liquid ensures the moist killing of the microbes.

* Once the sterilization phase is completed (which depends on the level of contamination of material inside), the pressure is released from the inside of the chamber through the whistle.

* The pressure inside the chamber is then restored back to the ambient pressure while the components inside remain hot for some time.
## Procedure for running an autoclave

In general, an autoclave is run at a temperature of 121° C for at least 30 minutes by using saturated steam under at least 15 psi of pressure. The following are the steps to be followed while running an autoclave:

1. Before beginning to use the autoclave, it should be checked for any items left from the previous cycle.

2. A sufficient amount of water is then put inside the chamber.

3. Now, the materials to be sterilized are placed inside the chamber.

4. The lid is then closed, and the screws are tightened to ensure an airtight condition, and the electric heater is switched on.

5. The safety valves are adjusted to maintain the required pressure in the chamber.

6. Once the water inside the chamber boils, the air-water mixture is allowed to escape through the discharge tube to let all the air inside to be displaced. The complete displacement can be ensured once the water bubbles cease to come out from the pipe.

7. The drainage pipe is then closed, and the steam inside is allowed to reach the desired levels (15 lbs in most cases).

8. Once the pressure is reached, the whistle blows to remove excess pressure from the chamber.

9. After the whistle, the autoclave is run for a holding period, which is 15 minutes in most cases.

10. Now, the electric heater is switched off, and the autoclave is allowed to cool until the pressure gauge indicates the pressure inside has lowered down to that of the atmospheric pressure.

11. The discharge pipe is then opened to allow the entry of air from the outside into the autoclave.

12. Finally, the lid is opened, and the sterilized materials are taken out of the chamber.

## Uses of Autoclave

Autoclaves are important devices to ensure the sterilization of materials containing water as they cannot be sterilized by dry heat sterilization. Besides, autoclaves are used for various other purposes.

✓ They are used to decontaminate specific biological waste and sterilize media, instruments and labware.
✓ Regulated waste that might contain bacteria, viruses, and other biological materials is recommended to be inactivated by autoclaving before disposal.

✓ Autoclaves are used to sterilize equipment, glassware, surgical equipment and medical wastes.

✓ Similarly, autoclaves are used for the sterilization of culture media, autoclavable containers, plastic tubes and pipette tips.

## Precautions

Although autoclaves are pretty simple to use, there are certain rules of precautions to be followed while operating an autoclave. Some of the important precautions to be followed while running an autoclave are:

1. Autoclaves should not be used to sterilize water-proof or water-resistant substances like oil or powders.

2. The autoclave should not be overcrowded, and the materials should be loaded in a way that ensures sufficient penetration of articles by the steam.

3. The items to be autoclaved should always be placed in a secondary container.

4. Only autoclavable bags are to be used to autoclave packaged waste.

5. To ensure sufficient penetration, articles should be wrapped in something that allows penetration by steam, and materials like **aluminum foils should not be used**.

6. The items placed inside the chamber should not touch the sides or top of the chamber.

7. The wastes and clean items should be autoclaved separately.

8. Attempts to open the lid when the autoclave is working should never be made.

9. Liquid components should never be autoclaved in sealed containers.

10. The liquid inside the containers should only be filled to a specific volume fraction of the total volume to prevent the spilling of the liquid.

11. Plastic or polyethylene trays or containers should not be used as they might melt and damage the autoclave.

12. Besides, never autoclave flammable, reactive, corrosive, toxic, or radioactive materials, household bleach, or paraffin-embedded tissue.

13. The paper should not be placed directly inside an autoclave as it is a combustible substance. It should be autoclaved in a waste bag on a bio bag setting to prevent fire.
# Preparation of Media, Types and Sterilization of Culture Media

## 1. Media

The media used in the laboratory have to be chosen to suit the nutritional requirements of the species of organism to be grown.

**Growth Media:** Media designed specifically to allow for the growth of microorganisms.

### A. On Consistency:

1. **Solid Media:** Solid media is used for the isolation of bacteria as pure culture. Advantages of solid media:

* Bacteria may be identified by studying the colony character

* Mixed bacteria can be separated

* **Agar:** is most commonly used to prepare solid media. Agar is **polysaccharide extract** obtained from seaweed.

* Agar is an **ideal solidifying agent** as it is:

* (a) Bacteriologically inert, i.e. no influence on bacterial growth,

* (b) It remains solid at 37°C, and

* (c) It is transparent.

2. **Liquid Media:** It is used for profuse growth, e.g. blood culture in liquid media. Here mixed organisms cannot be separated.

### B. On Chemical Composition

1. Routine Laboratory Media

2. Synthetic Media. These are chemically defined media prepared from pure chemical substances. It is used in research work.

**Routine Laboratory Media:** These are classified into six types:

(1) Basal media

(2) Enriched media

(3) Selective

(4) Indicator media

(5) Transport media and

(6) Storage media.

1. **BASAL MEDIA.** Basal media are those that may be used for growth (culture) of bacteria that do not need enrichment of the media.
2. **ENRICHED MEDIA.** The media are enriched usually by adding blood, serum or egg. Examples: Enriched media are blood agar and Lowenstein-Jensen media. Streptococci grow in blood agar media.

3. **SELECTIVE MEDIA.** These media favours the growth of a particular bacterium by inhibiting the growth of undesired bacteria and allowing growth of desirable bacteria. Examples: MacConkey agar and Lowenstein-Jensen media. Antibiotic may be added to a medium for inhibition.

4. **INDICATOR (DIFFERENTIAL) MEDIA.** An indicator is included in the medium. A particular organism causes change in the indicator, e.g. blood, neutral red, tellurite. Examples: Blood agar and MacConkey agar are indicator media.

5. **TRANSPORT MEDIA.** These media are used when speciemen cannot be cultured soon after collection. Examples: Cary-Blair medium, Amies medium, Stuart medium.

6. **STORAGE MEDIA.** Media used for storing the bacteria for a long period of time. Examples: Egg saline medium, chalk cooked meat broth.
# STERILIZATION OF CULTURE MEDIA

* The constituents of culture media, water and containers contribute to the contamination by vegetative cells and spores.

* The media must be free from contamination before use in fermentation.

* Sterilization of the media is most commonly achieved by applying heat and to a lesser extent by other means (physical methods, chemical treatment, and radiation).

## 1. HEAT STERILIZATION:

* Heat is the most widely used and the most useful method for the sterilization technique of nutrient media

* A number of factors influence the success of heat sterilization.

* The number and type of microorganisms, the composition of the media and its pH and size of the suspended particles are the important factors that influence the success of heat sterilization.

* Vegetative cells are rapidly eliminated at relatively low temperatures.

* In general, vegetative cells are destroyed at a lower temperature in a short time (around 60°C in 5-10 minutes).

* However, the destruction of spores requires higher temperature and relatively longer time (around 80°C for 15-20 minutes).

* Spores of *Bacillus stearothermophilus* are the most heat resistant.

* In fact, this organism is exploited for testing the sterility of fermentation equipment.

* Therefore they are used as assay organisms for testing the various procedures used to sterilize equipment.

<table><caption>Table: sterilization time and sterilization temperature of various groups of organisms</caption><thead><tr><td>Cells</td><td>Sterilization time min</td><td>Sterilization temperature °C</td></tr></thead><tbody><tr><td>Vegetative cells</td><td>5-10</td><td>60</td></tr><tr><td>Fungus spores/yeast spores</td><td>15</td><td>80</td></tr><tr><td><i>Streptomyces</i> spores</td><td>5-10</td><td>60-80</td></tr><tr><td>Bacterial spores (general)</td><td>5</td><td>121</td></tr><tr><td>Spores of <i>Bacillus stearothermophilus</i></td><td>15</td><td>121</td></tr></tbody></table>
## 2. Radiation (UV, X rays or γ rays):

* Although occasionally used in the food industry, these agents are not used in industrial fermentation.

## 3. Chemical methods:

* Although a number of chemical disinfectants are known, they cannot be used to sterilize nutrient media because there is a risk that inhibition of the fermentation organism could occur from the residual chemical.

* Note: The chemical methods (by using disinfectants) and radiation procedures (by using UV rays, γ rays, X-rays) are not commonly used for media sterilization.

## 4. Mechanical removal of organisms (Physical methods):

* The physical methods such as filtration, centrifugation, and adsorption (to ion-exchangers or activated carbon) are in use.

* Among these, filtration is the only method in practical use.

* Filter sterilization is often used for all components of nutrient solutions which are heat sensitive and would thus be denatured through the steam sterilization process normally used in industrial fermentations.

* Certain constituents (vitamins, blood components, antibiotics) of culture media are heat labile and therefore, are destroyed by heat sterilization.

* Such components of the medium are completely dissolved (absolutely essential or else they will be removed along with microorganisms) and then subjected to filter sterilization.

* Deep filters (plate filters) are sometimes used to filter complex nutrient solutions.

### Two main limitations of filtration technique:

1. Application of high pressure in filtration is unsuitable or undesirable for industrial practice.

2. Certain components of the media may be absorbed on filter material from the media during filtration.

* One approach which is cost effective is the filtration of just the water which is to be used in the preparation of the culture medium.

* Sometimes, a combination of filtration and heat sterilization is applied.

* For instance, the water used for media preparation is filtered while the concentrated nutrient solution is subjected to heat sterilization.

* The filtered water is now added for appropriate dilution of the media.
* For example, in steroid bioconversion processes, a concentrated nutrient solution is sterilized by heat in the fermenter and is then diluted to the normal concentration with water which has been filter sterilized.

<figure><img src="image_172.png" /></figure>
# Isolation, and Streaking Used in Microbiology Lab

## Isolation

Separation of desirable microorganism from whole microbial population is called isolation.

## Streaking

Transfer of pure culture from the slant of one culture to the slant of another culture is called streaking.

*Streak* literally means “*a long, thin line*”: and the streak plate method is a microbiological culture technique where a sample is spread in a petri dish in the form of a long, thin line over the surface of solid media.

## Types of Streak Plate Method

* **Quadrant Streaking**: It is the most commonly used and the most preferred method where four equal-sized sections of the agar plate are streaked.

* **T-Streaking**: It is another method of streaking where the agar Petri plate is divided into three sections and each section is streaked. Hence, this method is also known as the "three-sector streak" method.

* **Continuous Streaking**: It is another commonly followed method where an inoculum is evenly distributed in a single continuous movement from starting point to the center of the plate. There is no need to divide the plate and sterilize the loop during the process. It is easy and quick.

* **Radiant Streaking**: It is another method of streaking where the inoculum is first streaked at one edge and spread in vertical lines above the edge. Finally, the vertical lines are cross streaked diagonally. This method is suitable to propagate pure culture, and also in the case of a dilute specimen.

* **Semi-quantitative Streaking**: It is routinely followed in urine culture. It is a modified form of continuous streaking. In this method, a calibrated loop (usually a loop of 1 or 2µl) is used to streak a certain volume of the liquid specimen. A loopful of the specimen is streaked in a horizontal line in the middle of the Petri plate, and the specimen is spread all over the plate in a single continuous back and forth movement. This method allows us to approximately quantify the viable load (in a range, not an exact number) as well as get the pure culture in a single go.
o **Zigzag Streaking**: It is another form of continuous streaking where a loopful of the specimen is streaked all over the plate in a zigzag pattern in a single continuous movement. It is commonly done to propagate the pure culture and culture them in large quantities.

Streak Plate Method

<figure><img src="image_173.png" /></figure>
# Compositions of Different Media

## (i) Media for total bacterial count; Thornton's Medium (1922)

<table><thead><tr><td>Contents</td><td>weight</td></tr></thead><tbody><tr><td>K<sub>2</sub>HPO<sub>4</sub></td><td>1.00g</td></tr><tr><td>KNO<sub>3</sub></td><td>0.50 g</td></tr><tr><td>MgSO<sub>4</sub>·7 H<sub>2</sub>O</td><td>0.20 g</td></tr><tr><td>CaCl<sub>2</sub>·6H<sub>2</sub>O</td><td>0.10 g</td></tr><tr><td>NaCl</td><td>0.10 g</td></tr><tr><td>FeCl<sub>3</sub>·6 H<sub>2</sub>O</td><td>Trace</td></tr><tr><td>Asparagine</td><td>0.50 g</td></tr><tr><td>Mannitol</td><td>1.00 g</td></tr><tr><td>Agar</td><td>15.0 g</td></tr><tr><td>Distilled water</td><td>1000 mL</td></tr></tbody></table>

## (ii) Media for total fungal count; Rose-bengal Agar (Martin, 1950)

<table><thead><tr><td>Contents</td><td>weight</td></tr></thead><tbody><tr><td>Glucose</td><td>10.0 g</td></tr><tr><td>Peptone</td><td>5.0 g</td></tr><tr><td>KH<sub>2</sub>PO<sub>4</sub></td><td>1.0 g</td></tr><tr><td>MgSO<sub>4</sub>·7H<sub>2</sub>O</td><td>0.05 g</td></tr><tr><td>Streptomycin</td><td>30.0 mg</td></tr><tr><td>Agar</td><td>15.0 g</td></tr><tr><td>Rose-bengal</td><td>0.035g</td></tr><tr><td>Distilled water</td><td>1000 mL</td></tr></tbody></table>

## (iii) Media for total actinomycetes count

### Kenknight and Munaier's medium (Subba Rao, 2003)

<table><thead><tr><td>Contents</td><td>weight</td></tr></thead><tbody><tr><td>Dextrose</td><td>1.0 g</td></tr><tr><td>KH<sub>2</sub>PO<sub>4</sub></td><td>0.10g</td></tr><tr><td>NaNO<sub>3</sub></td><td>0.10 g</td></tr><tr><td>KCl</td><td>0.10 g</td></tr><tr><td>MgSO<sub>4</sub>·7H<sub>2</sub>O</td><td>0.10 g</td></tr><tr><td>Agar</td><td>15.0 g</td></tr><tr><td>Distilled water</td><td>1000 mL</td></tr></tbody></table>
(iv) Media for isolation of Azotobacter Jensen's Medium, (Lipman, 1904)

<table><thead><tr><td>Contents</td><td>Weight (g L<sup>-1</sup>)</td></tr></thead><tbody><tr><td>Sucrose</td><td>20.0 g</td></tr><tr><td>K<sub>2</sub>HPO<sub>4</sub></td><td>1.0 g</td></tr><tr><td>MgSO<sub>4</sub>·7H<sub>2</sub>O</td><td>0.50 g</td></tr><tr><td>NaCl</td><td>0.50 g</td></tr><tr><td>FeSO<sub>4</sub></td><td>0.10 g</td></tr><tr><td>Na<sub>2</sub>MoO<sub>4</sub>·2H<sub>2</sub>O</td><td>0.005 g</td></tr><tr><td>CaCO<sub>3</sub></td><td>2.0 g</td></tr><tr><td>Agar</td><td>15.0 g</td></tr><tr><td>Distilled water</td><td>1000 mL</td></tr></tbody></table>

pH adjusted to 7.0 ± 7.2

The pH of the medium was adjusted to 6.0 to 6.2 with H<sub>2</sub>SO<sub>4</sub> (5 % solution) before it was sterilized.

(v) Media for isolation of Azospirillum

(Semi solid medium; Bulow and Dobereiner, 1975)

<table><thead><tr><td>Contents</td><td>Weight (g L<sup>-1</sup>)</td></tr></thead><tbody><tr><td>K<sub>2</sub>HPO<sub>4</sub></td><td>0.10 g</td></tr><tr><td>KH<sub>2</sub>PO<sub>4</sub></td><td>0.40 g</td></tr><tr><td>MgSO<sub>4</sub></td><td>0.20 g</td></tr><tr><td>NaCl</td><td>0.10 g</td></tr><tr><td>CaCl<sub>2</sub></td><td>0.02 g</td></tr><tr><td>FeCl<sub>3</sub></td><td>0.01 g</td></tr><tr><td>Na<sub>2</sub>MoO<sub>4</sub></td><td>0.002 g</td></tr><tr><td>Sod. Malate</td><td>5.0 g</td></tr><tr><td>Bromothymcol Blue (0.05% Ethanol)</td><td>5.0 ml</td></tr><tr><td>Agar</td><td>1.75 g·0 g</td></tr><tr><td>Distilled water</td><td>1000 mL</td></tr></tbody></table>

The pH of the medium was adjusted to 6.80
(vi) Media for isolation of Acetobacter; LGI medium, (Lipman, 1904)

<table><thead><tr><td>Contents</td><td>Weight (g L<sup>-1</sup>)</td></tr></thead><tbody><tr><td>Sucrose</td><td>5.0 g</td></tr><tr><td>K<sub>2</sub>HPO<sub>4</sub></td><td>0.20 g</td></tr><tr><td>KH<sub>2</sub>PO<sub>4</sub></td><td>0.60 g</td></tr><tr><td>CaCl<sub>2</sub>·2H<sub>2</sub>O</td><td>0.02 g</td></tr><tr><td>MgSO<sub>4</sub>·7H<sub>2</sub>O</td><td>0.20 g</td></tr><tr><td>Na<sub>2</sub>MoO<sub>4</sub>·2H<sub>2</sub>O</td><td>0.002 g</td></tr><tr><td>FeEDTA (solution 16.4 g L<sup>-1</sup>)</td><td>4 mL</td></tr><tr><td>Bromothymol blue</td><td>(5 g L<sup>-1</sup> in 0.2 N KOH) 5 mL</td></tr><tr><td>Vitamin solution</td><td>1 mL</td></tr><tr><td>For semisolid medium- agar</td><td>add 1.6 to 1.80 agar L<sup>-1</sup></td></tr><tr><td>For solid medium- Agar</td><td>15.0 g</td></tr><tr><td>Distilled water</td><td>1000 mL</td></tr></tbody></table>

pH adjusted to 6.0 ± 6.2

The pH of the medium was adjusted to 6.0 to 6.2 with H<sub>2</sub>SO<sub>4</sub> (5 % solution) before it was sterilized.

(vii) Media for isolation of Rhizobium (YEMA medium)

<table><thead><tr><td>Contents</td><td>Weight (g L<sup>-1</sup>)</td></tr></thead><tbody><tr><td>Mannitol</td><td>10.0 g</td></tr><tr><td>K<sub>2</sub>HPO<sub>4</sub></td><td>0.50 g</td></tr><tr><td>MgSO<sub>4</sub>·7H<sub>2</sub>O</td><td>0.20 g</td></tr><tr><td>NaCl</td><td>1.0</td></tr><tr><td>Yeast Extract</td><td>0.1 g</td></tr><tr><td>Agar</td><td>1.75 g·0 g</td></tr><tr><td>Distilled water</td><td>1000 mL</td></tr></tbody></table>

The pH of the medium was adjusted to 6.80
(viii) Media for isolation of phosphate solubilizing fungi

Pikovskaya's medium (1948)

<table><thead><tr><td>Contents</td><td>Weight</td></tr></thead><tbody><tr><td>Glucose</td><td>10.0g</td></tr><tr><td>Tricalcium phosphate Ca<sub>3</sub>(PO<sub>4</sub>)<sub>2</sub></td><td>5.0 g</td></tr><tr><td>Ammonium sulphate</td><td>0.50 g</td></tr><tr><td>Sodium Chloride {NaCl}</td><td>0.2 g</td></tr><tr><td>Potassium chloride</td><td>0.20 g</td></tr><tr><td>MgSO<sub>4</sub>·7H<sub>2</sub>O</td><td>0.10 g</td></tr><tr><td>Yeast extract</td><td>0.50 g</td></tr><tr><td>MnSO<sub>4</sub></td><td>Trace</td></tr><tr><td>FeSO<sub>4</sub>·7H<sub>2</sub>O</td><td>Trace</td></tr><tr><td>Agar</td><td>15.0 g</td></tr><tr><td>Distilled water</td><td>1000 mL</td></tr></tbody></table>

pH adjusted to 7.0 ± 0.2

The pH of the medium was adjusted to 7 ± 0.2 before it was sterilized.

(ix) Media for isolation of K- Solubilizers

Aleksandrov medium composition

<table><thead><tr><td>Contents</td><td>Weight (g L<sup>-1</sup>)</td></tr></thead><tbody><tr><td>Glucose</td><td>5.00 g</td></tr><tr><td>Potassium alumino silicate</td><td>3.00 g</td></tr><tr><td>Magnesium sulphate MgSO<sub>4</sub>·7H<sub>2</sub>O</td><td>0.50 g</td></tr><tr><td>Ferric chloride FeCl<sub>3</sub></td><td>0.006</td></tr><tr><td>Tricalcium phosphate Ca<sub>3</sub>(PO<sub>4</sub>)<sub>2</sub></td><td>2.00 g</td></tr><tr><td>Calcium carbonate CaCO<sub>3</sub></td><td>0.10 g</td></tr><tr><td>Agar</td><td>20.0 g</td></tr><tr><td>Distilled water</td><td>1000 mL</td></tr></tbody></table>

The pH adjusted to about 7.2±0.2 by adding 1N NaOH (25°C).
(x) Media for isolation of Sulphur oxidizing Bacteria (SoB)

Isolation of sulphur oxidizing bacteria include modified thiosulphate broth (Parker, 1957)

<table><thead><tr><td>Contents</td><td>Weight (g L<sup>-1</sup>)</td></tr></thead><tbody><tr><td>Na<sub>2</sub>S<sub>2</sub>O<sub>3</sub></td><td>5.0 g</td></tr><tr><td>K<sub>2</sub>HPO<sub>4</sub></td><td>0.1 g</td></tr><tr><td>NaHCO<sub>3</sub></td><td>0.2 g</td></tr><tr><td>NH<sub>4</sub>Cl</td><td>0.1 g</td></tr><tr><td>Glucose</td><td>5.0g</td></tr><tr><td>Distilled water</td><td>1000 mL</td></tr></tbody></table>

Maintain Initial pH: 8.0.

(xi) Media for isolation of Zinc solubilizing bacteria (ZSB)

<table><thead><tr><td>Contents</td><td>Weight (g L<sup>-1</sup>)</td></tr></thead><tbody><tr><td>Glucose</td><td>10.0g</td></tr><tr><td>Peptone</td><td>1.0 g</td></tr><tr><td>K<sub>2</sub>HPO<sub>4</sub></td><td>0.1 g</td></tr><tr><td>MgCl<sub>2</sub></td><td>0.2 g</td></tr><tr><td>yeast extract</td><td>1.0 g</td></tr><tr><td>(NH<sub>4</sub>)<sub>2</sub>SO<sub>4</sub></td><td>0.5 g</td></tr><tr><td>Agar</td><td>15 g</td></tr><tr><td>Distilled water</td><td>1000 mL</td></tr></tbody></table>

Buffered to pH 7.5
# Sterilization Techniques for Glassware

## Introduction

Sterilization is a process of complete removal of all types of living organisms from certain surfaces, objects, or liquids to avoid the chances of contamination and spoilage. It is either done by physical methods like ultraviolet radiations, autoclaving, etc., or by chemical methods like using ethanol. It is different from disinfection in the fact that disinfection kills many types of microorganisms but not all. Sterilization is more effective as compared to disinfection.

## Need of Sterilization

There are some microorganisms that are very dangerous and a person who is working in close vicinity with such microorganisms is likely to fall sick when exposed to them. That is why it is important to stop the transmission of these microorganisms from such surfaces by sterilizing them. Also, sterilization prevents any kind of cross-contamination and spoilage of the media to be used.

## Sterilization of Glassware

Sterilization is the process of destruction of all microbes including bacterial and fungal spores by physical or chemical methods. The physical methods for sterilization include

1. By dry heat.

2. By moist heat.

### 1. By Dry Heat

**Flaming:**

Glassware like the mouth of the culture tubes, slides and cover slips are passed over Bunsen flame but is not allowed to become red hot. This method kills the bacteria.

**Hot air oven:**

Glassware like Petri dishes, test tubes, conical flasks, measuring cylinders, beakers, etc., are sterilized by this method. Here dry heat is used for the sterilization. The oven is heated with the help of electricity at a temperature of 1600C to 1700C and glassware is sterilized at this temperature for about 2 hours. The oven is fitted with fans that ensure the even distribution of heat. But the problem with a hot air oven is that air is a very poor conductor of heat hence the penetration is low therefore there are chances of inadequate sterilization.

### 2. By Moist Heat

**Autoclaving**
* The dry heat methods are not useful when it comes to the removal of bacterial and fungal spores. To overcome this limitation moist heat or steam is used. The use of steam for sterilization of pieces of equipment and media is done with the help of an autoclave.

* Autoclave works on the principle that steam having high latent heat has high penetration. It is an inexpensive and non-toxic method that can effectively kill microbes and spores rapidly.

* An autoclave produces heat by pressurized steam which damages the structure protein and enzymes of the microorganisms and causes their denaturation and coagulation. Ideal temperature used in the autoclaving process is 1210C for a period of 30 min.

* They are frequently used in laboratories, research facilities, medical centres and hospitals. They can also be used in other businesses as it is cheap, easy, and non-toxic. It is used in places where glassware has to be used again and again.

## Preservation of the Sterility

All the lab equipment that is being sterilized is maintained by keeping it in a package until it is used. It can be plastic or paper which is used for packaging.

## Conclusion

While working in laboratories, researchers and professionals are exposed to a number of microorganisms, some of them can be harmful and some can be harmless. Therefore, while working in such an environment it is highly essential to keep the working area and pieces of equipment free of these microbes which are brought about by sterilization.

While working with glassware it is preferable to either autoclave them or keep them in a hot air oven to remove any microbe from its surface. Doing so can protect the working professional as well as give correct results.