Scope of Microbiology

INTRODUCTION TO MICROBIOLOGY

Microbiology is the study of microscopic organisms, including bacteria, viruses, fungi, protozoa, and

algae. These microorganisms play important roles in various aspects of our lives, including our health,

food production, and the environment.

Microbiology focuses on the behavior, structure, and function of microorganisms, and how they interact

with each other and with their environment. Microbiologists use a variety of techniques, such as

microscopy and culturing, to study microorganisms and understand their behavior.

Some of the key areas of study within microbiology include:

1. Medical microbiology, which focuses on the role of microorganisms in human diseases and how

they can be treated and prevented.

2. Industrial microbiology, which studies how microorganisms can be used in industrial processes,

such as the production of food, beverages, and bioplastics.

3. Environmental microbiology, which focuses on the role of microorganisms in the environment

and how they affect and are affected by environmental factors.

4. Agricultural microbiology, which studies the role of microorganisms in agriculture, including the

use of microorganisms as biofertilizers and biopesticides.

5. Food microbiology, which focuses on the microorganisms present in food and their impact on

food safety and quality.

In conclusion, microbiology is a diverse and complex field that continues to expand our understanding of

the world and provides us with important tools and techniques for improving our lives.

Food Microbiology and Laboratory –

Food microbiology is a subfield of microbiology that focuses on the study of microorganisms in food and

their impact on food safety, quality, and preservation. A food microbiology laboratory is a specialized

laboratory equipped with the tools and techniques needed to study microorganisms in food and assess

their impact on food safety, quality, and preservation.

In a food microbiology laboratory, scientists can perform a variety of tests to identify and enumerate

microorganisms in food, including culturing, microscopy, and molecular biology techniques such as PCR

(Polymerase Chain Reaction). They can also study the growth of microorganisms in food, including the

conditions that promote or inhibit growth, and develop methods for preserving food and controlling the

growth of harmful microorganisms.

Some of the key activities that take place in a food microbiology laboratory include:

1. Microbial analysis of food, including the identification and enumeration of microorganisms in

food.

2. Testing for foodborne pathogens, including Escherichia coli (E. coli), Salmonella, and Listeria.

3. Studying the impact of preservation techniques, such as refrigeration, freezing, and canning, on

the growth of microorganisms in food.

4. Developing and evaluating the effectiveness of antimicrobial agents, such as preservatives and

decontaminants, for controlling the growth of harmful microorganisms in food.

5. Investigating the role of microorganisms in food spoilage and quality deterioration, and

developing strategies to prevent spoilage and maintain food quality.

A food microbiology laboratory is an essential resource for ensuring the safety and quality of the food we

consume. By conducting research and performing tests, food microbiologists help to prevent foodborne

illness and maintain the quality and freshness of food products.

Microbiology Lab Practices and Safety –

In a microbiology laboratory, it is important to follow strict laboratory practices and safety protocols to

prevent contamination of samples and to protect laboratory personnel from potential health risks

associated with handling microorganisms. Some specific microbiology laboratory practices and safety

protocols include:

1. Good laboratory practices (GLP): Adhering to GLP principles, such as maintaining a clean and

organized laboratory and following standard operating procedures (SOPs), helps to prevent

contamination and ensure the accuracy of results.

2. Personal hygiene: Requiring laboratory personnel to practice good personal hygiene, such as hand

washing, and to avoid eating, drinking, or using tobacco products in the laboratory helps to

prevent contamination of samples.

3. Protective clothing: Requiring laboratory personnel to wear protective clothing, such as lab coats

and gloves, when handling microorganisms and potentially hazardous materials helps to reduce

the risk of exposure and contamination.

4. Microbial decontamination: Implementing procedures for decontaminating surfaces, equipment,

and samples before and after laboratory work helps to reduce the risk of contamination and

spread of microorganisms.

5. Handling of hazardous microorganisms: Following established procedures for handling hazardous

microorganisms, such as pathogenic bacteria and viruses, and using appropriate safety measures,

such as PPE, helps to prevent exposure and spread of these microorganisms.

6. Disposal of hazardous waste: Properly disposing of hazardous waste, such as samples containing

hazardous microorganisms, and following established procedures for decontaminating

equipment and surfaces helps to prevent contamination of the environment and protect

laboratory personnel from exposure to hazardous substances.

7. Biosafety levels: Adhering to established biosafety levels and following specific protocols for

handling, storing, and disposing of hazardous microorganisms helps to reduce the risk of exposure

and spread of these microorganisms.

By following these microbiology laboratory practices and safety protocols, laboratory personnel can

ensure the safety and integrity of samples and reduce the risk of contamination and spread of

microorganisms. It is important for microbiology laboratories to have a comprehensive safety plan in

place, train laboratory personnel on safe practices, and regularly evaluate and update safety protocols as

needed.

LABORATORY QUALITY MANAGEMENT SYSTEMS

INTRODUCTION

A quality management system with both national and international recognition is critical to the successful

operation of food testing laboratories. Quality Management System is an organized structure of

responsibilities, activities, resource, and event that are integrated to ensure the capability of a laboratory

to meet quality requirement. The components of a functional Quality Management System include

interrelated documentation of the quality policies or objectives (what to do), procedures (how to do it),

and evidence of compliance (records).

Quality assurance is an inherent component of any Quality Management System. The objective of Quality

Assurance is to ensure the reliability of analytical information used in decision making.

Quality Assurance two major components:

1. Quality control

2. Assessment: Consists of audit activities whose purpose is to review the efficacy of quality control.

THE ROLE OF MANAGEMENT IN LABORATORY QUALITY ASSURANCE

Management must evaluate the risks associated with laboratory variability, including the cost of error and

the time required to address such errors, and the cost and benefits of reducing variability through a

Quality Assurance program.

The first step in developing a Quality Assurance program for the laboratory is to draft a quality manual

that includes a quality policy statement and outlines the requirements for the following:

 Organization and management

 Quality systems for media and reagent quality control, audit and review

 Personnel requirements and training

 Accommodation and environment critical to the integrity of test results, such as cleaning and

sanitation.

 Use and maintenance of equipment and reference materials

 Calibration of equipment and test materials suitable for the tests being performed, traceable to

national or international sources

 Calibration procedures

 Validated test methods, handling of calibration items (thermometers, weights, reference

cultures)

 Sample handling

 Records

 Certificates and reports

 Testing

 Complaints concerning the quality of the data

GENERAL ENVIRONMENT OF THE LABORATORY CONDUCIVE TO SAFTEY AND PROPER PRACTICES

The laboratory should be air-conditioned and well ventilated to minimize temperature variations. The air

conditioning unit with clean vent filters will reduce the amount of particulates in the air.

The laboratory should be designed with worker safety in mind. It needs to be spacious enough to include

all necessary equipment and have adequate workbench space for the staff.

Adequate storage is needed to minimize clutter, which allows for proper cleaning and sanitization of

surfaces. The laboratory should be will lit, with a maintained light intensity of approximately 50-1000

lumens. A dependence on natural light is discouraged during the day owing to high variability in intensity.

Direct sunlight should also be avoided as it can negatively affect media, reagents, and organisms.

Laboratory conditions should be comfortable for workers. It is recommended that the laboratory

atmosphere be at an ambient temperature between 21°C and 23°C, with a relative humidity of 45%-50%.

EQUIPMENT/INSTRUMENTATION

The laboratory is furnished with many items of equipment, including reference materials, required for the

correct performance of testing. Regularly scheduled maintenance of this equipment is essential to the

smooth operation of the laboratory. A lack of attention to this aspect of the laboratory quality

management system will lead to unexpected and expensive equipment failures. Besides performing

regularly scheduled maintenance of equipment, ISO/IEC 17025:2005 requires that laboratories have a

record of the maintenance plan and the maintenance performed.

SAMPLE MANAGEMENT

HANDLING OF SAMPLE IN THE LABORATORY

After receipt, samples must be stored to maintain their original condition until analyzed. Sample should

be tested as soon as possible after receipt. Facilities should be available for both short-term storage before

and during analyses and, when required for forensic reasons, long-term storage after analyses have been

completed.

It may be necessary for an individual in the laboratory to have the responsibility as sample custodian to

maintain accountability records for samples in the laboratory. This individual may

 Receive samples

 Record the date and time samples are received

 Initially verify the identity of samples

 Store samples according to the accompanying instructions

 Record the date and time when samples are delivered to analyst for examination, and the date

and time when they are returned to storage following analysis

 Maintain a long-term sample storage system

 Dispose of samples as necessary

CULTURE MEDIA AND REAGENT PREPARATION OR TEST KITS

MEDIA/REAGENTS

Because microbiological media and regents are critical materials that may affect the quality of analytical

data, each new lot of medium or reagent must undergo performance testing. The media performance

should be verified using national standards. All new batches of media, whether made internally or

purchased pre-made, should be tested for sterility, productivity, selectivity, and appearance. Then should

be verified at the time of use.

TYPES AND CLASSES OF MEDIA

Culturing Media

The survival and growth of microorganisms depend on available of nutrients and favorable growth

environment. So the nutrient preparation that are used for culturing M.O are called Media. This media

contain C source (sugar), N (peptone), metallic ions (Ca, Zn, Na, K, Cu, Mn, Mg and Fe), and non metallic

ions (sulfur and P), and water (70-90%).

Types of Media

The types and classes of microbiological media used in the examination of foods depend principally on

their purpose for use.

Media are used to maintain pure culture for further studies or for long-term storage of cultures. Such

media are generally simple in formulation do not generally contain selective agents, and do not contain

ingredients that allow luxurious growth of microorganisms. Addition of cryoprotectant (e.g, Glycerol) to

such media can allow frozen (-70°C to -80°C) storage of pure cultures.

Enrichment Media

Media are used to allow the growth of nutritionally fastidious microorganisms and may incorporate one

or more factors that provide essential nutrients or allow recovery of injured cells. An example of such a

medium would be tryptic soy agar supplemented with 5% sheep's blood.

Selective Enrichment Media

Enrichment media may be selective in nature (selective enrichment media) and often are liquid media

that contain nutrients for growth and one or more agents that suppress undesirable organisms.

Enrichment media, in particular selective enrichment media, are utilized when the numbers of a targeted

microorganism in a food sample are too low to be detected directly by cultural or molecular methods.

Isolation Media

Media are used to detect the growth of a certain microorganism from among other microorganism for

further characterization and study. Sample containing mixed cultures are plated onto the surfaces of solid

media and plates are then incubated to allow colonies to develop from inoculated cells.

CLASSES OF MEDIA

Media also fall into one or more classes, depending on their ingredients and purpose for use.

Non-selective media

Media contain ingredients that allow the growth of multiple types of microorganisms and do not contain

chemical selective or differential agents in their formulation. Non-selective media, however, can be

incubated in such a manner so as to select against the growth of some organisms based on inability to

survive exposure or storage under the condition of incubation. For example, standard methods agar can

be handled so as to inhibit the growth of obligate aerobic bacteria by the incubation of plates in an

anaerobic or modified atmosphere environment.

Selective Media

Media are those containing one or more agents that exert inhibitory activity against one or more

Microorganisms or classes of microorganisms. Such agents are added to suppress the growth of

undesirable microorganisms while simultaneously allowing the growth of targeted microorganisms.

Example of selective agent types include selective dyes (e.g, crystal violet), antibiotic (e.g, novobiocin),

chemical antimicrobials (e.g, tartaric acid), or other compounds that exhibit inhibitory activity under

selected conditions (e.g, lauryl sulfate).

Differential Media

Differential media are bacteriological growth media that contain specific ingredients to allow one to

distinguish selected species or categories of bacteria by visual observation. Differential Media are used to

distinguish between closely related organisms or groups of organisms. Differential media contain

compounds that allow some groups of microorganisms to be visually distinguished by the appearance of

the colony or the surrounding media. Due to the presence of certain dyes or chemicals in the media, the

organisms will produce characteristic changes or growth patterns that are used for identification or

differentiation. This macroscopic differentiation of bacteria is based on the capability of the bacteria to

carry out specific biochemical processes. Blood agar is one type of differential medium, allowing bacteria

to be distinguished by the type of hemolysis produced.

Selective and differential media

Media are those that combine the usage of both selective and differential agents along with nutrients and

other ingredients in the formulation. Many such media are used for the selective identification of bacterial

pathogen from foods, e.g. selective differentiation of Staphylococcus aureus on Baird-Parker Agar.

FORMS OF MEDIA

Dehydrated Media

Dehydrated media should be store in a cool, dry location protected from light and in sealed containers, or

as instructions by the manufacturer. If the storage environment is hot and humid, dehydrated media may

be stored in a refrigerator or freezer as preferred, provided manufacturer instructions for medium storage

allow the user to choose.

Once opened and after the first usage, the quality of the dehydrated medium may depend upon the

storage environment. Air and moisture entering an unsealed container can initiate reactions that result in

reduced quality and productivity of the medium. Many dehydrated media contain hygroscopic ingredients

that will absorb atmospheric or environmental moisture. Care should also be taken to avoid chemical or

microbiological contamination from occurring by the use of unclean spatulas used to weigh or transfer

media into other containers.

Prepared Media

The shelf life of prepared media, whether in tubes, bottles or petri dishes, depends on the type of medium

and conditions of storage. Prepared media should not be stored unless protected against water loss.

Prepared plates should be stored in moisture-proof containers to minimize moisture loss. It is best to store

prepared plates no more than one week prior to use upon receipt, and media in screw-capped tubes

should not be stored for more than six months prior to use.

All prepared media, whether in plates or capped tubes, should be stored between 2°C and 8°C unless

otherwise recommended by the manufacturer.

COMMON MEDIA COMPONENTS

Most media used in the maintenance, enrichment and isolation of microorganisms contain one or more

of the following components. Components used in a medium, especially in cases where a medium is

prepared from individual components rather than a dehydrated base, should be reagent grade unless

otherwise specified. Follow manufacturer instructions for storing reagents; ingredients and reagents

showing signs of deterioration, contamination, or excess hydration should be discarded if unable to be

reclaimed.

Gelling or Solidifying Agent

Many media contain a polymeric substance capable of forming a gel under inoculation and incubation

temperature. The most commonly used gelling agent in media is Agar, a polysaccharide extracted and

refined from marine algae. Agar can be purchased in powdered, flaked, or granulated form. Its gelling

properties and insensitivity to microbial hydrolytic enzymes make it very useful in bacteriological studies.

It does not solubilize in water at room temperature, allowing it to be washed to remove impurities. Once

solubilized in heated (85°C) water, it forms a reversible gel once cooled to below 40°C. However, repeated

melting of agar-containing media, or prolonged sterilization by heating, results in decreased gel strength.

Gelatin is used as a solidifying agent in some media, though less frequently compared to alginates or agar.

Some media specifically utilize gelatin to serve dual purposes (gelling and differentiation of gelatinaseproducing

microorganisms).

Protein Digestate

Microorganisms require protein to maintain life and carry out enzymatically driven processes. Thus, a

source of amino acid is included in media to sustain growth and replication of microorganism. Enzymatic

or acid hydrolysis of animal or vegetable protein results in a digestate containing free amino acid and

polypeptides of varying sizes, termed peptone. Source of protein include animal muscle, liver, blood,

casein, milk, and soy flour these may digested by proteases such as pepsin, trypsin or other proteolytic

enzyme.

Carbohydrates

Carbohydrates are added as a source of useful energy and can included mono-,di-,oligo-,and

polysaccharides; sugar alcohols (e.g. sorbitol, adonitol); alcohols; and glycosides (e.g. esculin, salicin).

Meat and Yeast Extracts

Beef heart, liver, muscle, and brain are commonly used extracts in microbiological media. These contain

water soluble organic bases, polypeptides and other products of protein degradation, vitamins and

minerals. Similarly, yeast extract is prepared from autolysed baker’s or brewer’s yeast; yeast extract is rich

in nutrients such as various amino acids and b-complex vitamins. These products do undergo the severe

hydrolysis processing used for peptones, allowing retention of greater nutrient content.

Blood

Blood from horse, cow, goat, rabbit, and sheep is some time used for its inherent nutrient value or for the

differentiation of hemolysis-producing microorganisms.

Buffering Agents

Buffering agents are added to media to provide pH buffering in excess of that provided by other

components, though most media are not heavily buffered.

Water

The principle ingredient in almost all media is water; water used for preparation of media should be

purified and should be free of trace metals and any substances potentially inhibitory to microorganisms.

Culture media will provide some protection from toxic agents that may be present in water, and so water

purity is most important in the preparation of dilution media.

BASIC STEPS IN MEDIA PREPARATION

 Confirm the balance to be used is resting on a flat, level surface and switched on if weight are

given on electronic screen. Many balances today incorporate a device to ensure the balance is

appropriately leveled.

 Tare the balance with a clean weigh-boat or weigh paper.

 Weigh carefully the required amount of dehydrated medium or constituent ingredients. If

preparing media from individual components rather than a commercial product, weigh

ingredients into separate weigh papers so as to prevent contamination or ingredients.

 Add the weighed material to a container that contains between 50% and 80% of the water

required for the batch of medium being prepared. Mix with a stirring rod. Gently pour the

remaining water, Being sure to pour a small amount of water over the weigh paper used so as to

capture all weighed media. Pour any remaining water into the container and continue to mix.

 If necessary, heat the solution to effect complete solubilization. Note that extensive heating can

irreversibly damage some reagents. Consult the manufacturer’s preparation instruction for

medium preparation in addition to this manual to ensure medium quality. Hot plates are

commercially available and allow the simultaneous heating and mixing of media. Agar containing

media will require boiling to ensure complete solubilization; prolonged boiling will result in

undesirable foaming. Once heating/solubilization have been completed, water lost to evaporation

should be replaced as necessary.

 Determine the medium pH and adjusting.

 Dispense medium into containers suitable for sterilization or into containers for use if sterilization

is not recommended or required.

 Sterilize the medium according to recommended procedures, using thermal or filtration-based

methods. When using steam sterilization, media should be sterilized by holding in pressurized

steam for 15 min at 121°C. Large volumes of media may require a longer sterilization process (20-

30 min) or a completion of multiple small batches of media as needed.

OR

 All glassware are sterilize and cleaned with ISOPROPANOL ALCOHOL or ACITONE. After

clean/wash, glassware have dried in oven having 170°C temperature.

 Always take the distilled water for making media. Media are taken in require amount.

 Mostly I had used HiMedia's Media for the different type of parameters and Mostly media are

autoclave for sterilization at 121°C on 15 lbs pressure for 15 minutes like PCA, CYGA, TBX etc,

some are at 115°C on 15 Ibs for 15 minutes like RVSM, Urea agar ete. and some are only for heat

to boil.

 Measure the pH of the all media as instructions are given on media container.

Adjusting Reaction (pH)

Determine the hydrogen ion concentration (pH) of culture media electrometrically at 25°C.

Determination made at 45°C to take advantage of the liquid state of agar-containing media are not

accurate, differing significantly from those obtained at the recommended temperature. The

temperature of the test solution and buffer should be the same room temperature 25°C is generally

used for convenience.

Sterilization and Storage

Before sterilization, bring an agar-containing medium to boiling temperature, stirring frequently. pH

of a medium may change during sterilization and because of possible browning reaction, it is

important not to exceed the recommended temperature and time. Reduce pressure with reasonable

promptness (but is no less than 15 min) to prevent undue changes in the nutritional properties of the

medium and remove from the autoclave when atmospheric pressure is obtained. For medium

containing agar, autoclave the medium in the flasks or bottle from which melted medium may be

poured into plates.

Prepare medium in such quantities that, if stored, it will be used before loss of moisture through

evaporation because evident. To prevent contamination and excessive evaporation of moisture from

a medium in flasks and bottles during storage, optionally fit pliable aluminum foil, rubber, plastic

securely over closures before autoclaving. Use of screwcap closures on containers appreciable

reduces contamination and evaporation. Media should be stored at 2°C to 8°C in a dry, dust-free area

and should not be exposed to direct sunlight.

Steam Sterilization

Steam-sterilize media, water, and materials such as rubber, cotton, paper, heat-stable plastic tubes,

and closures by autoclaving at 121°C for no less than 15 min. Autoclave media and dilution blanks

within one hour of preparation.

Hot-Air Sterilization

Sterilize equipment with dry heat in hot air sterilizers (hot air ovens) so that materials at the center

of the load are heated to no less than 170°C (air temperature) for no less than 1 hour.

Filter Sterilization

Commonly used filters are made of cellulose esters, nylon, or polytetrafluoroethylene (PTFE). These

filters may be sterilized by autoclaving, or obtained pre-sterilized from commercial suppliers. To

sterilize heat-labile solution, inject the liquid through a syringe containing a membrane filter with

pores or larger than 0.20 to 0.45 μm in diameter. Most bacteria become trapped in the filter, as their size

typically ranges from 0.8 to larger than 1.0 μm.

MICROBIOLOGICAL TECHNIQUES

MEDIA HANDLING

The shelf lives of prepared media vary depending on the stability of the components. Manufacturers’

instruction or other reference materials should be consulted. In general prepared media should not

be stored for longer than 1 month. Some media require refrigeration and storage in the dark place.

The can be accomplished by pouring 20ml of molten agar into 12 x 100 mm Petri dish.

STREAKING TECHNIQUE

To ensure the isolation of a pure culture of the target microorganism, it is essential that the streaking

technique will provide isolated colonies.

 Using a sterile inoculating loop, transfer a loopful of the enrichment culture to the surface of

the agar plate, near the edge of the plate. Streaks the loops back and forth over the top

quarter of the plate about five to ten times in a tight “Z” fashion. The streaking lines should

not cross each other.

 Re-sterilize the inoculating loop and allow it to cool streak the right quarter of the plate by

passing the loop through the original area of inoculation (top quarter of plate) and streaking

in a tight “Z” about five to ten times. The loop should not contact the original area of

inoculation after the first pass.

 Re-sterilize the inoculating loop and allow to cool. Streak the remainder of the plate in the

same fashion, beginning with a single pass through the second streaked area (right quarter of

plate).

POUR PLATE TECHNIQUE

 Sample Preparation : The bench area should be cleaned and sanitized. All possible source of

contamination should be removed or reduced to a minimal level.

 Labeling : Label all petri plates, tubes, and bottles where necessary, with the sample number,

dilution, date, and any other desired information.

 Dilutions : For an accurate count, dilution should be selected to ensure that plates containing the

appropriate number of colonies will be produced.

 Melting and Tempering Media : Melt agar media in a flowing steam or boiling water, avoiding

prolonged exposure to high temperatures. Temper the melted media promptly and maintain

between 44 and 46°C until used.

 Plating : When measuring diluted samples of a food into petri plates, lift the cover of the petri

plate just high enough to insert the pipette. Hold the pipette at about a 45°C angle with the tip

touching the inside bottom of the petri plate. Deposit the sample away from the center of the

plate to aid in mixing.

 Pouring Agar : After removing tempered agar medium from the water bath, blot the bottle dry

with clean towels to prevent water from contaminating the plates. Pour 12 to 15 mL of liquefied

medium at 44 to 46°C into each plate by lifting the cover of the petri plate just high enough to

pour the medium.

This can be accomplished by rotating the plate first in one direction and then in the opposite

direction, by tilting and rotating the plate. Allow agar to solidify on a level surface.

SPREAD PLATE TECHNIQUE

The spread plate method is a technique to plate a liquid sample containing bacteria so that the bacteria

are easy to count and isolate. A successful spread plate will have a countable number of isolated bacterial

colonies evenly distributed on the plate.

Classically a small volume of a bacterial suspension is spread evenly over the agar surface using a sterile

bent glass rod as the spreading device.

This technique is used for isolating and counting the total number of viable microorganisms (i.e.

calculating the colony-forming units per mL (CFU/mL) in the given sample.

PICKING COLONIES TECHNIQUE

Picking colonies is a technique used in microbiological analysis to select and isolate specific colonies of

microorganisms from a culture plate. This technique is used to identify and study different types of

microorganisms present in a sample, or to obtain pure cultures of specific microorganisms for further

testing or analysis.

The basic steps of the picking colonies technique are:

1. Prepare a culture plate by inoculating it with a sample of microorganisms.

2. Incubate the plate at the appropriate temperature and conditions for the microorganisms to

grow.

3. Observe the plate and select specific colonies of interest, usually by their shape, size, color, or

other characteristics.

4. Transfer the selected colonies to a new culture plate or tube using a sterile loop or toothpick.

5. Incubate the new plate or tube to confirm the growth and purity of the selected colonies.

6. Repeat the process to select and isolate additional colonies of interest.

The picking colonies technique is widely used in many fields such as food industry, medicine, or

environmental science to identify microorganisms and monitor the quality and safety of food products or

to diagnose and treat infections.

It's important to use sterile technique throughout the process, as contamination from other

microorganisms can compromise the results. The colonies that have been selected and transferred should

be confirmed by microscopy, biochemistry or molecular biology techniques, to ensure that they are the

desired microorganisms.

EQUIPMENT/INSTRUMENTATION

AUTOCLAVE

Steam sterilization of materials is a dependable procedure for the destruction of all forms of microbial

life. Steam sterilization generally denotes heating in an autoclave utilizing saturated steam under a

pressure of approximately 15 pounds per square inch (psi) to achieve a chamber temperature of at least

121ºC (250ºF) for a minimum of 15 minutes (for small loads). The time is measured after the temperature

of the material being sterilized reaches 121ºC (250ºF).

When using a steam autoclave, consider the following:

1. Never autoclave FLAMMABLE, REACTIVE, CORROSIVE, TOXIC, or RADIOACTIVE MATERIALS.

2. Always wear safety glasses, goggles or face shield, lab coat or apron, and heat-protective gloves

when opening door or removing items from autoclave.

3. Open door slowly beware of a rush of steam.

4. Open door only after chamber pressure returns to zero. Leave door open for several minutes to

allow pressure to equalize and for materials to cool.

5. Do not mix loads which require different exposure times and exhaust.

6. Materials that will melt (e.g., plastic lab wear) should be placed in a shallow stainless steel

autoclave pan.

Tape Indicators : Tape indicators are adhesive backed paper tape with heat sensitive, chemical indicator

markings. Commonly used heat sensitive markings include diagonal stripes (autoclave tape) and/or the

word “sterile.” These markings only appear when the tape has been exposed for a few minutes to normal

autoclave decontamination temperatures.

AUTOMATIC PIPETTORS

A pipette is a laboratory tool commonly used in chemistry, biology and medicine to transport a measured

volume of liquid, often as a media dispenser.

BALANCE

Microbiology laboratory weighing balance mainly used for media preparation,micro pipette calibration

and product testing purpose. Advanced micro,semi-micro,analytical and precision balances have now

been perfected to such a degree that, in general, no special weighing rooms are needed.

pH METER

A pH meter is a laboratory instrument that is used to measure the acidity or basicity (alkalinity) of a

solution. The pH scale ranges from 0 to 14, with 0 being the most acidic, 7 being neutral, and 14 being the

most basic. The pH meter works by measuring the electrical potential difference between a reference

electrode and a pH electrode, which is immersed in the solution being tested. The pH meter then uses

this measurement to calculate the pH of the solution. pH meters are commonly used in a wide range of

applications such as chemical, pharmaceutical, food and beverage, environmental monitoring and many

other fields. They can be found in various forms such as benchtop, portable, and even in handheld

versions.

WATER BATH

A water bath is laboratory equipment made from a container filled with heated water. It is used to incubate

samples in water at a constant temperature over a long period of time.

A water bath of appropriate size and thermostatically controlled is needed for holding melted media at 44 to

55°C.

A water bath is a laboratory equipment that uses heated water to provide a constant temperature

environment for a reaction or an experiment. It is a container that is filled with water that is heated to a

desired temperature by an external heating source. The container is insulated to maintain the

temperature of the water, and the sample is placed in a smaller container that is placed inside the water

bath. Water baths are commonly used in a wide range of applications such as chemistry, biology, and

microbiology. They can be used to heat samples, melt and mix compounds, perform enzymatic reactions,

or to maintain a constant temperature for experiments that require a specific temperature range. They

come in various sizes, and can be either digital or analog, which can be adjusted to the desired

temperature.

HOT PLATE

A hot plate is a laboratory equipment that is used to heat samples, reagents, and other materials. It is a

flat heating surface that is powered by electricity. The heat is generated by an electric heating element

that is located under the plate. The temperature of the hot plate can be adjusted by a thermostat or a

digital controller. Hot plates are commonly used in a wide range of applications such as chemistry, biology,

and microbiology. They can be used to heat samples, melt and mix compounds, perform enzymatic

reactions, sterilize equipment, or to maintain a constant temperature for experiments that require a

specific temperature range. They come in various sizes and can be either magnetic or non-magnetic, and

can be used for stirring or for heating only.

LAF/LAMINAR AIR FLOW & BSC/BIO SAFTEY CABINET

A laminar air flow is an equipment that is generally used in microbiology laboratories. It consists of a

chamber with an air blower attached to its rear side that allows the flow of air with a uniform velocity in

straight lines that are parallel to each other. The main purpose of a laminar flow cabinet/hood is to form

a contaminant-free work environment. For this purpose, it filters and captures all types of impurity

particles entering the cabinet. It makes use of a filter pad and a special filter system known as a highefficiency

particulate air filter or HEPA filter, which can remove the airborne impurity particles that are up

to 0.3 micrometers in size.

A biosafety cabinet —also called a biological safety cabinet or microbiological safety cabinet—is an

enclosed, ventilated laboratory workspace for safely working with materials contaminated with

pathogens requiring a defined biosafety level.

INCUBATOR

An incubator is a laboratory equipment that is used to create and maintain optimal conditions for the

growth and cultivation of cells, microorganisms, and other living specimens. It typically provides a

controlled environment with a specific temperature, humidity, and atmosphere. There are several types

of incubators, each designed for specific applications. Some common types of incubators include:

1. Environmental incubators, which are used to control temperature and humidity for cell and tissue

cultures, as well as for microorganism cultivation.

2. CO2 incubators, which are used to control the concentration of carbon dioxide, as well as

temperature and humidity, for cell and tissue cultures.

3. BOD incubators, which are used to control temperature and humidity for the measurement of

biological oxygen demand.

4. Shaker incubators, which are used to maintain a constant temperature while shaking the samples,

typically used in microbiology, biochemistry and molecular biology.

5. Cryogenic incubators, which are used to maintain samples at extremely low temperatures,

typically used in preserving biological samples.

Incubators are commonly used in a wide range of applications such as cell culture, microbiology,

biotechnology, and molecular biology. They can be found in various forms such as benchtop, walk-in or

portable.

HOT AIR OVEN

That equipment cannot be wet or material that will not melt, catch fire, or change form when exposed to

high temperatures are sterilized by using the dry heat sterilization method.

Hot air oven also known as forced air circulating oven.

Some examples of material which cannot be sterilized by employing a hot air oven such as surgical

dressings, rubber items, or plastic material.

We can sterilize Glassware (like petri dishes, flasks, pipettes, and test tubes), Powder (like starch, zinc

oxide, and sulfadiazine), Materials that contain oils, Metal equipment (like scalpels, scissors, and blades)

by using hot air oven.

To destroy microorganisms and bacterial spores, a hot air oven provides extremely high temperatures

over several hours.

The widely used temperature-time relationship in hot air ovens to destroy microorganisms are 170

degrees Celsius for 30 minutes, 160 degrees Celsius for 60 minutes, and 150 degrees Celsius for 150

minutes.

COLONY COUNTER

Colony counters count the number of colonies of microorganisms that have grown on an agar plate

prepared from a sample. The colony counters can use fluorescent labels or the contrast between light and

dark areas on the plates to make their count.

MICROSCOPE

A microscope is a laboratory instrument that is used to magnify and observe small objects or specimens.

It typically consists of an objective lens that is used to magnify the specimen, an eyepiece lens that is used

to further magnify the image, and a light source that is used to illuminate the specimen. Microscopes can

be divided into two main categories: optical microscopes and electron microscopes.

1. Optical microscope: This type of microscope uses visible light and lenses to magnify the specimen.

They are the most common type of microscope and are widely used in biology, medicine, and

other fields. They can be further divided into compound and stereo microscopes.

2. Electron microscope: This type of microscope uses a beam of electrons instead of light to examine

the specimen. They are able to achieve much higher magnifications and resolutions than optical

microscopes and are widely used in materials science, biology, and medicine. They can be further

divided into transmission electron microscope (TEM) and scanning electron microscope (SEM).

Both types of microscope have different features, applications, and advantages, depending on the

characteristics and properties of the specimen being examined and the experiment or research being

conducted.

MICROBIAL ANALYSIS (TESTING) OF FOOD PRODUCTS

Microbial analysis, also known as microbial testing, is a method used to detect and quantify

microorganisms present in food products. This can include bacteria, viruses, fungi, and other

microorganisms that can be harmful to human health or cause spoilage of the food product. Microbial

analysis is an important step in ensuring the safety and quality of food products for human consumption.

Microbiological analysis of food products is the use of biological, biochemical, molecular or chemical

methods for the detection, identification or enumeration of microorganisms in a material (e.g. food, drink,

environmental or clinical sample). It is often applied to disease causing and spoilage microorganisms.

STANDARD METHODS FOR THE EXAMINATION OF FOOD PRODUCTS

Standard methods for the examination of food products are established protocols that provide guidelines

for the detection, identification, and quantification of various food-related parameters, including physical,

chemical, and microbiological characteristics. These methods are used by food industry, regulatory

agencies and researchers to ensure the safety and quality of food products.

 BIS: Bureau of Indian Standard came on 23 December 1986

 ISO: International Organization for Standardization, founded on 23 February 1947

 APHA: American Public Health Association, founded in 1872

 FSSAI: Food Safety and Standards Authority of India formed in 2011

Microbial Enumeration Test

TOTAL BACTERIA COUNT (IS 5402)

The total bacteria count (TBC) of a substance is a quantitative estimate of the number of microorganisms

present in a sample. This measurement is represented by the number of colony-forming bacterial units

(CFU) per gram (or milliliter) in the sample.

Method-

 10gm of sample is weighed into a stomacher bag, and 90ml of the sterile diluent is added and

stomached in the stomacher and serially diluted.

 Dilutions were depending on the matrix.

 Transfer 1ml of diluted suspension to pair sterile Petri dishes from respective dilution with the

help of sterile pipette. Pour about 12 to 15ml of PCA at 44°C to 47°C which is already prepared.

 Carefully mix the inoculum (3-5 times) with the media and allow the plates to solidify. Invert and

Incubate the plates at 30°C ± 19°C for 72 hours ± 3 hours.

 Observe all paired plates, count and calculate the colonies by the Colony Count Equation on each

plate and report in CFU/ml.

Total

Bacteria Count on PCA

COLIFORM (IS 5401 Part-1)

Coliform bacteria are a group of bacteria commonly used as indicator organisms to determine the

presence of fecal contamination and the sanitation of water, food, and surfaces. Coliform bacteria are

defined as facultatively anaerobic, Gram-negative, non-spore-forming rods that ferment lactose

vigorously to acid and gas. The most commonly used coliform bacterium is Escherichia coli (E. coli). Testing

for coliform bacteria is an important part of food safety and water quality management.

Method-

 10gm of sample is weighed into a stomacher bag, and 90ml of the sterile diluent is added and

stomached in the stomacher and serially diluted.

 Dilutions were depending on the matrix.

 Transfer 1ml of diluted suspension to pair sterile Petri dishes from respective dilution with the

help of sterile pipette. Pour about 12 to 15ml of VRBA at 44°C to 47°C which is already prepared.

 Carefully mix the inoculum (3-5 times) with the media and allow the plates to solidify. Invert and

Incubate the plates at 37°C ± 1°C for 24 hours.

 Observe all paired plates, count and calculate the colonies by the Colony Count Equation on each

plate and report in CFU/ml.

Coliform Bacteria on VRBA

Typical Colonies: Purple Red Colonies (Sometimes surrounded by reddish zone of precipitated bile)

YEAST & MOULD (IS 5403)

Yeast and mold are types of fungi that can be found in various environments, including on food and

in the air. Yeast is a single-celled organism that can ferment sugars to produce carbon dioxide and

alcohol, making it important in the production of bread, beer, and wine. Mold is a multi-cellular fungus

that can grow on a variety of organic materials, including food, and can cause spoilage and health

problems. Molds can produce toxic compounds called mycotoxins. Yeasts are microscopic fungi

consisting of solitary cells that reproduce by budding. Molds, in contrast, occur in long filaments

known as hyphae, which grow by apical extension.

Method-

 10gm of sample is weighed into a stomacher bag, and 90ml of the sterile diluent is added and

stomached in the stomacher and serially diluted.

 Dilutions were depending on the matrix.

 Transfer 1ml of diluted suspension to pair sterile Petri dishes from respective dilution with

the help of sterile pipette. Pour about 12 to 15ml of CYGA at 44°C to 47°C which is already

prepared.

 Carefully mix the inoculum (3-5 times) with the media and allow the plates to solidify. Invert

and Incubate the plates at 25°C ±1°C for 5 days.

 Observe all paired plates, count and calculate the colonies by the Colony Count Equation on

each plate and report in CFU/ml.

ESCHERICHIA COLI (IS 5887 Part 1)

Escherichia coli (E. coli) is a gram-negative bacterium that is a normal part of the human gut microbiome.

However, certain strains of E. coli can cause serious illness. Many strains of E. coli are motile, meaning they

have the ability to move using flagella. E. coli can grow in the presence or absence of oxygen. It is widely

distributed in the environment and is a normal part of the human gut microbiome.

Method-

 10gm of sample is weighed into a stomacher bag, and 90ml of the sterile diluent is added and

stomached in the stomacher and serially diluted.

 Dilutions were depending on the matrix.

 Transfer 1ml of diluted suspension to pair sterile Petri dishes from respective dilution with the

help of sterile pipette. Pour about 12 to 15ml of TBX at 44°C to 47°C which is already prepared.

 Carefully mix the inoculum (3-5 times) with the media and allow the plates to solidify. Invert and

Incubate the plates at 44.5°C for 24 hrs.

 Observe all paired plates, count and calculate the colonies by the Colony Count Equation on each

plate and report in CFU/ml.

E.Coli on TBX Plate

Typical Colonies: Blue Green Color Colonies

ENTEROBACTERIACEAE (Enterobacter aerogenes) (ISO 21528-2 017)

Enterobacteriaceae is a family of gram-negative bacteria that includes many medically important species

such as Escherichia coli, Salmonella, Klebsiella, and Proteus. These bacteria are often found in the

gastrointestinal tract of humans and animals and can cause a variety of infections ranging from mild

gastrointestinal upset to severe sepsis.

Method-

 10gm of sample is weighed into a stomacher bag, and 90ml of the sterile diluent is added and

stomached in the stomacher and serially diluted.

 Transfer 1ml of diluted suspension to pair sterile Petri dishes from respective dilution with the

help of sterile pipette. Pour about 12 to 15ml of VRBGA at 44°C to 47°C which is already prepared.

 Dilutions were depending on the matrix.

 Carefully mix the inoculum (3-5 times) with the media and allow the plates to solidify. Invert and

Incubate the plates at 37°C for 24 hours.

 Observe all paired plates, count and calculate the colonies by the Colony Count Equation on each

plate and report in CFU/ml.

Enterobacteriaceae on VRBGA

Typical Colonies: Pink purple color colonies with zone

BACILLUS CEREUS (ISO 7932 2004)

Bacillus cereus is a facultatively anaerobic, toxin-producing gram-positive, spore-forming bacterium that

is commonly found in soil and on food surfaces. It has the ability to move using flagella. Bacillus cereus is

capable of producing two types of toxins, one of which causes vomiting and the other causes diarrhea,

which are the hallmark symptoms of food poisoning caused by this bacterium.

Method-

 10gm of sample is weighed into a stomacher bag, and 90ml of the sterile diluent is added and

stomached in the stomacher and serially diluted.

 Dilutions were depending on the matrix.

 Transfer 1ml (0.3, 0.3, 0.4ml) from each plate of diluted, Sterile Petri dishes from respective

dilution with the help of sterile pipette. Plating Media Mannitol yeast extract agar (MYPA),

Spreading the plate with the help of sterile spreader.

 Allow the plates to solidify. Invert and Incubate the plates at 30°C for 48 hours.

 Observe all paired plates, count and calculate the colonies by the Colony Count Equation on each

plate and report in CFU/ml.

Bacillus

cereus on MYPA

Typical Colonies: Dark pink colonies with lecithinase activity

STAPHYLOCOCCUS AUREUS (ISO 6888-1 2021)

Staphylococcus aureus is a gram-positive spherical bacteria commonly found on human skin and mucous

membranes. It can cause a range of infections including skin infections, food poisoning, toxic shock

syndrome, pneumonia and bacteremia. S. aureus is often resistant to multiple antibiotics, including

methicillin. S. aureus is known for its ability to produce exotoxins which contribute to the severity of

infections. It is a facultative anaerobe, which means it can grow in the presence or absence of oxygen.

Method-

 10gm of sample is weighed into a stomacher bag, and 90ml of the sterile diluent is added and

stomached in the stomacher and serially diluted.

 Transfer 1ml (0.3, 0.3, 0.4ml) from each plate of diluted, Sterile Petri dishes from respective

dilution with the help of sterile pipette. Plating Media Baird parker agar (BPA), Spreading the plate

with the help of sterile spreader.

 Allow the plates to solidify. Invert and Incubate the plates at 35-37°C for 48 hours.

 Observe all paired plates, count and calculate the colonies by the Colony Count Equation on each

plate and report in CFU/ml.

S.aureus on BPA

Typical Colonies: Black colonies with white margin and clear zone

Microbial Detection Test

Salmonella spp (ISO 6579-1 2017, AMD 2020)

The following are some of the characteristics of Salmonella bacteria:

1. Gram-negative: Salmonella is a gram-negative bacterium, which means it has a thin peptidoglycan

layer and a unique outer membrane.

2. Rod-shaped: Salmonella bacteria are rod-shaped, also known as bacilli.

3. Aerobic or facultative anaerobic: Salmonella can grow in the presence or absence of oxygen.

4. Non-spore-forming: Salmonella does not form spores.

5. Motile: Some strains of Salmonella can move by using flagella.

6. Facultative intracellular parasite: Salmonella can infect and replicate inside host cells.

7. Pathogenic: Salmonella can cause a range of diseases, including salmonellosis, typhoid fever, and

paratyphoid fever.

8. Temperature-tolerant: Salmonella can grow at a wide range of temperatures, with optimal

growth at 37°C (98.6°F).

9. Resistant to drying and acidic conditions: Salmonella can survive in a dry environment and can

also tolerate acidic conditions.

Method-

 25gm of sample into 225ml of buffered peptone water and incubate at 37°C for 18 ± 2hours. Keep

the quality control checks (Media control, Blank control, Positive control) along with sample.

 After incubation transfer 0.1ml of the culture to a tube containing 10ml of the Rappaport-

Vassiliiadis Medium (RVS) and 1ml to the 10 ml of Muller-Kauffmann Tetrathionate Novobiocin

Broth (MKTTn).

 Incubate RVS at 41.5°C for 24 hours and MKTTn at 37°C for 24 hours.

 After incubation loop full of the inoculum RVS and MKTTn in Streaked onto the selective agar

Xylose Lysine Deoxycholate Agar (XLDA) and Brilliant Green Agar (BGA), both the plates were

incubated at 37°C for 24 hours.

Typical Colonies:

 XLDA (Xylose Lysine Deoxycholate Agar) – Red color colonies with black center

 BGA (Brilliant Green Agar) – Light pink color colonies with media color red

Salmonella spp on NA Salmonella spp on XLDA Salmonella spp on

BGA

Biochemical Confirmation:

From NA (Nutrient Agar) / PAC, SCDA Typical/atypical colonies were picked and inoculated to Nutrient

Agar plates and incubated at 37°C for 24 Hours.

 Gram Staining – Perform the steps of gram staining and observe under 100x lens or oil

immersion. Salmonella will appear as gram-negative rods.

 TSI Test – Stab and streak suspected colonies on TSI slants. Incubate at 36°C ± 2°C for 24

Hours. Typical Salmonella culture show red slants (Lactose negative) with gas formation and

yellow (Glucose positive) but with blackening of the agar formation H2S.

 L-Lysine-Decarboxylation – Inoculate the specific medium and incubate at 36°C ± 2°C for 24

Hours. Salmonella gives a positive response which is manifested by formation of purple color.

 Urease Production – streak the suspected colonies on urea slants and incubate at 36°C ± 2°C

for 24 Hours presence of pink color indicates positive reaction. Salmonella sp. is negative

towards urease test.

 Serological Confirmation – Using O, H & Vi antisera go for serological identification on basis

of agglutination. If agglutinations happens is considered salmonella.

Place one drop of the saline solution onto a cleaned glass slide mix a single colony with the

saline to obtain the homogenous suspension and add drop of H,O antiserum and mix properly.

Rock the slide gently for 30-60sec and observed for the agglutination.

Listeria spp (ISO 11290-1 2017)

Listeria spp is a genus of bacteria that includes several species, including Listeria monocytogenes, which

is a significant human pathogen. Some key characteristics of Listeria spp include:

1. Gram-positive: Listeria spp are gram-positive, meaning they retain the violet crystal stain used in

the Gram staining procedure.

2. Motile: Listeria spp are motile, meaning they are capable of movement.

3. Rod-shaped: Listeria spp are rod-shaped, or bacillus, in appearance.

4. Facultative anaerobic: Listeria spp are facultative anaerobic, meaning they can grow in the

absence of oxygen.

5. Wide temperature range: Listeria spp can grow at refrigeration temperatures as well as at

warmer temperatures.

6. Resistant to heat: Listeria spp are relatively heat-resistant, which makes it difficult to kill them

through cooking.

7. Pathogenic: Listeria monocytogenes is a significant human pathogen that can cause a serious

foodborne illness called listeriosis.

Method-

 25gm of sample into 225ml of Half Fraser broth incubate at 30°C for 24 hours.

(Supplement FD1251 and FD141 to be added before addition of samples.

 After incubation transfer 0.1ml of the primary enrichment to 10ml of the Fraser broth (Secondary

enrichment medium).

 Incubate Fraser broth at 37°C for 24 hours.

 After incubation from primary enrichment loop full of the inoculum is streaked onto Listeria

Oxford Agar (OXA) & ALOA Agar.

 Incubate the plates at 37°C for 24-48hours.

Typical Colonies:

 OXA – Brown-grey color colonies with black center and black helo.

 ALOA – Blue-green color colonies with zone.

Listeria spp ALOA Listeria spp OXA

Escherichia coli O157 (ISO 16654:2001)

Escherichia coli (E. coli) O157 is a strain of bacteria that can cause serious foodborne illness. Some key

characteristics of E. coli O157 include:

1. Gram-negative: E. coli O157 is a gram-negative bacterium, meaning it has a thin peptidoglycan layer

and a lipopolysaccharide outer layer.

2. Rod-shaped: E. coli O157 is a rod-shaped bacterium.

3. Metabolism: Facultative anaerobe

4. Ferments lactose: E. coli O157 is a lactose-fermenting bacterium, meaning it can produce lactic acid

from lactose.

5. Pathogenic: E. coli O157 is a pathogenic bacterium, meaning it can cause disease in humans and

animals.

6. Produces Shiga toxins: E. coli O157 is a Shiga toxin-producing bacterium, meaning it can produce

toxins that can damage cells and cause serious illness, including bloody diarrhea, kidney failure, and

death.

7. Spread through food or water: E. coli O157 can spread to humans through contaminated food or

water.

Method-

 25gm sample into 200ml of 0.1% Peptone water, homogenized and mixed the sample.

 Then transfer 10ml primary enrichment to a tube containing 10ml MacConkey Broth (MCB)ds with

durham’s tube and 1ml to the 10ml MacConkey Broth (MCB)ss with durham’s tube.

 Incubate MCB(ds) and MCB(ss) at 37°C for 24 hours.

 After incubation if bubble observe in the durham’s tube then streak on MacConkey Agar (MCA)

or EMB (Eosin Methylene Blue) media plates with inoculation loop.

 Incubate the plates at 37°C for 24hours.

Typical Colonies:

 MCA – Pink color colonies.

 EMB – Green metallic shine colonies.

E. coli O157 on MacConkey Agar E. coli O157 on Eosin Methylene

Blue

GRAM STAINING

Grams Staining is used for differentiation of bacteria on the basis of their gram nature.

Principle And Interpretation –

The Gram stain is a differential staining technique most widely applied in all microbiology disciplines

laboratories. It is one of the most important criteria in any identification scheme for all types of bacterial

isolates. Different mechanisms have been proposed to explain the gram reaction. There are many

physiological differences between gram-positive and gram-negative cell walls

(1). Ever since Christian Gram has discovered Gram staining ,this process has been extensively investigated

and redefined. In practice ,a thin smear of bacterial cells is stained with crystal violet, then treated with

an iodine containing mordant to increase the binding of primary stain

(2). A decolourizing solution of alcohol or acetone is used to remove the crystal violet from cells which

bind it weakly and then the counterstain (like safranin) is used to provide a colour contrast in those cells

that are decolourized. The gram-positive organisms or cells have more mucopeptide in their cell walls as

compared to gramnegative ones. Gram-negative bacteria have more content of polysaccharides and lipoproteins

in their cell walls. The polymers of glycerol or ribitol phosphate called as teichoic acids are also

found in the cell walls of gram-positive organisms but are very less or almost not present in gram-negative

organisms. In a properly stained smear by gram staining procedure, the grampositive bacteria appear blue

to purple and gram negative cells appear pink to red.

Ingredients –

 Gram's Crystal Violet

 Gram's Decolourizer

 Gram's Iodine

 Safranin,0.5% w/v

Directions –

1. Prepare a thin smear on clear, dry glass slide.

2. Allow it to air dry and fix by gentle heat.

3. Flood with Gram's Crystal Violet (S012) for 1 minute. (If over staining results in improper

decolourization of known gramnegative organisms,use less crystal violet).

4. Wash with tap water.

5. Flood the smear with Gram’s Iodine (S013). Allow it to remain for 1 minute.

6. Decolourize with Gram's Decolourizer (S032) until the blue dye no longer flows from the smear.

(Acetone may be used as a decolourizing agent with caution, since this solvent very rapidly

decolourizes the smear).

7. Wash with tap water.

8. Counter stain with 0.5% w/v Safranin (S027). Allow it to remain for 1 minute.

9. Wash with tap water.

10. Allow the slide to air dry or blot dry between sheets of clean bibulous paper and examine under

oil immersion objective.

Microscopic examination –

Gram staining was carried out and observed under oil immersion lens.

Results –

Gram-positive organisms : Violet coloured

Gram-negative organisms : Pinkish red coloured