b sc i sem biotechnology/microbiology lab manual

MICROBIOLOGY LAB MANUAL 2016-17

By, Sardar Hussain, Assistant Professor &Head, Dept. Of Biotechnology, GSC, CTA.

Experiment No. 1

Safety measures in Microbiology laboratory

Rules & Regulations

Safety in a microbiology laboratory is important in the prevention of infection that might be

caused by the microorganisms being studied. In addition, many of the reagents, equipment,

and procedures used are potentially hazardous. Attention to proper procedures and prudent

laboratory practices are required for your safety and protection.

This course does not require the use of any organisms known to be highly virulent human

pathogens. However, some of the organisms used may be potentially pathogenic. This means

that, although they may not cause disease in a normal healthy human, they might if the body's

antimicrobial defense mechanisms are impaired. In addition, many of the organisms used in

this course will be uncharacterized strains isolated from the environment - these cultures

should be handled with care, because their virulence is unknown.

In addition to organisms, there are some chemicals used in this laboratory which are

potentially harmful. Finally, many procedures involve equipment, glassware, open flames, and

sharp objects which can cause injury if used improperly.

Although none of the organisms, procedures, or materials used in this laboratory are very

dangerous, proper safety techniques and precautions should be understood and become part

of your reflexive laboratory technique. The following laboratory rules and regulations should

be adhered to at all times, NO EXCEPTIONS.

1. 1) Read and understand each laboratory exercise before you come to class.

2. 2) Follow precautionary statements given in each exercise.

3. 3) You should only have the necessary work material on the laboratory bench. Coats,

backpacks, and other personal belongings will not be allowed on the laboratory bench top. Store

them in a place designated by your instructor or in your laboratory cabinet. This is to prevent

cluttering of the workspace and to avoid exposing them to permanent stains, caustic chemicals, and

microorganisms used in the exercises.

4. 4) Do not eat, drink, smoke, or chew pens in the laboratory.

5. 5) You must wear shoes while in the laboratory.

6. 6) Long hair should be tied back so that it does not catch fire in a Bunsen burner flame and

does not fall into sterile media.

7. 7) Wipe your bench top with disinfectant at the beginning and end of every laboratory period.

8. 8) Wash your hands thoroughly before leaving the laboratory.



9. 9) Spills should be cleaned immediately. Spills of reagents should be cleaned using paper

towels, followed by a complete rinse with water. If the chemical is marked 'danger' or 'caustic' you

should also notify the instructor.

10. 10) Immediately report all accidents such as spills, cuts, burns, or other injuries to the

instructor.

11. 11) Know the location of the fire extinguisher and eye wash station.

12. 12) Leave all laboratory facilities and equipment in good order at the end of each class.

13) Before leaving the laboratory, check to make sure the gas to the Bunsen burner is turned off. 14)

Never, under any circumstances, remove equipment, media, or microbial cultures from the

laboratory.

During the course of the semester in the laboratory you will be taught the methods used in the proper

handling of microorganisms. Although you will not be working with any that are human pathogens,

exercise caution in handling all material coming in contact with live microbial cultures. All cultures

should be handled with respect and good technique as if they were potential pathogens. Specific

instructions which should be followed are:

Cultures or reagents should not be mouth-pipetted; a pipette bulb or automatic pipettor

should be attached to the pipette.

Always keep cultures capped and in proper storage racks or small buckets when not being

used during an exercise.

Never place a contaminated pipette, inoculating loop, or any other contaminated material on

the bench top. Flame loops before and after each use. Place used pipettes in the buckets

containing disinfectant. Place all other contaminated materials in their designated containers.

Do not place or put anything containing live cultures in the sink.

Aerosols should be avoided by the use of proper technique for flaming inoculating loops and

by performing any mixing of cultures and reagents in such a way as to avoid splashing.

If you spill material containing live microorganisms, pour disinfectant on the spill and notify

the laboratory instructor immediately.

Cultures of live microorganisms and any material coming in contact with live cultures must be

properly sterilized after use in the laboratory. Your instructor will inform you of specific

procedures. Follow the general rules outlined below.

Glassware such as test tubes, bottles, flasks, and pipettes, is reused and washed after

sterilization. These are normally placed on a cart at the front of the laboratory after you have



finished an experiment or exercise. Be sure to remove labels before placing any glassware on

the cart. Your instructor will sterilize and then wash these items.

Some materials, such as plastic petri dishes, plastic pipettes, microscope slides, and swabs, are

considered disposable. These are used once and if they become contaminated by contact with

live microorganisms are sterilized and discarded. All of these disposable contaminated

materials should be placed in the designated waste container containing an orange biohazard

autoclave bag.

Clothing worn in the microbiology laboratory should not be subsequently worn in a facility

such as a hospital, clinic, or nursing home, or in an area of public food preparation.

Experiment No. 2 Study of a Compound Microscope Aim: To study the parts and use of compound microscope.

Introduction: A microscope is used by jewelers to deliver perfection to the jewelry they design. It is

used by geologists to study about micro-organisms in soil. It is used by a veterinarian as a tool to help

treat animal health issues. All of these studies are easier with the help of a microscope. In the recent

years, schools all over the world have included studies related to microorganisms and human cell

structure in their curriculum, exposing the students to microscopes at an early age which has, as a



result, enhanced the interest and knowledge of students helping them later on in professional

studies.

1. Eyepiece or Ocular lens: An eyepiece is a magnifying lens attached to the microscope which helps in magnifying the sample object. It is called an eyepiece as we need to place our eye near it in order to see the magnifying image of the sample.

2. Body Tube: A body tube is an integral part of the microscope as it holds the eye piece and

connects it to the objective.

3. Arm: The arm is the part of microscope that connects to the base and helps carry the microscope

easily. One can hold the arm with on hand and put another hand under the base of the microscope so

that it can be carried easily.

4. Base: The base is the bottom part of the microscope, usually made up of durable material as it

supports the microscope to stand and provides stability. The base is very important as stability is very

important to gain accurate results. With an unstable base, the results may not be as accurate as we

require.

5. Illuminator: An illuminator is a source of light usually situated at the bottom/ base of the

microscope. It is a low voltage halogen bulb of about 110 volts to provide steady light to the sample in

order to facilitate the experiment/study.

6. Stage: A stage is an indispensable part of the microscope. It is a flat surface where the slide with

the specimen is placed. A mechanical stage is a stage used when working with higher magnifications.

It is moved by using knobs as even the slightest moment can affect the results.

7. Stage Clip: Stage clips are used to hold the slides in place in the absence of a mechanical stage. It is

used in comparatively simpler experiments. But even in simpler experiments, the movement of slides

is crucial hence stage clips are used to provide stability to the slides.

8. Revolving Nosepiece or Turret: A nosepiece is the part of the microscope which holds two or more

objectives simultaneously to provide various magnifications in order to view the same specimen in

various dimensions.

9. Objective lens: Objective lens is the part of microscope responsible for magnifying the image of



specimen. Usually there are three objective lenses in a standard microscope of 10X, 40X and 100X.

Depending upon the aim of study and nature of the specimen, the most suitable objective lens can be

brought to use.

10. Rack Stop: It is a part of the microscope responsible for adjusting and determining the distance

between the objective lens and the specimen. It is very important as it avoids the ramming of

objective lens into the slide, which can result in destroying the slide and specimen.

11. Condenser Lens: The function of the condenser lens is to collect the light from the illuminator

and focus it on the specimen. A microscope with a condenser provides with a sharper and clearer

image than a microscope without a condenser.

12. Diaphragm or Iris: The diaphragm is used to control the amount of light reaching the specimen. In

a student scope it is a rotating disk under the stage and above the condenser. There are various holes

in the diaphragm in order to facilitate the variants in the experiments carried on.

13. Coarse adjustment knob: A coarse adjustment knob is a knob present on the arm of a

microscope. The main function of this knob is to move the specimen back or forth to adjust the slide

containing specimen in order to bring it to focus and show the best image possible. The coarse

adjustment should be carefully moved and adjusted to attain desired results.

14. Fine Adjustment Knob: This knob is a sub part of the Coarse adjustment knob. It is used to bring

the specimen into sharp focus.

15. Power Switch: A Power switch is an electrical switch present at the bottom of the microscope in

order to switch of the light source i.e., the illuminator. At times the researcher/user does not require

the light from illuminator. In such a case, the power switch can be used to turn off the illuminator.

16. Low Power Objective: Low Power objective is a short length objective, most widely used in the

microscopes to view slides. Usually the experiments carried, use low power objective until the study

of the specimen is very specific. Also due to the short length of the objective, it avoids ramming into

the slide and protecting it from breaking.

17. High Power Objective: High power objective, also known as high-dry objective is used to study a

specimen in very fine and detailed manner. It is a bit longer in length than the low power objective



and needs to be handled with care.

18. Specimen on the Glass slide:

A glass slide is a thin and flat piece of glass used in the microscope. The specimen is kept on the glass

slide and put under the objective in order to study it. A typical glass slide is of dimensions 75x 26mm

and about 1 mm thick. The specimen on the glass slide is further covered with a very thin and smaller

sheet of glass called a cover slip so that the specimen doesn’t spill on the glass slide.

19. Aperture: Aperture is a small hole in the stage through which the light is transmitted and passed

on to the slide.

Experiment No. 03: Sterilization techniques

Effective sterilization techniques are essential for working with isolated cell lines for obvious

reasons you don’t want bugs from the environment growing in your nice culture medium, and

equally, cultures must be sterilized before disposal.

WET HEAT (Autoclaving)

The method of choice for sterilization in most labs is autoclaving; using pressurized steam to

heat the material to be sterilized. This is a very effective method that kills all microbes, spores

and viruses, although for some specific bugs, especially high temperatures or incubation times

are required.

Autoclaving kills microbes by hydrolysis and coagulation of cellular proteins, which is

efficiently achieved by intense heat in the presence of water.

The intense heat comes from the steam. Pressurized steam has a high latent heat; at 100degC

it holds 7 times more heat than water at the same temperature. This heat is liberated upon

contact with the cooler surface of the material to be sterilized, allowing rapid delivery of heat

and good penetration of dense materials.

At these temperatures, water does a great job of hydrolyzing proteins… so those bugs don’t

stand a chance.

Autoclave

An autoclave is a pressure chamber used to carry out industrial processes requiring elevated

temperature and pressure different from ambient air pressure. Autoclaves are used in medical

http://en.wikipedia.org/wiki/Latent_heat



applications to perform sterilization and in the chemical industry to cure coatings and

vulcanize rubber and for hydrothermal synthesis.

Many autoclaves are used to sterilize equipment and supplies by subjecting them to high pressure

saturated steam at 121 °C (249 °F) for around 15–20 minutes depending on the size of the load and

the contents. The autoclave was invented by Charles Chamberland in 1879, although a precursor

known as the steam digester was created by Denis Papin in 1679, The name comes from Greek auto-,

ultimately meaning self, and Latin clavis meaning key, thus a self-locking device

Air removal

It is very important to ensure that all of the trapped air is removed from the autoclave before

activation, as trapped air is a very poor medium for achieving sterility. Steam at 134 °C can achieve in

three minutes the same sterility that hot air at 160 °C can take two hours to achieve.

Methods of achieving air removal include:

Downward displacement (or gravity-type): As steam enters the chamber, it fills the upper areas first as

it is less dense than air. This compresses the air to the bottom, forcing it out through a drain which

often contains a temperature-sensing device. Only when air evacuation is complete does the

discharge stop. Flow is usually controlled by a steam trapor a solenoid valve, but bleed holes are

sometimes used, often in conjunction with a solenoid valve. As the steam and air mix it is also

possible to force out the mixture from locations in the chamber other than the bottom.

Steam pulsing: air dilution by using a series of steam pulses, in which the chamber is alternately

pressurized and then depressurized to near atmospheric pressure.

Vacuum pumps: a vacuum pump sucks air or air/steam mixtures from the chamber.

Super atmospheric cycles: achieved with a vacuum pump. It starts with a vacuum followed by a steam

pulse followed by a vacuum followed by a steam pulse. The number of pulses depends on the

particular autoclave and cycle chosen.

Sub atmospheric cycles: similar to the super atmospheric cycles, but chamber pressure never exceeds

atmospheric pressure until they pressurize up to the sterilizing temperature.

Uses:

Sterilization autoclaves are widely used in microbiology, medicine, podiatry, tattooing, body

piercing, veterinary medicine, mycology, funeral homes, dentistry,

https://en.wikipedia.org/wiki/Vulcanization

https://en.wikipedia.org/wiki/Hydrothermal_synthesis

https://en.wikipedia.org/wiki/Sterilization_(microbiology)

https://en.wikipedia.org/wiki/Charles_Chamberland

https://en.wikipedia.org/wiki/Steam_digester

https://en.wikipedia.org/wiki/Denis_Papin

https://en.wikipedia.org/wiki/Steam_trap

https://en.wikipedia.org/wiki/Solenoid_valve

https://en.wikipedia.org/wiki/Vacuum_pump

https://en.wikipedia.org/wiki/Microbiology

https://en.wikipedia.org/wiki/Medicine

https://en.wikipedia.org/wiki/Podiatry

https://en.wikipedia.org/wiki/Tattooing

https://en.wikipedia.org/wiki/Body_piercing

https://en.wikipedia.org/wiki/Body_piercing

https://en.wikipedia.org/wiki/Veterinary_medicine

https://en.wikipedia.org/wiki/Mycology

https://en.wikipedia.org/wiki/Funeral_homes

https://en.wikipedia.org/wiki/Dentistry



and prosthetics fabrication. They vary in size and function depending on the media to be

sterilized.

A notable recent and increasingly popular application of autoclaves is the pre-disposal

treatment and sterilization of waste material, such as pathogenic hospital waste. It is

particularly useful for materials which cannot withstand the higher temperature of a hot air

oven.

Autoclaves are also widely used to cure composites and in the vulcanization of rubber. The

high heat and pressure that autoclaves allow help to ensure that the best possible physical

properties are repeatably attainable.

The aerospace industry and sparmakers (for sailboats in particular) have autoclaves well over

50 feet (15 m) long, some over 10 feet (3.0 m) wide.

Other types of autoclave are used to grow crystals under high temperatures and pressures.

Synthetic quartz crystals used in the electronic industry are grown in autoclaves.

Packing of parachutes for specialist applications may be performed under vacuum in an

autoclave which allows the parachute to be warmed and inserted into the minimum volume.

DRY HEAT (Flaming, baking)

Dry heating has one crucial difference from autoclaving. You’ve guessed it – there’s no water, so

protein hydrolysis can’t take place.

Instead, dry heat tends to kill microbes by oxidation of cellular components. This requires more

energy than protein hydrolysis so higher temperatures are required for efficient sterilization by dry

heat.

For example sterilization can normally be achieved in 15 minutes by autoclaving at 121degC, whereas

dry heating would generally need a temperature of 160degC to sterilize in a similar amount of time.

Hot air Oven

Hot air ovens are electrical devices which use dry heat to sterilize. They were originally

developed by Pasteur.

Generally, they can be operated from 50 to 300 °C, using a thermostat to control the

https://en.wikipedia.org/wiki/Prosthetics

https://en.wikipedia.org/wiki/Quartz_crystal

https://en.wikipedia.org/wiki/Electrical

https://en.wikipedia.org/wiki/Dry_heat

https://en.wikipedia.org/wiki/Sterilization_(microbiology)

https://en.wikipedia.org/wiki/Thermostat



temperature.

Their double walled insulation keeps the heat in and conserves energy, the inner layer being a

poor conductor and outer layer being metallic.

There is also an air filled space in between to aid insulation.

An air circulating fan helps in uniform distribution of the heat. These are fitted with the

adjustable wire mesh plated trays or aluminium trays and may have an on/off rocker switch,

as well as indicators and controls for temperature and holding time. The capacities of these

ovens vary.

Advantages and disadvantages

They do not require water and there is not much pressure build up within the oven, unlike

an autoclave, making them safer to work with. This also makes them more suitable to be used

in a laboratory environment.

They are much smaller than autoclaves but can still be as effective.

They can be more rapid than an autoclave and higher temperatures can be reached compared

to other means.

As they use dry heat instead of moist heat, some organisms like prions, may not be killed by

them every time, based on the principle of thermal inactivation by oxidation

FILTRATION

Filtration is a great way of quickly sterilizing solutions without heating. Filters, of course, work by

passing the solution through a filter with a pore diameter that is too small for microbes to pass

through.

Filters can be scintered glass funnels made from heat-fused glass particles or, more commonly these

days, membrane filters made from cellulose esters. For removal of bacteria, filters with an average

pore diameter of 0.2um is normally used.

But remember, viruses and phage can pass through these filters so filtration is not a good option if

these are a concern.

https://en.wikipedia.org/wiki/Energy

https://en.wikipedia.org/wiki/Thermal_insulation

https://en.wikipedia.org/wiki/Aluminium

https://en.wikipedia.org/wiki/Autoclave

https://en.wikipedia.org/wiki/Laboratory

https://en.wikipedia.org/wiki/Dry_heat

https://en.wikipedia.org/wiki/Moist_heat

https://en.wikipedia.org/wiki/Prions



RADIATION

UV, x-rays and gamma rays are all types of electromagnetic radiation that have profoundly damaging

effects on DNA, so make excellent tools for sterilization.

The main difference between them, in terms of their effectiveness, is their penetration.

UV has limited penetration in air so sterilization only occurs in a fairly small area around the lamp.

However, it is relatively safe and is quite useful for sterilizing small areas, like laminar flow hoods.

X-rays and gamma rays are far more penetrating, which makes them more dangerous but very

effective for large scale cold sterilization of plastic items (e.g. syringes) during manufacturing.

So those are some of the main methods for sterilization I can think of. If I’ve missed any, please feel

free to let me know in the comments section.

Laminar Air Flow:

A laminar flow cabinet or laminar flow closet or tissue culture hood is a carefully enclosed

bench designed to prevent contamination of semiconductor wafers, biological samples, or any

particle sensitive materials.

Air is drawn through a HEPA filter and blown in a very smooth, laminar flow towards the user.

The cabinet is usually made of stainless steel with no gaps or joints where spores might

collect.

Such hoods exist in both horizontal and vertical configurations, and there are many different

types of cabinets with a variety of airflow patterns and acceptable uses.

Laminar flow cabinets may have a UV-C germicidal lamp to sterilize the interior and contents

before usage to prevent contamination of experiment. Germicidal lamps are usually kept on

for 15 minutes to sterilize the interior and no contact is to be made with a laminar flow hood

during this time. (It is important to switch this light off during use, to limit exposure to skin

and eyes as stray ultraviolet light emissions can cause cancer and cataracts.

Incubator:

In biology, an incubator is a device used to grow and maintain microbiological cultures or cell

https://en.wikipedia.org/wiki/HEPA

https://en.wikipedia.org/wiki/Laminar_flow

https://en.wikipedia.org/wiki/Stainless_steel

https://en.wikipedia.org/w/index.php?title=Airflow_patterns&action=edit&redlink=1

https://en.wikipedia.org/wiki/UV-C

https://en.wikipedia.org/wiki/Germicidal_lamp

https://en.wikipedia.org/wiki/Cataracts

https://en.wikipedia.org/wiki/Biology

https://en.wikipedia.org/wiki/Microbiological_culture

https://en.wikipedia.org/wiki/Cell_culture



cultures.

The incubator maintains optimal temperature, humidity and other conditions such as

the carbon dioxide (CO2) and oxygen content of the atmosphere inside.

Incubators are essential for a lot of experimental work in cell

biology, microbiology and molecular biology and are used to culture both bacterial as well

as eukaryotic cells.

The simplest incubators are insulated boxes with an adjustable heater, typically going up to 60

to 65 °C (140 to 150 °F), though some can go slightly higher (generally to no more than 100

°C).

The most commonly used temperature both for bacteria such as the frequently used E. coli as

well as for mammalian cells is approximately 37 °C, as these organisms grow well under such

conditions.

For other organisms used in biological experiments, such as the budding yeast Saccharomyces

cerevisiae, a growth temperature of 30 °C is optimal.

More elaborate incubators can also include the ability to lower the temperature (via

refrigeration), or the ability to control humidity or CO2 levels. This is important in the

cultivation of mammalian cells, where the relative humidity is typically >80% to prevent

evaporation and a slightly acidic pH is achieved by maintaining a CO2 level of 5%

Incineration Method:

Incineration is a waste treatment process that involves the combustion of organic

substances contained in waste materials. This method also burns any organism to ash. It is used to

sterilize medical and other biohazardous waste before it is discarded with non-hazardous waste.

Bacteria incinerators are mini furnaces used to incinerate and kill off any microorganisms that may be

on an inoculating loop or wire.

Chemical method:

Chromic acid:

Chromic acid: The term chromic acid is usually used for a mixture made by adding

concentrated sulfuric acid to a dichromate, which may contain a variety of compounds,

https://en.wikipedia.org/wiki/Cell_culture

https://en.wikipedia.org/wiki/Temperature

https://en.wikipedia.org/wiki/Humidity

https://en.wikipedia.org/wiki/Carbon_dioxide

https://en.wikipedia.org/wiki/Oxygen

https://en.wikipedia.org/wiki/Cell_biology

https://en.wikipedia.org/wiki/Cell_biology

https://en.wikipedia.org/wiki/Microbiology

https://en.wikipedia.org/wiki/Molecular_biology

https://en.wikipedia.org/wiki/Bacteria

https://en.wikipedia.org/wiki/Eukaryote

https://en.wikipedia.org/wiki/Escherichia_coli

https://en.wikipedia.org/wiki/Saccharomyces_cerevisiae

https://en.wikipedia.org/wiki/Saccharomyces_cerevisiae

https://en.wikipedia.org/wiki/Carbon_dioxide

https://en.wikipedia.org/wiki/Humidity

https://en.wikipedia.org/wiki/PH

https://en.wikipedia.org/wiki/Sulfuric_acid

https://en.wikipedia.org/wiki/Dichromate



including solid chromium trioxide.

This kind of chromic acid may be used as a cleaning mixture for glass.

Chromic acid may also refer to the molecular species, H2CrO4 of which the trioxide is

the anhydride. Chromic acid features chromium in an oxidation state of +6 (or VI).

It is a strong and corrosive oxidizing agent.

To prepare a chromic acid wash mix 20 g of Sodium or Potassium Chromate with sufficient

distilled H2O to make a paste of the chromate salt. Add 300mL of concentrated Sulfuric Acid.

Make larger amounts by increasing the proportions. Use the mixture until it turns green in

color.

Alcohols

Alcohols, usually ethanol or isopropanol, are sometimes used as a disinfectant, but more often

as an antiseptic (the distinction being that alcohol tends to be used on living tissue rather than

nonliving surfaces).

They are non-corrosive, but can be a fire hazard.

They also have limited residual activity due to evaporation, which results in brief contact times

unless the surface is submerged, and have a limited activity in the presence of organic

material.

Alcohols are most effective when combined with distilled water to facilitate diffusion through

the cell membrane; 100% alcohol typically denatures only external membrane proteins.

A mixture of 70% ethanol or isopropanol diluted in water is effective against a wide spectrum

of bacteria, though higher concentrations are often needed to disinfect wet

surfaces. Additionally, high-concentration mixtures (such as 80% ethanol + 5% isopropanol)

are required to effectively inactivate lipid-enveloped viruses (such as HIV, hepatitis B, and

hepatitis C).

The efficacy of alcohol is enhanced when in solution with the wetting agent dodecanoic

acid (coconut soap). The synergistic effect of 29.4% ethanol with dodecanoic acid is effective

against a broad spectrum of bacteria, fungi, and viruses.

https://en.wikipedia.org/wiki/Chromium_trioxide

https://en.wikipedia.org/wiki/Acidic_oxide

https://en.wikipedia.org/wiki/Oxidation_state

https://en.wikipedia.org/wiki/Oxidising_agent

https://en.wikipedia.org/wiki/Alcohol

https://en.wikipedia.org/wiki/Ethanol

https://en.wikipedia.org/wiki/Isopropanol

https://en.wikipedia.org/wiki/Antiseptic

https://en.wikipedia.org/wiki/Dodecanoic_acid

https://en.wikipedia.org/wiki/Dodecanoic_acid



Experiment No. 4: Preparation of culture media

Aim: To prepare the culture media.

Microorganisms, like all other living organisms, require basic nutrients for the sustenance of life.

The food material on which microbes are grown in the laboratory are known as culture medium

and the growth itself is called a culture.

Culture media vary in form and composition, depending on the species to be cultivated

Some media contain solutions of inorganic salts and may be supplemented with one or more

organic compounds while other media contains complex ingredients such as extracts or digests

of plant and animal tissues.

These ingredients except for the agar, are used to prepare broth or liquid media.

On the basis of their composition there are three main type of culture media

1. Natural or empirical culture media: the exact composition is not known, it includes milk,

urine, diluted blood, vegetable juice, meat extract, peptone

2. Semi synthetic media: chemical composition is partially known. Any medium which

contains agar becomes semi synthetic media. Ex. potato dextrose agar

3. Synthetic or chemically defined culture media: complete chemical composition is known.

It is also known as selective media, differential media or assay media. Ex. Martin rose

Bengal media.

a) Preparation of basic liquid media (broth) for cultivation of bacteria.

Introduction: Bacteria in contrast to fungi, are often cultured in a liquid broth. The most common

constituent are beef extract (a source of organic carbon, nitrogen, vitamins and inorganic salts) and

peptone (a semi digested protein).

Requirements:

Peptone

Beef extract

HCL and NaOH solution

pH meter

Test tubes

Glass rod

Cotton

Procedure:

5g of peptone and 3 g of beef extract was weighed and added to a conical flask containing

500ml of distilled water.



Flask was heated to dissolve the contents.

The final volume was made up to 1 liter with distilled water.

pH of the medium was adjusted to 7.0 with the help of pH meter.

10ml of media was poured into the sterilized test tubes.

Cotton plug was applied.

The contents were autoclaved at 1210C, 15 lbs pressure for 15 minutes.

Allowed the autoclave to cool and removed the tubes containing the broth and stored at room

temperature for future use.

Precautions: store the media at low temperature at dust free zone and cotton plugs should be

kept loose when autoclaving

b) Preparation of Nutrient agar media for cultivation of microorganisms.

Introduction: liquid growth media containing nutrients are usually solidified by the addition of agar. The

most common constituent are agar (a complex polysaccharide consisting of 3,6anhydro-L-galactose and

D-galacto pyranose, beef extract (a source of organic carbon, nitrogen, vitamins and inorganic salts) and

peptone (a semi digested protein).

Requirements:

Peptone

Agar

Beef extract


pH meter

Test tubes

Glass rod

Cotton

Procedure:

5g of peptone and 3 g of beef extract was weighed and added to a conical flask containing

500ml of distilled water.

Flask was heated to dissolve the contents.

pH of the medium was adjusted to 7.0 with the help of pH meter.

In another beaker, 20g of agar-agar was added to 100ml of water.

Both the solutions were mixed and the final volume was made up to 1 liter with distilled water.

10ml of media was poured into the sterilized test tubes and was kept in slant position to make

agar slants.



Cotton plug was applied.

The media can also be kept in conical flask for sterilization, with cotton plug.

The contents were autoclaved at 1210C,15 lbs pressure for 15 minutes.




kept loose when autoclaving

c) Preparation of Potato dextrose agar media for cultivation of microorganisms (fungi).

Introduction: PDA medium is used for cultivation and storage of fungi. Potato tubers are used as the

main ingredient. Other constituent are agar (a complex polysaccharide consisting of 3,6anhydro-L-

galactose and D-galacto pyranose, dextrose.

Requirements:

Agar

Potato tubers

Dextrose


pH meter

Test tubes

Glass rod

Cotton

Procedure:

500 ml of water was taken in 1liter beaker, to this 200g of washed, peeled ans sliced potatoes

were added.

Potatoes were boiled gently for 30 minutes and filtered through the muslin cloth. The liquid was

squeezed out.

To this 20g of dextrose was added.

In another beaker 200ml of water was taken. To this 20g of agar was added and dissolved.

Agar was mixed with potato extract. Final volume was made up to 1000ml with distilled water.

The media was transferred to a conical flask for sterilization, and a cotton plug was made.

The contents were autoclaved at 1210C,15 lbs pressure for 15 minutes.






kept loose when autoclaving.

d) Preparation of Eosin methylene blue agar.

Introduction: Eosin methylene blue (EMB, also known as "Levine's formulation") is a

selective stain for Gram-negative bacteria. EMB contains dyes that are toxic for Gram positive bacteria

and bile salt which is toxic for Gram negative bacteria other than coliforms. EMB is the selective and

differential medium for coliforms. It is a blend of two stains, eosin and methylene blue in the ratio of

6:1. A common application of this stain is in the preparation of EMB agar, a differential microbiological

medium, which slightly inhibits the growth of Gram-positive bacteria and provides a color indicator

distinguishing between organisms that ferment lactose (e.g., E. coli) and those that do not

(e.g., Salmonella, Shigella). Organisms that ferment lactose display "nucleated colonies"—colonies with

dark centers.

Rapid lactose fermentation produces acids, which lower the pH. This encourages dye absorption

by the colonies, which are now colored purple-black.

Lactose non-fermenters may increase the pH by deamination of proteins. This ensures that the

dye is not absorbed. The colonies will be colorless.

Requirements: EMB contains the following ingredients: peptone, lactose, dipotassium

phosphate, eosin Y (dye), methylene blue (dye), and agar.

Procedure:

10g of peptone and 10g of lactose was added to a beaker containing 200ml of distilled water,

heated to dissolve the contents.

In 100ml of distilled water eosin and methylene was dissolved.

In 100ml of distilled water potassium hydrogen phosphate was dissolved.

Liquefied the 15g of agar in 500ml of distilled water by heating to 1000C.

All the contents were mixed and made up to 1000ml.

pH was adjusted to 7.2 by using pH meter.

Sterilized the contents by autoclaving it at 1210C,15 lbs pressure for 15 minutes.

Medium were allowed to cool at 50-600C.

The contents were poured in Petri plates and allowed to solidify.

Stored at room temperature for future use.

https://en.wikipedia.org/wiki/Staining_(biology)

https://en.wikipedia.org/wiki/Gram-negative

https://en.wikipedia.org/wiki/Eosin

https://en.wikipedia.org/wiki/Methylene_blue

https://en.wikipedia.org/wiki/Differential_media

https://en.wikipedia.org/wiki/Differential_media

https://en.wikipedia.org/wiki/Gram-positive

https://en.wikipedia.org/wiki/Lactose



https://en.wikipedia.org/wiki/Dipotassium_phosphate

https://en.wikipedia.org/wiki/Dipotassium_phosphate

https://en.wikipedia.org/wiki/Eosin

https://en.wikipedia.org/wiki/Methylene_blue

https://en.wikipedia.org/wiki/Agar



Experiment no. 5: Serial dilution technique.

Aim: Isolation of microorganisms from soil by serial dilution method.

Introduction: Soil is the richest source of different kinds of microorganisms. Microbes of all major

taxonomic groups- bacteria, fungi, algae, protists and viruses are found in soil, but bacteria are more

predominant than all other kinds of microbes. Soil microbes are important as decomposers in the

carbon cycle and all phases of the nitrogen cycle. The bacteria present in the soil includes autotrophs,

heterotrophs, aerobes, anaerobes. The soil contains bacteria that digest cellulose, protein, pectin,

butyric acid and urea.

The plate count method or serial dilution agar plating method is used for the enumeration of

bacteria from soil. It is performed by the dilution of the original sample in serial dilution tubes, followed

by the plating of aliquots of the prepared serial dilutions into appropriate agar plates by the pore plate

or spread plate technique.

Principle: The plate method is based on viable cell counts. When material containing microbes is

cultured each viable cell or spore cell develop into a colony that are counted as the number of colony

forming units (CFUs). This typical counting ranges are 25 to 250 CFU per standard plate count of agar.

The CFU and respective dilution factor are then multiplied to calculate the original number of microbes

per gram. This method is used primarily in the enumeration of sample with high microbes that do not

grow well in liquid media.

Sodium chloride (0.85%) is preferred as a diluent in place of sterile water for the preparation of

dilution of soil sample.

Requirements:

Soil sample

Nutrient agar media plates.

9ml of NaCl or sterile water

Sterile pipettes

Microscope

Bunsen burner

Procedure:

Petri plate and test tubes are labelled as dilutions.

To the first tube, labelled as 10-11gm of soil and 9ml of water or NaCl solution was added.

Mixed well.

From this tube 1ml of solution is transferred to the 10-2 labeled tube. Similarly next dilutions

are prepared.

0.1 ml of solution from each dilution tubes are transferred to the nutrient agar plate by pour

plate method or spread plate method.



The plates were incubated at 370C in bacteriological incubator for 3-4 days.

Result: The plates were observed after incubation period and number of colonies were

counted.

Aim: Isolation of microorganisms from water by serial dilution method.

Introduction: water is the richest source of different kinds of microorganisms. Microbes of all major

taxonomic groups- bacteria, fungi, algae, protists and viruses are found in water, but bacteria are more

predominant than all other kinds of microbes. The bacteria present in the water includes autotrophs,

heterotrophs, aerobes, anaerobes. The soil contains bacteria that digest cellulose, protein, pectin,

butyric acid and urea.

The plate count method or serial dilution agar plating method is used for the enumeration of

bacteria from water. It is performed by the dilution of the original sample in serial dilution tubes,

followed by the plating of aliquots of the prepared serial dilutions into appropriate agar plates by the

pore plate or spread plate technique.

Principle: The plate method is based on viable cell counts. When material containing microbes is

cultured each viable cell or spore cell develop into a colony that are counted as the number of colony

forming units (CFUs). This typical counting ranges are 25 to 250 CFU per standard plate count of agar.

The CFU and respective dilution factor are then multiplied to calculate the original number of microbes

per gram. This method is used primarily in the enumeration of sample with high microbes that do not

grow well in liquid media.

Sodium chloride (0.85%) is preferred as a diluent in place of sterile water for the preparation of

dilution of soil sample.

Requirements:

Soil sample

Nutrient agar media plates.


Sterile pipettes

Microscope

Bunsen burner

Procedure:

Petri plate and test tubes are labelled as dilutions.

To the first tube, labelled as 10-11 ml of water sample and 9ml of water or NaCl solution was

added. Mixed well.

From this tube 1ml of solution is transferred to the 10-2 labeled tube. Similarly next dilutions

are prepared.



0.1 ml of solution from each dilution tubes are transferred to the nutrient agar plate by pour

plate method or spread plate method.



counted.

Aim: Isolation of microorganisms from air.

Principle: Air is the medium for many bacteria and fungi, microbes present in air move from one place

to other by dust particles, these dust particles are influenced by, wind, rain, temperature, etc. The

presence of spore forming and resistant bacteria in air pose great health hazards to the human beings.

The plate exposure technique relies on the principle of gravity settle technique. Where a petri dish

containing suitable agar media is exposed to air, and the particles settle down on the media due to

gravitational force.

Requirements:

Nutrient agar / P.D.A. media plates.


Sterile pipettes

Microscope

Bunsen burner

Incubator.

Procedure:

Prepare sterilize and pour the molten media PDA or NA. into separate sterile Petri plates

Allow the plates to solidify.

Expose the air to air for five minutes at all different places such as classrooms, cinema

theatres, buses, toilets, hospitals.


Calculate the number of colonies/cubic feet as follows

No.of colonies /cubic feet = Total no.of colonies X diameter of plate / exposure time.



Site of

exposure

Time of

exposure

Colony

characters

No.of

colonies

No.of organisms/cubic

feet

Lab

Class room

Theatre

bus

Rest room


counted

Experiment no. 6: Pure culture techniques.

The microbial population in our environment- air, soil and water, infectious materials such as

pus, sputum, urine, stool and food is large and are composed of mixed population of bacteria, fungi,

protozoa, and other organisms. Mixed population is the culture that contains more than one kind of

microbes, whereas a pure culture contains only a single species of organisms. Pure culture methods are

very much essential for the study of cultural, morphological, physiological, immunological and other

characteristics of an individual organism.

There are three most commonly used pure culture techniques are,

1) Streak plate method

2) Pour plate method and

3) Spread plate method.

Streak plate method

Introduction: This method is one of the most common method for pure culture technique. It involves

spreading of a loop full of culture across the agar surface creating visible streaks. So that the progeny of

the single cell can be picked up from the surface and transferred easily to a sterile medium. Quadrant

streak, radiant streak and continuous streak are the three method used for streaking on the agar plate.

Principle: It is a rapid qualitative purification method, involves the diluting the bacterial cells which are

rubbed off the loop onto the medium in the form of streaks. Very fewer bacteria are deposited as the

streaking.

Requirements:

Cultures

Nutrient agar plates

Inoculation loop



Bunsen burner

Procedure:

Plates were labelled on the back side with organism names.

The sterilized nutrient agar media is poured in the petri plates in sterilized conditions.

The media is allowed to cool and solidify.

With the help of the sterile inoculation loop the single colony in the culture plate is taken and

streaked on the solidified agar plate.

Result:The plates are incubated and after incubation observed for the growth of colonies.

Pour plate method

Introduction: This method is also called as loop dilution method, of obtaining a pure culture involves

serial dilution, transferring to melted agar (450C), a specific volume of the dilution, followed by the

pouring of the suspension into a sterile petri plate resulting in the formation of isolated colonies on the

agar after 24-48 hrs.

This method is useful for culturing microaeroplies that cannot tolerate exposure to oxygen in the

air at the surface of the medium.

Principle: It is a rapid qualitative purification method, involves the dilution of the original sample by

serial dilution method then, diluted sample is poured on to a nutrient agar plate.

Requirements:

Cultures


Bunsen burner

Procedure:




The serially diluted sample is poured on the surface of the nutrient agar plate. The petri dish is

closed and rotated in such a way that the sample is spread on its surface.

Result: The plates are incubated and after incubation observed for the growth of colonies.

Spread plate method

Introduction: This method is one of the most common method for pure culture technique. It involves

spreading of a known amount of culture across the agar surface creating visible colonies. The sample is

spreaded with the help of the L shaped bent glass rod or a spreader.

Principle: It is similar to pour plate method. It is a rapid quantitative purification method, which

determines the number of bacteria in a sample. Due to the use of bent glass rod the cells are separated

from each other by a distance sufficient to allow the colonies that develop to be free from each other.



Requirements:

Cultures


L shaped glass rod/ spreader

Bunsen burner

Procedure:




The diluted culture is poured on the surface of the nutrient agar media and with the help of the

L- shaped glass rod, culture is spreaded on the medium.

Result: The plates were incubated and after incubation observed for the growth of colonies.

Experiment no. 7: Bacterial staining techniques:

Staining is an auxiliary technique used in microscopy to enhance contrast in

the microscopic image. Stains and dyes are frequently used in biology and medicine to highlight

structures in biological tissues for viewing, often with the aid of different microscopes. Stains may be

used to define and examine bulk tissues (highlighting, for example, muscle fibers or connective

tissue), cell populations (classifying different blood cells, for instance), or organelles within individual

cells.

Biological staining is also used to mark cells in flow cytometry, and to flag proteins or nucleic acids in gel

electrophoresis. Simple staining is staining with only one stain/dye. There are various kinds of multiple

staining, many of which are examples of counter staining, differential staining, or both, including double

staining and triple staining.

The staining techniques are,

Simple staining

Negative staining

Gram’s staining

Simple staining

Introduction: simple staining employs staining of bacterial smear with a single basic stain to observe size,

shape and arrangement of bacterial cells. The commonly used simple stains are, crystal violet, carbol

fuchsin, and safranin.

https://en.wikipedia.org/wiki/Microscopy

https://en.wikipedia.org/wiki/Microscope

https://en.wikipedia.org/wiki/Dye

https://en.wikipedia.org/wiki/Biology

https://en.wikipedia.org/wiki/Medicine

https://en.wikipedia.org/wiki/Biological_tissue

https://en.wikipedia.org/wiki/Microscope

https://en.wikipedia.org/wiki/Muscle_fiber

https://en.wikipedia.org/wiki/Connective_tissue

https://en.wikipedia.org/wiki/Connective_tissue

https://en.wikipedia.org/wiki/Cell_(biology)

https://en.wikipedia.org/wiki/Blood_cell

https://en.wikipedia.org/wiki/Organelle

https://en.wikipedia.org/wiki/Flow_cytometry

https://en.wikipedia.org/wiki/Protein

https://en.wikipedia.org/wiki/Nucleic_acid

https://en.wikipedia.org/wiki/Gel_electrophoresis

https://en.wikipedia.org/wiki/Gel_electrophoresis

https://en.wikipedia.org/wiki/Counterstain

https://en.wikipedia.org/wiki/Differential_staining



Principle: The basic dyes (or stains) which carry positive charge (cationic) color bearing ions

(chromophores) are attracted to the positive charge and color negatively charged bacterial cells. For

example in methylene blue (methylene + chloride-) the chromophore methylene +will bind to the

bacterium.

Requirements:

Simple stain- crystal violet/methylene blue/safranin etc.

Wash bottle

Blotting paper, glass slide, Bunsen burner, and microscope.

Procedure:

1. Clean and dry microscope slides thoroughly.

2. Flame the surface in which the smear is to be spread.

3. Flame the inoculating loop.

4. Transfer a loop full of tap water to the flamed slide surface.

5. Reflame the loop making sure the entire length of the wire that will enter the tube has been

heated to redness.

6. Remove the cotton plug with the fingers of the hand holding the loop.

7. Flame the tube mouth.

8. Touch the inoculating loop to the inside of the tube to make sure it is not so hot that it will

distort the bacterial cells; then pick up a pinhead size sample of the bacterial growth

without digging into the agar.

9. Reflame the tube mouth, replace the cotton plug.

10. Disperse the bacteria on the loop in the drop of water on the slide and spread the drop over

Slide. It should be a thin, even smear.

11. Reflame the inoculating loop to redness including the entire length that entered the tube.

12. Allow the smear to dry thoroughly.

13. Heat-fix the smear cautiously by passing the underside of the slide through the burner flame

two or three times. Test the temperature of the slide after each pass against the back of the

hand. It has been heated sufficiently when it feels hot but can still be held against the skin

for several seconds. Overheating will distort the cells.

14. Stain the smear by flooding it with one of the staining solutions and allowing it to remain

covered with the stain for some time



Methylene blue - 1 minute

Crystal violet - 30 seconds

Carbol fuchsin - 20 seconds

During the staining the slide may be placed on the rack or held in the fingers.

15. At the end of the designated time rinse off the excess stain with gently running tap water.

Rinse thoroughly.

16. Wipe the back of the slide and blot the stained surface with tissue paper.

17. Observe cells and draw as they appear.

Result: colored bacterial cells were observed against colorless background.

Result: colorless bacterial cells were observed against dark background.

Negative staining

Introduction: Negative staining or background staining technique is so named because the background

gets stained and the organism remains colorless. In this technique a negative stain (eg. Acid stains;

Indian ink, nigrosine, eosin) is used to color the background around the cells. Because this procedure

does not require heat fixing or staining the cells, which can cause some cell shrinkage, provides a more

accurate demonstration of the shape and size of the cell.

Principle: it is based upon the principle of using an acidic dye (a dye having anionic, or negatively

charged chromophore i.e color bearing ions) that will not stain bacteria due to electrostatic repelling

forces because bacteria are also negatively charged. For ex. Eosin (sodium + eosinate-) an acidic dye

having an anionic chromophore will not stain bacteria. Hence, cells appear transparent and unstained in

microscopic observation.

Requirements:

Negative stain: eosin, nigrosine etc.

Wash bottle

Blotting paper, glass slide, Bunsen burner, and microscope.

Procedure:

1. Place a very small drop of nigrosin near one end of a well-cleaned and flamed slide.

2. Remove a small amount of the culture from the slant with an inoculating loop and disperse it in

the drop of stain without spreading the drop.



3. Use another clean slide to spread the drop of stain containing the organism.

4. Rest one end of the clean slide on the center of the slide with the stain. Tilt the clean slide toward the

drop forming an acute angle and draw that slide toward the drop until it touches the drop and causes it

to spread along the edge of the spreader slide. Maintaining a small acute angle between the slides,

push the spreader slide toward the clean end of the slide being stained dragging the drop behind the

spreader slide and producing a broad, even, thin smear.

5. Allow the smear to dry without heating.

6. Focus a thin area under oil immersion and observe the unstained cells surrounded by the gray stain.

Gram staining:

Introduction: Gram staining, also called Gram's method, is a method of staining used to

differentiate bacterial species into two large groups (gram-positive and gram-negative). The name

comes from the Danish bacteriologist Hans Christian Gram, who developed the technique.

Gram staining differentiates bacteria by the chemical and physical properties of their cell

walls by detecting peptidoglycan, which is present in a thick layer in gram-positive bacteria. Gram-

positive bacteria retain the crystal violet dye, while a counterstain (commonly safranin or fuchsine)

added after the crystal violet gives all gram-negative bacteria a red or pink coloring.

Principle:

Gram-positive bacteria have a thick mesh-like cell wall made of peptidoglycan (50–90% of cell

envelope), and as a result are stained purple by crystal violet, whereas gram-negative bacteria have a

thinner layer (10% of cell envelope), so do not retain the purple stain and are counter-stained pink by

safranin.

Gram staining is based on the ability of microorganisms to retain the purple color of the crystal violet

during decolonization with alcohol. Crystal violet iodine (CVI) complex combines to form a larger

molecule which precipitates within the cell and does not leach out on treatment with alcohol. Because

of thick peptidoglycan layer in gram positive bacteria and remain purple. In gram negative bacteria the

alcohol being a lipid solvent dissolves the outer lipopolysaccharide layer, as result CVI complex leaches

out through the thin layer of peptidoglycan. Hence, when a counter stain (safranin) is added, they take

up the color and appears pink.

Requirements:

Bacterial culture (18 to 24 hrs.)

Stains: crystal violet, gram’s iodine, 95% alcohol, safranin.

https://en.wikipedia.org/wiki/Staining

https://en.wikipedia.org/wiki/Bacteria

https://en.wikipedia.org/wiki/Gram-positive_bacteria

https://en.wikipedia.org/wiki/Gram-negative_bacteria

https://en.wikipedia.org/wiki/Hans_Christian_Gram

https://en.wikipedia.org/wiki/Cell_wall

https://en.wikipedia.org/wiki/Cell_wall

https://en.wikipedia.org/wiki/Peptidoglycan

https://en.wikipedia.org/wiki/Crystal_violet

https://en.wikipedia.org/wiki/Safranin

https://en.wikipedia.org/wiki/Fuchsine

https://en.wikipedia.org/wiki/Peptidoglycan



Wash bottle

Blotting paper

Inoculation loop

Glass slides, Bunsen burner, microscope.

Procedure:

Prepare and heat fix the bacterial smear.

Cover the smear with crystal violet stain and allow it to stand for 20-60 seconds.

Wash off the stain for two seconds using distilled water.

Cover the smear with gram’s iodine and allow it to stand for 60 seconds.

Decolorize the smear with 95% ethyl alcohol (or acetone) for 10- 20 seconds or until no more

color flows from the smear.

Immediately wash the smear with distilled water for 2 seconds and drain.

Cover the smear with safranin stain and allow it to stand for 20 seconds.

Wash off the stain for two seconds using distilled water.

Blot dry the smear.

Let the stained slide air dry.

Examine the smear and record the observations.

Result: violet colored cocci were observed under 40x, hence they are Gram positive coccus

Experiment no. 8: Biochemical tests of microorganisms. Catalase test: Introduction:

Catalase is an enzyme, which is produced by microorganisms that live in oxygenated

environments to neutralize toxic forms of oxygen metabolites; H2O2. The catalase enzyme neutralizes

the bactericidal effects of hydrogen peroxide and protects them. Anaerobes generally lack the catalase

enzyme.



Catalase mediates the breakdown of hydrogen peroxide H2O2into oxygen and water. To find out

if a particular bacterial isolate is able to produce catalase enzyme, small inoculums of bacterial isolate is

mixed into hydrogen peroxide solution (3%) and the rapid elaboration of oxygen bubbles occurs.

Catalase-positive bacteria include strict aerobes as well as facultative anaerobes. They all have

the ability to respire using oxygen as a terminal electron acceptor.

Catalase-negative bacteria may be anaerobes, or they may be facultative anaerobes that only

ferment and do not respire using oxygen as a terminal electron acceptor (ie. Streptococci).

Percentage of H202 used on catalase test:

1. For routine testing of aerobes, 3% hydrogen peroxide is used.

2.15% H2O2 solution: For the identification of anaerobic bacteria

Catalase test is used to differentiate aero tolerant strains of Clostridium (catalase negative), from

Bacillus species (catalase positive).

3. The superoxol catalase test used for the presumptive speciation of certain Neisseria organisms

requires a different concentration of H2O2.

Principle: The enzyme catalase mediates the breakdown of hydrogen peroxide into oxygen and water.

The presence of the enzyme in a bacterial isolate is evident when a small inoculum is introduced into

hydrogen peroxide, and the rapid elaboration of oxygen bubbles occurs. The lack of catalase is evident

by a lack of or weak bubble production. The culture should not be more than 24 hours old.

Bacteria thereby protect themselves from the lethal effect of Hydrogen peroxide which is accumulated

as an end product of aerobic carbohydrate metabolism.

Procedure: Tube Method:

1. Pour 1-2 ml of hydrogen peroxide solution into a test tube.

2. Using a sterile wooden stick or a glass rod, take several colonies of the 18 to 24 hours test organism

and immerse in the hydrogen peroxide solution.

3. Observe for immediate bubbling.

Slide Method

1. Use a loop or sterile wooden stick to transfer a small amount of colony growth in the surface of a

clean, dry glass slide.

2. Place a drop of 3% H2O2 in the glass slide.



3. Observe for the evolution of oxygen bubbles.

Uses of catalase test:

The morphologically similar Enterococcus or Streptococcus (catalase negative)

and Staphylococcus (catalase positive) can be differentiated using the catalase test.

Also valuable in differentiating aerobic and obligate anaerobic bacteria.

Semi quantitative catalase test is used for the identification of Mycobacterium tuberculosis.

It is used to differentiate aero tolerant strains of Clostridium, which are catalase negative, from

Bacillus species, which are positive.

Catalase test can be used as an aid to the identification of Entero bacteriaceae.

Results:

Catalase Positive reactions: Evident by immediate effervescence (bubble formation)

Catalase Negative reaction: No bubble formation (no catalase enzyme to hydrolyze the

hydrogen peroxide)

Precautions while performing catalase test

1. Do not use a metal loop or needle with H2O2; it will give a false positive and degrade the metal.

2. If using colonies from a blood agar plate, be very careful not to scrape up any of the blood agar as

blood cells are catalase positive and any contaminating agar (Carryover of Red Blood Cells) could give

a false positive.

3. Because some bacteria possess enzymes other than catalase that can decompose hydrogen

peroxide, a few tiny bubbles forming after 20 to 30 seconds is not considered as positive test.

Indole test:

This test demonstrate the ability of certain bacteria to decompose the amino acid tryptophan to

indole, which accumulates in the medium. Indole production test is important in the identification of

Enterobacteria. Most strains of E. coli, P. vulgaris, P. rettgeri, M. morgani and Providencia species break

down the amino acid tryptophan with the release of indole. This is performed by a chain of a number of

different intracellular enzymes, a system generally referred to as “tryptophanase.” It is used as part of

the IMViC procedures, a tests designed to distinguish among members of the family Enterobacteriaceae.

A variation on this test using Ehrlich’s reagent (using ethyl alcohol in place of iso amyl alcohol,

developed by Paul Ehrlich) is used when performing the test on non-fermenters and anaerobes.



Principle:

Tryptophan is an amino acid that can undergo deamination and hydrolysis by bacteria that

express tryptophanase enzyme. Indole is generated by reductive deamination from tryptophan via the

intermediate molecule indole pyruvic acid. Tryptophanase catalyzes the deamination reaction, during

which the amine (-NH2) group of the tryptophan molecule is removed. Final products of the reaction

are indole, pyruvic acid, ammonium (NH4+) and energy. Pyridoxal phosphate is required as a coenzyme.

When indole is combined with Kovac’s Reagent (which contains hydrochloric acid and p-dimethyl amino

benzaldehyde in amyl alcohol) the solution turns from yellow to cherry red. Because amyl alcohol is not

water soluble, the red coloration will form in an oily layer at the top of the broth.

In the spot test, indole combines, in the filter paper matrix, at an acid pH with p-Dimethyl amino

cinnamaldehyde (DMACA) to produce a blue to blue-green compound. Indole Spot Reagent has been

reported to be useful in detecting indole production by members of the family Enterobacteriaceae and

certain anaerobic species.

Reagents Used in Indole Test

Ingredients per liter:

Indole Spot Reagent:

p-Dimethylaminocinnamaldehyde (DMACA) 10.0 gm

Hydrochloric Acid, 37% 100.0 ml

Deionized Water 900.0 ml

Indole Kovacs Reagent:

p-Dimethylaminobenzaldehyde 50.0 gm

Hydrochloric Acid, 37% 250.0 ml



Amyl Alcohol 750.0 ml

Procedure:

1. Take a sterilized test tubes containing 4 ml of tryptophan broth.

2. Inoculate the tube aseptically by taking the growth from 18 to 24 hrs culture.

3. Incubate the tube at 37°C for 24-28 hours.

4. Add 0.5 ml of Kovac’s reagent to the broth culture.

5. Observe for the presence or absence of ring.

Result:…………………………………………………………………………………………………………………………………………………

Citrate utilization test:

This test is among a suite of IMViC Tests (Indole, Methyl-Red, Vogues-Proskauer, and Citrate)

that are used to differentiate among the Gram-Negative bacilli in the family Enterobacteriaceae.

Principle:

Citrate agar is used to test an organism’s ability to utilize citrate as a source of energy. The

medium contains citrate as the sole carbon source and inorganic ammonium salts (NH4H2PO4) as the

sole source of nitrogen.

Bacteria that can grow on this medium produce an enzyme, citrate-permease, capable of

converting citrate to pyruvate. Pyruvate can then enter the organism’s metabolic cycle for

the production of energy. Growth is indicative of utilization of citrate, an intermediate metabolite in

the Krebs cycle.

When the bacteria metabolize citrate, the ammonium salts are broken down to ammonia, which

increases alkalinity. The shift in pH turns the bromthymol blue indicator in the medium from green to

blue above pH 7.6.

Christensen developed an alternative citrate test medium that does not require the organism to use

citrate as a sole carbon source. Christensen’s medium contains both peptone and cysteine. Thus citrate-

negative bacteria can also grow on this medium. A positive reaction shows that the organism can use

citrate but not necessarily as the sole carbon source.



Media used in Citrate Utilization Test

Simmon’s Citrate Agar: Composition

Sodium Chloride 5.0 gm

Sodium Citrate (dehydrate) 2.0 gm

Ammonium Dihydrogen Phosphate 1.0 gm

Dipotassium Phosphate 1.0 gm

Magnesium Sulfate (heptahydrate) 0.2 gm

Bromothymol Blue 0.08 gm

Agar 15.0 gm

Deionized water = 1,000 ml

Final pH 6.9 +/- 0.2 at 25 degrees C.

Preparation

1. Dissolve above salts in deionized water.

2. Adjust pH to 6.9.



3. Add agar and Bromothymol blue.

4. Gently heat, with mixing, to boiling until agar is dissolved.

5. Dispense 4.0 to 5.0 ml into 16-mm tubes.

6. Autoclave at 121 degree C under 15 psi pressure for 15 minutes.

7. Cool in slanted position (long slant, shallow butt).

8. Tubes should be stored in a refrigerator to ensure a shelf life of 6 to 8 weeks.

9. The uninoculated medium will be a deep forest green due to the pH of the sample and the

bromothymol blue.

Procedure of Citrate Utilization Test

1. Streak the slant back and forth with a light inoculum picked from the center of a well-isolated

colony.

2. Incubate aerobically at 35 to 370C for up to 4-7 days.

3. Observe a color change from green to blue along the slant.

Result:……………………………………………………………………………………………………………………………………………..



Diagrams: COMPOUND MICROSCOPE



Hot air oven



Autoclave



SERIAL DILUTION TECHNIQUE PURE CULTURE TECHNIQUES



BIOCHEMICAL TESTS



OBSERVATION AND SKETCHES

b sc i sem biotechnology/microbiology lab manual

Science