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An Assignment on

MICROBIOLOGY
LABORATORY

Written by

Md. Abu Nayeem

B.Sc. Fisheries (Hons)
M.S. In Fisheries Technology
BAU, Mymensingh
Nayeem
nayeem.worldfish@gmail.com
 Microbiology
Microbiology (from Greek , mkros, "small"; , bios, "life"; and -, -logia) is
the study of microorganisms, which are unicellular or cell-cluster microscopic organisms.
This includes eukaryotes such as fungi and protists, and prokaryotes. Viruses, though not
strictly classed as living organisms, are also studied. In short; microbiology refers to the study
of life and organisms that are too small to be seen with the naked eye. Microbiology typically
includes the study of the immune system, or Immunology. Generally, immune systems
interact with pathogenic microbes; these two disciplines often intersect which is why many
colleges offer a paired degree such as "Microbiology and Immunology".

Microbiology is a broad term which includes virology, mycology, parasitology, bacteriology

and other branches. A microbiologist is a specialist in microbiology.

Microbiology is researched actively, and the field is advancing continually. We have

probably only studied about one percent of all of the microbe species on Earth. Although
microbes were directly observed over three hundred years ago, the field of microbiology can
be said to be in its infancy relative to older biological disciplines such as zoology and botany.

History
Ancient

The existence of microorganisms was hypothesized for many centuries before their actual
discovery in the 17th century. In 600 BCE, the ancient Indian surgeon Susruta held microbes
responsible for several diseases and explained in Sushruta Samhita that they can be
transmitted through contact, air or water. Theories on microorganisms was made by Roman
scholar Marcus Terentius Varro in a book titled On Agriculture in which he warns against
locating a homestead in the vicinity of swamps:

...and because there are bred certain minute creatures which cannot be seen by the eyes, which float in
the air and enter the body through the mouth and nose and there cause serious diseases.

This passage seems to indicate that the ancients were aware of the possibility that diseases
could be spread by yet unseen organisms.

In The Canon of Medicine (1020), Ab Al ibn Sn (Avicenna) stated that bodily secretion is
contaminated by foul foreign earthly bodies before being infected. He also hypothesized on
the contagious nature of tuberculosis and other infectious diseases, and used quarantine as a
means of limiting the spread of contagious diseases.

When the Black Death bubonic plague reached al-Andalus in the 14th century, Ibn Khatima
hypothesized that infectious diseases are caused by "minute bodies" which enter the human
body and cause disease.

In 1546 Girolamo Fracastoro proposed that epidemic diseases were caused by transferable
seedlike entities that could transmit infection by direct or indirect contact or even without
contact over long distances.

1
All these early claims about the existence of microorganisms were speculative in nature and
not based on any data or science. Microorganisms were neither proven and observed, nor
correctly and accurately described until the 17th century. The reason for this was that all
these early inquiries lacked the most fundamental tool in order for microbiology and
bacteriology to exist as a science, and that was the microscope.

Antonie van Leeuwenhoek, the first microbiologist and the first to observe microorganisms
using a microscope. Known as the 'Father of Microbiology'. Whilst he did not invent the
microscope, he greatly developed it.

Modern

Bacteria, and other microorganisms, were first observed by Antonie van Leeuwenhoek in
1676 using a single-lens microscope of his own design. In doing so Leeuwenhoek made one
of the most important discoveries in biology and initiated the scientific fields of bacteriology
and microbiology. The name "bacterium" was introduced much later, by Ehrenberg in 1828,
derived from the Greek meaning "small stick". While Van Leeuwenhoek is often
cited as the first microbiologist, the first recorded microbiological observation, that of the
fruiting bodies of molds, was made earlier in 1665 by Robert Hooke.

The field of bacteriology (later a subdiscipline of microbiology) is generally considered to

have been founded in the 19th century by Ferdinand Cohn, a botanist whose studies on algae
and photosynthetic bacteria led him to describe several bacteria including Bacillus and
Beggiatoa. Cohn was also the first to formulate a scheme for the taxonomic classification of
bacteria. Louis Pasteur and Robert Koch were contemporaries of Cohns and are often
considered to be the founders of medical microbiology. Pasteur is most famous for his series
of experiments designed to disprove the then widely held theory of spontaneous generation,
thereby solidifying microbiologys identity as a biological science. Pasteur also designed
methods for food preservation (pasteurization) and vaccines against several diseases such as
anthrax, fowl cholera and rabies. Koch is best known for his contributions to the germ theory
of disease, proving that specific diseases were caused by specific pathogenic microorganisms.
He developed a series of criteria that have become known as the Koch's postulates. Koch was
one of the first scientists to focus on the isolation of bacteria in pure culture resulting in his
description of several novel bacteria including Mycobacterium tuberculosis, the causative
agent of tuberculosis.

While Pasteur and Koch are often considered the founders of microbiology, their work did
not accurately reflect the true diversity of the microbial world because of their exclusive
focus on microorganisms having direct medical relevance. It was not until the late 19th
century and the work of Martinus Beijerinck and Sergei Winogradsky, the founders of
general microbiology (an older term encompassing aspects of microbial physiology,
diversity and ecology), that the true breadth of microbiology was revealed. Beijerinck made
two major contributions to microbiology: the discovery of viruses and the development of
enrichment culture techniques.[11] While his work on the Tobacco Mosaic Virus established
the basic principles of virology, it was his development of enrichment culturing that had the
most immediate impact on microbiology by allowing for the cultivation of a wide range of
microbes with wildly different physiologies. Winogradsky was the first to develop the
concept of chemolithotrophy and to thereby reveal the essential role played by

2
microorganisms in geochemical processes. He was responsible for the first isolation and
description of both nitrifying and nitrogen-fixing bacteria.

Fields
The field of microbiology can be generally divided into several subdisciplines:

Microbial physiology: The study of how the microbial cell functions biochemically. Includes
the study of microbial growth, microbial metabolism and microbial cell structure.

Microbial genetics: The study of how genes are organized and regulated in microbes in
relation to their cellular functions. Closely related to the field of molecular biology.

Cellular microbiology: A discipline bridging microbiology and cell biology.

Medical microbiology: The study of the pathogenic microbes and the role of microbes in
human illness. Includes the study of microbial pathogenesis and epidemiology and is related
to the study of disease pathology and immunology.

Veterinary microbiology: The study of the role in microbes in veterinary medicine or

animal taxonomy.

Environmental microbiology: The study of the function and diversity of microbes in their
natural environments. Includes the study of microbial ecology, microbially-mediated nutrient
cycling, geomicrobiology, microbial diversity and bioremediation. Characterisation of key
bacterial habitats such as the rhizosphere and phyllosphere, soil and groundwater ecosystems,
open oceans or extreme environments (extremophiles).

Evolutionary microbiology: The study of the evolution of microbes. Includes the study of
bacterial systematics and taxonomy.

Industrial microbiology: The exploitation of microbes for use in industrial processes.

Examples include industrial fermentation and wastewater treatment. Closely linked to the
biotechnology industry. This field also includes brewing, an important application of
microbiology.

Aeromicrobiology: The study of airborne microorganisms.

Food microbiology: The study of microorganisms causing food spoilage and foodborne
illness. Using microorganisms to produce foods, for example by fermentation.

Pharmaceutical microbiology: the study of microorganisms causing pharmaceutical

contamination and spoil

Agricultural microbiology: The study of agriculturaly important microorganisms.

Fishery Microbiology: Fisheries microbiology is that branch of science which deals with the
study of a vast collection of microscopic, unicellular and largely undifferentiated life from of
microscopic living organism and their effect on the fish and fish culture.

3
 Aquatic Bacteria
The bacteria whose proper home is in water and which can developed optimally only in
water.

Other sources of Bacteria in Water

Besides genuine aquatic bacteria, a number of bacteria from other habitates are also found.

Soil: Water in close contact with soil

Air: A constant rain of bacteria falls from the air on to surface waters
Plants, animals and human

Thw bacteria living in the sea are different from those in fresh water and amongst the latter
those of the rivers are different from those in lakes.

General characteristics of aquatic bacteria

Majority of aquatic bacteria are heterophilic, i.e. they live on organic substances.
Some are photo and chemoautotrophic needing only inorganic nutrients.
Morphologically, most aquatic bacteria are similar to the basic types of terrestrial
bacteria.
In most water predominant bacteria are gram (-).
The majority of aquatic bacteria are motile, as a rule by means of flagella.
Genuine aquatic bacteria are distinguished by their ability to utilize very small
concentration of nutrients.
Bacteria may live in the water free or growing on some solid substratum.
Systematically aquatic bacteria are not a homogenous group, their representatives are
found in almost all orders of the class of Bacteria.
There are great biological differences between inland and marine bacteria.

Bacteria in inland waters

There are relationships between the bacterial flora of inland waters and that of the soil.

Ground water
Ground water is poor in micro-organisms and in nutrients. Ground water from different parts
of the world examined included the microorganisms belonging to the genera:-

Achromobacter, Flavobacterium, Micrococcus, Nocardia, Cytophaga, Hypomicrobium,

Planctomyces, Gallionella, Caulobacter, Agrobacterium, Clostridium etc.

The ground water of oil-bearing rocks contains large number of bacteria which decompose
hydrocarbons.

4
Spring Water
Spring water contains more or less similar type of bacteria as mentioned above. Other
bacteria added are iron containing water contain iron bacteria (Gallionella ferrugina,
Leptothrix ochrcea etc.), thermal springs contain thermotolerant and thermophilic species
(Sulfolobus acidocaldarius, Leptothrix thermalis, Thermus aquaticus etc.).

Streams
In streams which are poor in nutrients, gram negative non sporeing rod shaped predomonate
which include- Psedomonas, Flavobacterium, Acinetobacter, Moraxella etc. with increasing
eutrophication Bacillus and Enterobacteria gain importance.

River
Depending on the sewage load, rivers carry more or less numerous sewage bacteria which
include Escherichia coli, Proteus vulgaris, Salmonella sp, Clostridium, Desulfovibrio
desulfuricans etc.

Lakes
Non sporing rod shaped bacteria predominant in the lakes of temperate climate. Looking
through the literature, it can be gathered that bacteria particularly of the genera
Achromobacter, Flavobacterium, Vibrio, Brevibacterium, Spirillum, Microccus, Sarcina,
Bacillus, Pseudomonas, Nocardia, Streptomyces and Cytophaga occur widely in lakes.
Majority of bacteria living in salt lakes are halophilic and halotolerance forms
(Halobacterium and Halococcus).

5
 General Suggestion and Safety in a Microbiology
Laboratory
Laboratory Philosophy
Laboratories are places for practical work by professional scientists, technicians and students.
Laboratory coats, stout shoes and often safety spectacles should be worn by all to enter.
Recreational and refreshment areas should be provided away from the laboratories and food
and drink for consumption should never be brought to the laboratory. Smoking, eating,
drinking and application of cosmetics should not be allowed in the laboratory. All personnel
should be aware that if something does go wrong as a result of failure to follow safe
procedures there will be criticism from all quarters. In short laboratories are specialized areas
that require careful planning, sufficient resources and rigorous supervision.

General Suggestion
1. Before each laboratory period, read over the exercise to be done and plan your works
carefully and know how exercise to be done. Few trials should be done before
commencement of the actual experiment.

2. Each laboratory meeting will begin with a short discussion and instruction period. Do
not begin work until you have received your instruction.

3. Properly record all observation at the time when they are made. Laboratory
examination will cover both information given by your laboratory teacher and that
contain in the manual as well as your own observation and education. Drawing should
be made where it is necessary.

Microbiology Lab Practices and Safety Rules

1. Outdoor cloths and bags should be left outside the laboratory, preferably in a locker.

2. Laboratory coats and aprons should be worn at all time in the microbiology laboratory

3. Top of the laboratory desk must be sponge off the germicide solution at both the
beginning and close of each laboratory period.

4. Eating, drinking or smoking inside the laboratory is strictly prohibited.

5. Cuts and abrasions should be covered with a waterproof dressing before entering the
laboratory.

6. Work in a microbiology laboratory should not be done unsupervised.

7. A safety bulb should always be used during transferring liquids, mouth pipetting is
prohibited.

8. Contaminated pipettes should be placed in to discard jar containing disinfected.

6
 9. If contaminated materials are accidentally spill on to the bench or floor, it should be
disinfected with an appropriate disinfectant like Lysol.

10. Any type of accident like cuts, abrasion or breaking of glassware with bacterial
culture should be reported to a supervisor.

11. Contaminated materials should be placed on the designated place.

12. Wash your hands with disinfectant soap when you arrive at the lab and again before
you leave.

13. Absolutely no food, drinks, chewing gum, or smoking is allowed in the laboratory. Do
not put anything in your mouth such as pencils, pens, labels or fingers. Do not store
food in areas where microorganisms are stored.

14. Purchase a lab coat and safety glasses, bring them to class, and use them.
Alternatively, a long sleeved shirt that buttons or snaps closed is acceptable protective
clothing. This garment must cover your arms and be able to be removed without
pulling it over your head. Leave protective clothing in the lab and do not wear it to
other non-lab areas.

15. Avoid loose fitting items of clothing. Wear appropriate shoes (sandals are not
allowed) in the laboratory.

16. Keep your workspace free of all unnecessary materials. Backpacks, purses, and coats
should be placed in the cubbyholes by the front door of the lab. Place needed items on
the floor near your feet, but not in the aisle.

17. Disinfect work areas before and after use with 70% ethanol or fresh 10% bleach.
Laboratory equipment and work surfaces should be decontaminated with an
appropriate disinfectant on a routine basis, and especially after spills, splashes, or
other contamination.

18. Label everything clearly.

19. Replace caps on reagents, solution bottles, and bacterial cultures. Do not open Petri
dishes in the lab unless absolutely necessary.

20. Inoculating loops and needles should be flame sterilized in a Bunsen burner before
you lay them down.

21. Turn off Bunsen burners when not is use. Long hair must be restrained if Bunsen
burners are in use.

22. When you flame sterilize with alcohol, be sure that you do not have any papers under
you.

23. Treat all microorganisms as potential pathogens. Use appropriate care and do not take
cultures out of the laboratory.

7
 24. Wear disposable gloves when working with potentially infectious microbes or
samples (e.g., sewage). If you are working with a sample that may contain a pathogen,
then be extremely careful to use good bacteriological technique.

25. Sterilize equipment and materials.

26. Never pipette by mouth. Use a pipetting aid or adjustable volume pipettors. [In the
distant past, some lab personnel were taught to mouth pipette. This practice has been
known to result in many laboratory-acquired infections. With the availability of
mechanical pipetting devices, mouth pipetting is strictly prohibited.

27. Consider everything a biohazard. Do not pour anything down the sink. Autoclave
liquids and broth cultures to sterilize them before discarding.

28. Dispose of all solid waste material in a biohazard bag and autoclave it before
discarding in the regular trash.

29. Familiarize yourself with the location of safety equipment in the lab (e.g., eye-wash
station, shower, sinks, fire extinguisher, biological safety cabinet, first aid kit,
emergency gas valve).

30. Dispose of broken glass in the broken glass container.

31. Dispose of razor blades, syringe needles, and sharp metal objects in the sharps
container.

32. Report spills and accidents immediately to your instructor. Clean small spills with
care (see instructions below). Seek help for large spills.

33. Report all injuries or accidents immediately to the instructor, no matter how small
they seem.

Laboratory Safety Equipment

Biological Safety Cabinet

A biological safety cabinet (BSC) is used as a primary barrier against exposure to infectious
biological agents. A BSC has High Efficiency Particulate Air (HEPA) filters. The airflow in a
BSC is laminar, i.e. the air moves with uniform velocity in one direction along parallel flow
lines. Depending on the design, a BSC may be vented to the outside or the air may be
exhausted into the room. BSCs are not chemical fume hoods. A percentage of the air is
recirculated in most types of BSCs. HEPA filters only trap particulates, allowing any
contaminant in non-particulate form to pass through the filter.

Proper Use of BSCs:

1. Operate the cabinet for five minutes before and after performing any work in it in order to
purge airborne contaminants.

2. Before and after use, wipe the surface of the BSC with a suitable disinfectant, e.g., 70%
alcohol or a 10% bleach solution.

8
3. Place everything you will need inside the cabinet before beginning work, including a
waste container. You should not have to penetrate the air barrier of the cabinet once work
has begun.

4. Do not place anything on the air intake grills, as this will block the air supply.

5. You should prevent unnecessary opening and closing of door because this will disrupt the
Airflow of the cabinet.

6. Always wear a lab coat while using the cabinet and conduct your work at least four inches
Inside the cabinet.

7. Place burners to the rear of the cabinet to reduce air turbulence.

8. Do not work in the BSC while the ultraviolet light is on. Ultraviolet light can quickly
injure the eye.

9. When finished with your work procedure, decontaminate the surfaces of any equipment.

10. Remove the equipment from the cabinet and decontaminate the work surface.

11. Thoroughly wash your hands and arms.

Eyewash and shower

Fire Extinguisher

First Aid Kit

Emergency Gas Valve

Cleaning Small Spills

First, contact your instructor or the Biology Department Safety Officer. If it is a small spill of
a low hazard microorganism or sample, then you should clean the spill yourself.
The proper procedures for cleaning small spills of microorganisms or samples (BSL1 and
BSL2 levels):
1. Wear a lab coat, disposable gloves, safety glasses or a face shield, and if needed, approved
respiratory equipment.
2. Soak a paper towel(s) in an appropriate disinfectant (70% ethanol or fresh 10% bleach
solution) and place around the spill area.
3. Working from the outer edges into the center, clean the spill area with fresh towels soaked
in the disinfectant. Be sure to decontaminate any areas or surfaces that you suspect may
have been affected by the spill. Allow 10 minutes contact time.
4. Place the paper towels and gloves into a biohazard bag and autoclave these materials to
sterilize them.
5. Dispose of any contaminated clothing properly.
6. Wash your hands with a disinfectant soap.

If it is a large spill and your instructor and the Biology Department Safety Officer are not
available, and then call the UMD Department of Environmental Health and Safety. Each lab
is equipped with a spill response kit.

9
Biosafety Levels and Practices

The Centers for Disease Control (CDC) and the National Institutes of Health (NIH) have
developed standard procedures providing protection against biological hazards. The
publication, Biosafety in Microbiological and Biomedical Laboratories provides specific
descriptions of microbiological practices, laboratory facilities, and safety equipment, and
recommends their use in four biosafety levels (BSLs). Biosafety levels are selected to provide
the end-user with a description of the minimum containment required for handling different
microorganisms safely in a laboratory setting and reduce or eliminate exposure to potentially
hazardous agents. Containment refers to safe methods for managing infectious material in the
laboratory environment. These biosafety levels are applicable to facilities such as diagnostic,
research, clinical, teaching, and production facilities that are working at a laboratory scale.
The four biosafety levels are described as:

BSL Agents Practices Safety Equipment Facilities

1 Not known to cause Standard None required Open bench top
disease in healthy microbiological practice Hand washing sink
adults required
2 Associated with BSL-1 plus Class I or II BSC or BSL-1 plus
human disease; Limited access to lab other physical Autoclave available
main hazard is Biohazard warning contamination devise
percutaneous injury, sign used for all
ingestion and Sharps precaution manipulations of agents
mucous membrane Biosafety manual that cause splasher or
exposure defining waste decon aerosols of infectious
and medical materials. PPE lab
surveillance policies coats, gloves,
respiratory protection as
needed
3 Indigenous or exotic BSL-2 plus Class I or II BSC or BSL-2 plus
agents with Controlled access other physical Physical separation
potential for aerosol Decon of all waste containment device from access
transmission. Decon of lab clothing used for all open corridors
Disease may have before laundering manipulations of Self-closing, double
serious or lethal Baseline serums agents. PPE: lab coats; door access
consequences obtained gloves, respiratory Exhaust not
protection as needed recirculated
Negative airflow
into BSL-3 lab
4 Dangerous/exotic BSL-3 plus All procedures Lab in a separate
agents which pose a Clothing change conducted in class III building or
risk of life before entering BSC or class I or II isolated zone
threatening disease, Shower upon exit BSC in combination Dedicated supply
aerosol-transmitted All material with full body, air and exhaust
lab infections, or decontaminated upon supplied, positive system
related agents with exit from facility pressure personnel suit. Other as outlined
unknown risk of in text
transmission

Biosafety Level 1 (BSL1)

Examples of BSL1 Agents: Bacillus subtilus, Naegleria gruberi, many Escherichia coli,
Infectious Canine Hepatitis Virus

10
BSL1 containment is suitable for work involving well-characterized agents not known to
cause disease in healthy adult humans, and of minimal potential hazard to laboratory
personnel and the environment. A BSL1 lab requires no special design features beyond those
suitable for a well-designed and functional laboratory. Biological safety cabinets (BSCs) are
not required. Work may be done on an open bench top, and containment is achieved through
the use of practices normally employed in a basic microbiology laboratory.

Biosafety Level 2 (BSL2)

Examples of BSL2 Agents: Bacillus anthracis, Bordetella pertussis, Brucella spp.,
Cryptococcus neoformans, Clostridium botulinum, Clostridium tetani, Helicobacter pylori,
most Salmonella spp., Yersinia pestis, Mycobacterium leprae, Shigella spp., Human
Immunodeficiency Virus, Human blood

The primary exposure hazards associated with organisms requiring BSL2 are through the
ingestion, inoculation and mucous membrane route. Agents requiring BSL2 facilities are not
generally transmitted by airborne routes, but care must be taken to avoid the generation of
aerosols (aerosols can settle on bench tops and become an ingestion hazard through
contamination of the hands) or splashes. Primary containment devices such as BSCs and
centrifuges with sealed rotors or safety cups are to be used as well as appropriate personal
protective equipment (i.e., gloves, laboratory coats, protective eyewear). As well,
environmental contamination must be minimized by the use of hand washing sinks and
decontamination facilities (autoclaves).

Biosafety Level 3 (BSL3)

Examples of BSL3 Agents: Myobacterium tuberculosis, Salmonella typhi, Vesicular
Stomatitis Virus, Yellow Fever Virus, Francisella tularensis, Coxiella burnetti

Laboratory personnel have specific training in handling these pathogenic and potentially
lethal agents and are supervised by scientists who are experienced in working with these
agents. These agents may be transmitted by the airborne route, often have a low infectious
dose to produce effects and can cause serious or life-threatening disease. BSL3 emphasizes
additional primary and secondary barriers to minimize the release of infectious organisms
into the immediate laboratory and the environment. Additional features to prevent
transmission of BSL3 organisms are appropriate respiratory protection, HEPA filtration of
exhausted laboratory air and strictly controlled laboratory access.

Biosafety Level 4 (BSL4)

Examples of BSL5 Agents: smallpox virus, Ebola virus, hemorrhagic fever viruses

This is the maximum containment available and is suitable for facilities manipulating agents
that are dangerous/exotic agents, which post a risk of life threatening disease. These agents
have the potential for aerosol transmission, often have a low infectious dose and produce very
serious and often fatal disease; there is generally no treatment or vaccine available. This level
of containment represents an isolated unit, functionally and, when necessary, structurally
independent of other areas. BSL4 emphasizes maximum containment of the infectious agent
by complete sealing of the facility perimeter with confirmation by pressure decay testing;
isolation of the researcher from the pathogen by his or her containment in a positive pressure
suit or containment of the pathogen in a Class III BSC line; and decontamination of air and
other effluents produced in the facility.

11
 Important Terminology Used in Microbiology
Laboratory
Sterilization
The complete destruction or removal of all living microorganisms or their spores by any
physical, chemical or mechanical means is called sterilization. Sterilization can be
accomplished by using heat, filtration and gases.

Sterilization by heat
Heat sterilization can be accomplished by several ways:-

Red heat incineration

Instruments such as inoculating loop, wire etc. are sterilize by holding them in flame of spirit
lamp of Bunsen burner until they are red-hot.

Flaming

It is method commonly used for decontamination of the mouth of bottles, flasks, tubes, glass
slide etc. by passing through a flame without allowing to become red-hot.

Hot air oven

This method is used to sterilize several partials such as glass, petri dishes, flasks, pipettes,
metal instruments etc. The temperature used for sterilization in an oven is generally 1700C
and heating time is about one hour.

Moist heat
bacteria are more readily killed by moist heat than by dry heat because steams kill bacteria by
denaturing protein. For this purpose 1210C temperature is used for 15 to 20 minutes under 15
lb/inch2 pressures. Sterilization by moist heat normally carried out in autoclave.

Tyndallization
Tyndallization is a heating process in which some heat sensitive media or reagent are
sterilized by steaming (1000C) for 32-35 minutes on three successive days to kill spores
completely. On the first occasion vegetative cells of microorganisms are killed. Any spore
that germinates in the media over night producing vegetative forms are killed by the second
or third steaming.

Ionizing irradiation
This method is used in industry for the sterilization of disposable materials for using in the
hospital or laboratory. For purpose X-rays or gamma rays from a radio active source is used.

12
Sterilization by filtration
Certain sugars, blood serum etc which are destroyed by heating are sterilized by filtration
method by using different types of filtration devises, This method is not suitable to remove
mycoplasma and viruses. For filtration of bacteria, following filters are used:-

Sintered glass filter

Seitz asbestos filter
Membrane filter (Nitrocellulose filter)
Chamberland filter
Mander filter etc.

Gaseous sterilization: It is a modern and common method of sterilization which is done by

ethylene oxide. Ethylene oxide is a highly toxic material for bacteria, fungi and viruses and
also for most heat resistant spores. Mixture of 10% ethylene oxide 90% or freyon gas is used
for this purpose and most effective.

Disinfection: It denotes the use of chemical substances or agent to destroy the infectious
microorganisms or their infectivity in a substrate.

Disinfectant: The chemical substances which are used for disinfection of lifeless object is
called disinfectant. For example:- ethanol, phenol, benzoic acid, carbonic acid, sodium
hypochlorite etc.

Antiseptic

Antiseptics (from Greek - anti, '"against" + - septikos, "putrefactive") are

antimicrobial substances that are applied to living tissue/skin to reduce the possibility of
infection, sepsis, or putrefaction. Antiseptics are generally distinguished from antibiotics by
their ability to be transported through the lymphatic system to destroy bacteria within the
body, and from disinfectants, which destroy microorganisms found on non-living objects.
Some antiseptics are true germicides, capable of destroying microbes (bacteriocidal), whilst
others are bacteriostatic and only prevent or inhibit their growth. Antibacterials are
antiseptics that have the proven ability to act against bacteria. Microbicides which kill virus
particles are called viricides or antivirals.

Some common antiseptics

Alcohols
Most commonly used are ethanol (6090%), 1-propanol (6070%) and 2-
propanol/isopropanol (7080%) or mixtures of these alcohols. They are commonly
referred to as "surgical alcohol". Used to disinfect the skin before injections are given,
often along with iodine (tincture of iodine) or some cationic surfactants
(benzalkonium chloride 0.050.5%, chlorhexidine 0.24.0% or octenidine
dihydrochloride 0.12.0%).

Quaternary ammonium compounds

Also known as Quats or QAC's, include the chemicals benzalkonium chloride (BAC),
cetyl trimethylammonium bromide (CTMB), cetylpyridinium chloride (Cetrim, CPC)
and benzethonium chloride (BZT). Benzalkonium chloride is used in some pre-
operative skin disinfectants (conc. 0.050.5%) and antiseptic towels. The

13
 antimicrobial activity of Quats is inactivated by anionic surfactants, such as soaps.
Related disinfectants include chlorhexidine and octenidine.

Boric acid
Used in suppositories to treat yeast infections of the vagina, in eyewashes, and as an
antiviral to shorten the duration of cold sore attacks. Put into creams for burns. Also
common in trace amounts in eye contact solution. Though it is popularly known as an
antiseptic, it is in reality only a soothing fluid, and bacteria will flourish comfortably
in contact with it.

Brilliant Green
A triarylmethane dye still widely used as 1% ethanol solution in Eastern Europe and
ex-USSR countries for treatment of small wounds and abscesses. Efficient against
gram-positive bacteries.

Chlorhexidine Gluconate
A biguanidine derivative, used in concentrations of 0.54.0% alone or in lower
concentrations in combination with other compounds, such as alcohols. Used as a skin
antiseptic and to treat inflammation of the gums (gingivitis). The microbicidal action
is somewhat slow, but remanent. It is a cationic surfactant, similar to Quats.

Hydrogen peroxide
Used as a 6% (20 Vols) solution to clean and deodorize wounds and ulcers. More
common 3% solutions of hydrogen peroxide have been used in household first aid for
scrapes, etc. However, even this less potent form is no longer recommended for
typical wound care as the strong oxidization causes scar formation and increases
healing time. Gentle washing with mild soap and water or rinsing a scrape with sterile
saline is a better practice.

Iodine
Usually used in an alcoholic solution (called tincture of iodine) or as Lugol's iodine
solution as a pre- and post-operative antiseptic. No longer recommended to disinfect
minor wounds because it induces scar tissue formation and increases healing time.
Gentle washing with mild soap and water or rinsing a scrape with sterile saline is a
better practice. Novel iodine antiseptics containing povidone-iodine (an iodophor,
complex of povidone, a water-soluble polymer, with triiodide anions I3-, containing
about 10% of active iodine) are far better tolerated, don't affect wound healing
negatively and leave a deposit of active iodine, creating the so-called "remanent," or
persistent, effect. The great advantage of iodine antiseptics is the widest scope of
antimicrobial activity, killing all principal pathogenes and given enough time even
spores, which are considered to be the most difficult form of microorganisms to be
inactivated by disinfectants and antiseptics.
Mercurochrome
Not recognized as safe and effective by the U.S. Food and Drug Administration
(FDA) due to concerns about its mercury content. Other obsolete organomercury
antiseptics include bis-(phenylmercuric) monohydrogenborate (Famosept).

Manuka Honey
Recognized by the U.S. Food and Drug Administration (FDA) as a medical device for
use in wounds and burns. Active +15 is equal to a 15% solution of phenol.

14
 Octenidine dihydrochloride
A cationic surfactant and bis-(dihydropyridinyl)-decane derivative, used in
concentrations of 0.12.0%. It is similar in its action to the Quats, but is of somewhat
broader spectrum of activity. Octenidine is currently increasingly used in continental
Europe as a QAC's and chlorhexidine (with respect to its slow action and concerns
about the carcinogenic impurity 4-chloroaniline) substitute in water- or alcohol-based
skin, mucosa and wound antiseptic. In aqueous formulations, it is often potentiated
with addition of 2-phenoxyethanol.

Phenol (carbolic acid) compounds

Phenol is germicidal in strong solution, inhibitory in weaker ones. Used as a "scrub"
for pre-operative hand cleansing. Used in the form of a powder as an antiseptic baby
powder, where it is dusted onto the navel as it heals. Also used in mouthwashes and
throat lozenges, where it has a painkilling effect as well as an antiseptic one.
Example: TCP. Other phenolic antiseptics include historically important, but today
rarely used (sometimes in dental surgery) thymol, today obsolete hexachlorophene,
still used triclosan and sodium 3,5-dibromo-4-hydroxybenzenesulfonate (Dibromol).

Sodium chloride
Used as a general cleanser. Also used as an antiseptic mouthwash. Only a weak
antiseptic effect, due to hyperosmolality of the solution above 0.9%.

Sodium hypochlorite
Used in the past, diluted, neutralized and combined with potassium permanganate in
the Daquin's solution. It is now used only as disinfectant.

Calcium hypochlorite
Used by Semmelweis, as "chlorinated lime", in his revolutionary efforts against
childbed fever.

Sodium bicarbonate (NaHCO3)

has antiseptic and disinfectant properties.

Terpenes
are the main type of compound found in essential oils, and some have reasonably
strong antibacterial, antifungal and antiviral properties. For example Terpinen-4-ol is
found in Tea tree oil.

Bactericide

The agents or substances which cause killing of bacteria are called bactericide or a
bactericide or bacteriocide is a substance that kills bacteria and, ideally, nothing else.
Bactericides are either disinfectants, antiseptics or antibiotics.

Bactericidal Disinfectants

The most used disinfectants are those applying

active chlorine (i.e., hypochlorites, chloramines, dichloroisocyanurate and

trichloroisocyanurate, wet chlorine, chlorine dioxide etc.),

15
 active oxygen (peroxides, such as peracetic acid, potassium persulfate, sodium
perborate, sodium percarbonate and urea perhydrate),
iodine (iodpovidone (povidone-iodine, Betadine), Lugol's solution, iodine tincture,
iodinated nonionic surfactants),
concentrated alcohols (mainly ethanol, 1-propanol, called also n-propanol and 2-
propanol, called isopropanol and mixtures thereof; further, 2-phenoxyethanol and 1-
and 2-phenoxypropanols are used),
phenolic substances (such as phenol (also called "carbolic acid"), cresols (called
"Lysole" in combination with liquid potassium soaps), halogenated (chlorinated,
brominated) phenols, such as hexachlorophene, triclosan, trichlorophenol,
tribromophenol, pentachlorophenol, Dibromol and salts thereof),
cationic surfactants, such as some quaternary ammonium cations (such as
benzalkonium chloride, cetyl trimethylammonium bromide or chloride,
didecyldimethylammonium chloride, cetylpyridinium chloride, benzethonium
chloride) and others, non-quaternary compounds, such as chlorhexidine,
glucoprotamine, octenidine dihydrochloride etc.),
strong oxidizers, such as ozone and permanganate solutions;
heavy metals and their salts, such as colloidal silver, silver nitrate, mercury chloride,
phenylmercury salts, copper sulfate, copper oxide-chloride etc. Heavy metals and
their salts are the most toxic, and environment-hazardous bactericides and therefore,
their use is strongly oppressed or canceled; further, also
properly concentrated strong acids (phosphoric, nitric, sulfuric, amidosulfuric,
toluenesulfonic acids) and
alkalis (sodium, potassium, calcium hydroxides),

such as of pH < 1 or > 13, particularly under elevated temperature (above 60C), kills
bacteria.

Bactericidal Antiseptics

As antiseptics (i.e., germicide agents that can be used on human or animal body, skin,
mucoses, wounds and the like), few of the above mentioned disinfectants can be used, under
proper conditions (mainly concentration, pH, temperature and toxicity toward man/animal).
Among them, important are some

properly diluted chlorine preparations (f.e. Daquin's solution, 0.5% sodium or

potassium hypochlorite solution, pH-adjusted to pH 7 - 8, or 0.5 - 1% solution of
sodium benzenesulfochloramide (chloramine B)), some
iodine preparations, such as iodopovidone in various galenics (oinment, solutions,
wound plasters), in the past also Lugol's solution,
peroxides as urea perhydrate solutions and pH-buffered 0.1 - 0.25% peracetic acid
solutions,
alcohols with or without antiseptic additives, used mainly for skin antisepsis,
weak organic acids such as sorbic acid, benzoic acid, lactic acid and salicylic acid
some phenolic compounds, such as hexachlorophene, triclosan and Dibromol, and
cation-active compounds, such as 0.05 - 0.5% benzalkonium, 0.5 - 4% chlorhexidine,
0.1 - 2% octenidine solutions.

Others are generally not applicable as safe antiseptics, either because of their corrosive or
toxic nature.

16
Bactericidal Antibiotics

Bactericidal antibiotics kill bacteria; bacteriostatic antibiotics only slow their growth or
reproduction.

Antibiotics that inhibit cell wall synthesis: the Beta-lactam antibiotics, (penicillin derivatives
(penams), cephalosporins (cephems), monobactams, and carbapenems) and vancomycin.
There may be others.

Also bactericidal are daptomycin, fluoroquinolones, metronidazole, nitrofurantoin, co-

trimoxazole. There may be others.

Aminoglycosidic antibiotics are usually considered bactericidal, although they may be

bacteriostatic with some organisms

Bacteriostatic agent

Bacteriostatic antibiotics limit the growth of bacteria by interfering with bacterial protein
production, DNA replication, or other aspects of bacterial cellular metabolism.

Bacteriostatic antibiotics inhibit growth and reproduction of bacteria without killing them;
killing is done by bactericidal agents. Bacteriostatic agents must work with the immune
system to remove the microorganisms from the body. However, there is not always a precise
distinction between them and bactericides; high concentrations of some bacteriostatic agents
are also bactericidal, whereas low concentrations of some bacteriocidal agents are
bacteriostatic.

This group includes the

tetracyclines
sulphonamides
spectinomycin
trimethoprim
chloramphenicol
macrolides
lincosamides

Fungicide

Fungicides are chemical compounds or biological organisms used to kill or inhibit fungi or
fungal spores. Fungi can cause serious damage in agriculture, resulting in critical losses of
yield, quality and profit. Fungicides are used both in agriculture and to fight fungal infections
in animals. Chemicals used to control oomycetes, which are not fungi, are also referred to as
fungicides as oomycetes use the same mechanisms as fungi to infect plants.

Fungicides can either be contact or systemic. A contact fungicide kills fungi by direct
contact; a systemic fungicide has to be absorbed by the affected organism.

Most fungicides that can be bought retail are sold in a liquid form. The most common active
ingredient is sulfur, present at 0.08% in weaker concentrates, and as high as 0.5% for more
potent fungicides. Fungicides in powdered form are usually around 90% sulfur and are very

17
toxic. Other active ingredients in fungicides include neem oil, rosemary oil, jojoba oil, and
the bacterium Bacillus subtilis.

Fungicide residues have been found on food for human consumption, mostly from post-
harvest treatments. Some fungicides are dangerous to human health, such as vinclozolin,
which has now been removed from use.

Sepsis

Contamination, Infection or putrefaction by unwanted microorganisms is called sepsis.

Asepsis

The technique which is used to keep all the unwanted microbes from the field of work of
observation for the prevention of sepsis called asepsis.

Antibiotic
Any chemical substance produced by a microorganism or prepared synthetically having
antimicrobial action is called antibiotic.

Antimicrobial agent
Any chemical substances or agents which have antimicrobial action is called antimicrobial
agent but all antimicrobial agents are not antibiotics.
Sanitation
Sanitation refers to a procedure which is used to reduce bacterial load from food and utensils.
Microbial contamination
It is defined as the attack on food or any other substance by undesirable microorganisms.
Spoilage
Undesirable changes in food caused by microorganisms is called spoilage
Inoculums
The microbial sample which is taken from a food materials, water, soil, plant, animal or
microbial culture in order to culture them in a medium is called an inoculum.
Inoculation
It is a process technique by which an inoculum is transferred in to the culture medium by
using an inoculating loop or needle.

Culture media
A culture medium is a substance having favorable condition such as moisture, food, pH, O2
content and growth factors to support the growth of microorganisms.

18
 Culture Media
Culture media
Any nutrient substance or a mixture of nutrient substances used for the artificial culture of
microorganisms in the laboratory is termed as culture media.

Performance tests on culture media

Culture media may be prepared from the individual ingredients or may be prepared from
dehydrated powders available commercially. The important points in QC of media are listed
here:
Do not over-stock the media. Store the required quantities only which can be used in 6-12
months.
Store the media away from moisture by securing the caps of all the containers tightly.
Store in a dark, cool and well-ventilated place.
Keep a record of the receipt, and opening of the media container.
Discard all dehydrated media that are either darkened or caked. Rotate the stock of media,
following the principle of "first in, first out".
For preparation of media adhere strictly to the manufacturers instructions.
Prepared media should be protected from sunlight and heat.

Types of culture media:

According to the nature of their constituents culture media may be divided into two major
groups:

Natural media

Media which contains infusion of natural substances, the chemical composition of which
varies from time to time.

Synthetic or defined media

Media which contains ingredients of known chemical composition. These media usually
comprise solution of mineral salts to which the substrate to be tested is added.

On the basis of nutrition and purpose media may be of several types:

Ordinary media:
This media contains minimum amount of nutrient substances for the growth and propagation
of microorganisms, for example, nutrient agar and nutrient broth.

Enriched or isolation media

These may be sample nutrient media containing sufficient amount of essential constituents
for the growth of bacteria, such as blood agar medium and serum agar medium

19
Selective or inhibitory media
These media contain such substance or substances which help the growth of only one type of
bacteria and suppress the growth of others

Indicator media
This media generally consist of a sample but nutritionally adequate base to which a substrate
and an indicator are added to show that a change in reaction has occurred. This helps in the
identification of bacteria.

Besides these four groups of media there are many special purpose media like, isolation
media, media for maintenance of culture, media for bacteriological characterization, media
for determining nutritional requirement, screening media, media for microbiological assay of
vitamins and amino acids etc.

On the basis of consistency media can be classified into three groups

Solid media

These media are widely used in the laboratory due to the advantage of their use. One can see
the bacterial colony on the agar medium and colony characteristics help in the determination
and identification of many bacteria

Liquid Media
This media called broth. Bacterial colony can not be observed in this media but this media is
used for the study of many biochemical characteristics of bacteria. Besides that samples of
pathogens can be directly placed in this media.

Semi-liquid media
This type of media is not frequently used in the laboratory. But bacterial samples can be
transported from one place to another by pacing in it. This media is also used for the
preservation of bacterial isolates.

Media constituents

Agar

This can be obtained as shreds, flakes, granules or powder and is made from certain types of
seaweed. Its gelling properties is used for the preparation solid and semi-solid culture media.
It is insoluble in cold water but soluble when heated at about 900C. It becomes solidified at
or below 450C.

20
Peptone

It is a product of varying composition made by acid or enzymatic hydrolysis of animal or

vegetable protein, from material such as muscle, liver, blood, milk, casein, lactalbumin,
gelatin and soybean. The exact composition depends on the raw material and the method of
manufacture.

Meat Extract

Commercial meat extracts contain soluble organic bases, protein degradation products,
vitamins and minerals. Beef, beef heart, liver, brain, spleen fish muscle etc are used to
prepare meat extract. These extracts are readily available and easy to use

Yeast Extract
Yeast extract is made from bakers or brewers yeast and rich source of amino acids and
vitamins of B-Complex. In culture media it is used to supplement or replaced meat extracts.

Blood agar plate (BAP)

The choice of blood is often a matter of convenience and may depends on the animals kept by
a laboratory. Hoarse blood from commercial sources is commonly used, but the blood from
other species (Man, cow, goat, rabbit, sheep) may be necessary for special purposes.
Mammalian blood (usually sheep or horse), typically at a concentration of 510%. BAP are
an enriched, differential media used to isolate fastidious organisms and detect hemolytic
activity. -hemolytic activity will show lysis and complete digestion of red blood cell
contents surrounding colony. Examples include Streptococcus haemolyticus. -hemolysis
will only partially lyse(the cells are either lysed or not- it is the digestion that may be
incomplete) the hemoglobin and will appear green. An example of this would be
Streptococcus viridans. -hemolysis (or non-hemolytic) is the term referring to a lack of
hemolytic activity.

Red blood cells on an agar plate are used to

diagnose infection. On the left is a positive
Staphylococcus infection, on the right a positive
Streptococcus culture.

Plasma
Plasma is used for determining coagulate activity. In medical bacteriology laboratories,
human plasma is usually preferred but rabbit plasma is also used.

Serum
It is separated from blood. Collected without addition of an anti coagulant, by removal of
liquid that separates when the clot contracts.

21
Bile salts

Commercial bile salt are prepared by extracting dried ox or gig bile with ethanol,
decolorizing the extract with charcoal and precipitating the bile salts with ether to form a
water soluble yellowish-brown hygroscopic powder. It is used in some selective media.

Gelatin

It is the protein obtained by extraction on collagenous material from animal tissues and is
available as sheets, shreds, granules or powder. Gelatin dissolves in water at above 250C and
the solution gels on cooling to below 250C. Gelatin has little nutritive value but is used in
culture media as a substrate for detecting gelatinase activity.

Carbohydrates

Carbohydrates, collectively called sugars are usually used to enrich media to promote growth
of pigmentation, and to determine whether organisms can produce acid or acid and gas from
them. Concentration of carbohydrate in oxidation and fermentation studies is usually .5 to 1
%.

Nutrient agar
Nutrient agar is usually used for growth of non-fastidious organisms and observation of
pigment production. It is safe to use in school science laboratories because it does not
selectively grow pathogenic bacteria.

Aspergillus niger growing in

potato dextrose agar

Indicators
Indicators are incorporated in some culture media to give visual evidence of pH or other
changes occurring during the growth of bacteria.

22
Culture of Bacteria In An Agar Plate By Streak Plate
Method
Introduction
The streaking is process by which microorganisms are spread on the surface of the agar
medium by an inoculating loop making streak marks. The bacterial colonies are produced
along the streak mark. The plate which is prepared by streaking is called streak plate. Streak
plate method is used for obtaining pure culture from mixed population. Streak plate gives
well separated and isolated colonies of bacteria which can easily be isolated preserved as pure
culture. This method can also be used for the study of various characteristics of bacteria.

Materials
Agar plates
Bacterial Sample
Inoculating Loop
Spirit Lamp
Incubator

Procedure
Preparation of an agar plate
Sterilization of an inoculating loop by read heat
Cooling of the loop by jabbing in the agar plate
Small amount of bacterial sample inoculated on the agar surface by marking parallel
streak marks
Loop sterilizes again and cooled and streaking continued from the end point of
previous streak mark
This process repeated several times
Incubate the streak plates at 350C for 24 to 48 hrs

Observation

Bacterial growth could be observed along the streak marks and separated colonies on the later
streak marks

An agar plate streaked with microorganisms

isolated from a deep-water sponge. Individual
colonies may be seen center right.

23
Streaking (microbiology)

A plate which has been streaked. Note the colonies thinning in progressive regions of the
plate. The last region near the top of the picture contains pure colonies each grown from a
single bacterium.

Streaking is a technique used in

microbiology to isolate a pure strain from a
single species of microorganism, often
bacteria. A microbiological culture can be
grown so that the organism can be
identified, studied, or tested. A sterile cotton
swab or inoculation loop is sterilized and
dipped in a broth or patient specimen
containing many species of bacteria. The
loop is then spread across one quadrant of
an agar plate containing a growth medium
which has been sterilized in an autoclave.
This introduces a solution of the bacteria or
fungi to a substrate which provides them
nutrients. Choice of which growth medium
to use depends on which microorganism is
being cultured, and which are being selected A plate which has been streaked. Note the
for, if any. Growth media are usually based colonies thinning in progressive regions of the
on agar, a gelatinous substance derived from plate. The last region near the top of the picture
contains pure colonies each grown from a single
seaweed. bacterium.

The loop is re-sterilized and dragged across the inoculated quadrant of the streak plate. This
is done to collect some bacteria on the loop. The loop is spread around another fourth of the
plate much like the previous step. The loop is sterilized and the procedure is repeated. Each
time the loop gathers fewer and fewer bacteria until it gathers just one single bacterial cell
that can grow into a colony.

The streak plate is then incubated, usually for 24 to 36 hours, to allow the bacteria to
reproduce. At the end of incubation there should be enough bacteria to form visible colonies
in the areas touched by the inoculation loop. From these mixed colonies, single bacterial or
fungal species can be identified based on their morphological (size/shape/colour) differences,
and then sub-cultured to a new media plate to yield a pure culture for further analysis.

24
 Culture of Bacteria in Nutrient Broth
Introduction
Nutrient broth medium contain sufficient nutrient and favorable environment for the growth
of majority of bacteria. Nutrient broth is suitable for the growth of aerobic, anaerobic,
facultative, oxidative and fermentative bacteria. Broth culture is used for the enrichment and
study of many biochemical characteristics of bacteria.

Materials
Test tube
Nutrient broth medium (Sterilized)
Micro-pipette
Bacterial sample (pond water)
Spirit lamp
Incubator

Procedure

Preparation of broth tube (transfer of 5.0 ml nutrient broth by micro-pipette)

Inoculation of broth tube with bacterial sample
Incubation at 350 C until next class

Observation
Bacterial growth was indicated by-

Cloudiness of the medium

Pellicle or firm formation on the surface of the medium
Sediment formation on the bottom of the tube

25
 Culture of Bacteria in an Agar Plate by Pour Plate
Method
Introduction
This is a method by which bacteria are cultured throughout the agar medium. Although the
solidified agar medium restricts the movement of bacteria, the medium is soft enough for the
bacterial growth inside the medium. This method is used for the quantitative estimation of
bacteria, isolation of bacteria. Study of colony characteristics and many biochemical
characteristics of bacteria.

Materials
Sterilized Petridis
Melted agar medium
Bacterial sample
Pipette
Spirit lamp
Incubator

Procedure
Transfer of bacterial sample into Petridis (0.5-1.0 ml)
Pouring of melted agar in the Petridis (12-15 ml)
Mixing the agar with bacterial sample by rotating the Petridis
Place the agar plate on a level surface
Let the agar to be solidified and then incubate the plate in an inverted position in
incubator at 350 C for 24-48 hrs

Observation
Bacterial colonies formed throughout the medium
Colony colour and shape were observed

26
Culture of Bacteria in an Agar Plate by Spread Plate
Method

Introduction
This is a method by which bacteria are cultured on the surface of solidified agar medium.
This method is used for the quantitative estimation of bacteria, isolation of bacteria, study of
colony characteristics and many biochemical characteristics of bacteria.
Materials

Agar plates (previously prepared)

Bacterial sample
Pipette
L-shaped glass rod
Spirit lamp
Incubator

Procedure

Transfer of bacterial sample (0.1 ml) on the agar surface in the Petridis
Spreading of bacterial sample with the help of L-shaped glass rod by pushing it back
and forth while rotating the agar plate
Spreading continued until drying of the sample on the agar surface
Incubation of the agar plate in an inverted position at 300 C until next class

Observation
Clear and well separated bacterial colonies formed on the agar surface
Colony colour and shape were observed

27
 Culture of Bacteria in Agar Tube by Stabbing

Introduction

This is a method by which bacteria is cultured deep inside the agar medium in a test tube. The
stabbing method is used for the culture of aerobic, anaerobic and facultative bacteria as well
to study many biochemical characteristics and motility of bacteria.

Materials

Nutrient agar tube

Bacterial sample
Inoculating needle
Spirit lamp
Incubator

Procedure

About 8-10 ml agar medium in a test-tube was sterilize in an autoclave.

Test tube cooled in a vertical position until the medium solidified.
An inoculating needle red hot sterilized and cooled.
A small portion of bacterial sample inoculated in the agar by vertically inserting deep
inside the agar medium in the tube.
Agar tube incubated at 350 C for 24-48 hrs.

Observation
Bacterial growth could be observed on the surface of the agar and inside the agar through to
the bottom.

28
 Culture of Bacteria on Agar Slant

Introduction

Agar slant is a test-tube containing solidified agar medium in a slanting position; this method
is used for the culture of aerobic and facultative bacteria. Agar slant is useful for the
preservation of stock culture in the laboratory. This method is also used for the determination
of biochemical characteristics of bacteria.

Materials

Agar medium
Test tube
Bacterial sample
Inoculating loop
Spirit lamp
Incubator

Procedure

About 8-10 ml agar medium sterilize in an autoclave.

Test tube cooled in a slanting position until the medium solidified.
An inoculating needle red hot sterilized and cooled.
A small portion of bacterial sample inoculated on the agar surface by inoculating loop
streaking in a zigzag motion
Agar slant incubated at 350 C for 24-48 hrs.

Observation
Bacterial growth could be observed along the streak mark on the surface of the agar slant in
the test tube.

29
 Culture of Bacteria in Selective Media (Violate Red
Bile Agar and Eosine-Methylene Blue Agar) by
Spread Plate Method
Selective or inhibitory media contain such substance or substances which help the growth of
only ane type of bacteria and suppress the growth of others. VRBA, EMB (Eosin Methylene
blue) agar, SS agar, TSI (Triple sugar iron) agar, Sodium azide agar etc are some of the
selective media.

Violate red bile agar (VRBA)

It is selective medium for the detection enumeration of coliform organism. This medium is
recommended for the direct plate count of coliform bacteria in water, milk, dairy and other
food products. Coliform organisms produce dark-red colonies which are surrounded by
reddish zone.

Composition of VRBA
Ingredients Amount/litre
Yeast extract 3.0 g
Peptone 7.0 g
Bile salt 1.5 g
Lactose 10.0 g
NaCl 5.0 g
Bacto agar 15.0 g
Neutral red 0.03 g
Crystral violet 0.002 g

The pH of this selective medium at 250 C is 7.4 + 0.2

Culture Characteristics:
Organism
E. coli
Enterobacter aerogenes
Salmonella enterotidis
Growth Colour of colonies
Good to excellent Reddish with dark red centre
Good to excellent Pink
Good to excellent Colour less

Eosine Methylene Blue Agar (EMB Agar)

EMB agar is differential plating medium, used for the isolation and differentiation of gram
negative enteric bacilli.

30
Composition of EMB Agar

Ingredients Amount/litre
Peptone 10.0 g
Lactose 5.0 g
Sucrose 5.0 g
Dipotassium 2.0 g
phosphate
Sodium thiosulfate 8.5 g
Eosine Y 0.4 g
Methylene blue 0.065 g
Agar 13.5 g

The pH of this selective medium at 250 C is 7.4 + 0.2

Culture Characteristics
Organism Growth Colour of colonies
E. coli Luxurient Purple with black centre and
greenish metallic sheen
Enterobacter aerogenes Luxurient Pink, no sheen
Salmonella typhimorium Luxurient Colorless
Klebsiella pneumonia Luxurient Dark centers and greenish
Proteus mirabilis Luxurient Colorless
Staphylococcus aureus Inhibited -

Materials and Equipments

Sterile Petri dish
Selective agar medium
Bacterial sample
Pipette
L-shaped glass rod
Incubator

Procedure
VRBA and EMB agar powders were suspended in a definite amount of distilled water. They
were boiled to completely dissolve the ingredients. VRBA was not sterilized but EMB agar
was sterilized at 15 lbs pressure at 1210C for 15 minutes. Both the agars were cooled to 500C
and 12-15 was poured into the sterile Petri dish in order to prepare VRBA and EMB agar
plates. After solidification of the agar plates they were inoculated with bacterial samples and
incubated at 350C for 18-24 hrs. After incubation bacterial growth was observed in the plates
and recorded in the note book.

31
 Study of the Bacterial Morphology by Grams
Staining Method
The Grams staining is the most important staining procedure used in bacteriology. It is a
differential staining method. It differentiate bacteria into gram (+) and gram (-). In this
procedure bacterial sample divided into two groups:- first group stain into purple to violet
color while the second group pink to red. The organism staining violet is called gram (+) and
the organism staining to red is called gram (-).

Materials
Bacterial sample
Grease free clean glass slide
Inoculating loop
Staining reagent
Slide rack
Reagent tanks
Immersion oil
Microscope
Spirit lamp
Distilled water

Preparation of reagents

1. Crystal violet solution

Solution A: Crystal violet- 20.0g (90% dye content)
Ethyl alcohol- 20 ml (95%)

Solution B: Ammonium oxalate- 0.85g

Distilled water- 80 ml

Mix solution A and solution B

2. Grams Iodine solution (Lugols iodine)

Iodine crystal 5g
Potassium iodide 10g
Distilled water- 10 ml

Make the 100 ml stock solution in distilled water to make the final volume 300 ml.

3. Acetone-Alcohol solution (Decolorizing agent)

45% acetone 30 ml
45% ethanol 70 ml

32
4. Safranin solution (Counter stain)
Safranin 2g (dry powder)
Distilled water 100 ml

Procedure for Grams stain

Prepare a smear:- Put a drop of distilled water or saline on a non greasy clean glass slide.
Aseptically add a little of the colony for staining. Mix and spread well in the saline or water.
Air dry. Fix by passing the slide 2-3 times through a spirit lamp flame.

Place the slide in crystal violet for 60 secs.

Wash in tap water

Place in Lugols iodine 60 sec.

Treatment with Acetone-Alcohol for 2-3 sec.

Wash in tap water.

Place in safranin for 30 sec.

Wash in tap water.

Air dry

Observed under oil immersion objective lens on microscope

Observation
Observation was made on the following

a. Gram reaction
b. Cell shape
c. Cell arrangement
d. Spore formation

33
Gram staining

Gram-positive anthrax bacteria (purple rods) in cerebrospinal fluid sample. If present, a

Gram-negative bacterial species would appear pink. (The other cells are white blood cells).

Staphylococcus aureus (Gram positive)

Escherichia coli (Gram negative)

Gram staining (or Gram's method) is an empirical method of differentiating bacterial

species into two large groups (Gram-positive and Gram-negative) based on the chemical and
physical properties of their cell walls. The Gram stain is almost always the first step in the
identification of a bacterial organism. While Gram staining is a valuable diagnostic tool in
both clinical and research settings, not all bacteria can be definitively classified by this
technique, thus forming Gram variable and Gram indeterminant groups as well.

34
The method is named after its inventor, the Danish scientist Hans Christian Gram (1853
1938), who developed the technique in 1882 and published it in 1884 to discriminate between
two types of bacteria with similar clinical symptoms: Streptococcus pneumoniae (also known
as the pneumococcus) and Klebsiella pneumoniae bacteria.

The word Gram is always spelled with a capital, referring to the name of the inventor of the
Gram staining.

Uses

Gram staining is used to differentiate bacterial species into two large groups (Gram-positive
and Gram-negative) based on the physical properties of their cell walls. Gram staining is not
used to classify archaea, since these microorganisms yield widely varying responses that do
not follow their phylogenetic groups.

Research

Gram staining is a bacteriological laboratory technique. The technique is used as a tool for
the differentiation of Gram-positive and Gram-negative bacteria, as a first step to determine
the identity of a particular bacterial sample.

The Gram stain is not an infallible tool for diagnosis, identification, or phylogeny, however.
It is of extremely limited use in environmental microbiology, and has been largely superseded
by molecular techniques even in the medical microbiology lab. Some organisms are Gram-
variable (that means, they may stain either negative or positive); some organisms are not
susceptible to either stain used by the Gram technique. In a modern environmental or
molecular microbiology lab, most identification is done using genetic sequences and other
molecular techniques, which are far more specific and information-rich than differential
staining.

Staining mechanism

Gram-positive bacteria have a thick mesh-like cell wall made of peptidoglycan (50-90% of
cell wall), which stains purple while gram-negative bacteria have a thinner layer (10% of cell
wall), which stains pink. Gram-negative bacteria also have an additional outer membrane
which contains lipids, and is separated from the cell wall by the periplasmic space. There are
four basic steps of the Gram stain, which include applying a primary stain (crystal violet) to a
heat-fixed smear of a bacterial culture, followed by the addition of a trapping agent (Gram's
iodine), rapid decolorization with alcohol or acetone, and counterstaining with safranin.[7]
Basic fuchsin is sometimes substituted for safranin since it will more intensely stain
anaerobic bacteria but it is much less commonly employed as a counterstain.

Crystal violet (CV) dissociates in aqueous solutions into CV+ and chloride (Cl ) ions. These
ions penetrate through the cell wall and cell membrane of both gram-positive and gram-
negative cells. The CV+ ion interacts with negatively charged components of bacterial cells
and stains the cells purple.

Iodine (I or I3 ) interacts with CV+ and forms large complexes of crystal violet and iodine
(CVI) within the inner and outer layers of the cell. Iodine is often referred to as a mordant,

35
but is a trapping agent that prevents the removal of the CV-I complex and therefore color the
cell.

When a decolorizer such as alcohol or acetone is added, it interacts with the lipids of the cell
membrane. A gram-negative cell will lose its outer membrane and the lipopolysaccharide
layer is left exposed. The CVI complexes are washed from the gram-negative cell along
with the outer membrane. In contrast, a gram-positive cell becomes dehydrated from an
ethanol treatment. The large CVI complexes become trapped within the gram-positive cell
due to the multilayered nature of its peptidoglycan. The decolorization step is critical and
must be timed correctly; the crystal violet stain will be removed from both gram-positive and
negative cells if the decolorizing agent is left on too long (a matter of seconds).

After decolorization, the gram-positive cell remains purple and the gram-negative cell loses
its purple color. Counterstain, which is usually positively charged safranin or basic fuchsin, is
applied last to give decolorized gram-negative bacteria a pink or red color.

Some bacteria, after staining with the Gram stain, yield a Gram-variable pattern: a mix of
pink and purple cells are seen. The genera Actinomyces, Arthobacter, Corynebacterium,
Mycobacterium, and Propionibacterium have cell walls particularly sensitive to breakage
during cell division, resulting in Gram-negative staining of these Gram-positive cells. In
cultures of Bacillus, Butyrivibrio, and Clostridium a decrease in peptidoglycan thickness
during growth coincides with an increase in the number of cells that stain Gram-negative. In
addition, in all bacteria stained using the Gram stain, the age of the culture may influence the
results of the stain.

Examples

Gram-negative bacteria

The proteobacteria are a major group of Gram-negative bacteria. Other notable groups of
Gram-negative bacteria include the cyanobacteria, spirochaetes, green sulfur and green non-
sulfur bacteria.

These also include many medically relevant Gram-negative cocci, bacilli and many bacteria
associated with nosocomial infections.

Gram-positive bacteria

In the original bacterial phyla, the Gram-positive forms made up the phylum Firmicutes, a
name now used for the largest group. It includes many well-known genera such as Bacillus,
Listeria, Staphylococcus, Streptococcus, Enterococcus,Diplococcus pneumoniae and
Clostridium. It has also been expanded to include the Mollicutes, bacteria like Mycoplasma
that lack cell walls and so cannot be stained by Gram, but are derived from such forms.

36
Reagents necessary for different tests

The reagents were prepared in the laboratory as per direction of Cowan and Steel's Manual
for the Identification of Medical Bacteria (edited by Barrow and Felthham, 1993) and with
the help of DIFCO, Manual of Dehydrated Culture Media and Reagents, 9th edition, 1964.

Brief descriptions of reagent preparation and their use are given below:

Reagents for Gram's staining (Lillie, 1928)

Ammonium oxalate-crystal violet solution
Solution A

Crystal violet (C.I. No. 49555) : 10 g

95% Ethanol : 150 ml
Solution B
Ammonium oxalate : 1% aqueous solution

For use, 20 ml of solution A was mixed with 80 ml of solution B.

Lugol's iodine

Iodine : 5.0 g
Potassium iodide : 10 g
Distilled water : 100 ml

Iodide and potassium iodide were dissolved in small amount of water and then distilled
water was added to make it 100 ml.

Safranine solution

Safranine : 0.25 g
95% Ethanol : 10 ml
Distilled water : 90 ml

Acetone/Alcohol mixture

Ethanol : 950 ml
Acetone : 50 ml

37
Reagent for Oxidase test (Kovacs. 1956; modified by Cowan, 1974)

It is used to detect cytochrome C oxidase activity of bacteria.

N'N'N'N' - Tetramethyl-p-phenylenediamine Dihydrochloride

(C10H18Cl2N2) : 1.0 g
Distilled water (Sterile) : 100 ml

A solution of tetramethyl-p-phenylenediamine dihydrochloride was prepared fresh each

time before use. Solution was discarded if it was turned blue. Rapid autoxidation of the
reagent was retarded by the addition of 1% ascorbic acid.

Gram's staining
A small portion of bacterial culture was taken on a clear glass slide with a sterile
inoculating loop and prepared smear on the slide. The smear was then dried in air and
fixed by passing the slide 3 to 4 times over a spirit lamp flame. Then it was allowed to
cool and flooded with crystal violet solution for approximately 1 minute. Then it was
washed with tap water and treated with Gram's iodine solution. After 1 minute the
iodine solution was tipped off from the slide by tap water. Then the decolorizing agent,
95% acetone or ethanol was applied to the slide and left for 8-10 seconds and was
thoroughly washed with water. The smear was flooded with counter strain, safranine for
about 1 minute and washed with water. The smear was air dried and microscopically
examined under the oil immersion objectives.

Oxidase test
A filter paper disc in a sterile petridish lid was moistened with few drops of the Kovacs'
reagents. Then a loopfull of sample from fresh growth of Gram negative, rod shaped
bacterial culture was smeared over the moist filter paper with a sterile platinum (not
nichrome) loop. The appearance of a dark purple colour on the paper within 30 seconds
denotes a positive reaction.

Hugh and Leifson's (OF) test

An individual isolate was taken from the fresh Gram negative and oxidase negative
bacterial culture of 8-24 hours by scraping single colony off the plates with a sterile loop
and was inoculated into freshly prepared tubes of medium by a single stab.

The medium contained bromothymol blue pH indicator to indicate the formation of acid
from the breakdown of lactose. A control tube was maintained without inoculation. The
tubes were then incubated at 25C and examined daily for 7-10 days.

38
Detection

Tube observed Results

Yellow Oxidation (O)

Yellow with bubble Fermentation (F)

Identification of bacterial isolates

Different types of colonies which grew on surface of different media were identified
by studying their morphological and biochemical characteristics. It was done
according to method suggested in Cowan and Steels manual for the identification of
medical bacteria (edited by Barrow and Feltham, 1993) and Flow Chart for the
Presumptive Identification of Selected Bacteria from Fishes (Emmet, B and Shotts, Jr,
1994). For bacterial identification, the flow charts given in Flow chart-1, 2, 3 and 4
were used.

39
 Gram stain of 3% KOH
treatment of unknown colony

Gram reaction-Purple of blue Gram reaction- Pink or red

KOH-Bacterial suspension KOH-Bacterial suspension
Not viscous (slimy) or stringy viscous and stringy

Gram-positive bacteria Gram-negative bacteria

Flow chart-01

40
 Aerobic Gram-positive Bacteria

Cocci Rods

+ Catalase + Endospore

+ Pinpointed Colonies

Bacillus Other gram

Positive rod
+ Obligate Aerobic

Micrococcus Staphylococcus Hemolysis on Blood agar

+ Growth anearobically

Streptococcus Aerococcus

(Alpha) or (Beta) (Gamma)

Carnobacterlum Corynebacterium
or
Lactobacillus

Flowchart-02

41
 Gram Negative-Rod shaped Bacteria

Yellow Pigment +

Cytophage/Flavobecterium

Kovacs Oxidase test +

Glucose utilization

Oxidative acid but None or alkali formed Fermentative

no gas from glucose

Non-fluorescent No acid from

Pseudomonas ssp.
Non-green
Pseudomonas ssp. Non-green Pseudomenas

Acid but no from glucose Acid but much gas from glucose

Sensitive to compound 0.129 Vibrio sp. Aeromonas sp.

Flowchart-03

42
 Gram Negative, Oxidase-Negative, Rod-shape bacteria

+ Lactose

+ Indole Phenylalanine +
Deaminase
Citrate +

Escherichia + Motility Morgenelly/Proteus/

Providencia

Enterobacter Klebsiella

+ Citrate

+ Motility at 350C

+ H2S Hafnia / Edwarsiella Yersinia

Salmonella + Motility

Serratia Shigella

Figure 05. Flow chart-04

43
Autoclave
An autoclave is a device to sterilize equipment and supplies by subjecting them to high
pressure steam at 121 C or more typically for 15 to 20 minutes depending on the size of the
load and the contents. It was invented by Charles Chamberland in 1879, although a precursor
known as the steam digester was created by Denis Papin in 1679.

Picture: Inner and outer view an autoclave

Types

There are two main types of Autoclaves:

Stove top autoclaves actually resemble a culinary pressure cooker. Each unit comes
complete with a bolt-down lid and a pressure gauge on the outside. These units
require an outside heat source and can be extremely dangerous in untrained hands.
They should only be used by experienced professionals.

Front loading autoclaves are more widely used for their convenience, but must also be
handled with great care. The units are box-shaped and self contained, equipped with a
heating unit to turn water into vapor for sterilization. The autoclave's controls allow
the operator to set the desired temperature, and determine how long the machine will
remain in operation. There is also a gauge to track chamber temperature/pressure.

Uses

Autoclaves are widely used in microbiology, medicine, body piercing, veterinary science,
mycology, dentistry and podiatry.

Typical loads include glassware, medical waste, utensils, animal cage bedding, and Lysogeny
broth.

A notable growing application of autoclaves is in the treatment and sterilization of waste,

such as pathogenic hospital waste. Machines in this category largely operate under the same
principles as the original autoclave in that they are able to neutralize potentially infectious
agents by utilizing pressurized steam and superheated water. A new generation of waste
converters is capable of achieving the same effect without any pressure vessels to sterilize
culture media, rubber material, gowns, dressing, gloves etc. It is particularly useful for

44
materials which cannot withstand the higher temperature of hot air oven. For all glass
syringes, hot air oven is a better sterilizing method.

Air removal

It is very important to ensure that all of the trapped air is removed, as hot air is very poor at
achieving sterility. Steam at 134 C can achieve in 3 minutes the same sterility that hot air at
160 C takes two hours to achieve.[5] Methods of achieving air removal include:

Downward displacement (or gravity type) - As steam enters the chamber, it fills the upper
areas as it is less dense than air. This compresses the air to the bottom, forcing it out through
a drain. Often a temperature sensing device is placed in the drain. Only when air evacuation
is complete should the discharge stop. Flow is usually controlled through the use of a steam
trap or 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 - Vacuum pumps to suck air or air/steam mixtures from the chamber.

Superatmospheric - This type of cycle uses a vacuum pump. It starts with a vacuum
followed by a steam pulse and then a vacuum followed by a steam pulse. The number of
pulses depends on the particular autoclave and cycle chosen.

Subatmospheric - Similar to superatmospheric cycles, but chamber pressure never exceeds

atmospheric until they pressurize up to the sterilizing temperature.

Autoclaves Use in Microbiology Lab

Stovetop autoclaves - the simplest of autoclaves

A autoclave is a device that uses steam to

sterilize equipment and other objects. This
means that all bacteria, viruses, fungi, and
spores are inactivated. However, prions, like
those associated with Creutzfeldt-Jakob
disease, may not be destroyed by autoclaving
at the typical 134 C for 3 minutes or 121 C
for 15 minutes. Also, some recently-
discovered organisms, such as Strain 121, can
survive at temperatures above 121 C.
Autoclaves are found in many medical settings and other microbiological laboratories that
need to ensure sterility of an object. Many procedures today use single-use items rather than
sterilized, reusable items. This first happened with hypodermic needles, but today many
surgical instruments (such as forceps, needle holders, and scalpel handles) are commonly
single-use items rather than reusable. See waste autoclave.

45
Because damp heat is used, heat-labile products (such as some plastics) cannot be sterilized
this way or they will melt. Some paper or other products that may be damaged by the steam
must also be sterilized another way. In all autoclaves, items should always be separated to
allow the steam to penetrate the load evenly.

Autoclaving is often used to sterilize medical waste prior to disposal in the standard
municipal solid waste stream. This application has grown as an alternative to incineration due
to environmental and health concerns raised by combustion byproducts from incinerators,
especially from the small units which were commonly operated at individual hospitals.
Incineration or a similar thermal oxidation process is still generally mandated for pathological
waste and other very toxic and/or infectious medical wastes.

Incubator (microbiology)
In microbiology, an incubator is a device for controlling the temperature, humidity, and other
conditions in which a microbiological culture is being grown. 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). More elaborate
incubators can also include the ability to lower the temperature (via refrigeration), or the
ability to control humidity or CO2 levels.

Most incubators include a timer; some can also be programmed to cycle through different
temperatures, humidity levels, etc. Incubators can vary in size from tabletop to units the size
of small rooms.

Incubators also contain certain features such as the shake speed, measured by revolutions per
minute. As for temperature, most commonly used is approximately 36 to 37 degrees Celsius.
Most bacteria, especially the frequently used E. Coli, grow well under such conditions. For
other experimental organisms, such as the budding yeast Saccharomyces cerevisiae, a growth
temperature of 30 C is optimal.

Picture: A Bacteriological incubator (Inner and Outer view)

46
Colony counter
A colony counter is an instrument used to count colonies of bacteria or other
microorganisms growing on an agar plate. Early counters were merely lighted surfaces on
which the plate was placed, with the colonies marked off with a felt-tipped pen on the outer
surface of the plate while the operator kept the count manually. More recent counters attempt
to count the colonies electronically, by identifying individual areas of dark and light
according to automatic or user-set thresholds, and counting the resulting contrasting spots.

Microorganism enumeration

Such counters are used to estimate the density of microorganisms within a liquid culture. An
appropriate dilution, or several dilutions within the estimated appropriate range, is spread
using sterile technique on the agar plate, which is then incubated under the appropriate
conditions for growth until individual colonies appear. Each colony marks the spot where a
single organism was originally placed, thus the number of colonies on the plate equals the
number of organisms within the volume of liquid spread on the plate. That concentration is
then extrapolated by the known dilution from the original culture, to estimate the
concentration of organisms within that original culture.

The maximum number of colonies which may be effectively counted on a single plate is
somewhere between 100 and 1,000, depending on the size of the colony and the type of
organism.

Picture: An electronic bacterial colony counter

Picture: Colony counter genetally use in

microbiology Laboratory

47
Microscope
A microscope (from the Greek: , mikrs, "small" and , skopen, "to look" or
"see") is an instrument to see objects too small for the naked eye. The science of investigating
small objects using such an instrument is called microscopy. Microscopic means invisible to
the eye unless aided by a microscope.

Types

"Microscopes" can be separated into optical theory microscopes (Light microscope), electron
microscopes (e.g.,TEM), and scanning probe microscopes (SPM). Optical microscopes
function through the optical theory of lenses in order to magnify the image generated by the
passage of a wave through the sample, or reflected by the sample. The waves used are
electromagnetic (in optical microscopes) or electron beams (in electron microscopes). Types
are the compound light, stereo, and the electronic microscope.

Optical microscope

Optical microscopes, using visible wavelengths of light, are the simplest and most used.
Optical microscopes have refractive glass and occasionally of plastic or quartz, to focus light
into the eye or another light detector. Mirror-based optical microscopes operate in the same
manner. Typical magnification of a light microscope, assuming visible range light, is up to
1500x with a theoretical resolution limit of around 0.2 micrometres or 200 nanometers.
Specialized techniques (e.g., scanning confocal microscopy, Vertico SMI) may exceed this
magnification but the resolution is diffraction limited. The use of shorter wavelengths of
light, such as the ultraviolet, is one way to improve the spatial resolution of the optical
microscope, as are devices such as the near-field scanning optical microscope.
Sarfus, a recent optical technique increases the sensitivity of standard optical microscope to a
point it becomes possible to directly visualize nanometric films (down to 0.3 nanometer) and
isolated nano-objects (down to 2 nm-diameter). The technique is based on the use of non-
reflecting substrates for cross-polarized reflected light microscopy. The traditional optical
microscope has been recently modified into a digital microscope, where instead of directly
viewing the object, a charge-coupled device (CCD) camera projects the image to a monitor.

Electron microscopes
Three major variants of electron microscopes exist:

Scanning electron microscope (SEM): looks at the surface of bulk objects by scanning
the surface with a fine electron beam and measuring reflection. May also be used for
spectroscopy. See also environmental scanning electron microscope
Transmission electron microscope (TEM): passes electrons completely through the
sample, analogous to basic optical microscopy. This requires careful sample
preparation, since electrons are scattered so strongly by most materials.This is a
scientific device that allows people to see objects that could normally not be seen by
the naked or unaided eye.
Scanning Tunneling Microscope (STM): is a powerful technique for viewing surfaces
at the atomic level.

48
 Microscope use in laboratory

Vortex mixer
A vortex mixer is a simple device used commonly in laboratories to mix small vials of
liquid. It consists of an electric motor with the drive shaft oriented vertically and attached to a
cupped rubber piece mounted slightly off-center. As the motor runs the rubber piece
oscillates rapidly in a circular motion. When a test tube or other appropriate container is
pressed into the rubber cup (or touched to its edge) the motion is transmitted to the liquid
inside and a vortex is created. Most vortex mixers have variable speed settings and can be set
to run continuously, or to run only when downward pressure is applied to the rubber piece.

Vortex mixers are quite commonplace in

bioscience laboratories. In cell culture and
microbiology laboratories they may be used to
suspend cells. In a biochemical or analytical
laboratory they may be used to mix the reagents
of an assay or to mix an experimental sample
and a dilutant.

Picture: A Whirlimixer brand vortex mixer

The vortex mixer was invented by the Kraft brothers (Jack A. Kraft and Harold D. Kraft)
while working for Scientific Industries (a laboratory equipment manufacturer). A patent was
filed by the Kraft brothers on April 6, 1959 and granted on October 30, 1962 (US patent
3,061,280). Scientific Industries still makes a version of this original vortex mixer.

An alternative to the electric vortex mixer is the "finger

vortex" technique in which a vortex is created manually by
striking a test tube in a forward and downward motion with
one's finger or thumb. This generally takes longer and
often results in inadequate suspension, although it may be
suitable in some cases when a vortex mixer is unavailable
or the forces involved in vortexing would damage the
sample, but this technique is not recommended when
caustic substances are involved. The technique is better
suited to accelerate the mixture of solutions which do not
require the kinetic energy input needed to create Picture: Vortex mixture use in
suspensions. laboratory

49
Hot air oven
Hot air ovens are electrical devices used in sterilization. The oven uses dry heat to sterilize
articles. Generally, they can be operated from 50 to 300 C. There is a thermostat controlling
the temperature. These are digitally controlled to maintain the 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. Power supply needs vary from country to country, depending on the voltage
and frequency (hertz) used. Temperature sensitive tapes or other devices like those using
bacterial spores can be used to work as controls, to test for the efficacy of the device in every
cycle.

Usage

A complete cycle involves heating the oven to the required temperature, maintaining that
temperature for the proper time interval for that temperature, turning the machine off and
cooling the articles in the closed oven till they reach room temperature. The standard settings
for a hot air oven are:

1.5 to 2 hours at 160 C (320 F)

6 to 12 minutes at 190 C (374 F)

plus the time required to preheat the chamber before beginning the sterilization cycle. If the
door is opened before time, heat escapes and the process becomes incomplete. Thus the cycle
must be properly repeated all over.

These are widely used to sterilize articles that can withstand high temperatures and not get
burnt, like glassware and powders. Linen gets burnt and surgical sharps lose their sharpness.

Picture: Oven use in mocrobiology laboratory

50
Electric heater
Electric heating is any process in which electrical energy is converted to heat. Common
applications include heating of buildings, cooking, and industrial processes.

An electric heater is an electrical appliance that converts electrical energy into heat. The
heating element inside every electric heater is simply an electrical resistor, and works on the
principle of Joule heating: an electric current through a resistor converts electrical energy into
heat energy.

Alternatively, a heat pump uses an electric motor to

drive a refrigeration cycle, drawing heat from a
source such as the ground or outside air and
directing it into the space to be warmed. Such
systems can deliver two or three units of heating
energy for every unit of electricity purchased.

Refrigerator
A refrigerator (often called a "fridge" for short) is a cooling appliance comprising a
thermally insulated compartment and a heat pumpchemical or mechanical meansto
transfer heat from it to the external environment, cooling the contents to a temperature below
ambient. Refrigerators are extensively used to store foods which spoil from bacterial growth
if not refrigerated. A device described as a "refrigerator" maintains a temperature a few
degrees above the freezing point of water; a similar device which maintains a temperature
below the freezing point of water is called a "freezer." The refrigerator is a relatively modern
invention among kitchen appliances. It replaced the icebox, which had been a common
household appliance for almost a century and a half prior. For this reason, a refrigerator is
sometimes referred to as an "icebox".

Outer view of a refrigerator A typical refrigerator with its door open

Generally this type of refrigerator are widely use in the laboratory as well as
microbiology laboratory to preserve various chemical and liquids

51
Blender
A blender (occasionally liquidiser in British English and
occasionally vitamiser in Australian English) is a kitchen
appliance used to mix ingredients or puree food. Blenders
are also used to prepare emulsions, such as mayonnaise,
and cream soups. Blenders are to be distinguished from
lower-speed hand-powered or electric mixers that are used
for mixing applications. The term typically refers to a
stationary, upright electrical device, but hand-held
immersion blenders have become common in recent years.
Blenders are also used in laboratory applications.

The blending container can be made of glass, plastic, stainless steel, or porcelain, and often
has graduated markings for approximate measuring purposes. At the top of the container is a
lid to prevent ingredients from escaping during operation. At the bottom is a blade assembly,
sometimes removable for cleaning purposes. In cases where the blades are removable, the
container should have an o-ring or gasket between the body of the container and the base to
seal the container and prevent the contents from leaking. The blending container is generally
shaped in a way that encourages material to circulate through the blades, rather than simply
spinning around.

The container rests upon a base that contains a motor

for turning the blade assembly and has controls on its
surface. Most modern blenders offer a number of
possible speeds. Low-powered blenders require some
liquid to be added for the blender to operate correctly.
This is because the liquid is used to move the solids
around the jar and bring it in contact with the blade as
the "whirlpool" fluid movement brings items from the
top to the bottom. High-powered blenders are capable
of milling grains and crushing ice without such
assistance.

The hand-held immersion blender has no container of its own, but instead has a mixing head
with rotating blades that can be immersed in a container. Immersion blendors are convenient
for homogenizing volumes that are too large to fit in the bowl of a stationary blender or, as in
the case of soups, are too hot to be safely poured into the bowl.

Some of the functions of blenders have been taken over by food processors. In particular,
thicker mixtures such as mayonnaise and hummus are conveniently made in food processors.

Applications

Blenders are used both in home and commercial kitchens for various purposes, such as to:

Mix and crush ice in cocktails such as the Zombie, Pia Colada and frozen margaritas
Crush ice and other ingredients in non-alcoholic drinks such as Frappucinos and
smoothies
Emulsify mixtures

52
 Make smooth pures of semi-solid ingredients, such as cooked vegetables and meat
Reduce small solids such as spices and seeds to powder or nut butters
Blend mixtures of powders, granules, and/or liquids thoroughly
Help dissolve solids into liquids

Blenders also have a variety of applications in microbiology and food science. In addition to
standard food-type blenders, there is a variety of other configurations of blender for
laboratories.

Development

It is popularly believed that Dr. Oliver Johnson

Schofield, an English engineer and entrepreneur,
invented the first electric blender in 1921. However,
Polish-American Stephen J. Poplawski, owner of
the Stevens Electric Company, began designing
drink mixers in 1919 under contract with Arnold
Electric Company,[1] and patented the drink mixer
in 1922 which had been designed to make Horlicks
malted milk shakes at soda fountains. He also
introduced the liquefier blender in 1922. Stevens
Electric was sold to Oster Manufacturing, a
manufacturer of barber equipment, in 1946. Oster
commercialized the liquefier blender under the
trademark Osterizer. Oster was bought by Sunbeam
Products in 1960.

In 1935, Fred Osius invented another kind of blender. He approached Fred Waring, a popular
musician who financed and promoted the "Miracle Mixer", which was commercialized in
1937 by Waring Products, now part of Conair. Waring popularized the smoothie in the 1940s.
Waring long used the spelling "blendor" for its product.

With the rising popularity of smoothies, Frappucinos and other frozen drinks prepared in
front of the customer, new models of commercial blenders often include a sound-reducing
enclosures and computerized controls.

Specialized blenders for making

smoothies are becoming popular,
chiefly resembling an ordinary model
with a spigot added for quick serving.
Some models also feature a gimballed
stirring rod mounted on the lid,
constructed so that mixtures can be
stirred whilst the machine is running
with no chance of the stirrer fouling
the blades. The powertrain of a blender.

53
Mechanical operation

A blender consists of a housing, motor, blades, and food container. A fan-cooled electric
motor is secured into the housing by way of vibration dampers, and a small output shaft
penetrates the upper housing and meshes with the blade assembly. Usually, a small rubber
washer provides a seal around the output shaft to prevent liquid from entering the motor.
Most blenders today have multiple speeds. As a typical blender has no gearbox, the multiple
speeds are often implemented using a motor with multiple stator windings and/or
multitapped stator windings; in a blender with electromechanical controls, the button (or
other electrical switching device or position) for each different speed connects a different
stator winding/tap or combination thereof. Each different combination of energized windings
produces a different torque from the motor, which yields a different equilibrium speed in
balance against the drag (resistance to rotation) of the blade assembly in contact with the
material inside the food container.

Laboratory Distilled water plant

Laboratory Distilled water plant is the water distiller that has virtually all of water
impurities removed through distillation. Distillation involves boiling the water and then
condensing the steam into a clean container, leaving most if not all solid contaminants
behind.

54
Petri Dish
A Petri dish (or cell culture dish) is a shallow glass or plastic cylindrical lidded dish that
biologists use to culture cells. It was named after German bacteriologist Julius Richard Petri,
who invented it when working as an assistant to Robert Koch. Glass Petri dishes can be
reused by sterilization (for example, in an autoclave or by dry heating in a hot air oven at
160C for one hour). For experiments where cross-contamination from one experiment to the
next can become a problem, plastic Petri dishes may have to be disposed of after one use.

Modern Petri dishes often have rings on the lids and bases which allow them to be stacked so
that they do not slide off one another. Multiple dishes can also be incorporated into one
plastic container to create what is called a "multi-well plate".

Microbiology

For microbiology, agar plates are very frequently used. The dish is partially filled with warm
liquid agar along with a particular mix of nutrients, sheep blood, salts, sugars, dyes,
indicators and amino acids and, optionally, antibiotics. After the agar solidifies, the dish is
ready to receive a microbe-laden sample. Petri plates are incubated upside down (agar on top)
to keep the weight in the lid for sterility, and so excess water accumulates away from the
bacterial colonies.

Other uses

Other Petri dish uses do not involve agar; for instance, cell culture.

As well as making agar plates, empty Petri dishes may be used to observe plant germination
or small animal behavior, or for other day-to-day laboratory practices such as drying fluids in
an oven and carrying and storing samples.

Picture: Petri dish use in microbiology Physcomitrella patens plants

laboratory growing axenically on an agar
plate (9 cm diameter)

55
Bunsen burner
A Bunsen burner is a common piece of laboratory equipment that produces a single open
gas flame, which is used for heating, sterilization, and combustion.

History

When the University of Heidelberg hired Robert Bunsen in

1852, the authorities promised to build him a new
laboratory building. Heidelberg had just begun to install
coal-gas street lighting, so the new laboratory building was
also supplied with illuminating gas. Illumination was one
thing; a source of heat for chemical operations something
quite different. Previous laboratory lamps left much to be
desired regarding economy and simplicity, as well as the
quality of the flame for a burner lamp, for it was desirable
to maximize the temperature and minimize the luminosity.
While his building was still under construction late in 1854,
Bunsen suggested certain design principles to the
universitys talented mechanic, Peter Desaga, and asked
him to construct a prototype. (Similar principles had been
used in an earlier burner design by Michael Faraday as well
as in a device patented in 1856 by the gas engineer R W
Elsner.) The Bunsen/Desaga design succeeded in generating
a hot, sootless, non-luminous flame by mixing the gas with
air in a controlled fashion before combustion. Desaga
created slits for air at the bottom of the cylindrical burner,
the flame igniting at the top. By the time the building
opened early in 1855, Desaga had made fifty of the burners
for Bunsen's students. Bunsen published a description two
years later, and many of his colleagues soon adopted the
design. Bunsen burners are now used in schools all around
the world.

Operation

Different flame types of Bunsen burner

depending on flow through the throat holes
(holes on the side of the Bunsen burner -- not to
be confused with the needle valve for gas flow
adjustment). 1. air hole closed (Safety flame) 2.
air hole slightly open 3. air hole half open 4. air
hole almost open (roaring blue flame)

The device in use today safely burns a

continuous stream of a flammable gas such as
natural gas (which is principally methane) or a
liquefied petroleum gas such as propane, butane,
or a mixture of both.

56
The hose barb is connected to a gas nozzle on the lab bench with rubber tubing. Most lab
benches are equipped with multiple gas nozzles connected to a central gas source, as well as
vacuum, nitrogen, and steam nozzles. The gas then flows up through the base through a small
hole at the bottom of the barrel and is directed upward. There are open slots in the side of the
tube bottom to admit air into the stream via the Venturi effect, and the gas burns at the top of
the tube once ignited by a flame or spark. The most common methods of lighting the burner
are using a match or a spark lighter.

The amount of air (or rather oxygen) mixed with the gas stream affects the completeness of
the combustion reaction. Less air yields an incomplete and thus cooler reaction, while a gas
stream well mixed with air provides oxygen in an equimolar amount and thus a complete and
hotter reaction. The air flow can be controlled by opening or closing the slot openings at the
base of the barrel, similar in function to the choke in a car's carburetor.

If the collar at the bottom of the tube is adjusted so more air can mix with the gas before
combustion, the flame will burn hotter, appearing blue as a result. If the holes are closed, the
gas will only mix with ambient air at the point of combustion, that is, only after it has exited
the tube at the top. This reduced mixing produces an incomplete reaction, producing a cooler
but brighter yellow which is often called the "safety flame" or "luminous flame". The yellow
flame is luminous due to small soot particles in the flame which are heated to incandescence.
The yellow flame is considered "dirty" because it leaves a layer of carbon on whatever it is
heating. When the burner is regulated to produce a hot, blue flame it can be nearly invisible
against some backgrounds. The hottest part of the flame is the tip of the inner flame, while
the coolest is the whole inner flame. Increasing the amount of fuel gas flow through the tube
by opening the needle valve will of course increase the size of the flame. However, unless the
airflow is adjusted as well, the flame temperature will decrease because an increased amount
of gas is now mixed with the same amount of air, starving the flame of oxygen. The blue
flame in a Bunsen burner is hotter than the yellow flame. The hottest part of the blue flame is
just above the unburnt gas. The hottest part of the yellow flame is the chimney.

The burner will often be placed on a suitable heatproof mat to protect the lab bench surface.

Picture: General Bunsen burner commonly use in microbiology laboratory

57
Test tube
Two small test tubes in a test tube rack

A test tube, also known as a culture tube or sample tube,

is a common piece of laboratory glassware consisting of a
finger-like length of glass or clear plastic tubing, open at the
top, usually with a rounded U-shaped bottom. A large test
tube designed specifically for boiling liquids is called a
boiling tube.

Test tubes are available in a multitude of lengths and widths, typically from 10 to 20 mm
wide and 50 to 200 mm long. The top often features a flared lip to aid pouring out the
contents; some sources consider that the presence of a lip is what distinguishes a test tube
from a culture tube. Some test tubes have a flat bottom; some are made so as to accept a
ground glass stopper or a screw cap. They are often provided with a small ground glass or
white glaze area near the top for labeling with a pencil.

Uses

Test tubes are widely used by chemists to hold, mix, or

heat small quantities of solid or liquid chemicals,
especially for qualitative experiments and assays. Their
round bottom and straight sides minimize mass loss
when pouring, make them easier to clean, and allow
convenient monitoring of the contents. The long,
narrow neck slows down the spreading of vapors and
gases to the environment. A test tube filled with water
and upturned into a water-filled beaker is often used to
capture gases, e.g. in electrolysis demonstrations.

Culture tubes are often used in biology for handling and culturing all kinds of live organisms,
such as molds, bacteria, seedlings, plant cuttings, etc.; and in medicine and forensics to store
samples of blood or other fluids.

A test tube with a stopper is often used for temporary storage of chemical or biological
samples.

Test tubes are usually held in special-

purpose racks, clamps, or tongs. Some racks
for culture tubes are designed to hold the
tubes in a nearly horizontal position, so as to
maximize the surface of the culture medium
inside.

Test tubes are sometimes put to casual uses outside of lab environments, e.g. as flower vases
or containers for spices.

58
Beaker (glassware)
A beaker is a simple container for stirring, mixing and
heating liquids commonly used in many laboratories.
Beakers are generally cylindrical in shape, with a flat
bottom and a lip for pouring. Many also have a small spout
to aid pouring as shown in the picture. Beakers are available
in a wide range of sizes, from one millilitre up to several
litres.

Beakers of several sizes

Structure

Standard or "Low-form" beakers typically have a height about 1.4 times the diameter. The
common low form with a spout has been called the Griffin form. "Tall form" beakers have a
height about twice the diameter. These are sometimes called Berzelius beakers.

A beaker is distinguished from a flask by having sides which are straight rather than sloping.
The exception to this definition is a slightly conical sided beaker called a Phillips beaker.

Materials

Beakers are commonly made of glass (today usually borosilicate glass), but can also be in
metal (such as stainless steel or aluminium) or certain plastics, (notably polythene,
polypropylene, PTFE). A common use for polypropylene beakers is gamma spectral analysis
of liquid and solid samples.

Shape

Beakers are often graduated, that is, marked on the side with lines indicating the volume
contained. For instance, a 250 mL beaker might be marked with lines to indicate 50, 100,
150, 200, and 250 mL of volume. These marks are not intended for obtaining a precise
measurement of volume (a graduated cylinder would be a more appropriate instrument for
such a task), but rather an estimation.

The presence of a lip means that the beaker cannot have a lid. However, when in use, beakers
may be covered by a watch glass to prevent contamination or loss of the contents, but
allowing venting via the spout.

59
Measuring cylinder
A measuring cylinder or graduated cylinder is used to measure the volume of liquids. A
measuring cylinder is used for measuring solutions, liquids and also water. For example, a
solution made up of salt and water could be measured.

Erlenmeyer or conical flask

An Erlenmeyer flask, commonly known as a conical flask or E-flask, is a widely used type
of laboratory flask which features a flat base, a conical body, and a cylindrical neck. The
flask is named after the German chemist Emil Erlenmeyer, who created it in 1861.

The Erlenmeyer flask is usually marked on the side (graduated) to indicate the approximate
volume of their contents, and has a spot of ground glass or rough white enamel where it can
be labeled with a pencil. It differs from the beaker by its tapered body and narrow neck.

Original drawing of the Erlenmeyer flask. A conical flask

The opening usually has a slight rounded lip so that the flask can be easily stoppered using
rubber bungs or cotton wool. Alternatively, the neck may be fitted with a female ground glass
joint to accept a glass stopper. The conical shape allows the contents to be swirled or stirred

60
during an experiment, either by hand or by a shaker or magnetic stirrer; the narrow neck
keeps the contents from spilling. The smaller neck also slows evaporative loss better than a
beaker. The flat bottom of the conical flask makes it unlikely to tip over, unlike the Florence
flask.

Uses

Conical flask used in a Titration setup.

Erlenmeyer flasks are used in chemistry labs for titration, especially of pH.

Erlenmeyer flasks are often used to heat liquids, e.g. with a

Bunsen burner. For that purpose, the flask is usually placed
on a ring held to a ring stand by means of a ring clamp. A
wire gauze mesh or pad is usually placed between the ring
and the flask to prevent the flames from directly touching
the glass. An alternative way to set up the apparatus is to
hold the flask by the neck with a test tube clamp fixed to the
stand.

If the flask is to be heated in an oil or water bath, a 'C' shaped lead or iron weight may be
placed over the conical part of the flask to prevent it from floating in the bath.

Erlenmeyers are also used in microbiology for the preparation of microbial cultures. Plastic
Erlenmeyer flasks used in cell culture are pre-sterilized and feature closures and vented
closures to enhance gas exchange during incubation and shaking.

61
Pipette
A pipette (also called a pipet, pipettor or chemical dropper) is a laboratory instrument used to
transport a measured volume of liquid.

A selection of pipettes

Use and variations

Pipettes are commonly used in molecular biology as well as medical tests. Pipettes come in
several designs for various purposes with differing levels of accuracy and precision, from
single piece glass pipettes to more complex adjustable or electronic pipettes. Many pipettes
types work by creating a partial vacuum above the liquid-holding chamber and selectively
releasing this vacuum to draw up and dispense liquid.

Pipettes that dispense between 1 and 1000 l are termed micropipettes, while macropipettes
dispense a greater volume of liquid. Two types of micropipettes are generally used: air-
displacement pipettes and positive-displacement pipettes. In particular, piston-driven air-
displacement pipettes are micropipettes which dispense an adjustable volume of liquid from a
disposable tip. The pipette body contains a plunger, which provides the suction to pull liquid
into the tip when the piston is compressed and released. The maximum displacement of the
plunger is set by a dial on the pipette body, allowing the delivery volume to be changed.
Whereas for larger volumes cylindrical pipettes, such as volumetric or graduated pipettes are
used and driven by a pipette aid.

Piston-driven air displacement pipettes

These pipettes are the most precise and accurate type of pipette, they operate by piston-driven
air displacement. A vacuum is generated by the vertical travel of a metal or ceramic piston
within an airtight sleeve. As the piston moves upward, driven by the depression of the
plunger, a vacuum is created in the space left vacant by the piston. Air from the tip rises to
fill the space left vacant, and the tip air is then replaced by the liquid, which is drawn up into
the tip and thus available for transport and dispensing elsewhere. These micropipette were
invented at the University of Wisconsin-Madison in 1972 by several people , primarily
inventor Warren Gilson and Henry Lardy, hence the biggest producer is the original company
called Gilson Inc., as a result they are colloquially refered to as Gilsons.

Several different type of air displacement pipettes exist:

adjustable or fixed
volume handled
Single-channel, multi-channel or repeater
conical tips or cylindrical tips
standard or locking

62
 manual or elecronic
manufacturer

Positive displacement pipette

These are similar to air displacement pipettes, but are less commonly used and are used to
avoid contamination and for volatile or viscous substances at small volumes, such as DNA.
The major difference is that the disposable tip is a microsyringe (plastic), composed of a
plunger which directly displaces the liquid.

positive displacement the chuck which will be used to move the an early pipette
pipette plunger

Vacuum assisted pipette

serological pipettes are used for large volumes and for sterile work, but require a pipette aid

Non-piston driven vacuum assisted pipettes are hollow narrow cylinders which work like a
straw and require the use of some kind of additional suction device. Originally pipettes were
made of pyrex glass, but currently are made of polystyrene. It is more commonly used in
chemistry, with aqueous solutions. There are two types. One type, the volumetric pipette, has
generally a large bulge with a long narrow portion above with a mark as it is calibrated for a
single volume. Typical volumes are 10, 25, and 50 mL. Alternatively, graduated pipettes are
straight-walled, and graduated for different volumes such as 5 mL in 0.5 mL increments. The
single volume pipette is usually more accurate, with an error of 0.1 or 0.2 mL.

The pipette is filled by dipping the tip in the volume to be measured, and drawing up the
liquid with a pipette filler past the inscribed mark. The volume is then set by releasing the
vacuum using the pipette filler or a damp finger. While moving the pipette to the receiving
vessel, care must be taken not to shake the pipette because the column of fluid may "bounce".

Volumetric pipettes

Volumetric pipettes allow the user to measure a volume of solution extremely accurately and
then add it to something else. They are commonly used to make laboratory solutions from a
base stock as well as prepare solutions for titration. They are typically marked to indicate one
single volume in a particular size pipette (as are volumetric flasks). Many different sizes are
available.

63
Graduated pipettes

Graduated pipettes use a series of marked lines (as on a graduated cylinder) to indicate
different calibrated volumes. These also come in a variety of sizes. These are used much like
a burette, in that the volume is found by calculating the difference of the liquid level before
and after liquid is dispensed. Typically the precision of a graduated pipette is not as great as
that of a volumetric pipette. Two types of graduated pipettes exist:

Mohr pipettes or drain-out pipettes have a 0ml mark before the start of the conical
end, which is a dead volume.
serological or blow-out pipettes have no 0ml mark as that corresponds to an empty
pipette.

Pipette aids

Various methods exist to handle the liquids inside a pipette. Before the advent of more
sophisticated pipette aids, it was common practice to "mouth pipette" i.e. to aspirate fluid into
the pipette by applying suction with one's mouth. the main three types of pipette aid are the
bulb filler, pipette pump and the electronic controller or helper.

bulb filler are the simplest
cylindrical pipette controllers,
where valves are opened or
closed by squeezing the rubber
pipette pumps allow a more electronic controllers are
accurate handling of the expensive (200-300GB) but
volumes inside a cylindrical allow an accurate and easy
pipette handling of volumes inside a
cylindrical pipette

Pipette accessories

Pipette fillers are used to fill the pipette easily, avoiding the need for mouth pipetting.
Pipette helpers are battery-operated and are designed to be used with disposable
pipette tubes. These pipettes cannot be calibrated and their accuracy is determined by
that of the printed graduations on the disposable tubes.
Light-guided pipetting systems are pipetting accessories which are computer based.
They utilize flat screen LCD monitors or LED arrays to light up source and
destination wells in microplates or vials for accurate well to well pipetting. Some of
these systems use text to speech to alert the operator during plate or volume changes
when pipetting lab protocols.
Pipette tips. The pipettors and injection molded plastic disposable tips form together a
reliable pipetting system. It is recommended to use original manufacturers tips to
guarantee the precision and accuracy of the pipettes. The precision-made pipettor tips
provide excellent reproducibility and accuracy. Pipettor tips are available in
autoclavable boxes, refills and bulk packaging. Non-sterile, pre-sterilized and filtered
tips are usually available in single trays as RNase, DNase and endotoxin certified free.

64
Pasteur pipette

Dispensable pipettes

Pasteur pipettes, also known as droppers, are plastic or glass pipettes

used to transfer small amounts of liquids, but are not graduated or
calibrated for any particular volume. Transfer pipettes, also known as
Beral pipettes, are similar to Pasteur pipettes. However, they are made
from a single piece of plastic and their bulb can serve as the liquid-
holding chamber.A commercial variant of the pasteur pipette is the
dispensable pipettes which are often made of plastic and intended to be
used to administer medicine into the eye or ear of a patient (see image).

Micropipette
A micropipette is one of two different instruments used in science laboratories.

A Gilson multichannel adjustable micropipette. This one has only four channels in use.

Adjustable micropipette

Pipettes are used to accurately measure and dispense small volumes of liquid. The capacity of
a micropipette can range from less than 1l to 1000l (1ml), while macropipettes can
measure volumes greater than 1ml.

Micropipettes brands include: Eppendorf, Hamilton, Rainin, Drummond, BrandTech, Oxford,

Hirschmann, Biohit, Labnet, Nichiryo, Gilson, Corning, VistaLab, Thermo, Jencons, Vertex,
Handypett, Pricisexx

Glass micropipette

These are used to physically interact with microscopic samples, such as in the procedures of
microinjection and patch clamping. Most micropipettes are made of borosilicate,
aluminosilicate or quartz with many types and sizes of glass tubing being available. Each of
these compositions has unique properties which will determine suitable applications.

65
Glass micropipettes are fabricated in a micropipette puller.

Borosilicate glass micropipette pulled with a Flaming/Brown micropipette puller P-97

Stirring rod/Glass rod

A stirring rod is a piece of laboratory equipment used
to mix chamicals and liquids for laboratory purposes.
They are usually made of solid glass, about the
thickness and slightly longer than a drinking straw

Microscope Slide
A microscope slide is a thin flate piece of glass typically 75
by 25 mm (3 by 1 inches) and about 1 mm thick, used to
hold object for examination under a microscpoe. Typically
the object is placed or secured (mounted) on the slide,
and then both are inserted together in the microscope for
viewing. This arrangement allows several slide-mounted
objects to be quickly inserted and removed from the
microscope, labeled, transported and stored in appropriate
slide cases or folders.

Microscope slide are often used together with a cover slip or cover glass, a smaller thinner
sheet of glass that is placed over the specimen. Slides are held in place on the microscopes
stage by slide clips or slide clamps.

An old box of microscope slides Slide use in microbiology lab

with various mounted specimens for teaching use.

66
Scoopula
A scoopula is a utensil used primarily in chemistry lab settings to transfer solids: to a weigh
paper for weighing, to a cover slip to measure melting point, or to a watch glass from a flask
or beaker through scraping. Usually made of metal. It is often used to transfer solids from a
container into a weighting vessel.

Inoculation loop
An inoculation loop, also called a smear loop,
inoculation wand or microstreaker, is a simple
tool used mainly by microbiologists to retrieve an
inoculum from a culture of microorganisms. Its tip
is a wire made of platinum or nichrome, the latter
being inferior but less expensive. The wire forms a
small loop with a diameter of about 5 mm. This
loop is handy for taking an inoculum from a liquid
by using the phenomenon of surface tension.

An inoculating loop

The loop is used to cultivate microbes in Agar jelly and to use the streak manouvre to transfer
microbes.

The inoculation loop is always sterilized in a flame until it becomes red hot before and after
each use. By doing this, the same tool can be reused in different experiments without fear of
cross-contamination. After flame sterilization, the loop must be cooled so that the next cells
to touch the loop don't instantly die.

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

Most wash bottles are made up of polyethylene, which is a petroleum based plastic.

Usually, wash bottles are filled with deionized water. Wash bottles may also be filled with
detergent solutions and rinse solvents such as acetone and ethanol.

67
When pressure is applied to the bottle, the water inside becomes pressurized and as a result it
is forced out the nozzle into a narrow stream of liquid.

Lab Wash Bottles

Plastic wash bottlers for ethanol and water

Other names Squeeze Bottle

To clean lab glassware and other

Uses
lab equipment. They are filled
with water or other cleaning
liquids, and poured over the tool
that needs to be cleaned.

Notable The wash bottle is generally used

experiments in the clean-up phase of many
experiments

Class II cabinet
A Class II Cabinet or microbiological safety cabinet is a piece of laboratory equipment that
provides a safe working area for people handling material potentially contaminated with
pathogens.

The principle of operation involves using a fan

mounted in the top of the cabinet to draw a curtain
of sterile air over the products that are being
handled. The air is then drawn underneath the
work surface and back up to the top of the cabinet
where it passes through filters (HEPA filters) that
are capable of removing harmful bacteria and
viruses. Seventy percent of the air is continuously
recirculated in this way, while 30 percent is
exhausted through another HEPA filter in the top
of the cabinet. The air that is exhausted is made
up by air being drawn into the front of the cabinet A class II safety cabinet used for
underneath the worksurface. The air being drawn handling
in acts as a barrier to potentially contaminated air
coming back out to the operator.

"Class II" refers to the NSF classification of this type of cabinet. Subdivisions within this
class based on airflow patterns inside the cabinet and acceptable uses include A1, A2, B1,
B2. Older pre-2002 NSF designations include A/B3 and others.

68
Test Tube Holder
Test tube Holder generally use in the microbiology laboratory to put test tube with or without
samples for various purpose and to keep safety from broken.

Picrure: Wooden made test tube holders are widely in microbiology lab to put testtube in safe

Slide Holder
Slide Holder generally use in the microbiology
laboratory to put slide with or without samples for
various purpose and to keep safety from broken.

Aluminium foil
Aluminium foil (known as aluminum foil in North
America) is aluminium prepared in thin metal leafs,
with a thickness less than 0.2 mm / 0.008 in,
although much thinner gauges down to 0.006 mm
are commonly used. Foils are commonly gauged in
Mils. The foil is extremely pliable, and can be bent
or wrapped around objects with ease. However, thin
foils are fragile and easily damaged, and are often
laminated to other materials such as plastics or
paper to make them more useful. It replaced tin foil
in the mid 20th century.

Aluminium foil is sometimes known as al-foil or alu-foil. It is also often called Reynolds
wrap after Reynolds Metals, the leading manufacturer in North America. Metallised films are
sometimes mistaken for aluminium foil, but are actually polymer films coated with a thin
layer of aluminium.

Generally in microbiology laboratory aluminium foil is widely use for wrapping laboratory
equipment like glass ware and several chemical where the sample is subject to biomarker
analysis.

69
 Picture shows the use of aluminium foil in the microbiology laboratory for various purpose

Personal protective equipment

Disposable PPE

Personal protective equipment (PPE) is specialized clothing or equipment worn by a

worker for protection against a hazard. The hazard in a health care setting is exposure to
blood, saliva, or other bodily fluids or aerosols that may carry infectious materials such as
Hepatitis C, HIV, or other blood borne or bodily fluid pathogen. PPE prevents contact with a
potentially infectious material by creating a physical barrier between the potential infectious
material and the healthcare worker.

Components of Personal protective equipment (PPE) include gloves, gowns, bonnets, shoe
covers, face shields, CPR masks, goggles, and respirators. How many components are
used and how the components are used is often determined by regulations or the infection
control protocol of the facility in question. Many or most of these items are disposable to
avoid carrying infectious materials from one person to another and person who closely
contact with various laboratory materials and chemicals.

Goggles hade and face mask

Hand gloves

70
DCA agar
DCA agar - Deoxycholate Citrate Agar is a solid bacteriological growth medium.

A microbiological culture of
Salmonella sp.
bacteria on DC agar culture
medium

Uses

It is particularly useful for the isolation of organisms that cause bacilliary dysentery,
salmonella strains that cause food poisoning and Salmonella Paratyphi. It is not so selective
for Salmonella Typhi. This growth medium is inhibitory to most gut bacteria, in particular
species of the genus Proteus, although these species do survive on DCA agar. It is therefore
essential that suspected pathogens must be subcultured on a less inhibitory medium prior to
identification. Salmonella spp appear to be yellow or colourless colonies, often with a dark
centre. As there are many bacteria that also look like Salmonella on DCA, it is widely
recommended that more selective agars are used for the identification of Salmonella, namely
xylose lysine deoxycholate (XLD) agar. This growth medium is heat-sensitive and should be
poured and cooled as soon as possible after addition of the deoxycholate, otherwise it tends to
become very soft and difficult to handle. It has a pH of approximately 7.3, and when poured
and cooled, appears light to dark pink in colour.

TSI slant
TSI agar slant results: (from left) preinoculated (as control), P. aeruginosa, E. coli,
Salmonella Typhimurium,Shigella flexneri

The Triple Sugar Iron or TSI test is a microbiological test roughly named for its ability to
test microorganism's ability to ferment sugars and to produce hydrogen sulfide. It is often
used in the selective identification of enteric bacteria including but not limited to Salmonella
and Shigella.

71
Composition

The TSI slant is a test tube that contains agar, a pH-

sensitive dye (phenol red), 1% lactose, 1% sucrose,
0.1% glucose, as well as sodium thiosulfate and
ferrous sulfate or ferrous ammonium sulfate.All of
these ingredients are mixed together and allowed to
solidify in the test tube at a slanted angle. The slanted
shape of this medium provides an array of surfaces
that are either exposed to oxygen-containing air in
varying degrees (an aerobic environment) or not
exposed to air (an anaerobic environment).

TSI agar medium was developed based

on Kligler's iron agar, which had been
used for the determination of lactose
fermentative bacteria, by addition of
sucrose to be able to detect also sucrose
fermentative bacteria.

Eosin Methylene Blue Agar

Eosin methylene blue (EMB) is a selective stain for Gram-negative bacteria. 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 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.

Lactose fermentation produces acids, which lower the pH. This

encourages dye absorption by the colonies, which are now
coloured purple-black.

Lactose non-fermenters may increase the pH by deamination of

proteins. This ensures that the dye is not absorbed.

On EMB if E.coli is grown it will give a distinctive metallic

green sheen (due to the metachromatic properties of the dyes,
E. coli movement using flagella, and strong acid end-products
of fermentation). Some species of Citrobacter and Enterobacter
will also react this way to EMB.

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Eosin
Eosin B

Eosin Y

Eosin is a fluorescent red dye resulting from the action of bromine on fluorescein. It can be
used to stain cytoplasm, collagen and muscle fibers for examination under the microscope.
Structures that stain readily with eosin are termed eosinophilic.

Methylene blue

Methylene blue is a heterocyclic aromatic chemical compound with molecular formula:

C16H18N3SCl. It has many uses in a range of different fields, such as biology and chemistry.
At room temperature it appears as a solid, odorless, dark green powder, that yields a blue
solution when dissolved in water. The hydrated form has 3 molecules of water per molecule
of MB. Methylene blue should not be confused with methyl blue, another histology stain,
new methylene blue, nor with the methyl violets often used as pH indicators.

The terms "methylthioninium chloride" and "methylene blue" are sometimes used
interchangeably.

XLD agar/SS Agar

Xylose lysine deoxycholate agar (XLD agar) is a selective growth medium used in the
isolation of Salmonella and Shigella species from clinical samples and from food. It has a pH
of approximately 7.4, leaving it with a bright pink or red appearance due to the indicator
phenol red. Sugar fermentation lowers the pH and the phenol red indicator registers this by
changing to yellow. Most gut bacteria, including Salmonella, can ferment the sugar xylose to
produce acid; Shigella colonies cannot do this and therefore remain red. After exhausting the
xylose supply Salmonella colonies will decarboxylate lysine, increasing the pH once again to
alkaline and mimicking the red Shigella colonies. Salmonellae metabolise thiosulfate to
produce hydrogen sulfide, which leads to the formation of colonies with black centers and
allows them to be differentiated from the similarly coloured Shigella colonies.

73
 Other Enterobacteria such as E. coli will ferment
the lactose and sucrose present in the medium to
an extent that will prevent pH reversion by
decarboxylation and acidify the medium turning
it yellow.

Salmonella species: red colonies, some with

black centers. The agar itself will turn red due to
the presence of Salmonella type colonies.

Shigella species: red colonies.

Coliforms: yellow to orange colonies.

Pseudomonas aeruginosa: pink, flat, rough

colonies. This type of colony can be easily
mistaken for Salmonella due to the colour
similarities.

XLD agar contains: Yeast extract-3g/l, L-Lysine-5g/l, Xylose-3.75g/l, Lactose-7.5g/l,

Sucrose-7.5g/l, Sodium deoxycholate-1g/l, Sodium chloride-5g/l,, Sodium thiosulfate-6.8g/l

Plate count agar

Plate count agar (PCA) is a microbiological growth medium commonly used to assess or to
monitor "total" or viable bacterial growth of a sample. PCA is not a selective medium.

The composition of plate count agar may vary, but typically it contains (w/v):

0.5% peptone

0.25% yeast extract

0.1% glucose

1.5% agar

pH adjusted to neutral at 25 C.

Plate count broth does not contain agar.

74
Nutrient Broth

Of Basal Medium

Sodium hydroxide Pellets purified

Sodium hydroxide (NaOH), also known as lye and
caustic soda, is a caustic metallic base. It is used in
many industries, mostly as a strong chemical base in the
manufacture of pulp and paper, textiles, drinking water,
soaps and detergents and as a drain cleaner. World-wide
production in 1998 was around 45 million tonnes.
Sodium hydroxide is a common base in chemical
laboratories.

Pure sodium hydroxide is a white solid; available in pellets, flakes, granules and as a 50%
saturated solution. It is hygroscopic and readily absorbs water from the air, so it should be
stored in an airtight container. It is very soluble in water with liberation of heat. It also
dissolves in ethanol and methanol, though it exhibits lower solubility in these solvents than
does potassium hydroxide. Molten sodium hydroxide is also a strong base, but the high
temperature required limits applications. It is insoluble in ether and other non-polar solvents.
A sodium hydroxide solution will leave a yellow stain on fabric and paper.

75
1. Physical properties

H dissolution for diluted aqueous -44.45 kJ / mol;

From aqueous solutions at 12.3-61.8C, it crystallizes in monohydrate, with a melting point

65.1 C and density of 1.829 g/cm 3;

H form -734.96 kJ / mol;

Monohydrate from -28 to -24C;

Heptahydrate from -24 to -17.7C;

Pentahydrate from -17.7 to -5.4C;

Tetrahydrate (- changed), at -5 , 4 - 12.3C Also know metastable - NaOH 4* H2O. Which

above 61.8C are crystallized.

2. Chemical properties

Sodium hydroxide is completely ionic, containing sodium cations and hydroxide anions. The
hydroxide anion makes sodium hydroxide a strong base which reacts with acids to form water
and the corresponding salts, e.g. with hydrochloric acid, sodium chloride is formed:

NaOH(aq) + HCl(aq) NaCl(aq) + H2O(l)

In general such neutralization reactions are represented by one simple net ionic equation:

OH(aq) + H+(aq) H2O(l)

This type of reaction with a strong acid releases heat, and hence is referred to as exothermic.
Such acid-base reactions can also be used for titrations, which is a common method to
determine the concentration of acids.

Another type of reaction that sodium hydroxide is involved in is with acidic oxides. The
reaction of carbon dioxide has already been mentioned, but other acidic oxides such as sulfur
dioxide (SO2) also react completely. Such reactions are often used to "scrub" harmful acidic
gases (like SO2 and H2S) and prevent their release into the atmosphere.

2 NaOH + CO2 Na2CO3 + H2O

Sodium hydroxide slowly reacts with glass to form sodium silicate, so glass joints and
stopcocks exposed to NaOH have a tendency to "freeze". Flasks and glass-lined chemical
reactors are damaged by long exposure to hot sodium hydroxide, and the glass becomes
frosted. Sodium hydroxide does not attack iron since iron does not have amphoteric
properties. A few transition metals, however, may react with sodium hydroxide in a vigorous
way.

In 1986, an aluminium road tanker in the UK was mistakenly used to transport 25% sodium
hydroxide solution, causing pressurization of the contents and damage to the tanker. The

76
pressurization was due to the hydrogen gas which is produced in the reaction between sodium
hydroxide and aluminium:

2 Al(s) + 6 NaOH(aq) 3 H2(g) + 2 Na3AlO3(aq)

Unlike NaOH, the hydroxides of most metals are insoluble, and therefore sodium hydroxide
can be used to precipitate metal hydroxides. One such hydroxide is aluminium hydroxide,
used as a gelatinous flocculant to filter out particulate matter in water treatment. Aluminium
hydroxide is prepared at the treatment plant from aluminium sulfate by reacting with NaOH.
This reaction is highly profitable, and is hence an important synthesis reaction.

Sodium hydroxide reacts readily with carboxylic acids to form their salts and is even a strong
enough base to form salts with phenols. NaOH can be used for the base-driven hydrolysis of
esters (as in saponification), amides and alkyl halides. However, the limited solubility of
NaOH in organic solvents means that the more soluble KOH is often preferred.

Ethanol
Ethanol, also called ethyl alcohol, pure alcohol,
grain alcohol, or drinking alcohol, is a volatile,
flammable, colorless liquid. It is a somewhat potent
psychoactive drug, best known as the type of
alcohol found in alcoholic beverages and in modern
thermometers. Ethanol is one of the oldest
recreational drugs. In common usage, it is often
referred to simply as alcohol or spirits.

Ethanol is a straight-chain alcohol, and its molecular formula is C2H5OH. Its empirical
formula is C2H6O. An alternative notation is CH3CH2OH, which indicates that the carbon
of a methyl group (CH3) is attached to the carbon of a methylene group (CH2), which is
attached to the oxygen of a hydroxyl group (OH). It is a constitutional isomer of dimethyl
ether. Ethanol is often abbreviated as EtOH, using the common organic chemistry notation of
representing the ethyl group (C2H5) with Et.

The fermentation of sugar into ethanol is one of the earliest organic reactions employed by
humanity. The intoxicating effects of ethanol consumption have been known since ancient
times. In modern times, ethanol intended for industrial use is also produced from by-products
of petroleum refining.

Ethanol has widespread use as a solvent of substances intended for human contact or
consumption, including scents, flavorings, colorings, and medicines. In chemistry, it is both
an essential solvent and a feedstock for the synthesis of other products. It has a long history
as a fuel for heat and light, and more recently as a fuel for internal combustion engines.

77
Glucose
D-Glucose

Glucose (Glc), a monosaccharide (or simple sugar) also known as grape sugar, blood sugar,
or corn sugar, is a very important carbohydrate in biology. The living cell uses it as a source
of energy and metabolic intermediate. Glucose is one of the main products of photosynthesis
and starts cellular respiration in both prokaryotes (bacteria and archaea) and eukaryotes
(animals, plants, fungi, and protists).

The name "glucose" comes from the Greek word glukus (), meaning "sweet", and the
suffix "-ose," which denotes a sugar.

Two stereoisomers of the aldohexose sugars are known as glucose, only one of which (D-
glucose) is biologically active. This form (D-glucose) is often referred to as dextrose
monohydrate, or, especially in the food industry, simply dextrose (from dextrorotatory
glucose). This article deals with the D-form of glucose. The mirror-image of the molecule, L-
glucose, cannot be metabolized by cells in the biochemical process known as glycolysis.

Structure

Glucose (C6H12O6) contains six carbon atoms, one of which is part of an aldehyde group.
Therefore glucose is an aldohexose. In solution, the glucose molecule can exist in an open-
chain (acyclic) form and a ring (cyclic) form (in equilibrium). The cyclic form is the result of
a covalent bond between the aldehyde C atom and the C-5 hydroxyl group to form a six-
membered cyclic hemiacetal. At pH 7 the cyclic form is predominant. In the solid phase,
glucose assumes the cyclic form. Because the ring contains five carbon atoms and one
oxygen atom (like pyran), the cyclic form of glucose is also referred to as glucopyranose. In
this ring, each carbon is linked to a hydroxyl side group with the exception of the fifth atom,
which links to a sixth carbon atom outside the ring, forming a CH2OH group. Glucose is

78
commonly available in the form of a white powder or as a solid crystal. It can also be
dissolved in water as an aqueous solution. Its solubility level is very high.

The chain form of D- -D-

glucose -D-
glucopyranose glucopyranose
The Fischer projection of the
chain form of D-glucose

Chain form: ball-and-stick Chain form: space-

model -D-
filling model -D-
glucopyranose glucopyranose

Production

Natural

1. Glucose is one of the products of photosynthesis in plants and some prokaryotes.

2. In animals and fungi, glucose is the result of the breakdown of glycogen, a process
known as glycogenolysis. In plants the breakdown substrate is starch.
3. In animals, glucose is synthesized in the liver and kidneys from non-carbohydrate
intermediates, such as pyruvate and glycerol, by a process known as gluconeogenesis.
4. In some deep-sea bacteria glucose is produced by chemosynthesis.

Commercial

Glucose is produced commercially via the enzymatic hydrolysis of starch. Many crops can be
used as the source of starch. Maize, rice, wheat, cassava, corn husk and sago are all used in
various parts of the world. In the United States, cornstarch (from maize) is used almost
exclusively.

Glucose tablets

79
Function

Glucose metabolism and various forms of it in the process.

-Glucose-containing compounds and isomeric forms are digested and taken up by the body in
the intestines, including starch, glycogen, disaccharides and monosaccharides.
-Glucose is stored in mainly the liver and muscles as glycogen.
-It is distributed and utilized in tissues as free glucose.

Scientists can speculate on the reasons why glucose, and not another monosaccharide such as
fructose (Fru), is so widely used in organisms. One reason might be that glucose has a lower
tendency, as compared to other hexose sugars, to non-specifically react with the amino
groups of proteins. This reaction (glycation) reduces or destroys the function of many
enzymes. The low rate of glycation is due to glucose's preference for the less reactive cyclic
isomer. Nevertheless, many of the long-term complications of diabetes (e.g., blindness, renal
failure, and peripheral neuropathy) are probably due to the glycation of proteins or lipids. In
contrast, enzyme-regulated addition of glucose to proteins by glycosylation is often essential
to their function.

As an energy source

Glucose is a ubiquitous fuel in biology. It is used as an energy source in most organisms,

from bacteria to humans. Use of glucose may be by either aerobic respiration, anaerobic
respiration, or fermentation. Carbohydrates are the human body's key source of energy,
through aerobic respiration, providing approximately 3.75 kilocalories (16 kilojoules) of food
energy per gram. Breakdown of carbohydrates (e.g. starch) yields mono- and disaccharides,
most of which is glucose. Through glycolysis and later in the reactions of the citric acid cycle
(TCAC), glucose is oxidized to eventually form CO2 and water, yielding energy sources,
mostly in the form of ATP. The insulin reaction, and other mechanisms, regulate the
concentration of glucose in the blood. A high fasting blood sugar level is an indication of
prediabetic and diabetic conditions.

Glucose is a primary source of energy for the brain, and hence its availability influences
psychological processes. When glucose is low, psychological processes requiring mental
effort (e.g., self-control, effortful decision-making) are impaired.

80
Cleaning agent
Cleaning Agents are substances, usually liquids, that are used to remove dirt, including dust,
stains, bad smells and clutter on surfaces. Purposes of cleaning agents include health, beauty,
absence of offensive odor, avoidance of shame, and to avoid the spreading of dirt and
contaminants to oneself and others. Some cleaning agents can kill bacteria and clean at the
same time.

Types

Cleaning agents normally water solutions that might be acidic, alkaline or neutral depending
on the use. Cleaning agents may also be solvent based or solvent containing and is then called
degreasers.

Acidic

Acidic washing agents are mainly used for removal of inorganic deposits like scaling. The
active ingredients are normally strong mineral acids and chelants. They are often added
surfactants and corrosion inhibitors.

Alkaline

Alkaline washing agents contains strong bases like sodium hydroxide and/or potassium
hydroxide. The alkali dissolves grease, oils, fats and protein based deposits. They are often
added dispersing agents and/or chelants to prevent redeposition of dissolved dirt.

Neutral

Neutral washing agents are pH-neutral and based on non-ionic surfactants that disperses
different types of dirt.

Degreaser

Cleaning agents specially made for removal of grease are called degreasers. These may be
solvent based or solvent containing and may also have surfactants as active ingredients. The
solvents have a dissolving action on grease and similar dirt. The solvent containing degreaser
may have an alkaline washing agent added to a solvent to promote further degreasing.
Degreasing agents may also be made solvent free based on surfactants.

81
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