microbiology

1. INCUBATOR , BUNSEN BURNER AND
PIPETTES
Dr. sarah abdulsalam
1st STAGE / LEC9/ Lab Instrumentation
theory

2. INCUBATOR:
 Incubator, in microbiology, is an insulated and enclosed
device that provides an optimal condition of temperature,
humidity, and other environmental conditions required for
the growth of organisms.
 An incubator is a piece of vital laboratory equipment
necessary for the cultivation of microorganisms under
artificial conditions.
 An incubator can be used for the cultivation of both
unicellular and multicellular organisms.

3. PRINCIPLE/ WORKING OF INCUBATOR
 An incubator is based on the principle that microorganisms require a particular set
of parameters for their growth and development.
 All incubators are based on the concept that when organisms are provided with
the optimal condition of temperature, humidity, oxygen, and carbon dioxide levels,
they grow and divide to form more organisms.
 In an incubator, the thermostat maintains a constant temperature that can be read
from the outside via the thermometer.
 The temperature is maintained by utilizing the heating and no-heating cycles.
 During the heating cycle, the thermostat heats the incubator, and during the no-
heating period, the heating is stopped, and the incubator is cooled by radiating
heat to the surrounding.
 Insulation from the outside creates an isolated condition inside the cabinet, which
allows the microbes to grow effectively.
 Similarly, other parameters like humidity and airflow are also maintained through
different mechanisms that create an environment similar to the natural
environment of the organisms.
 Similarly, they are provided with adjustments for maintaining the concentration of
CO2 to balance the pH and humidity required for the growth of the organisms.
 Variation of the incubator like a shaking incubator is also available, which allows
for the continuous movement of the culture required for cell aeration and solubility
studies.

4. PROCEDURE FOR RUNNING AN INCUBATOR
1. Once the cultures of organisms are created, the culture plates are to be placed inside an
incubator at the desired temperature and required period of time. In most clinical
laboratories, the usual temperature to be maintained is 35–37°C for bacteria.
2. The following are the steps to be followed while running an incubator:
3. Before using the incubator, it should be made sure that no remaining items are present in
the incubator from the previous cycles. However, in some cases, if the same incubator is
being used for multiple organisms, and they require the same set of parameters, they can
be placed together in the same incubator.
4. The door of the incubator is then kept closed, and the incubator is switched on. The
incubator has to be heated up to the desired temperature of the growth of the particular
organism. The thermometer can be used to see if the temperature has reached.
5. In the meantime, if the organism requires a particular concentration of CO2 or a specific
humidity, those parameters should also be set in the incubator.
6. Once all the parameters are met, the petri dish cultures are placed on the perforated
shelves upside down, i.e., media uppermost. This is necessary because if the plates are
incubated normally, condensation collects on the surface of the medium and prevents the
formation of isolated colonies.
7. If it is necessary to incubate Petri dish cultures for several days, the plates are sealed with
adhesive tapes or are placed in plastic bags or plastic food containers.
8. Now, the door is locked, and the plates are kept inside for the required time before taking
them out.

5. TYPES OF INCUBATORS:-
 On the basis of the presence of a particular parameter or the
purpose of the incubator, incubators are divided into the
following types:
 Benchtop incubators
 This is the most common type of incubator used in most of the
laboratories.
 These incubators are the basic types of incubators with
temperature control and insulation.
 CO2 incubators
 CO2 incubators are the special kinds of incubators that are
provided with automatic control of CO2 and humidity.
 This type of incubator is used for the growth of the cultivation of
different bacteria requiring 5-10% of CO2 concentration.
 For humidity control, water is kept underneath the cabinet of the
incubator.

6.  Cooled incubators
 For incubation at temperatures below the ambient, incubators are fitted
with modified refrigeration systems with heating and cooling controls.
 This type of incubator is called the cooling incubator.
 In the cooling incubator, the heating and cooling controls should be
appropriately balanced.
 Shaker incubator
 A thermostatically controlled shaker incubator is another piece of
apparatus used to cultivate microorganisms.
 Its advantage is that it provides a rapid and uniform transfer of heat to
the culture vessel, and its agitation provides increased aeration, resulting
in acceleration of growth.
 This incubator, however, can only be used for broth or liquid culture
media.
 Portable incubator
 Portable incubators are smaller in size and are used in fieldwork, e.g.
environmental microbiology and water examination.

7. USES OF INCUBATOR
 incubators have a wide range of applications in various areas
including cell culture, pharmaceutical studies, hematological studies,
and biochemical studies.
 Some of the uses of incubators are given below:
1. Incubators are used to grow microbial culture or cell cultures.
2. Incubators can also be used to maintain the culture of organisms to
be used later.
3. Some incubators are used to increase the growth rate of organisms,
having a prolonged growth rate in the natural environment.
4. Specific incubators are used for the reproduction of microbial colonies
and subsequent determination of biochemical oxygen demand.
5. These are also used for breeding of insects and hatching of eggs in
zoology.
6. Incubators also provide a controlled condition for sample storage
before they can be processed in the laboratories.

8. PRECAUTIONS
 The following precautions are to be followed while running an
incubator:
1. As microorganisms are susceptible to temperature change, the
fluctuations in temperature of the cabinet by repeatedly
opening the door should be avoided.
2. The required parameters growth of the organism should be
met before the culture plates are placed inside the cabinet.
3. The plates should be placed upside down with the lid at the
bottom to prevent the condensation of water on to the media.
4. The inside of the incubators should be cleaned regularly to
prevent the organisms from settling on the shelves or the
corners of the incubator.
5. While running the incubator for an extended period of time,
sterile water should be placed underneath the shelves to
prevent the culture media from drying out.

10. BUNSEN BURNER
 Bunsen burner is a standard tool used in laboratories, named
after Robert Bunsen.
 It is a gas-fueled single open flame.
 Working Principle:
 This burner is made with a metal tube on a flat base with a gas
inlet at the bottom of the tube, which may have an adjustable
valve. On the sides of the tube are openings which can be
adjusted with a collar to control the amount of air that can enter.
 Once the burner is connected to a gas source, the gas is forced
by the gas pressure so that the gas reaches the top where the
flame is ignited with a match or a lighter.
 Uses:
 It is commonly used for processes like sterilization, combustion,
and heating. In medical or microbiology laboratories, it is
commonly used for micro-loop sterilization.

12. TYPES OF PIPETTES :
 Pipette Filler (Manual)
 A manual pipette filler is somewhat simpler
in design than a bulb filler, but there is a
maximum capacity . This type of filler can
be used with all types of pipettes and the
size of the barrel is proportional to the
capacity of the pipette that is used with the
filler.
 Beral pipette (commonly called a
disposable pipette)
 A Beral (disposable) pipette is typically
made of plastic and is used for non-
quantitative transfer of solutions (filling a
cuvette for example). Sometimes these do
have graduations in the stem, but
generally should never be used when
precise volume recordings are needed.

13.  Pasteur pipette
 A thin glass transfer pipette, similar
in use and limitations of a Beral
pipette. A Pasteur pipette requires a
disposable rubber bulb to draw up
solutions and transfer them. The
thin stem allows for more accuracy
than a Beral pipette in small volume
transfers.
 Bacteriologic pipet
 Glass or polypropylene
 Use for transport of bacterial
specimen esp. pus
 Sealed by flame and cotton plug
 Provide maximum sterility

14. MICROPIPETTE
 A micropipette is a common yet an
essential laboratory instrument
used to accurately and precisely
transfer volumes of liquid in the
microliter range. Micropipettes are
available in single channel and
multi channel variants. While the
single channel micropipettes are
used in labs that perform research
related to molecular biology,
microbiology, immunology, cell
culture, analytical chemistry,
biochemistry and genetics,
the multichannel micropipettes are
recommended for ELISA
(diagnostic test), molecular
screening, kinetic studies and
DNA amplification.

15.  Micropipets types:
 1. Air displacement
 2. Positive displacement
 3. Dilutor or dispenser