autoclave, oven, centrifuge, ph meter, microscope basic details

Sem 1 Ex 1
by- Dr. Rutvesh Borisagar

Microscope
The word microscope is derived from two Greek words 'miero'
meaning small and 'scope' meaning to view. In other words a
microscope is an instrument used for visual examination of small
objects which can not be properly examined by the unaided eye.
SIMPLE MICROSCOPE
The simplest form of microscope is nothing more than a magnifying
glass'. It consists of two parts:1. The optical part or viewing part, and
2. The mechanical part whose function is to hold the slide or object
in the proper position.
Dissecting microscope is a type of simple microscope which is fitted
with mirror to focus bright light and a stage for placing the object.

Dissecting microscope

COMPOUND MICROSCOPE
It differs from the simple microscope in that, it has two separate lens systems. Component parts of the
microscope, their locations and functions are as follows.
Functions of Various Parts of Compound Microscope
1. Base or stand: the U' shaped or square foundation which gives stability to the instrument.
2. Arm or handle: by means of which it may be moved or carried, to which are attached the magnifying
and adjustment systems.
3. Stage: a platform which provides a surface for the placement of slide with its specimen overthe
opening. In addition to the fixed stage, most microscopes have a mechanical stage thatcan be moved
vertically or horizontally by means of adjustment knobs.
4. Clips: the set of removable spring clips which hold in place the slide with object.
5. Mirror: the light reflecting device placed under stage and employed to illuminate the object on the
stage. Modern microscopes have been replaced by inbuilt source of light.
6. Condenser: a system of lenses placed under stage: its function is to focus a strong beam of light upon
the object being examined.
7. Iris diaphragm: a device placed beneath the condenser and capable of manipulation so as to adjust the
quantity of light.
8. Body tube: a hollow cylindrical tube of the magnification system through which light passes from the
objective lenses at its bottom to the eyepiece lenses at its top.
9. Nosepiece: a revolving disk at the bottom of the body tube to which the objectivesattached

10. Objectives a system of small lenses which constitute primary
magnifying mechanism and are attached to the nose piece. There
are usually three objectives, low power, high power,
11. Eye piece or ocular: a combination of lenses, placed in the
upper portion of the body tube,which magnifies the image formed
by the objective lens system. Two eye pieces -5X and- 10X are
generally provided.
12. Coarse and fine adjustment the screw mechanisms by which
the body tube and magnification system may be raised or lowered
quickly over a wide range to bring the objectiu approximately imo
focus. Fine adjustment moves the body tube slowly, and over a very
limited range, by means of which the object is brought into sharp or
exact focus. (Coaxial focus: Moderfocusing system that has both the
coarse and fine focusing knobs mounted on the same enUsually the
coarse knob is larger and on the outside and the fine knob is smaller
and on the inside

MAGNIFICATION
The total magnification is the product of the separate
magnification of the objective the eyepiece, and depends on
three factors whose relationship is as follows. Total
magnification =length of the body tube X eve piece
magnification /focal length of objectiveis
RESOLVING POWER
the ability to show two adjacent objects as discrete entities
(separate objects). dependent on the wavelength of light used
and the numerical aperture of the objective lens.
This is expressed by the following formula,
Resolving power = Wave length of light / 2 NA (numerical
aperture)

Thus, to have the greater resolving power; the shorter wave length and the larger numerical aperture
values are desirable.
Numerical Aperture (NA): The angle subtended by the optical axis and the outermost rays still covered
by the objective is the measure of the aperture of the objective: it is the half aperture angle. The
magnitude of this angle is expressed as a sine value. The sine value of the half-aperture angle multiplied
by the refractive index (71) of the medium filling the space between the front lens and the cover slip
gives the numerical aperture:
Numerical aperture = n sin Ø
The use of mineral oil [which has the same refractive index (n) as glass), as the mounting medium
increase the amount of light entering the objective and thereby also increase the value of numerical
aperture. Several other substances used as the mounting media are as follows.
Mounting Media Refractive index (n)
Paraffin oil 1.47
Canada balsam 1.53
Cedarwood oil 1.51
Sandalwood oil 1.51

Autoclave
AUTOCLAVEHeat may be used for sterilization in three
different ways by steam or hot water (moist heat) by
prolonged baking in the oven (dry heat), and by complete
burning Cincineration)
Moist heat readily kill viruses, bacteria, and fungi. Exposure
to boiling water for 10 minutesis sufficient to destroy
vegetative cells and eucaryotic spores. However this
temperature is norhigh enough to destroy bacterial
endospores which may survive hours of boiling
Thereforeboiling may be used for disinfection of drinking
water etc. but boiling does not sterilize.

Autoclave

Principle
The temperature of saturated steam at normal atmospheric pressure is 100 PC.
Compressed steam (steam under pressure) is hotter than boiling water or free-
flowing steam which is used in Tyndallization. Higher the steam pressure, higher
the temperature will be. If the pressureis increased inside a closed vessels like
autoclave or cooker, the temperature at which the water boils will also raise
above 100 C.
 Relationship between pressure and temperature is as follows.
 Thus the air present in autoclave will adversely affect steam penetration.
Hence all air that surrounds and permeates the load must first be removed
before steam sterilization begins.
psi ºc
05 109
10 115
15 121
20 126
25 130
30 135

Hot air oven
 HOT AIR OVENA though the sterilization by dry heat is not
as effective as moist heat, certain materials can not be
sterilized either by autoclave or by inspissator.
 Principle it is based on the principle that the dry-heat or
hot air destroys microorganisms by oxidizing their
chemical constituents and denaturation of proteins.
Normally a temperature of 165-170 °C (329–338 "F) for 2-3
hours is sufficient to kill the live spores by dry heat.

 Working
 1. Load materials to be sterilized in the hot air oven, It
requires different workout for different materials .e.g.,
glasswares are wrapped in craft paper, and tied with
string, pipettes are placedin a metal container with the
metal lid kept loose.
 2. Switch on the oven and set the thermostat to the
required temperature of 165 "C. Maintainthis temperature
for 2-3 hours.
 3. Switch off the current and allow incubator to cool
slowly (this prevents the glasswares from cracking as a
result of rapid fall in temperature).

Incubators
 INCUBATORSIncubators are the cabinets in which any desired
temperature may be constantly maintained
 Principle-Most of the modern incubators are heated electrically
with the help of heaters provided usually at the bottom of the
cabinet. As air at the bottom is heated up it gets lighter and
rises up while the cold air is pushed down at the bottom this
generates convection currents for air circulation. Cycle
continues till the temperature inside the cabinet is same
throughout. The incubator is set to the desired temperature
which is normally the optimum temperature of most
microorganisms.

 Use- For most purposes laboratory incubators are used
for providing constant temperature required for the
experiment. Since all organisms have optimum
temperature requirement for the growth, cultivation
of such organisms is carried out in incubators which is
adjusted to the required optimum temperature
 > Normally the temperature of incubator is constantly
maintained at 37 °C. this is due to the fact that; (A)
most of the routine bacteria studied are mesophiles, &
(B) human pathogen grow optimally at this
temperature (because the normal human body
temperature is also 37°C)

Biological filters
 FILTERS-Filtration is a method used to separate
microorganisms from a liquid for gas) in which they are
dispersed. This method is particularly useful for
sterilization of heat labile liquids and solutions which can
not be heated.
 Principle-Filters remove bacteria by two different
mechanisms:
1. By mechanical sieve like action of the minute pores of
filters whose pore size is smaller than that of bacteria.
2. Due to differences in the electrical charges between
bacteria and the filter, bacteria are adsorbed on to the
positively charged surfaces of the filter passages.

 Types of Filters> Filters are of various types, and are made from
various materials. Some of the common ones are as follows
Filters
Berkefeld Kieslguhr-diatomaceous
earth
Chamberland
Sintered glass
Seitz
Membrane
Unglazed porcelain
Fused glassparticles
compressed asbestos
Cellulose acetate

brkefeld filter

Chamberland

sintered glass filter

Seitz
Membrane

Rotary shaker
 ORBITAL SHAKEROrbital shakers are mainly of two types: 1) Environmental
shakers, and 2) Open air bench top shakers. They are primarily used for
cultivation of aerobic microorganisms, study of OTR. some solubility studies,
extraction procedures and washing procedures
 Environmental incubator shakers are designed to maintain constant
temperature and provideswirling or agitation to tasks, and tubes for a variety
of applications. It is primarily usedfor cultivation of aerobic microorganisms.
It has the facilities for maintaining the uniform Temperature required gaseous
environments and continuous variable shaking speed (RPM).
 > Open air shakers have no temperature control so they are ideal for
protocols that only require ambient temperatures. Open air bench top shakers
are cheap as there is no cost associated with compressors or heating
mechanisms

 Use-It is primarily used for cultivation of microorganisms. Due to optimum
temperature, aeration and agitation rapid growth is obtained in a very short
time. It can also be used for mixing of certain liquids.

pH meter
 pH METER As a convenient way of expressing hydrogen ion concentration, the
symbol pH was introduced by S. P. L.Sorensen in 1909. pH is defined as the
negative value of the logarithm to the base 10 of the hydrogen ion
concentration, or the logarithm (to the base 10) of the reciprocal of the
hydrogen-ion concentration’.
 pH = -log10[H+] = log10 1/[H+], or (H+] = 10-pH
 The most reliable way to measure the pH of a solution is to use pH meter. A
pH meter measures the differences in potential (e.m.f.) between two
solutions of different pH value.

 Principle-When two solutions containing hydrogen ion are separated by the bulb
of a glass pH electrode that is hydrogen sensitive, an electrical potential is
developed across the thin glass separating the two solutions. If the solution
inside the bulb is of fixed hydrogen ion concentration, the potential across the
glass will change as the hydrogen ion concentration of the other solution varies.
This difference in potential can be measured by making an electrical connection
between the internal element of the glass electrode and a reference electrode
(potential of which is known).The reference electrode is usually a calomel
electrode that hus an electrical potential of 0.2415 volts at 25 "C (calibrated
against the standard hydrogen electrode which has an electrode potential of
zero). It consists of a mercury electrode covered with mercurial chloride
(calomel and solution of mercury in saturated potassium chloride. The calomel
electrode will maintain its specified potential providing its calomel and mercury
content remain intact and its potassium chloride solution is kept saturated.

Spectrophotometer
 Principle-It is based on the Beer-Lamberts' law.Accordingly, when
monochromatic light passes through a transparent (or colored) medium the
amount of light adsorbed (1.e. the decrease in intensity) is directly
proportional to the concentration of the substance and the length through
which the light passes.
 > Since the standard and test solutions are always compared in identical
cuvettes or tubes the same light path or thickness of solution is used; the rate
of absorption oflight is directly proportional to concentration of the
substance.
 > The concentration of test solution can be derived by the following formula:
Concentration of test = absorbance of test X concentration of
standard/absorbance of standard

 Light from a low voltage lamp passes through a selected filter, which
generates a monochromatic light. It then passes through sample test
tube (cuvette) containing colored solution. Some of the light is
absorbed by the colored solution and the remaining falls on a
photosensitive cell. The current generated by this photocell deflects
the galvanometer needle Light passing through a colored solution is
absorbed, the amount of absorption is proportional to the
concentration of solution. The more light absorbed, the less light is
transmitted to the photoelectric cell and the smaller the current
generated.
 > Most spectrophotometers are provided with two scales, one from 0
to 100 indicating per cent transmission and the other to a showing
absorbance [also commonly known as optical density). The 100
percent transmission always corresponds to zero absorbance and a on
absorbance scale corresponds to zero transmission.

CENTRIFUGE
 Centrifuge is a device used for separation of particulate
matter (having different densities) from a liquid medium
by spinning it at high speed. As a result the centrifugal
force of spinning pushes the solid particles of higher
density outwards' which settle at the bottom of the
centrifuge tube and form a pellet.
 Priciple-Normally a particulate material suspended in the
liquid phase settles down under a force of gravitation (G).
If particles are extremely minute the time required for
sedimentation is very high, in order to speed up the
sedimentation, the G is artificially increased by
centrifugation.

 > The speed of centrifuge is normally measured in revolutions per minute
(rpm) but this is not very standardized, because the centrifugal force that is
created by spinning depends on the Size of the centrifuge head. The relative
centrifugal force' (RCF) which is measured in gravity G (dynes/cm) can be
expressed as:
 RCF (G) =r×(rpm)2 ×118×10-7 where
r= radius in centimeters (between bucket and the axis of rotation) andWhere,
rpm = revolutions per minute.
Other factors which influence the rate of sedimentation are the shape, size, and
the density of the particles, as well as the viscosity and density of the liquid.

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