Chapter 1 1

INTRODUCTION AND STUDY

OF DIFFERENT EQUIPMENTS
AND PROCESSING
LAMINAR FLOW (LAF) 2
 Microbiology studies should be performed in an aseptic room
 One of the equipment/instrument to keep the area aseptic is LAF.
 The air supplied to clean room must be filtered through High Efficiency Particulate Air (HEPA)
filters. The HEPA filter must be positioned at the inlet to the clean room and pre-filter may be
fitted upstream of the HEPA filter to prolong the life of the final filter.
 Laminar air flow equipment can deliver clean air in a vertical, horizontal or curvilinear direction.
 Room air is sucked into the equipment and passes through a pre-filter which removes large sized
air contaminants. This prefiltered air passes through a second filter system in the laminar air flow
unit called a High Efficiency Particulate Air (HEPA) filter.
 The air emitted from laminar air flow equipment is 99.97% free of contaminants. Removes
particles of size 0.3µm and larger. Air velocity at all parts of the filter should be 90 ± 20 feet per
minute (0.54m/sec)
 Dioctylphalate is a volatile liquid which converts to vapor or smoke, under pressure having a size
range 0.3 to 0.4µm (challenge test)
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ASEPTIC HOOD 5
 It is usually same like the laminar air flow bench.
 Hood designed with laminar air flow system is called as Aseptic hood.
INCUBATOR

6
An Incubator is similar to an oven in construction and consists of an insulated cabinet fitted with heating element.
 The temperature of the incubator is maintained at the desired level by a thermostat.
 It is ventilated by perforated shelves.
 Used for maintaining a constant temperature for growth of various micro-organisms.
 It should not be overloaded.
AUTOCLAVE

7
Steam under pressure can be effectively utilized for sterilization by using special apparatus called
Autoclave.
Principle:
 Water boils when its vapour pressure (VP) is equal to surrounding atm.
 When VP inside closed system ↑, temp at which water boils also ↑
 Saturated steam has greater penetration power.
 Steam comes in contact with cooler surface, it condenses to water and gives heat to the surface.
 This condensed water ensures moist conditions for killing the microbes present in the sample.
Table. Temperature and time for Autoclave
Pressure (lb/sq.inch) Temperature (oC) Holding Time (min)
10 115.5 30
15 121.5 20
20 126.5 15
Procedure: 8
 Object packed- material which will not create any obstacle for steam penetration and removal of air.
 Materials should be packed in tubes, bottles and flasks closed with cotton stoppers or loosely applied
screw caps.
 Place sufficient water into required level in chamber and place materials on the perforated tray.
Close the door and seal it completely.
 Open the manual discharge tap and the heating starts.
 Safety valve adjusted to required pressure. The steam air mixture is allowed to escape freely till all
the air has been displaced. Holding time indicated in table.
 When the holding time is over, heater turned off and allowed to cool till the pressure inside equals
the atmospheric pressure. Discharge tap opened and finally the autoclave is unloaded.
Advantages:
9
 Great penetrating power. Thus, micro-organisms destroyed more efficiently than dry heat.
 This method is applicable for the wide variety of materials and for a large load.
 Spores are easily destroyed by moist heat sterilization.
Disadvantages:
 During autoclaving the pH of an aqueous solution gets changed due to water loss.
 Oils do not get sterilize . Being hydrophobic in nature they do not allow steam to penetrate them
 Autoclave sterilization is not used for thermolabile sub., powders and plastic that melts.
Applications:
 To sterilize aqueous solution, saline solution, bacteriological media like nutrient broth.
 To sterilize surgical dressings and instrument, rubber gloves and different glassware (pipette,
petriplate, flask, metal instruments)
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HOT AIR STERILIZER 11
 It is an equipment used for dry heat sterilization by hot air under atmospheric pressure.

Table. Temp-time relationship for hot air oven

Temperature (ºC) Time (hrs)
140 3
150 2.5
160 2
170 1
180 0.5

Applications:
 Used to sterilize glassware, forceps, all-glass syringes, petri dishes, testtubes, pipettes etc.
 Used for sterilization of fixed oil, powders, glycerin, waxes, liquid paraffin and other articles that are
spoiled or not effectively sterilized by the moist heat.
Construction and principle: 12
 Consists of double walled chamber of Al or stainless steel separated from the outer case by
thick layer of insulation.
 Heating is affected by electrical heating elements and thermostat automatically controls
temp.
 Sterilization is accompanied by dry heat or hot air.
 Dry heat is less effective as compared to moist heat (because of better penetrating power).
 Microbes are destroyed by the process of protein denaturation and oxidation.
 Operation- For normal sterilization work-
Temp- 160ºC
Time- 2 hrs.
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REFRIGERATOR 14
 It is a basic requirement in a microbiology laboratory. It is used for storing stock cultures of micro-
organisms at 4ºC.
 It is also used to store sterile media to prevent dehydration and to serve as a repository for thermolabile
solution, serums, and biochemical reagents.
 It is divided into two compartments:
a) Freezing compartment (-10 to -20ºC) DEEP FREEZER
Used for keeping subs. in frozen state (eg- Sera, antibiotics)
b) Cooling compartment (4 to 8ºC)
Used for storing micro-organisms.
MICROSCOPE
1. Compound Microscope 15
A microscope may be defined as an optical instrument consisting of a lens or a combination of lens, for making
enlarged or magnified image of minute objects.
Depending on the number of lenses, microscopes are classified into two types such as simple microscope and
depending on the number of eyepiece, microscopes are classified into two types as monocular microscope and binocular
microscope, microscopes can be classified as light microscope and electron microscope, depending upon source of
illumination.
a) Depending on number of lenses microscope:
1) Simple microscope
2) Compound microscope
b) Depending on source of illumination microscope:
1) Light Microscope –
i) Bright-field Microscope
ii) Fluorescence Microscope
iii)Phase-contrast Microscope
2) Electron Microscope –
i) Transmission Electron Microscope
ii) Scanning Electron Microscope
 The compound microscope consists of three major systems: 16
1) Support system
2) Illumination system
3) Magnification system
 Parts of Compound Microscope:
Oculars: A series of lenses that magnify the object and correct some of the defects of the objective.
Objectives: It is the most important lens on a microscope because its properties make the final image.
It consists of low power, high power and oil immersion with magnification of 10x, 45x, 100x.
Three types- achromatic, fluorite and apochromatic.
Condenser: Found directly under the stage and contains two set of lenses that collects and concentrates light
passing upward from the light source into the lens system.
Iris Diaphragm: It controls the intensity of light entering the condenser. Adjusts the light intensity.
Illumination: the light source is positioned in the base of the instrument.
Body Tube: Above the stage and attached to the arm of the microscope is the body tube. Upper end contains
eyepiece lens and lower portion contains movable nosepiece with objective lens.
Revolving Nose Piece: A base in which the objectives are fixed.
Focus Adjustable Knobs: There are two focus adjustment knobs, a coarse adjustment (brings object into
focus) and a fine adjustment (fine and clear focus of specimen).
Mechanical Stage: It is a platform on which the specimen to be viewed is placed. Contains clips to hold the glass.
  Important terms in Microscopy:
1. Magnification:
17
 The ability of microscope to enlarge an image is called the magnification of the instrument.
Denoted by X.

The total magnification of

compound microscope
Magnification Total magnification
Ocular lens/ eye piece Objective lens
5x 10x
50x
10x 45x 450x
15x 40x 600x
10x 100x 1000x

2. Resolving Power:
 It is the ability to reveal closely adjacent points as separate and distinct. Formula- d=λ/2*N.A.

Where, d= limit of resolution; λ= wavelength of light; N.A.= Numerical aperture.

3. Working distance: 18
It is defined as the distance between the front
lens of the objective and the object on the slide.