STUDY OF DIFFERENT TYPES OF EQUIPMENT AND PROCESSING USED IN EXPERIMENTAL MICROBIOLOGY.

S.Y B.PHARM SEMESTER III

PHARMACEUTICAL MICROBIOLOGY PRACTICAL

EXPERIMENT NO. 1

 STUDY OF DIFFERENT TYPES OF EQUIPMENT AND PROCESSING USED IN EXPERIMENTAL MICROBIOLOGY.

AIM:  STUDY OF DIFFERENT TYPES OF EQUIPMENT AND PROCESSING USED IN EXPERIMENTAL MICROBIOLOGY.

REFERENCE: 

1.       Pharmaceutical Microbiology (Experiments & Techniques) by Chandrakant Kokare published by Career Publication fourth edition Page No. 19 – 35

2.      Practical pharmaceutical microbiology book by Prof Md. Rageeb, K.D Baviskar, N.G Patil, Published by S. Vikas And Company (Medical Publisher), Edition 2018, Page No 13, 14.

List of equipment’s:

     1.       Microscope

     2.      Hot air oven

     3.      Autoclave

     4.      Incubator

     5.      Refrigerator

     6.      Aseptic hood

     7.      Laminar air flow

1.     Microscope:

Definition: A microscope may be defined as an optical instrument, consisting of a lens or a combination of lenses, for making enlarged or magnified images of minute objects.

Classification:

Depending on the number of lenses, microscopes are classified into two-types such as simple microscope and compound microscope and

Depending on number of eyepiece, microscopes are classified into two types as monocular microscope (single eyepiece) and binocular microscope (two eyepiece).

Depending on source of illumination microscope may be classified as light microscopes and electron microscope.

The compound microscope consists of three major systems.

1)      Support system: It comprises of base. Stage and body tube.

2)     Illumination system: It throws light on the object for proper viewing. It comprises of light source or mirror; iris diaphragm and condenser. The light source may be a concave or plain mirror or electrically illuminated by a tungsten filament lamp or a halogen lamp. Mirror and electric light source are generally interchangeable.

3)     Magnification system: It includes a set of lenses aligned in such a manner so that a magnified real image can be viewed. The objective is a set of lenses placed near the object. It partially magnifies the object, which can be observed through the eyepiece in a more magnified form.

Parts of compound microscope:

Oculars / Eyepiece: A series of lenses (5x, 6x, 10x, 15x) that magnify the object and corrects some of the defects of the objective. Huygenian, Ramsden and compensating oculars are commonly used in microscopy.

Objectives: The objective is the most important lens on a microscope because its properties make the final image. The objective lenses generally equipped with microscope are low power, high power and oil immersion lens having magnification of 10x, 40x or 45x and 100x, respectively. Functions of the objective lens are to gather the light rays coming from any point of the object and to unite the light at a point of the image and magnify the image. There are three types of objectives like achromatic, fluorite and apochromatic.

Condenser: This component is found directly under the stage and contains two sets of lenses that collect and concentrate light passing upward from the light source into the lens system. There are several different types of condensers, depending upon the type of microscopy. e.g. Abbe condenser, variable-focus condenser and achromatic condenser.

Iris diaphragm: It is equipped with a condenser. It controls the intensity of light entering the condenser. A lever is equipped with it to adjust the light intensity.

Illumination (light source): The light source is positioned in the base of the instrument some microscopes are equipped with a built-in light source to provide direct illumination. Others are provided with a mirror, with one side flat and the other concave. An external light source, such as a lamp, is placed in front of the mirror to direct the light upward into the lens system. The flat side of the mirror is used for artificial light and the concave side for sunlight.

Body tube: Above the stage and attached to the arm of the microscope is the body tube. The upper end of the tube contains the ocular or eyepiece lens. The lower portion consists of a movable nose piece containing the objective lenses. It also provides sufficient space for image formation.

Revolving nose piece: A base in which the objectives are fixed and it holds 2 to 4 objectives and which can be revolved to align the required objective.

Focus adjustment knobs: There are two focus adjustment knobs, a coarse adjustment and a fine adjustment. Coarse adjustment knob is used to bring the object into focus and fine adjustment knob is used for fine and clear focus of specimen.

Mechanical stage: It is a platform on which the specimen to be viewed is placed. Some stages have clips to hold the glass slide. Others have a mechanical stage, which makes it possible to move the slide across the stage.

2.   Hot Air Oven:

A hot air oven is equipment used for dry heat sterilization by hot air under atmospheric pressure.

Construction and Principle: The hot air ovens consist of a double-walled chamber of aluminum or stainless steel separated from the outer case by a thick layer of insulation made of fiber glass. Insulation is also filled in the hollow flanged door, which carries an asbestos jacket that provides 4 tight seal. Heating is affected by electrical heating elements and thermostat automatically controlled temperature.

An oven is based on the principle where sterilization is accomplished by dry heat or hot air. Dry heat is less effective as compared to moist heat because in presence of moisture, proteins are easily coagulated and moist heat has greater penetrating power than dry heat. By the process of protein denaturation and oxidation, microbes are destroyed by dry heat. For normal sterilization work, ‘ne oven should be operated at 160°C for 2 hours.

A time temperature relationship:

Temperature (0C)	Time (hrs)
140	3
150	2.5
160	2
170	1
180	0.5

Applications: Hot air oven is used to sterilize glassware, forceps, scissors, scalpels, all-glass syringes, Cotton swabs, Petri dishes, test tubes, pipettes etc.

Hot air oven is also used for sterilizations of fixed oils, powders (zinc oxide, talc), glycerin, liquid paraffin, propylene glycol, waxes and other articles that are either spoiled or not effectively sterilized by the moist heat.

3.   Autoclave:

Steam under pressure can be efficiently utilized for sterilization by using special apparatus called ‘Autoclave’. Autoclaves are available in different types, simple non-jacketed laboratory autoclave (horizontal and vertical) and steam jacketed autoclave with automatic air and condense discharge.

Construction: The autoclave is a double walled cylindrical metallic vessel, made of thick stainless steel or copper. The autoclave lid is provided with a pressure gauge for noting the pressure and steam cock for air exhaustion of the chamber. The autoclave is provided with control for adjusting the pressure and temperature, and a safety valve to avoid explosions. The articles to be sterilized are kept loosely in a basket, provided with holes for the free circulation of the steam.

Principle: The principle of the autoclave or steam sterilizer is that water boils when its vapor pressure equals that of the surrounding atmosphere. Hence when pressure inside a closed vessel increases, the temperature at which water boils also increases. Saturated steam has greater penetration power. When-steam comes into contact with a cooler surface it condenses to water and gives up its latent heat to that surface. The condensed water ensures moist conditions for killing the microbes present.

All microbial cells are destroyed by denaturation process and coagulation of proteins.

The Temperature pressure relationship of Autoclave:

Pressure (lb/sq.inch)	Temp (0C)	Holding Times (Minutes)
10	115.5	30
15	121.5	20
20	126.5	15

Procedure:  The object to be sterilized should be packed in a material which will not create any obstacle to steam penetration and removal of air. Materials can be packed in tubes, bottle closed with cotton Stoppers, flask plugged with cotton or loosely applied screw caps. Wrapping cloth or plastic or kraft paper may be used for wrapping of instruments.

Place sufficient water up to the required level in the chamber and place the materials on the perforated tray just above it. Close the door to seal it completely. Open the manual discharge tap and the heating starts. The safety valve is adjusted to the required pressure. The steam-air mixture is allowed to escape freely till all the air has been displaced. Close the discharge tap and wait till the pressure is reached to the required level. The holding times needed for sterilizations are indicated in given above Table in terms of the temperatures and pressures. When the holding period is over, the heater is turned off and the autoclave allowed to cool till the pressure gauge indicates that the pressure inside is equal to atmospheric pressure. The discharge tap is opened slowly and the air allowed to enter the autoclave, finally the autoclave is opened and unloaded.

Advantages:

1.       Because of greater penetrating power of the steam under pressure, microorganisms are destroyed more efficiently than the dry heat. |

2.      The method is applicable for a wide variety of materials and for a large load.

3.      Spores are easily destroyed by moist heat sterilsation.

Disadvantages:

1.       During autoclaving the pH of a aqueous solutions gets changed due to water loss.

2.      Oils do not get sterilsed in the autoclave as they are hydrophobic in nature and they do not allow the steam to penetrate them.

3.      Autoclave sterilizations is not used for thermo labile substances, powders and plastics that melt.

Applications:

1.       To sterilize aqueous solutions, saline solutions, bacteriological media like nutrient broth, Sabouraud's agar.

2.      To sterilize surgical dressings, rubber gloves, plastic fabrics and surgical instruments.

3.      Sterilize different glassware like pipettes, Petri plates, flasks and different metal instruments.

4.   Incubator:

An incubator is similar to an oven in construction and consists of an insulated cabinet fitted with a heating element. The temperature of the incubator (Plate 2.5) is maintained at the desired level by a thermostat. The incubator is properly ventilated by the perforated shelves. They are provided with double doors, the inner one made of glass so that the contents of the incubator, may be viewed without admitting outside air.

Application: An incubator is used for maintaining a constant temperature suitable for growth of various microorganisms.

5.    Refrigerator:

The refrigerator, a basic requirement in a microbiology laboratory, is used for storing stock cultures of microorganisms at 4°C. When cultures are stored at low temperatures, microorganisms are fairly inactive and will not suffer damage due to the evaporation of the medium. It is also used to store sterile media to prevent dehydration and to serve as a repository for thermolabile solutions, serums, antibiotic discs and biochemical reagents. Live cultures on a culture medium can be successfully stored in refrigerators or cold rooms. This method is only used for short time preservation of cultures and subculturing is necessary if-the period-exceeds four weeks. The refrigerator is divided into two compartments as the freezing compartment and the cooling compartment. The freezing compartment is used for keeping substances.

6.   Laminar Air Flow (LAF):

A laminar flow cabinet or laminar flow closet or tissue culture hood is a carefully enclosed ' bench which prevents contamination of semiconductor wafers, biological samples, or any particle sensitive materials. Air is drawn through a HEPA filter and blown in a very smooth laminar flow towards the user. The cabinet is usually made of stainless steel no gaps joints where spores might collect. Such hoods exist in both horizontal and vertical configurations, and there are many different types of cabinets with a variety of airflow patterns.

Laminar flow cabinets always equipped with UV light to sterilize the interior and contents before usage to prevent contamination during process. UV lamps are usually kept on for 15 minutes to sterilize the interior and no contact is to be made with a laminar flow hood during this time.

HEPA stands for high-efficiency particulate air. A HEPA filter is a type of mechanical air filter; it works by forcing air through a fine mesh that traps harmful particles such as pollen, pet dander, dust mites, and tobacco smoke. Its efficiency is rated at least 99.97% efficient on particles of 0.3 microns in size.