Laboratory Apparatus
INCUBATORS
Incubators 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.
Construction
Cabinets are made up of non corrosive metals which are lined inside with asbestos to prevent the loss of heat by radiation.The metallic door opens at the front,behind which there is an another glass door provided,so that the experiment may be watched undisturbed from outside without opening it,thus minimizing the loss of radiation.
Cabinet is provided with a temperature control system which is controlled by the regulator knob on sides. Pilot lamp will light when heating continues and will be off when the cabinet is heated to a previously set temperature.Temperature can be read by inserting a thermometer from the hole provided at the top of the cabinet.Incubators have a wide temperature ranges;varying from slightly over ambient temperature to more than 60 ˚C.
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 tem. of incubator is constantly maintained at 37 ˚C,this is due to the fact that; [i] most of the routine bacteria studied are mesophiles,and [ii] the human pathogens grow optimally at this tem. [because the normal human body tem. is also 37 ˚C]
Figure : Bacteriological Incubator
WATERBATH
Water baths are of two tyes – incubator waterbath and boiling waterbath.
INCUBATOR WATERBATH
Incubator Water bath has a constant tem. device which is electrically operated,and is controlled by a thermostat.The tem. is maintained constantly at a desired value with the help of automatic thermostat.
Principle
Water bath maintains better control of the tem. than that of the incubator.It also offers an additional advantage of an agitation and mixing due to the formation of convectional current in a liquid which is being incubates.Due to this reason most serological procedures require an incubation in waterbath rather than in incubator.
Working
- Waterbath is filled with deionized water [to prevent the deposition of mineral salts resent in tab water].Leave of water should be such that the test tubes to be immersed in the waterbath do not get drowned or float u.
- Place thermometer in the waterbath,and check that it always remains immersed in water.
- Switch on the waterbath and raise the tem. by turning knob of thermostat clockwise;the indicator lamp will start to glow.
- Watch the thermometer until tem. reaches the desired value.
- Turn the knob of thermostat counter clockwise till the indicator lam is off.
- Allow the tem. to be maintained for an hour or so before the waterbath is used.adjust the thermostat during this period [if required] as stated earlier.
- Once the tem. is set,do not change the setting of thermostar knob even after use.
Note: As a part of maintenance,keep the waterbath clean,and relace the water frequently when it appears dirty.Check the water level before starting the instumen,and never allow the instrument to dry up.Alsocheck the tem. frequently.
Use
Incubator water-baths are used for serological experiments involving
- Inactivation of complement at 56 ˚C.
- Agglutination test at 55 ˚C,(Widal test).
- Complement-fixation (Wasserman test) and recipitation reaction (Kahn tube test) at 37 ˚C.
pH METER
As a convenient way of expressing hydrogen ion concentration,the symbol pH was introduced by S.P.L.Sorensen in 1990. ‘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 = -log 10 [H+] = log 10 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 solution of different pH value.
Principle
When two solution containing hydrogen ion are separated by the bulb of a glass pH electrode that is hydrogen sensitive,an electrical otential is develoed across the thin glass searating the two solutions.If the solution inside the bulb is of fied 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 has an electrical potential of 0.2415 volts at 25 C (calibrated against the standard hydrogen electrode which has electrode potential of zero).It consists of a mercury electrode covered with mercurial chloride (calomel) and a solution of mercury in saturated otassium chloride.The calomel electode will maintain its specified potential roviding its calomel and mercury content remain intact and its potassium chloride solution is kept saturated.
Figure : pH meter
Working
- Switch on and allow the instrument to warm up;whilst this is taking place,make sure that requisite buffer solutions for calibration of the pH meter are available,and if necessary prepare any required solutios: this is conveniently done by dissolving an appropriate ‘buffer tablet’ (commercially available) in the specified volume of distilled water.
- Set the tem. value to the ambient (or tem. of the solution whose pH is to bemeasured) tem. using thetem. Control knob provided for it.
- Insert the electode in the first standard buffer solution contained in the small beaker,and set the selector knob of the instrument to read pH.
- Adjust the ‘Set Buffer’ control until the pH meter reading agrees with the known pH of the buffer solution.
- Remove the electrode,rinse in distilled water,and place into a small beaker containing thesecond solution buffer solution.If the meter reading does not agree with the known pH,adjust the ‘Slope’ control until the required reading is obtained.
- Remove the electrode,rinse in distilled water,replace in the first buffer solution and confirm that the correct pH reading is shown on the meter: if not,repeat the calibration.
- If the calibration is satisfactory,rinse the electrode in distilled water,and introduce into thetest solution contained in the small beaker.Read of the pH of the solution.
- Remove the electrode,rinse in distilled water,and leave standing in distilled water.
CENTRIFUGE
Centrifuge is a device used for separarion of particulate matter [having different densities] from a liquid medium by spinning it at high speed.As a result the centrifugal force of sinning ushes the solid particlesof higher density ‘outwards’ which settle at the bottom of the centrifuge tube and form a pellet.
Principle
Normally a particulate material suspended in the liquid hase settles down under a force of gravitation [G]. If particles are extremely minute the time required for sedimentation is very high;in order to seed u 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 sinning 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 × [rmp]² × 118 × 10-7
Where,
r = radius in centimeters [between bucket and the ais of rotation] and
rpm = revolutions per minute.
Other factors which influence the rate of sedimentation are the sha,size,and the densityof the particles;as well as the viscosity and density of the liquid.
Construction
Most laboratory centrifuges are angle head type in which the tubes are held in rigid position at a fied angle of 45˚.The machine consist of a steel bucket having the centrifuge head or a rotor which is fied to the shaft.The shaft rotates at very driven by a hidden motor.Electrical centrifuges have a rheostat to regulate the speed and some of them are also eduied with a tachometer which indicates the speed of sinning.Many centrifuges are provided with timer which helps in automatic switching off of the instrument after a specified time period.
The centrifuge has a lid which must be closed during the running of the centrifuge.Centrifuge tubes are made u of glass or plastic with either round bottom or conical endings.Centrifuge tubes are placed in sockets [provided in rotor] which should always contain soft rubber cushions,otherwise tube may break during oeration.
The common laboratory centrifuge develos a maximum field of about 5000 g;while for that of ultracentrifuge is about 500000 g.
Figure : Centrifuge
Working
- Place the centrifuge on the skid free padding so that it does not slide away while running.
- Label centrifuge tube befor filling them with solution.
- Centrifuge must be roerly balanced befor running ,Tubes placed in oosite sockets must be of equal weight.Otherwise instrument will vibrate and tubes may break during operation.
- Balancing is done by taking two similar centrifuge tube and filling it up with the equal volumes of fluid.However,if the fluids are of different densities,matching by volume is not necessary;rather weight them on balance.
- Put balance tubes in oosite sockets having cushions.
- Close the lid and start the motor and gradually increase the seed till desired seed is reached.
- Put on the timer or the alarm clock.
- Stop the centrifuge after it has run for the desired time period,and let the centrifuge slow down and stop gradually.
- Remove tubes slowly and carefully without disturbing the sediment or pellet.
COLONY COUNTER
The instrument is use when large number of colony counts are to be made. It consist of a metal box, the format function of which is cut, and fitted with a circular glass calibrated into different rulings. The Petri dish rests on this glass and is illuminated by a strong light source placed inside the metal cabinate. A simple magnifying lens [5X] is fitted at the top corner which can be adjust vertically as well as radially.
To count colony place the open dish with glass side up, over the illuminated screen. Count colony using the magnifying lens and a hand held counter. Mark the glass above each colony with a felt-tip pen. For large work loads semi- or fully automatic counters are essential. In semiautomatic counters, the pen used to mark the glass above the colonies, is connected to an electronic counter which displays the number counted on a small screen. In the fully automatic models a TV camera or laser bear scans the plate and the result are displayed or recorded on the screen.
Total numbers of colonies or colony forming units per milliliter [ cfu/ml ] can be calculated by multiplying the average number of colonies per countable plate by the reciprocal of the dilution.
Figure : Colony counter
REFRIGERATOR
A refrigerator is necessary in any microbiology laboratory. It is an instrument which maintains the tem. of a material at a lower lever than the tem. of the surrounding environment.
Principle
A refrigerator is used in the laboratory mainly for preservation of cultures and for the storage of perishable materials of biological origin. This is due to the fact that low tem. slows down the biochemical activities of the cell and exerts a microbiostatic effect. Hence the growth of the organism is inhibited.
Construction
For smaller laboratories, household refrigerators are satisfactory, whereas in large laboratories commercial refrigerators or deep-freeze or walk-in cold rooms are provided.
Mechanical refrigerator operates on the reversed heat engine cycle, where the heat is pumped from the lower level to the higher level of the tem. and rejected at the higher level of tem.. The modern refrigerators use various types of coolants like vaors of ammonia, carbon dioxide, sulphur dioxide, freons etc..
Routine refrigerator is vertical and has rectangular cabinet shape. It has arrangement for the compressor-motor-fan, condenser, and receiver at the base. The pipe carrying the liquid refrigerant passes through the body which cools the refrigerator by heat exchange.
The refrigerator is divided into two compartments; the freezing compartment and the cooling compartment. The freezing compartment is used for keeping the substances in the frozen state [sera, cartain antibiotic etc.]. The tem. of the freezing compartment is usually between -15 ˚C to -20 ˚C. The cooling compartment outside the freezing compartment should have the tem. of 4-6 ˚C.
Use
Refrigerator is used in the laboratory for preservation of culture. It is also used for storage of bacteriological media, reagents, blood, antisera, antibiotics, vaccines, and many other heat-labile substances.
SIMPLE STAINING
(MONOCHROME)
INTRODUCTION :
Staining procedures that use only one stain are called simple stains. It is usually a single step process. A simple stain that stains the bacteria is called monochrome stain, and a simple stain that stains the background, but leaves the bacteria unstained is a negative stain. In such simple staining all cells are stained in the similar way, however, differences in the size, shape and the arrangement can be easily visualized. In few cases structural features can also be observed.
MONOCHROME STAINING METHOD :
Principle :
It is believed that the process of staining involves ion-exchange reactions between the staIn and active sites at the surface of or within the cell. For example, the colored ions of the dye (chromophore) ions of the dye may replace other ions on cellular components. Certain chetnical groupings of cell proteins or nucleic acids may be involved in salt formation with positively charged ions such as Na+ or K+.
It it is assumed that the surface of a bacterial cell has an overall acidic characteristic because of large amount of carboxyl groups located on the cell surface due to acidic amino acids. Therefore, when ionization of carboxyl groups takes place it imparts negative charge to the cell surface as per the following equation.
COOH Ionization > COO- + H+
in nature, H+ is replaced by another posjtive charged ion e.g., Na+, or K+, and H+ bonds with oxygen to form water. .
Basic dye are usually salt of acids, e.g., methylene blue; the dye which is actually methylene blue chloride, (it has the positively charged colored ion (a cation), and if this ion is repre sented by the symbol, Mb, may be represented as Mb+CI-). When methylene blue rehydrates,it ionizes as follows.
Mb.CI Ionization > Mb+ + CI-
The ionic exchange taking place during staining may be represented by the following equation, in which the Mb+ cation replaces the Na+ cation in the cell:
Na+ Na+ Na+Mb+ Mb+ Mb+
Na+Na++ (MB+)+(CI):Mb++(Na+)(CI)
Na+ Na+ Na+ Mb+ Mb+ Mb+
Thus, when coloring agent forms ionic bond with cell or cell components, it will resuit into the staining of cell. :
Other basic stains like, crystal violet, safranin, malachite green, gentioh violet, basic fuchsin or carbol fuchsin can also be used for staining bacterial cells which have net negati.ve charge.
Requirements
1. Young cultures of Escherichia coli, Bacillus subtilis & Staphylococcus aureus
2. Methylene blue staining solution.
Procedure
1. Prepare a heat fixed smears of the above cultures.
2. Cover the smear with methylene blue stain for 3-5 minutes.
3. Wash the slide in tap water (without draining the stain), drain, blot and air
dry the smear.
4. Examine under oil-immersion objective.
Results
The monochrome staining shows organisms stained blue in color.
Note :Staining time differs with the type, the concentration, and the composition of the staining solution. For example, crystlal violet requires approximately 30 to 60 seconds for staining; while carbol fuchsin takes approximately 15 to 30 seconds for the similar staining. It also depends on the type of the organism to be stained.
SIMPLE STAINING
(NEGATIVE)
INTRODUCTION
Negative staining is a type of simple staining and is so called because the dye does not stain the cell instead it imparts contrasting background, thereby making the organism visible.
NEGATIVE STAINING METHOD :
Principle
Negative staining use acidic dyes like nigrosine, eosin or Congo red. Acidic dyes have negatively charged chromogen which will not bind (react) with bacterial cells because of the similar acidic (negative) charge on the surface of bacterial cells. The stain does not stain the bacteria because of ionic repulsion. Therefore cells are unstained and can be easily visualized against the contrasting background. Practical applications of negative staining are:
1. Natural shape and size of the cell is not distorted because the heat fixation is not required during the staining.
2. it is possible to .observe those bacteria which are difficult to stain, such as spirochetes.
Requirements
I. Young cultures of Escherichia coli, Bacillus subtilis & Staphylococcus aureus.
2. Nigrosine staining solution (10%).
Procedure :
I. Place a small drop of nigrosine at the end of the slide. For cultures on solid media, add a loopful of distilled water and emulsify a small amount of the culture in the nigrosine-water drop. For broth cultures, mix a loopful of the culture into the drop of nigrosine. Do not spread the drop or let dry.
2. Using the end edge of another slide, spread the drop out to produce a smear varying from opaque black to gray. The angle of spreading slide will determine the thickness of the smear.