1. Hot Air Oven for Sterilization:

It is used for sterilization of glassware’s, such as test tubes, pipettes and petri dishes. Such dry sterilization is done only for glassware’s. Liquid substances, such as prepared media and saline solutions cannot be sterilized in oven, as they lose water due to evaporation.

 

 

The glassware’s are sterilized at 180°C for 3 hours. An oven (Figure 3.2) has a thermostat-control, using which the required constant temperature can be obtained by trial and error. The thermostat dial reading is approximate and the exact temperature is read by introducing a thermometer into the oven or on a built-in L-shaped thermometer.

In a modern oven (Figure 3.3), there is a digital temperature display and automatic temperature controller to set the desired temperature easily. Time is set by a digital timer. After loading the glassware’s, the door is closed and oven switched on.

The required temperature is set. After the oven attains the set temperature, the required time of sterilization is set on the timer. The oven switches off automatically after the set time. The oven is opened, only after its temperature comes down near to room temperature. Otherwise, if the door is opened, while the inside of the oven is still very hot, cold air may rush in and crack the glassware’s.

2. Drying Oven:

For preparation of certain reagents, the glassware’s, after proper cleaning and rinsing with distilled water, are required to be dried. They are dried inside the drying oven at 100°C till the glassware’s dry up completely.

3. Autoclave:

Autoclave is the nucleus of a microbiology laboratory. It is used not only to sterilize liquid substances such as prepared media and saline (diluents) solutions, but also to sterilize glassware’s, when required.

It has the same working principle as a domestic pressure cooker. The maximum temperature that can be obtained by boiling water in an open container is 100°C (boiling point of water).

This temperature is sufficient to kill only the non-spore formers, but it is difficult to kill the spore-forming bacteria at this temperature, as they escape by forming heat resistant spores. It takes very long time to kill the spores at this temperature.

On the other hand, when water is boiled in a closed container, due to increased pressure inside it, the boiling point elevates and steam temperature much beyond 100°C can be obtained. This high temperature is required to kill all the bacteria including the heat resistant spore-formers. Steam temperature increases with increase in steam pressure (Table 3.1).

Table 3.1: Temperatures attainable at different steam pressures:

 

In operating a standard vertical autoclave, (Figure 3.4) sufficient water is poured into it. If water is too less, the bottom of the autoclave gets dried during heating and further heating damages it.

 

If it has in-built water heating element, (Figure 3.5) water level should be maintained above the element. On the other hand, if there is too much water, it takes long time to reach the required temperature.

The materials to be sterilized are covered with craft paper and arranged on an aluminium or wooden frame kept on the bottom of the autoclave, otherwise if the materials remain half-submerged or floating, they tumble during boiling and water may enter. The autoclave is closed perfectly airtight only keeping the steam release valve open.

Then, it is heated over flame or by the in-built heating element. Air inside the autoclave should be allowed to escape completely through this valve. When water vapour is seen to escape through the valve, it is closed.

Temperature and pressure inside goes on increasing. The pressure increase is observed on the pressure dial. Usually sterilization is done at 121 °C (a pressure of 15 pounds per square inch i.e. 15 psi) for 15 minutes. The required time is considered from the point, when the required temperature-pressure is attained.

Once required temperature-pressure is attained, it is maintained by controlling the heating source. After the specified time (15 minutes), heating is discontinued and steam release valve slightly opened. If fully opened immediately, due to sudden fall in pressure, liquids may spill out from the containers.

Gradually, the steam release is opened more and more, so as to allow all steam to escape. The autoclave is opened only after the pressure drops back to normal atmospheric pressure (0 psi). The autoclave should never be opened, when there is still pressure inside. The hot sterilized materials are removed by holding them with a piece of clean cloth or asbestos- coated hand gloves.

In case of a steam-jacketed horizontal autoclave, a boiler produces the steam (Figure 3.6). It is released at a designated pressure, into the outer chamber (jacket). Air is allowed to escape and then its steam release valve is closed.

The hot jacket heats the inner chamber, thereby heating the materials to be sterilised. This prevents condensation of steam on the materials. Now, steam under pressure is released from the jacket into the inner chamber and air is allowed to escape from it.

Then, its steam release valve is closed. The steam under pressure in the inner chamber reaches temperatures in excess of 100°C, which can sterilise the materials kept inside it. The autoclave also has automatic shutting system i.e. unless temperature and pressure comes down near to room conditions, the door cannot be opened.

Besides the pressure dial, it also has separate temperature dial to indicate the temperature inside the inner chamber. Moreover, the autoclave maintains the temperature and pressure automatically and switches off after the set time of sterilization.

4. Microbiological Incubator:

Profuse growth of microbes is obtained in the laboratory by growing them at suitable temperatures. This is done by inoculating the desired microbe into a suitable culture medium and then incubating it at the temperature optimum for its growth.

Incubation is done in an incubator (Figure 3.7), which maintains a constant temperature specifically suitable for the growth of a specific microbe. As most of the microbes pathogenic to man grow profusely at body temperature of normal human being (i.e. 37°C), the usual temperature of incubation is 37°C.

The incubator has a thermostat, which maintains a constant temperature, set according to requirement. The temperature reading on the thermostat is approximate. Accurate temperature can be seen on the thermometer fixed on the incubator. Exact temperature, as per requirement, is set by rotating the thermostat knob by trial and error and noting the temperature on the thermometer.

Most of the modern incubators (Figure 3.8) are programmable, which do not need trial and error temperature setting. Here, the operator sets the desired temperature and the required period of time.

The incubator automatically maintains it accordingly. Moisture is supplied by placing a beaker of water in the incubator during the growth period. A moist environment retards the dehydration of the media and thereby, avoids spurious experimental results.

5. BOD Incubator (Low Temperature Incubator):

Some microbes are to be grown at lower temperatures for specific purposes. The BOD low temperature incubator (Figure 3.9), which can maintain temperatures from 50°C to as low as 2-3°C is used for incubation in such cases.

The constant desired temperature is set by rotating the knob of the thermostat. Rotation of the thermostat knob moves a needle on a dial showing approximate temperature. Exact required temperature is obtained, by rotating the knob finely by trial and error and noting the temperature on the thermometer fixed on the incubator.

Most of the modern BOD incubators (Figure 3.10) are programmable, which do not need trial and error temperature setting. Here, the operator sets the desired temperature and the required period of time. The incubator automatically maintains it accordingly.

6. Fridge (Refrigerator):

It serves as a repository for thermo labile chemicals, solutions, antibiotics, serums and biochemical reagents at cooler temperatures and even at sub-zero temperatures (at less than 0°C). Stock cultures of bacteria are also stored in it between sub-culturing periods. It is also used for the storage of sterilized media, so as to prevent their dehydration.

7. Deep-fridge:

It is used to store chemicals and preserve samples at very low sub-zero temperatures.

8. Electronic Top-pan Balance:

It is used for weighing large quantities of media and other chemicals, where precise weighing is not of much importance.

9. Electronic Analytical Balance:

It is used to weigh small quantities of chemicals and samples precisely and quickly.

10. Double-pan Analytical Balance:

It is used to weigh chemicals and samples precisely. Weighing takes more time, for which it is used in emergency only.

11. Distilled Water Plant:

Water is used in the preparation of media and reagents. If the media are prepared using tap water, the chemical impurities present in it may interfere with the growth of the microorganisms in the media. Moreover, the higher is the bacteria content of the media, the longer is the time required for their sterilization and greater is the chance of survival of some bacteria.

Distilled water, though not bacteria- free, contains less number of bacteria. That is why; it is preferred in the preparation of microbiological media. It is also used in the preparation of reagents, because the chemical impurities present in tap water may interfere with the proper functioning of the reagent chemicals.

As manufacture of distilled water by Liebig condenser is a time-taking process, in most laboratories, it is prepared by ‘distilled water plants’. Usually a distilled water plant is made of steel or brass. It is also called distilled water still.

It has one inlet to be connected to the water tap and two outlets, one for distilled water to drop into a container and the other for the flow out of hot cooling water into the sink. The still is installed on the wall. It is heated by in-built electric heating elements (immersion heater).

The still works efficiently, when the water in-flow is so adjusted that, the temperature of the cooling water flowing out from the still into the sink is neither too high nor too low i.e., warm water should flow out. The distilled water may contain traces of metals corroded from the steel or brass container.

To get metal-free distilled water, glass distillation apparatus is used and still better is quartz distillation apparatus. However, for a microbiology laboratory, a steel or glass distillation apparatus is sufficient. For precision analyses, double- or triple- distilled water is used.

12. Ultrapure Water Purification System:

For precision analytical works, now-a-days, instead of using double- or triple-distilled water, micro- filtered water is used. In case of distilled water, there is chance that, few volatile substances present in the water get volatilized during heating of the water and subsequently get condensed into the distilled water collected.

Thus, there may be traces of such substances in the distilled water. To overcome this, ultrapure water is used. Here, water is allowed to pass through very fine microscopic pores, which retain the microscopic suspended particle including the microbes.

Then, the water passes through two columns of ion exchange resins. The anion exchange resin adsorbs the captions present in the water, while the caption exchange resin adsorbs the anions. The water that comes out is extremely pure.

13. Homogeniser:

For microbiological analysis, liquid samples are directly used, whereas solid samples have to be mixed thoroughly with a diluents (usually physiological saline), so as to get a homogenous suspension of bacteria. This suspension is assumed to contain bacteria homogenously.

The mixing of solid samples and diluents is done by a homogenizer, in which a motor rotates an impeller with sharp blades at high speed inside the closed homogenizer cup containing the sample and the diluents. It has a speed regulator for controlling the speed of rotation of the impeller.

In some laboratories mixing is done manually by sterilized pestle and mortar. In modern laboratories, a disposable bag is used, inside which the solid sample and liquid diluents are put aseptically and mixed mechanically by peristaltic action of a machine on the bag. This machine is called stomacher.

14. pH Meter:

A pH meter is an instrument for determining the pH of liquid media, liquid samples and buffers. It has a glass pH electrode. When not in use, it should be kept half immersed in water contained in a small beaker and preferably be covered by a bell jar to avoid dust accumulation in the water and loss of water through evaporation.

Before use, the meter is calibrated using two standard buffers of known pH. Usually buffers of pH 4.0, 7.0 and 9.2 are available commercially. The instrument is switched on and left for 30 minutes to warm up. The temperature calibration knob is rotated to the temperature of the solutions whose pH is to the measured.

Then, the electrode is dipped into the buffer (pH 7.0). If the reading is not 7.00, the pH calibration knob is rotated till the reading is 7.00. Then, the electrode is dipped in another buffer (pH 4.0 or 9.2).

If the reading is same as the pH of the buffer used, the instrument is working properly. Otherwise, the electrode is activated by dipping in 0.1 N HC1 for 24 hours. After calibration, the pH of samples is determined by dipping the electrode into them and noting the reading.

Every time, before dipping into any solution, the electrode should be rinsed with distilled water. The samples should not contain any suspended sticky materials, which may form a coating on the tip of the electrode and reduce its sensitivity.

The old model pH meters have double electrodes (one of them acting as reference electrode), while new models have single combined electrode. Moreover, to overcome the problem of temperature correction, now pH meters with automatic temperature correction are available.

Here, another ‘temperature electrode’ is also put into the solution along with the pH electrode, which measures the temperature of the solution and automatically corrects the influence of temperature variations.

Sophisticated pH meters have single gel electrode. Such electrodes have very little chance of breakage, as they are almost completely enclosed in a hard plastic casing except at the tip. The tip has both pH and temperature sensors.

Moreover, they are easy to maintain, as they do not require constant dipping in distilled water, because the electrode tip is closed with a plastic cap containing saturated solution of potassium chloride, when not in use. However, in preparation of microbiological media, pH is determined by narrow-range pH papers and is adjusted to the required pH by adding acids or alkalis as required.

15. Hot Plate:

Hot plate is used to heat chemicals and reagents. The hot plate is made of an iron plate, which gets heated by an electric heating element from below. The required degree of heating is obtained by a regulator.

16. Shaking Water Bath:

Sometimes, heating at very precise temperatures is required. Such precise temperatures cannot be obtained in an incubator or oven, in which temperature fluctuates, though slightly. However, precise temperatures can be maintained in a water bath, which provides a stable temperature.

A water bath consists of a container containing water, which is heated by electric heating elements. The required water temperature is obtained by increasing or decreasing the rate of heating by rotating the thermostat by trial and error.

In a shaking water bath, the substance is heated at the required temperature and at the same time, it is shaken constantly. Shaking is done by a motor, which rotates and moves the containers to and fro in each rotation. The rate of shaking is again controlled by a regulator. Shaking agitates the substance and enhances the rate of the process.

Most modern water baths are programmable and do not need trial and error temperature setting. A desired water temperature can be maintained over a desired period of time by programming accordingly. It is used for cultivation of bacteria in broth medium at a specific temperature.

17. Quebec Colony Counter:

In enumeration of bacteria in samples, it is assumed that a single bacterium gives rise to a single visible colony, when grown on a plate of solidified nutrient medium. Thus, by counting the number of colonies, the number of bacteria in a sample can be estimated.

Sometimes, colonies are very small and too much crowded making it difficult to count. Counting becomes easy, when a mechanical hand counter, called Quebec colony counter (Figure 3.11), is used. It divides the plate into several square divisions and the colonies are magnified 1.5 times by a magnifying glass, which makes counting easy.

18. Electronic Colony Counter:

Electronic colony counter is of two types:

(1) Hand-held electronic colony counter and

(2) Table-top electronic colony counter.

The hand-held electronic colony counter is a pen-style colony counter with an inking felt-tip marker. For counting of colonies of bacteria grown in a petri dish, it is kept in an inverted position, so that the colonies are visible through the bottom surface of the petri dish.

The colonies are marked by touching the glass surface of the petri dish with the felt-tip of the colony counter. Thus, each colony is marked by a dot made by the ink of the felt-tip on the bottom surface of the petri dish. In a single motion, the electronic colony counter marks, counts and confirms with a beep sound.

The cumulative count of colonies is displayed on a four-digit LED display. In case of table-top electronic colony counter, the petri dish containing the colonies of bacteria is placed on an illuminated stage and the count bar is depressed. The precise number of colonies is instantly displayed on a digital read out.

19. Magnetic Stirrer:

In the preparation of solutions, certain chemicals require stirring for long time, to be dissolved in certain solvents. Magnetic stirrer is used to dissolve such substances easily and quickly. A small teflon- coated magnet, called ‘stirring bar’, is put into a container containing the solvent and the solute.

Then, the container is placed on the platform of the magnetic stirrer, below which a magnet rotates at high speed by a motor. Attracted by the rotating magnet, the teflon-coated magnet rotates inside the container and stirs the contents. Now, the solute dissolves quickly.

The teflon coating prevents the magnet from reacting with the solution, which comes in contact with it. After complete dissolution, the teflon-coated magnet is removed from the solution by mean of a long retriever, called ‘stirring bar retriever’.

20. Sonicator:

It is used to rupture cells using high frequency waves.

21. Vortex Mixer:

It is an instrument used for thorough mixing of liquids in test tubes. It has a rotor, whose speed can be controlled. On the tip of the rotor is a foam-rubber top. When the bottom of a test tube is pressed upon this foam-rubber top, the rotor starts rotating, thereby rotating the bottom of the test tube at high speed.

Due to centripetal force, the solution gets mixed thoroughly. It is particularly helpful during serial dilution in enumeration of bacteria, which needs homogenous suspension of bacteria cells.

21. Laminar Flow Chamber:

It is a chamber (Figure 3.12) used for aseptic transfer of sterilized materials, as well as for inoculation of microbes. Dust particles floating in the air harbour microbes. These microbe-laden dust particles may enter into the sterilized media and contaminate them, when they are opened for short periods of time during inoculation of microbe or transfer from one container to another.

To overcome this, when inoculation is done in open air, the air of the small inoculating area is sterilized by the flame of a bunsen burner. The heated air becomes light and moves upwards, thereby preventing the dust particles from falling on the media during the short opening process.

To further reduce the chance of contamination by the microbe-laden air, a laminar flow chamber is used. It is a glass-fitted cuboidal chamber. An air blower blows air from the surrounding and passes it through a HEPA filter (High Efficiency Particulate Air filter), so as to make it dust free (microbe-free).

This microbe-free air passes through the chamber in a laminar manner and comes out from the chamber through the open front door. This laminar flow of microbe-free air from the chamber to outside through the open door prevents the outside air from entering into the chamber.

Thus, the chamber does not get contaminated with the microbes present in the outside air, though the door is kept opened during inoculation or transfer of media. An UV lamp fitted inside the chamber sterilizes the chamber before operation.

It has a stainless steel platform with provision for gas pipe connection for a bunsen burner. Before use, the platform is cleaned and disinfected with lysol, the bunsen burner is connected and then the glass door is closed.

The UV light is switched on for 10 minutes to sterilise the environment inside the chamber and then switched off. The glass door should never be opened when the UV light is on, because UV light has detrimental effect on skin and vision. The blower is switched on and then the glass door is opened.

Now, the bunsen burner is lighted and media transfer or inoculation is carried out in the chamber aseptically. If extremely hazardous microbes are to be handled, a laminar flow chamber with gloves projecting into the chamber from the front glass door is used, as inoculation has to be done keeping the front door closed.

22. Electronic Cell Counter:

It is used to directly count the number of bacteria in a given liquid sample. An example of electronic cell counter is the ‘Coulter counter’. In this equipment, a suspension of bacteria cells is allowed to pass through a minute orifice, across which an electric current flows.

The resistance at the orifice is electronically recorded. When a cell passes through the orifice, being non-conductor, it increases resistance momentarily. The number of times resistance increases momentarily is recorded electronically, which indicates the number of bacteria present in the liquid sample.

23. Membrane Filtration Apparatus:

Certain substances like urea disintegrate and lose their original properties, if sterilized by heat. Such substances are sterilized by membrane filtration apparatus. In this apparatus, the solution of the substance to be sterilized is filtered through a membrane filter, which does not allow bacteria cells to pass down. Filtration is done under suction pressure to increase the rate of filtration (Figure 2.19, page 30).

24. Microscopes:

Different types of microscopes are used for visual observation of morphology, motility, staining and fluorescent reactions of bacteria.

25. Computers:

Computers are generally used for analysis of results. They are also used for identification of bacteria easily within few hours. Otherwise, identification of bacteria is a tedious process and takes days together to identify one bacteria species.

The computers used for identification of bacteria are Apple II, IBM PC and TRS-80 and their modern variants. Each research personnel of the laboratory should be provided with a computer, along with internet facility.

26. Spectrophotometer:

It is an instrument for measuring the differences in color intensities of solutions. A beam of light of a particular wavelength is passed through the test solution and the amount of light absorbed (or transmitted) is measured electronically.

A simple visible spectrophotometer can pass light with wavelengths within visible range, whereas a UV-cum-visible spectrophotometer can pass light with wavelengths in ultraviolet as well as in visible range. In microbiology lab, it is used for direct counting of bacteria in suspension as well as for other purposes.

27. Electrical Devices:

A fluctuation of electric voltage in the laboratory is one of the most important reasons, which reduces the longevity of the equipments and sometimes damage them. Therefore, all the voltage-sensitive equipments should be provided with voltage protection devices like stabilizers, servo stabilizers or constant voltage transformers (CVT) as per the recommendations of the manufacturers of the equipments.

Computers, balances and some sophisticated equipments should be connected through uninterrupted power supply (UPS), as any breakdown in the electric power supply during their operation may severely damage some of their sensitive components.

The laboratory should have a high capacity generator to supply electric current to the whole laboratory in case of power failure. This is because, power failure not only brings the activities of the laboratory to a standstill, it also brings about undesirable irreversible changes in the samples stored in the deep-fridges and refrigerators.

28. Automatic Bacteria Identification System:

It is an instrument used for automatic computer-assisted identification of bacteria (Figures 3.13 and 3.14). The conventional method of identification of bacteria is very lengthy and cumbersome.

It mainly involves staining, motility test, cultural characteristics, a series of biochemical tests and finally searching the name of the bacteria in ‘Bergey’s Manual of Determinative Bacteriology’ by matching the results with those available in the manual. The automatic bacteria identification system automatically identifies the bacteria in very short time.

The system, like VITEK 2 (Figure 3.14) uses disposable cards. One card is required for the identification of one bacteria. The system can accommodate a series of cards, which can be arranged on a cassette, thus enabling the identification of several bacteria at a time.

Each card has several rows of wells. Usually there are 8 rows of 8 wells each (8X8 =64 wells). The wells contain different dehydrated media required for different biochemical tests. A capillary tube is fixed to each card, which sucks the suspension of bacteria to be identified and dispenses into all the wells.

The dehydrated media in the wells become hydrated by the suspension liquid, thereby allowing growth of the bacteria. After a prescribed period of incubation, the colour changes in all the wells are recorded automatically in the system.

The results of the color changes go to a computer attached to the system. The computer automatically compares the results with those available in its library for different bacteria and finally gives the name of the bacteria with a definite probability.

For identification, the given bacteria, grown as isolated colony on a plate or as pure culture grown on a slant are taken. A lapful of the bacteria is transferred aseptically into sterile saline solution in a test tube and a suspension of the bacteria is made.

The suspension should contain a prescribed density of bacteria, as determined by a densitometer. The test tube is fixed to the cassette and a card is fixed near it, such that the tip of the suction capillary tube of the card remains deeply submerged in the suspension.

Several such test tubes and cards are fixed to each cassette, depending on the number of bacteria to be identified. The cassette is put in the vacuum chamber of the system. A high vacuum is created inside the chamber, which forces the bacteria suspension to be sucked into the capillary tubes and dispensed into the wells of the cards.

The cassette is taken out and put inside the incubation and analysis chamber. Here, the capillary tubes are cut and the cut ends sealed automatically. Then, the incubation process starts at a prescribed temperature for a prescribed period of time, which is programmed by the control panel. During incubation, in every 15 minutes, each card automatically goes to the color reader, which reads the color changes in the wells and records them.

The recorded results go to the computer, which automatically compares them with those, available in its library for different bacteria. Finally, it gives the names of the bacteria with definite probabilities. The used cards fall into the waste disposal chamber of the system for removal and final disposal after sterilization.

The renowned automatic bacteria identification systems are VITEK 2 and API. While VITEK 2 works on the above principle, the API (Analytical Profile Indexing) system (Figure 3.13) uses a slightly different method for the automatic identification of bacteria, which involves manual inoculation and external incubation.

29. PCR Thermocycler, Refrigerated Centrifuge, Ultra-centrifuge, Gas Chromatography (GC), High Performance Liquid Chromatography (HPLC), Thin Layer Chromatography (TLC), Paper Chromatography, Column Chromatography and Electrophoresis Unit:

These are instruments used for isolation, purification and identification of biochemical substances, such as bacterial DNA, plasmids, microbial toxins etc. Polymerase chain reaction (PCR) is an important tool in nucleic acid based methods. It is a workhorse in modern microbiology and biotechnology laboratories.