MICROBIOLOGY LAB EQUIPMENTS
Hot Air Oven
It is used for the sterilization of glassware’s, such as test tubes, pipettes and Petri dishes. Such dry sterilization is done only for glassware. Liquid substances, such as prepared media and saline solutions cannot be sterilized in the oven, as they lose water due to evaporation. The glassware’s are sterilized at 180°C for 3 hours. An oven 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, 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 the 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 glasswares.
An 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 a 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 an increase in steam pressure In operating a standard vertical autoclave, sufficient water is poured into it. If water is too little, the bottom of the autoclave gets dried during heating and further heating damages it. If it has an in-built water heating element, the water level should be maintained above the element. On the other hand, if there is too much water, it takes a long time to reach the required temperature.
The materials to be sterilized are covered with craft paper and arranged on 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 a flame or by the in-built heating element. The 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 go on increasing. The pressure increase is observed on the pressure dial. Usually, sterilization is done at 121 °C (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 the required temperature pressure is attained, it is maintained by controlling the heating source. After the specified time (15 minutes), heating is discontinued and the steam release valve is slightly opened. If fully opened immediately, due to a 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 the case of a steam-jacketed horizontal autoclave, a boiler produces the steam. 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 the condensation of steam on the materials. Now, steam under pressure is released from the jacket into the inner chamber and the 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 an automatic shutting system i.e. unless temperature and pressure come down near to room conditions, the door cannot be opened.
Besides the pressure dial, it also has a 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.
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, 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 the body temperature of a 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. The 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 settings. 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.
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. The 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 are programmable, which do not need trial and error temperature settings. Here, the operator sets the desired temperature and the required period of time. The incubator automatically maintains it accordingly.
It serves as a repository for thermolabile 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 dehydration.
For microbiological analysis, liquid samples are directly used, whereas solid samples have to be mixed thoroughly with a diluent (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 the peristaltic action of a machine on the bag. This machine is called a stomacher.
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 settings. The desired water temperature can be maintained over a desired period of time by programming accordingly. It is used for the cultivation of bacteria in a broth medium at a specific temperature.
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 countermarks, counts and confirms with a beep sound. The cumulative count of colonies is displayed on a four-digit LED display. In the case of the 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
Laminar Flow Chamber
It is a chamber used for the aseptic transfer of sterilized materials, as well as for the 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 the 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 surroundings 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 the 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 open during inoculation or transfer of media. A 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 a 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.
Electronic Cell Counter
It is used to directly count the number of bacteria in a given liquid sample. An example of an 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 a 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.