Practical 01: Introduction to microbiology laboratory and its equipment

Aim: Introduction to microbiology laboratory and its equipment

Instruments used in microbiology lab are as follows

1. Hot Air Oven for Sterilization:

• It is used for the sterilization of glassware, such as test tubes, pipettes, and Petri dishes.

• Liquid substances, such as prepared media and saline solutions cannot be sterilized in the oven, as they lose water due to evaporation.

• These glasswares are sterilized at 180°C for 3 hours.

2. Drying Oven:

• For the preparation of certain reagents, the glasswares, 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 dries up completely.

3. Autoclave:

• An Autoclave is an essential component 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 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 this instrument, 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 and pressure are attained.

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, 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.

5. BOD Incubator (Low-Temperature Incubator):

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

6. Fridge (Refrigerator):

• 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.

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

10. 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 the greater is the chance of survival of some bacteria.

• Distilled water, though not bacteria-free, contains fewer 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.

• To get metal-free distilled water, glass distillation apparatus is used, and still better is the 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.

11. Ultrapure Water Purification System:

• For precision analytical works, nowadays, instead of using double- or triple-distilled water, microfiltered water is used.

• In the case of distilled water, there is a 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.

12. Homogenizer:

• For microbiological analysis, liquid samples are directly used, whereas solid samples have to be mixed thoroughly with 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 the peristaltic action of a machine on the bag. This machine is called a stomacher.

13. 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.

14. Hot Plate:

• A 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.

15. 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.

16. Colony Counter:

• In the 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.

Electronic Colony Counter:

Electronic colony counter is of two types:

(1) Hand-held electronic colony counter and

(2) Table-top electronic colony counter.

17. Magnetic Stirrer:

• In the preparation of solutions, certain chemicals require stirring for a long time, to be dissolved in certain solvents.

• A 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.

18. Sonicator: It is used to rupture cells using high-frequency waves.

19. Vortex Mixer:

• It is an instrument used for the 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 the enumeration of bacteria, which needs the homogenous suspension of bacteria cells.

20. 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 harbor 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 sterilize 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.

21. Microscopes:

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

22. 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 the microbiology lab, it is used for direct counting of bacteria in suspension as well as for other purposes.