The instruments used in microbiology laboratories include so many different kinds of instruments that are very necessary for a lot of different processes or practicals conducted within those laboratories.
Now we are going to study some Instruments, their principle, and uses,
- Hot air sterilizer/oven
- pH meter
- Clinical Centrifuge
- Laminar Air Flow
- Distillation Unit
- Chemical Balance
- Bright Field Microscope
Hot air sterilizer/oven
This is a dry air type of sterilizer/oven.
This instrument is less efficient than the wet heat type of sterilization.
It is constructed with three walls and two air spaces.
The outer wall is covered with thick asbestos to reduce the radiation of heat.
The burner manifold runs along both sides and rear between the outside and the intermediate wall space and interior of the oven, and the products of combustion escape through an opening in the top.
The instrument is usually used for drying autoclaved materials, powders, waxes, metal equipment.
Thermal insulation, continuous heat elements, thermostat, display unit, perforated metal sheets are the main components of dry heat sterilizer.
This instrument is operated at the temperature of 160 to 180 o C for 1 to 11⁄2 hr.
Wrapped materials are kept in the sterilizer.
Temperature is adjusted and kept for the prescribed time.
It is an apparatus with a chamber used to provide controlled environmental conditions (such conditions as temperature and humidity) usually for the cultivation of microorganisms.
Temperature and humidity are adjusted to provide optimum conditions for the growth of the microorganisms being handled.
For bacteria from the human body grow at 37oC whereas for soil microorganisms temperatures lesser than this can be employed.
Other temperatures for incubation are 30 o C used for the cultivation of leptospires and some bacteria, 25 – 28 o C used for many fungi, and 22oC for some fungi and gelatin cultures.
It has a double-walled metal cabinet with insulating material. The thermostable glass/fiber door of the inner cabinet is fitted with a rubber gasket.
The temperature is critically regulated by the thermostat and heating elements. For large-sized cabinets, Fan is fitted for circulation of air inside.
The temperature can be checked by inserting a thermometer through the opening on the top.
The incubators used for culturing animal and plant tissues are provided with inlets for different gases such as CO2, O2, N2, etc.
The humidity is maintained by keeping sterilized water in a tray at the bottom of the incubation cabinet.
Another modification of the conventional incubator is the incubator shaker, in which the incubator chamber is facilitated with a mechanical shaking platform.
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It is a cylindrical metal vessel having double walls except at the opening and can withstand high pressure with superheated steam.
Water boils at about 100 oC, depending upon the vapor pressure of the atmosphere. If the vapor pressure is increased, the temperature will increase.
As the steam pressure inside the autoclave is increased to 15 lb per sq in (2 atm absolute), the temperature rises to 121 oC.
The autoclave is operated usually at 15 lb (121 oC) steam pressure for a period of 15 min. The things to be sterilized such as glassware, media are kept inside the inner vessel.
The vessel is closed and the inner vessel is allowed to saturate with the superheated steam by keeping the exhaust valve open.
Observing the continuous stream of steam the exhaust valve is closed to reach the pressure of 15 psi (pounds per square inch). The exhaust valve so adjusted that the pressure.
An autoclave remains constant for 15 min after which the instrument is put off and pressure is allowed to come down to zero.
The lid is opened and sterilized materials are taken out. These operation conditions are true only if the air in sterilizing chamber is evacuated.
The autoclave is used for sterilizing media, solid and liquid heat-stable liquids, heat resistant instruments; glassware, etc. autoclave is infrequently used for oils and not used for volatile oils, powders, heat-sensitive fluids such as serum.
The colorimeter is especially used in the measurement of transmittance or absorbance by using visible light wavelengths.
Illuminator, glass field gelatin filters (of certain wavelength or range of wavelengths), glass cuvettes, Photo-detector, analyzer, and display unit are the components of the colorimeter.
The working of the instrument is simply based on Beer and Lambert’s law. The instrument measures the amount of transmitted light through the substance is measured when kept in a beam of light.
Transmission of light depends upon the concentration of the solution and path length (through the substance) traveled by the light.
The ratio of the intensities of transmitted (I) and incident light (Io) gives transmittance (T, usually expressed in %). 100% transmittance means no light is absorbed by a substance.
The value that lies in between is known as absorbance and is given by the logarithm of the reciprocal of transmittance.
- A = log (1 / T) = log (Io / I) = єλ c l
- Where, єλ = Molar absorbance coefficient for the absorber at wavelength,
- c = Concentration of absorbing solution,
- l = Path length of the light through the solution.
The reference liquid (solution or suspension) is poured in the tubes and kept in the cuvette holder.
Absorbance (A) / optical density (OD) is adjusted to zero. The test liquid (solution or suspension) is then kept in the cuvette holder and the ‘A’ or ‘OD’ value is recorded.
This instrument is used for fast, accurate measurement of Hydrogen ion (H+) concentration of the medium, (usually aqueous and hydrophilic media) with maximum precision.
The pH range that can be measured is 0.00 to 14.00 along with the resolution of 0.1 – 0.01 with such a fine accuracy. The concentration of H+ ions (in arithmetic or exponential scale) is correlated with the pH scale (a logarithmic scale) in the processor.
For example, the concentration of the H+ ions in pure water is 10-7 mole/liter. As the pH is the negative logarithm of H+ ions present in the medium, therefore the pH of pure water is seven.
The pH below seven is referred to as acidic and the pH above seven is alkaline.
The main components of the pH meter are the sensing electrode, reference electrode, voltmeter, and digital or analog display.
Sophisticated instruments include auto-calibration, auto- buffer recognition, auto temperature recognition, and temperature compensation, etc.
The pH meter actually measures the potential developed at the surface of the pH sensing electrode, which depends upon Hydrogen ions [H+] of the solution, and a reference electrode, which provides a constant potential, which is independent of the concentration of hydrogen ions [H+ ].
This difference in the potential is proportional and correlated to the pH of the medium. Usually, the pH meter is calibrated prior to measuring the pH of the medium.
First, an electrode is calibrated at pH 7 buffer, followed by pH 4 (if the sample is expected to be acidic) or a pH 9 buffer (if the sample is expected to be basic).
Once the calibration is done, the electrode is simply be immersed in the solution, whose pH is to be measured and a rapid and accurate estimate of pH can be made.
A laboratory centrifuge is one of the important laboratory equipment, driven by a motor, spinning liquid samples at high speed.
There are different types of centrifuges, depending on the size and strength of the sample.
Laboratory centrifuges operate by the law of sedimentation, where centripetal acceleration is used to separate objects of large and small sizes.
There are various types of centrifugation:
- Differential centrifugation is often used to separate certain organelles from whole cells for further analysis of specific parts of cells.
- Isopycnic centrifugation is often used to isolate nucleic acids such as DNA.
- Sucrose gradient centrifugation is often used to purify enveloped viruses and ribosomes, and also to separate cell organelles from crude cellular extracts.
There are different types of laboratory centrifuges:
- Microcentrifuges (small tube devices from 0.2 ml to 2.0 ml (microtubes), up to 96 well plates, compact design, small footprint; up to 30,000 g.)
- Clinical centrifuges (devices used for clinical applications such as blood collection tubes, low-speed devices).
Centrifugation protocols generally specify the acceleration rate to be used in the sample, rather than specifying the rotational speed such as changes per minute.
This difference is important because two rotors with different diameters operating at the same rotational speed will give the samples different speeds.
Acceleration can be calculated as a product of the radius and square of its angular velocity.
Relative centrifugal force (RCF) is nothing but the measurement of the force applied to a sample during centrifugation.
This can be calculated from the speed (RPM) and rotation radius (cm) using the following calculation.
g = RCF = 0.00001118 × r × N²
- g = Relative centrifuge force
- r = rotational radius (centimetre, cm)
- N = rotating speed (revolutions per minute, r/min)
Laminar Air flow
A laminar flow cabinet or laminar flow closet or tissue culture hood or laminar airflow is a carefully enclosed bench that is designed to prevent contamination of biological samples or any particle-sensitive device.
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 with no gaps or joints where spores might collect.
Such hoods are available in straight and vertical locations, and there are many types of cabinets with a variety of ventilation and acceptable use.
Laminate flow cabinets can have a UV-C disinfectant lamp to incorporate shell and content when not in use. (It is important to switch this light off during use, as it will quickly give any exposed skin sunburn and may cause cataracts.)
Laboratory scale distillation almost exclusively works as batch distillation.
The device used in distillation, sometimes referred to as a still, consists at a minimum of a reboiler or pot in which the source material is heated, a condenser in which the heated vapor is cooled back to the liquid state, and a receiver in which the concentrated or purified liquid, called the distillate, is collected. Several laboratory scale techniques for distillation exist.
Analytical balances, especially the four or five-place, digital, or substitution balances, are capable of measuring the weight of an object to 0.0001 or even 0.00001 grams-, i.e.(0.1 mg or 0.01 mg).
These balances if properly weighed and maintained may be the most accurate and accurate measurement tool available in any laboratory.
General Operating instructions for chemical balance are as follows –
- Before turning it on, make sure that the doors are closed and that the pan does not touch the ring being repaired.
- Turn on by pressing down on the gray bar and quickly release.
- Push again if the digital reading does not show 0,0000 g value.
- Place the object between the center of the pan and the door.
- Allow the display to stabilize; (g) will appear after the numerical display.
- Read (a record) result, remove the object from pan, leave doors closed, and re-zero if
A spectrophotometer is an instrument used for counting the population of bacteria, based on the principle of turbidity determination.
Turbidity or optical density is the cloudiness of the suspension. The more intense the suspension, the less light will be transmitted through it.
In other words, the amount of light that is concentrated and dissipated is equal to the number of cells in the path of light. As the bacteria grow in the broth, the clear broth becomes turbid.
As the number of cells increases; Turbidity also Increases, this indicator is used as an indicator of bacterial density present in the broth. Turbidity is also used for standardizing the population densities of bacterial cultures of clinical significance.
Bright Field Microscope
Microbiology is concerned with the study of microorganisms. To study microorganisms certain tools are required.
A microscope is one of the primary and most essential tools of a microbiologist. The microscope is a lens system that gives a magnified image of the object.
The microscope may be defined as an optical instrument, consisting of a lens or a combination of lenses, for making enlarged or magnifies images of minute objects.
A slide or a film projector, a photographer’s enlarger, or simply a concave mirror can magnify images.
It provides magnification which enables one to see the organism and structures invisible to the naked eye.
Microscopes, on the other hand, are used more for their resolving power rather than their magnification.
A film projector can project a 70 mm film onto a 35-meter wide screen, thus giving a magnification comparable to a light microscope. We cannot, however, use a film projector to observe microorganisms since their resolution is low.
The ability to differentiate between two closely placed points is termed resolving power. If two points are placed 1 mm apart we can see them as two separate points. If the distance is reduced to 0.1 mm we may still be able to ‘resolve’ them apart.
If the distance is reduced further to 0.05 mm they seem to merge with each other when viewed with the naked eye. Under a microscope, however, they can be seen as two separate objects.
A more useful concept is ‘minimum resolvable distance’, which is the minimum distance at which two points can be clearly resolved.
d = (0.5 x λ) / (n sin θ)
d = Wavelength / 2 N.A.
- d = the minimum distance between the two points that reveals them as separate entities.
- λ = wavelength of light used to illuminate the specimen
- n sin θ = Numerical aperture (N. A.)
- n = refractive index of the medium between the objective front lens and the object
- θ = angular aperture
Structure of bright field compound microscope
The optical components
- A compound microscope has three sets of lens systems. Condenser system: It gathers light and illuminates the object.
- Objective lens system: It creates a real inverted magnified image of the object. Most microscopes are equipped with three objectives with different magnifying power, low power (lOx), high power or high-dry (45x), and oil immersion lens (100x).
- Ocular lens system (Eyepiece): It converts the real image into a virtual image with further magnification that can be viewed by an observer looking through it.
The mechanical components
The body of a microscope that supports the optical, system consists of a foot on which the microscope rests; the stage holds the object and condenser and illumination system below it, i.e. arm which supports the body tube and focusing mechanisms and drawtube.
- https://en.wikipedia.org/wiki/Laminar_flow_cabinet – 3%
- https://microbiologynote.com/spectrophotometer-principle/ – 2%
- http://www.faqs.org/espionage/Lo-Mo/Microbiology-Applications-to-Espionage-Intelligence-and-Security.html – 2%