MICROSCOPE:

A microscope is an instrument used to see objects that are too small to be seen by the naked eye. Microscopy is the science of investigating small objects and structures using such an instrument.

There are a number of different types of microscopes and each of them solves unique problems. Below you will find information on the five different types of microscopes along with the applications for each microscope and just who might use each instrument. Below each description of the microscope and its use is an image that was captured using that particular microscope.

5 Different Types of Microscopes:

1. Stereo Microscope

2. Compound Microscope

3. Inverted Microscope

4. Metallurgical Microscope

5. Polarizing Microscope

CENTRIFUGE:

A centrifuge is a device used to separate components of a mixture on the basis of their size, density, the viscosity of the medium, and the rotor speed.

• In a centrifuge, the sample is kept in a rotor that is rotated about a fixed point (axis), resulting in strong force perpendicular to the axis.
• There are different types of centrifuge used for the separation of different molecules, but they all work on the principle of sedimentation.
• These centrifuges operate at the maximum speed of 4000-5000 rpm.

STERILIZATION:

AUTOCLAVE

Autoclaves are also known as steam sterilizers, and are typically used for healthcare or industrial applications. An autoclave is a machine that uses steam under pressure to kill harmful bacteria, viruses, fungi, and spores on items that are placed inside a pressure vessel.

WHAT IS THE AUTOCLAVE TEMPERATURE RANGE?

Commonly recommended temperatures for steam sterilization are 250° F (121° C), 270°F (132°C) or 275°F (135° C). To kill any microorganisms present, the items being sterilized must be exposed to these temperatures for the minimum time recommended by the manufacturer of the device being processed.

WHAT IS AUTOCLAVABLE?

Devices must be compatible with the autoclave process. Autoclavable items must be compatible with conditions of high heat and moisture and should be processed per the manufacturer's written instructions for use. Medical devices that have contact with sterile body tissues or fluids are considered critical items. These items may include surgical instruments, implanted medical devices and surgical drapes and linens. These items should be sterile when used because any microbial contamination could result in infection transmission. Steam is often the sterilant of choice for sterilization of heat and moisture stable items because it is reliable, consistent, and lethal to microorganisms while being safe for staff who operates the autoclave

A hot air oven is a type of dry heat sterilization. Dry heat sterilization is used on equipment that cannot be wet and on material that will not melt, catch fire, or change form when exposed to high temperatures.

COMPARE HOT AIR & AUTOCLAVE

Moist heat sterilization uses water to boil items or steam them to sterilize and doesn't take as long as dry heat sterilization. Examples of items that aren't sterilized in a hot air oven are surgical dressings, rubber items, or plastic material.

Items that are sterilized in a hot air oven include:

• Glassware (like petri dishes, flasks, pipettes, and test tubes)
• Powders (like starch, zinc oxide, and sulfadiazine)
• Materials that contain oils
• Metal equipment (like scalpels, scissors, and blades)

The commonly-used temperatures and time that hot air ovens need to sterilize materials is 170 degrees Celsius for 30 minutes, 160 degrees Celsius for 60 minutes, and 150 degrees Celsius for 150 minutes.

INTRODUCTION TO MICROTOMES

Microtome is an instrument with the help of which sections of tissues are cut and the process of cutting thin sections is known as Microtomy. The thickness of sections produced during microtomy may be between fractions of 50-100 nm, in ultramicrotomy, to several 100 microns. The common range is between 5-10m but both the maximum and minimum thickness is limited by the consistency of relation of the thickness of sections to the nature of tissues. These sections are stained using suitable staining techniques followed by observing them under the microscope.

TYPES OF MICROTOMES –

1. Rotary microtome

The Rotary microtome is so-called because of a Rotary action of the handwheel responsible for the cutting moment. The block holder is mounted on a steel carriage, which makes up and down in groves this type of instrument is the most ideal for routine and research work it is excellent for cutting serial sections.

Parts of the rotary microtomes

• Block holder
• Knife clamp screw
• Knife clamps
• Block adjustment
• Thickness gauge
• The angle of tilt adjustment
• Operating handle

Here the feed mechanism is activated by turning a wheel on one side of the machine. The knife is fixed with its edge fixed upwards and the object is moved against the knife rising and falling vertically.

One rotation of the operating wheel produces a complete cycle downwards cutting stroke and an upward return stroke and activation of the advanced mechanism. It is often modified to cut ultrathin sections between 50Å – 200Å

The wheel may be electrically operated or manually. In the former case the hands may be made free for tissue maintenance, makes it available for incorporation in automated cryostats.

Advantages of the Rotary microtome

• Heavy and stable.
• Ideal for serial sections in large numbers.
• Paraffin-embedded tissues are cut by a rotary microtome.
• The knife holder is movable.
• The sections are cut are flat.
• It is useful for routine and research papers.

2. Sliding or Base Sledge Microtome

This is a large heavy instrument with a fixed knife beneath which the object moves mounted on a heavy sliding base containing the feed mechanism used primarily for cutting the sections of cellulose nitrate embedded tissues with an obliquely set knife.

Parts of Base-sledge microtome

• Angular tilt adjustment
• Knife clamps
• Block holder
• Coarse feed adjustment
• Operating handle
• Thickness gauge
• Adjustment locking nut
• Block adjustment screw
• Split nut clasp

The blocks holder is mounted on a steel carriage which slides backward and forwards on groups against a fixed horizontal knife this microtome is heavy and very stable. The block is raised towards the knife at a predetermined thickness. This type of microtome is designed for cutting sections of very large blocks of tissues for example whole brain, this microtome has become popular for routine use.

Advantages of Base-sledge microtome

• It is useful for cutting extremely hard blocks and large sections.
• The microtome is heavy and stable.
• The knife used is sledge shaped which requires less honing.

3. Cambridge rocking microtome

The instrument is so named because the arm has to move in a rocking motion while cutting the sections. The instrument was invented by Sir Horace Darwin in 1881 was developed by Cambridge company hence it is called the Cambridge rocking microtome. It is a simple machine in which the knife is held by means of microtome thread. The rocking microtome was designed primarily for cutting paraffin wax sections but in an emergency use frozen section by inserting a wooden block in which the tissue is frozen.

Parts of the rocking microtomes

• Knife holder
• Block holder or chuck
• Upper arm
• Screw
• Lever
• Pawl
• Ratchet wheel
• Mil head microtome screw
• Sleeve
• Lower Arm
• Scale

It cuts the sections between 1 to 20 microns. The knife is fixed with the edge, while the object is moved against this knife circularly, producing a sharply curved surface to the block with each stroke the tissue holder automatically moves vertically towards the life. Cutting stroke is Spring operated and is easy to handle. The microtome must be placed on a solid non-slippery surface to allow a better hold

Advantages of Cambridge rocking microtomes

• The cost of a knife and microtome is low.
• Celloidin embedded tissues can be sectioned easily.

4. Freezing microtomes

This type has been designed for the production or preparation of frozen sections of fluid and non-fluid tissues usually without preliminary embedding. The object stage is connected to the cylinder of compressed carbon dioxide for the rapid cooling of the tissues and provisions are also made for the cooling of the knife.

Part of freezing type microtome

• Knife clamps
• Operating handle
• Thickness gauge
• Stage
• Stage valve
• Coarse adjustment

The movement of the knife takes place horizontally across the surface of the tissues. Ribbon sections cannot be prepared using this microtome. All freezing microtomes have the feature of employing a non-movable tissue block and cooling system.

Advantages of Freezing microtome

• It is used for sections required for Rapid diagnosis
• It cuts non-dehydrated fresh tissue in a frozen state.
• The method is useful for Rapid histopathological diagnosis during operation
• This type of microtome is also used when lipids, enzymes, and neurological structures are to be demonstrated.

Nowadays, the most commonly used type of microtome is a Rotary microtome which is easy to operate and ideal for routine use for diagnosis and research purposes.

WORKING PRINCIPLE OF ROTARY MICROTOME –

⇒ It is used for slicing paraffin tissue sections of uniform thickness.

⇒ This method is designed to cut 1-60 micron thick sections.

⇒ A knob on the device (typically at the backside) is used to modify the thickness of the sections.

⇒ A knife is constant inside the knife holder and clamped tightly.

⇒ The tissue block is drawn throughout the knife-edge and it is mechanically advanced. The top and bottom of the block have to be parallel and horizontal and as a minimum 1mm of paraffin has to be present in all aspects beyond the tissue.

⇒ The trimming of the edges of the block is usually completed with a single-sided razor blade and the block face is trimmed with the microtome knife.

⇒ The technician decides the type of section to be made in line with the nature of tissue and instructions received from the pathologist.

⇒ At some stage in section slicing, as the wheel of the microtome turns, sections are cut and slide on the knife. A ribbon of sections is produced.

⇒ The ribbon of sections is transferred to warm water inside the tissue floatation bath to put off any wrinkles present in the section.

⇒ The best quality section that is free from any scratches and cracks can be decided on from the tissue ribbons. The tissue ribbons are then taken on smooth glass slides with a respective identification number.

⇒ The slides are pulled from the water and the preferred sections are positioned flat on the surface of glass slides. The slides with the sections are positioned on a rack in a hot air oven to dry.

SAHALI’S HEMOGLOBIN METER (HEMOCYTOMETER)

An manual instrument that is used to determine the quantity of hemoglobin in the blood. In practice the hemoglobinometer proposed in 1902 by the Swiss scientist H. Sahli is used. It is based on comparison of the color of the tested blood, which is treated with hydrochloric acid, with the color of standards. In the USSR the GS-2 model of hemoglobinometer is produced. It consists of two-color standards and test tubes with two calibrations to determine hemoglobin in gram percent and percent (100 percent corresponds to 16-gram percent; each gram corresponds to 6 percent). In many countries hemoglobinometers are used in which 100 percent on the scale corresponds not to 16-gram percent, but to 14.8-gram percent, 17.3-gram percent, and so forth. When blood is treated with a hydrochloric acid solution, the hemoglobin is converted to hematin hydrochloride, and the solution turns brown. The solution in the test tube is diluted by gradually adding distilled water until the color of the solution is the same as that of the standard. The quantity of hemoglobin is determined by reading the level of the solution on the scale of the test tube.

WHAT IS COLORIMETER

A colorimeter is a device that is used in Colorimetry. It refers to a device which helps specific solutions to absorb a particular wavelength of light. The colorimeter is usually used to measure the concentration of a known solute in a given solution with the help of the Beer-Lambert law. The colorimeter was invented in the year 1870 by Louis J Duboscq.

Check out the derivation of Beer-Lambert law here.

Principle of Colorimeter

It is a photometric technique which states that when a beam of incident light of intensity Io passes through a solution, the following occur:

• A part of it is reflected which is denoted as Ir
• A part of it is absorbed which is denoted as Ia
• Rest of the light is transmitted and is denoted as It

Therefore, Io = Ir + Ia + It

To determine Ia the measurement of Io and It is sufficient therefore, Ir is eliminated. The amount of light reflected is kept constant to measure Io and It.

Colorimeter is based on two fundamental laws of photometry. We have discussed them below:

Beer’s law:

According to this law the amount of light absorbed is proportional to the solute concentration present in solution.

Log10 Io/It = asc

where, as is absorbency index c is the concentration of solution

Lambert’s law:

According to this law the amount of light absorbed is proportional to the length as well as thickness of the solution taken for analysis.

A = log10 Io/It = asb

Where, A is the test absorbance of test as is the standard absorbance b is the length / thickness of the solution

Working of Colorimeter

Step 1: Before starting the experiment, it is important to calibrate the colorimeter. It is done by using the standard solutions of the known solute concentration that has to be determined. Fill the standard solutions in the cuvettes and place it in the cuvette holder of colorimeter.

Step 2: A light ray of a certain wavelength, which is specific for the assay is in the direction of the solution. The light passes through a series of different lenses and filters. The coloured light navigates with the help of lenses, and the filter helps to split a beam of light into different wavelengths allowing only the required wavelength to pass through it and reach the cuvette of the standard test solution.

Step 3: When the beam of light reaches’ cuvette, it is transmitted, reflected, and absorbed by the solution. The transmitted ray falls on the photodetector system where it measures the intensity of transmitted light. It converts it into the electrical signals and sends it to the galvanometer.

Step 4: The electrical signals measured by the galvanometer are displayed in the digital form.

Step 5: Formula to determine substance concentration in test solution.

A = ∈cl

For standard and test solutions

∈ and l are constant

AT = CT ….. (i)

AS = CS ….. (ii)

From the above two equations,

AT × CS = AS × CT

CT = (AT/AS) × CS

Where,

• CT is the test solution concentration
• AT is the absorbance/optical density of test solution
• CS is the standard concentration
• AS is the absorbance / optical density of standard solution

Uses of Colorimeter

• It is used in laboratories and hospitals to estimate biochemical samples such as urine, cerebrospinal fluid, plasma, serum, etc.
• It is used in the manufacturing of paints.
• It is used in textile and food industry.
• It is used in the quantitative analysis of proteins, glucose, and other biochemical compounds.
• It is used to test water quality.
• It is used to determine the concentration of hemoglobin in the blood.

Advantages and disadvantages of Colorimeter

Some benefits are as follows:

It is an inexpensive method, widely used in the quantitative analysis of coloured samples, easy to carry, and transport.

Some disadvantages are as follows:

Analysis of colourless compounds is not possible, does not work in IR and UV regions.

LAMINAR FLOW

A laminar flow hood/cabinet is an enclosed workstation that is used to create a contamination-free work environment through filters to capture all the particles entering the cabinet.

• These cabinets are designed to protect the work from the environment and are most useful for the aseptic distribution of specific media and plate pouring.
• Laminar flow cabinets are similar to biosafety cabinets with the only difference being that in laminar flow cabinets the effluent air is drawn into the face of the user.
• In a biosafety cabinet, both the sample and user are protected while in the laminar flow cabinet, only the sample is protected and not the user.

Components/ Parts of Laminar flow hood

A laminar flow cabinet consists of the following parts:

1. Cabinet

• The cabinet is made up of stainless steel with less or no gaps or joints preventing the collection of spores.
• The cabinet provides insulation to the inner environment created inside the laminar flow and protects it from the outside environment.
• The front of the cabinet is provided with a glass shield which in some laminar cabinets opens entirely or in some has two openings for the user’s hands to enter the cabinet.

2. Working station

• A flat working station is present inside the cabinet for all the processes to be taken place.
• Culture plates, burner and loops are all placed on the working station where the operation takes place.
• The worktop is also made up of stainless steel to prevent rusting.

3. Filter pad / Pre-filter

• A filter pad is present on the top of the cabinet through which the air passes into the cabinet.
• The filter pad traps dust particles and some microbes from entering the working environment within the cabinet.

4. Fan/ Blower

• A fan is present below the filter pad that sucks in the air and moves it around in the cabinet.
• The fan also allows the movement of air towards the HEPA filter sp that the remaining microbes become trapped while passing through the filter.

5. UV lamp

• Some laminar flow hoods might have a UV germicidal lamp that sterilizes the interior of the cabinet and contents before the operation.
• The UV lamp is to be turned on 15 minutes before the operation to prevent the exposure of UV to the body surface of the user.

6. Fluorescent lamp

• Florescent light is placed inside the cabinet to provide proper light during the operation.

7. HEPA filter

• The High-efficiency particulate air filter is present within the cabinet that makes the environment more sterile for the operation.
• The pre-filtered air passes through the filter which traps fungi, bacteria and other dust particles.
• The filter ensures a sterile condition inside the cabinet, thus reducing the chances of contamination.

Principle/ Working of Laminar flow hood

• The principle of laminar flow cabinet is based on the laminar flow of air through the cabinet.
• The device works by the use of inwards flow of air through one or more HEPA filters to create a particulate-free environment.
• The air is taken through a filtration system and then exhausted across the work surface as a part of the laminar flow of the air.
• The air first passes through the filter pad or pre-filter that allows a streamline flow of air into the cabinet.
• Next, the blower or fan directs the air towards the HEPA filters.
• The HEPA filters then trap the bacteria, fungi and other particulate materials so that the air moving out of it is particulate-free air.
• Some of the effluent air then passes through perforation present at the bottom rear end of the cabinet, but most of it passes over the working bench while coming out of the cabinet towards the face of the operator.
• The laminar flow hood is enclosed on the sides, and constant positive air pressure is maintained to prevent the intrusion of contaminated external air into the cabinet.

Procedure for running the laminar flow cabinet

The procedure to be followed while operating a laminar flow cabinet is given below:

1. Before running the laminar flow cabinet, the cabinet should be checked to ensure that nothing susceptible to UV rays is present inside the cabinet.
2. The glass shield of the hood is then closed, and the UV light is switched on. The UV light should be kept on for about 15 minutes to ensure the surface sterilization of the working bench.
3. The UV light is then switched off, and a time period of around 10 minutes is spared before the airflow is switched on.
4. About 5 minutes before the operation begins, the airflow is switched on.
5. The glass shield is then opened, and the fluorescent light is also switched on during the operation.
6. To ensure more protection, the working bench of the cabinet can be sterilized with other disinfectants like 70% alcohol.
7. Once the work is completed, the airflow and florescent lamp both are closed and the glass shield is also closed.

Types of laminar flow cabinet

Depending on the direction of movement of air, laminar flow cabinets are divided into two types:

1. Vertical laminar flow cabinet

• In the vertical flow cabinets, the air moves from the top of the cabinet directly towards the bottom of the cabinet.
• A vertical airflow working bench does not require as much depth and floor space as a horizontal airflow hood which makes it more manageable and decreases the chances of airflow obstruction or movement of contaminated air downstream.
• The vertical laminar flow cabinet is also considered safer as it doesn’t blow the air directly towards the person carrying out the experiments.

2. Horizontal laminar flow cabinet

• In the horizontal laminar flow cabinets, the surrounding air comes from behind the working bench, which is then projected by the blower towards the HEPA filters.
• The filtered air is then exhausted in a horizontal direction to the workplace environment.
• One advantage of this cabinet is that airflow parallel to the workplace cleanses the environment with a constant velocity.
• The elluent air directly hits the operator, which might reduce the security level of this type of laminar flow cabinets.

Uses of Laminar flow hood

The following are some common uses of a laminar flow cabinet in the laboratory:

1. Laminar flow cabinets are used in laboratories for contamination sensitive processes like plant tissue culture.
2. Other laboratories processes like media plate preparation and culture of organisms can be performed inside the cabinet.
3. Operations of particle sensitive electronic devices are performed inside the cabinet.
4. In the pharmaceutical industries, drug preparation techniques are also performed inside the cabinet to ensure a particulate-free environment during the operations.
5. Laminar flow cabinets can be made tailor-made for some specialized works and can also be used for general lab techniques in the microbiological as well as the industrial sectors.

Precautions

While operating the laminar airflow, the following things should be considered:

1. The laminar flow cabinet should be sterilized with the UV light before and after the operation.
2. The UV light and airflow should not be used at the same time.
3. No operations should be carried out when the UV light is switched on.
4. The operator should be dressed in lab coats and long gloves.
5. The working bench, glass shield, and other components present inside the cabinet should be sterilized before and after the completion of work.

A hemocytometer is a specialized slide which is used for counting cells. It is actually a glass slide which has a 3×3 grid etched into it. Carved in it are intricate, laser-etched lines that form a grid. It also has its own coverslip.

Every person having anything to do with microbiology, biotechnology, pathology or other related fields will be required to know their way around a hemocytometer. Used for the count of various micro-particles or microorganisms, a hemocytometer is a special slide, and much more expensive than your average glass slide! It can be used to count the number of red blood cells in a sample, as well as white blood cells, microbes like yeast, and many others.

HEMOCYTOMETER

A hemocytometer looks, from a distance, like an average glass slide, just heavier. However, it is much more than that. Carved in it are intricate, laser-etched lines that form a grid. It also has its own coverslip, which is different from a regular coverslip. On the slide, there are marked grooves that appear like an ‘H’. The horizontal line of the ‘H’ separates the 2 grids for counting. Therefore, each slide has two identical grids for counting cells. The depth of these 2 grids is a fixed 0.1mm

Hemocytometer calculation

Each grid is a square with the dimensions of 3×3 mm2. This square has three equidistant vertical and horizontal lines. These divide it into 9 smaller squares of 1×1 mm2 each. These are separated from each other by triple-ruled lines. Of these 9 squares, the 4 corner squares are used for the counting of bigger cells, like WBCs, while the center square is used to count smaller cells, such as RBCs.

The 4 corner squares of the main grid are further divided into 16 smaller cells. The centre square of the main grid is divided into 25 smaller squares, each of which is again divided into 16 smaller squares. The sample to be counted is loaded onto the slide after the coverslip has been placed. Excess fluid drains into the grooves on the side. However, the person loading the sample must be extremely careful while loading.

This covers the structure and design of the hemocytometer, but to understand how counting and calculation is done, let’s consider the example of counting WBCs for the corner squares, and RBCs for the center square.

Hemocytometer counting

RBCs, being smaller in size and larger in number, are counted in the center square. This has a greater number of divisions and therefore makes counting easier. As mentioned above, the center square contains 25 smaller squares. The area of each of these is 1/25 mm2, which is 0.04 mm2. Once the sample is loaded, not all the cells are counted. Out of 25, any 5 squares are picked for the counting. The division of each of these 0.04 mm2 squares into 16 smaller ones makes it easier for the person to count the number of cells, rather than just having to count in an empty square. There are a number of patterns to select the 5 squares that should be counted. The corner 4 and center square can be picked, or any of the diagonal lines of squares.

Once the number of cells in 5 squares has been counted, their mean is taken. Let ‘n’ be the mean. Therefore, the average number of cells in each of the tiny 0.04 mm2 squares is n. The volume of each of these cells is 0.04 x 0.1 = 0.004 mm3. The number of cells in 1 mm3 is n/0.004. Therefore, the total number of cells in 1ml is (n/0.004) x 1000. We multiply by one thousand as 1000 mm3 = 1 cm3; and 1 cm3 = 1 mL

WBC Count

When WBCs are counted, the calculation is much easier. WBCs are counted in the 4 corner squares of the main grid. These squares have an area of 1 mm2 each. To get the WBC count, the number of cells in each square are counted, and their mean is then calculated. Let the mean be ‘n’. The volume of each square is 1 x 0.1 = 0.1 mm3. The number of cells in 1 mm3 is n/0.1. Therefore, the total number of cells in 1ml is (n/0.1) x 1000. We multiply by one thousand as 1000 mm3 = 1 cm3; and 1 cm3 = 1 mL

Rules to be followed

While counting cells, certain things require attention. Some cells may not lie either inside or outside the square. Rather, they may fall on the border. Therefore, a simple practice of including cells that fall on the top and left border and excluding cells that fall on the bottom and right border is followed. However, this is not a rule. The basic principle is that any 2 adjacent borders should be counted, and the remaining 2 borders should be rejected. Also, this selection criteria must apply to all the squares being counted.

Sometimes the solution of the sample may be too concentrated. If it is too highly concentrated, the cells will overlap and thus the counting will be wrong. Therefore, such concentrated cell solutions need to be diluted with an appropriate solution. This dilution also needs to be factored into the calculations. For instance, if the sample has been diluted by 10x, the final answer obtained from the calculations must be multiplied by 10.