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Microscope is the primary instrument of a microbiology laboratory and it produces enlarged images of small objects which are invisible by naked eye. With a microscope the operator can get a close view of minute structures which helps in examination and analysis. Examination of minute objects with a microscope is called ‘microscopy’. In microbiology, measurement of the microbial dimension observed under microscope is known as ‘micrometry’.

Types of microscopes

There are two types of microscopes i.e. light / optical and electron microscope depending on the sources of energy to create the image. The optical microscopes use the lenses and light (artificial or sunlight) to make an image. Following types of optical microscopes are available.

Normal bacterial flora is the bacteria which live on another living being (human or animals) or inanimate object without causing infection

Opportunistic bacteria are part of normal bacterial flora which can produce infection during lowered immunity (immunosuppression) of host or when transferred into other parts of the body

Saprophytic bacteria live on dead or decaying organic matter Infection

Infection can be defined as a pathological condition occurred due to multiplication of microbes.

Intoxication can be defined as pathological condition due to ingestion of microbial toxin (e.g. aflatoxin produced by Aspergillus fungus).

Mycetism is the pathological condition due to ingestion of fungus (e.g. toxic mushroom) which produces toxicity within the body of the host

Place of Microorganisms in Living World

Haeckel’s kingdom Protista

E.H. Haeckel, 1866 proposed this concept. Apart from plant and animal kingdom, the third kingdom “Protista” was developed by him. The protista includes unicellular organisms that are typically neither plant nor animal. These organisms, the protists, include bacteria, algae, fungi and protozoa. Bacteria are referred as lower protists and others are called as higher protists.

Whittaker’s five kingdom concept

R.H.Whittaker (1969) proposed the concept of five kingdom. He divided the living things in to 5 kingdoms.

The pathogenicity of an organism varies form mild attack to severe fatal attacks. The host has many mechanisms to resist or to avoid the pathogens apart from immune systems. One or few of these will be activated when the pathogen enters into the body. Following are some of the listed natural host defense mechanisms.

ALL LIVING ORGANISMS ARE MADE UP OF UNITS CALLED CELLS

All living creatures are made from 1 or more cells

All cells are produced from previously existing cells (no spontaneous generation)

All cells appear to be descended from the first cell which existed about 4 billion years ago

For a species to exist its reproductive cells must be potentially immortal (no aging)

Our bodies start from a single cell and contain about 100,000,000,000,000 (10^14) cells at maturity

MOST OF THE ENERGY FOR LIFE COMES FROM SUNLIGHT

Total rate of energy delivery from sun to earth (at the top of the atmosphere) = 175,000 terrawatts

Terrawatt = 10^12 watts

Rate of energy capture by plants from the sun = net primary productivity (NPP) = 100 terrawatts

Only ~ 0.06% of sunlight is captured

Captured in chemical bonds, especially in sugar

Small amounts of energy are available to specialized microorganisms from chemical reactions not linked to sunlight

It is estimated that 99% of the energy used by living cells comes from the sun

Incorporation of sunlight into chemical bonds occurs through the process of photosynthesis

“Invented” by cyanobacteria about 2 billion years ago

The word ‘Mycoplasma’ is originated from the Greek word mykes (fungus) and plasma (formed). It was first used by Albert Bernhard Frank (1889), a German biologist. He thought it was a fungus. An older name for Mycoplasma was pleuropneumonia-like organisms (PPLO), referring to organisms similar to the causative agent of contagious bovine pleuropneumonia (CBPP).It was later found that the fungus-like growth pattern of Mycoplasma mycoides is unique to the species. It was first isolated in 1898 by Edmond Nocard and E. Roux from bovine lungs. Mycoplasma pneumoniae was considered as a virus previously. Eaton and his co-workers cultured the causative agent of human primary atypical pneumonia (PAP) or ‘walking pneumonia’. It was known as ‘Eaton’s agent’. In the early 1960s, Leonard Hayflick, in Philadelphia, was first to isolate and cultivate the causative agent of primary atypical pneumonia and identified the agent as a Mycoplasma pneumoniae.

Properties of genus

1 Morphology: Mycoplasma have no cell wall and therefore are resistant to the antibiotics used to inhibit the cell wall synthesis like penicillin, cephalosporin and vancomycin. The bacteria are the smallest prokaryote (0.3-0.8µm). They are pleomorphic due to absence of the rigid cell wall. The different shapes include ring, globules, filaments and elementary bodies. Due to their plasticity in shape and size, they can be passed through the membrane filter with pore size of 0.45µm. The cell membrane is constituted of trilaminar structure enriched with phosphoprotein, lipoprotein, glycolipid, phospholipid and sterol. So Mycoplasma require sterol for their growth.

Helminths include the nematodes (roundworms), trematodes (flukes) and the cestodes (tapeworms). The adult worms live in intestine and discharge the eggs or larvae that comes out with the faeces. Therefore, diagnosis is based on the detection of eggs or larvae in faecal samples.

Overview of Nematode infections in humans and animals

The phylum Nematoda, (roundworms), is the second largest phylum in the animal kingdom, encompassing up to 500,000 species. Members of Nematoda are elongated, with bilaterally symmetric bodies that contain an intestinal system and a large body cavity. According to a report approximately 60 species of roundworms parasitize humans. Intestinal roundworm infections constitute the largest group of helminthic diseases in humans. According to 2005 report of World Health Organization (WHO), approximately 0.807-1.221 billion humans suffers from ascariasis, 604-795 million from trichuriasis and 576-740 million from hookworm infections worldwide.

Collection of faecal samples: To diagnose gastro-intestinal parasites of ruminants or other animals, the parasites or their eggs/larvae must be recovered from the digestive tract of animal or human or from the faecal/stool material.
 
The faecal samples for parasitological examination should be collected from the rectum of animals. If rectum samples cannot be obtained, fresh faecal samples may be collected from the pastures. Several samples should be collected and immediately dispatched to the diagnostic laboratory in suitable containers such as:

I. Definitions

Transpiration - evaporation of water from a plant surface

Evapotranspiration - evaporation of water from a plant surface and the soil (and abiotic surroundings.

Take-home-lesson: Plants loose a lot of water by transpiration. See text for some specific examples.

A. Gram’s Staining
Introduction
The most widely used staining procedure in microbiology is the Gram stain, discovered by the Danish scientist and physician Hans Christian Joachim Gram in 1884. Gram staining is a differential staining technique that differentiates bacteria into two groups: gram-positives and gram-negatives. The procedure is based on the ability of microorganisms to retain color of the stains used during the gram stain reaction. Gram-negative bacteria are decolorized by the alcohol, losing the color of the primary stain, purple. Gram positive bacteria are not decolorized by alcohol and will remain as purple. After decolorization step, a counterstain is used to impart a pink color to the decolorized gramnegative organisms.
The Gram stain procedure enables bacteria to retain color of the stains, based on the differences in the chemical and physical properties of the cell wall.
1. Gram positive bacteria: Stain dark purple due to retaining the primary dye called Crystal Violet in the cell wall. Example: Staphylococcus aureus
2. Gram negative bacteria: Stain red or pink due to retaining the counter staining dye called Safranin

A. Enumeration of Different Microflora of Soil
Introduction
Agriculturally, soil is the region which supports the plant life by providing mechanical support and nutrients required for growth. From the microbiologist view point, soil is one of the most dynamic sites of biological interactions in the nature. Soil is an admixture of five major components viz. organic matter, mineral matter, soil-air, soil water and soil microorganisms/living organisms. The amount/ proposition of these components varies with locality and climate.

The soil organisms are broadly classified in two group’s viz. soil flora and soil fauna, the detailed classification of which is as follows.
1) Soil flora
a) Microflora Bacteria, Fungi, Molds, Yeast, Mushroom, Actinomycetes, Streptomyces, Algae e.g. BGA, Yellow Green algae, Golden brown algae.
b) Macroflora

Microorganisms, being ubiquitous, are found in soil, air, water, food, sewage, on body surfaces as well as on degrading and decomposing substances. Their survival and continued growth mostly depend on an adequate supply of nutrients and favourable growth environments. The culture media (nutrients) consisting of chemicals support the growth of microorganisms in a given condition. They are devised in such a way that the organism should get all the nutritional requirements. These are usually prepared in laboratory by weighing and dispensing the individual ingredients. Generally, the common media contain both inorganic and organic nutrients; however, for the cultivation of large number of microorganisms, specialized media are required.

Basically the culture media are of three types: (1) Natural, (2) Semi synthetic and (3) Synthetic.

10.1 Transformation of E. coli by electroporation Principle

Preparation of electro-competent bacteria is considerably easier than preparing cells for transformation by chemical methods. Bacteria are simply grown to mid –log phase, chilled, centrifuged, washed extensively with ice cold buffer containing 10% glycerol. DNA may be introduced immediately into the bacteria by exposing them to a short high voltage electrical discharge. Alternatively, the cell suspension may be snap frozen and stored at-70^oC for 6 months before electroporation, without loss of transforming efficiency.

Since E.coli cells are small, they require very high strengths (12.5- 15KV cm^-1) for electroporation as compared to those used to introduce DNA into eukaryotic cells. Electroporation is temperature dependent and is best carried out at 0-4°C.

The highest efficiency of transformation is obtained when the concentration of input DNA is high (1-10 μg/ml) and when the length and intensity of the electrical pulse are such that only 30-50% of the cells survive in the procedure. Under these conditions, as many as 80% of the surviving cells may be transformed. Higher frequencies of transformation (colonies/ molecules input DNA) are obtained when the DNA concentration is low (?10pg/ml).

The method outlined in this protocol is followed with most strains of E.coli and with plasmids <15kb in size. However, substantial variation in the efficiency of transformation between the strains of E. coli. has been reported. As in case with chemical transformation, linear plasmid DNAs introduced into E.coli by a pulsed electrical discharge transform very efficiently from 10 to 1000 fold but less efficiently than the corresponding closed circular DNA – perhaps because the exposed terminal of linear DNA are susceptible to attack by intracellular nucleases.

In any organism and biomolecules (enzyme, proteins, etc.), most biological activities could be seen only within a narrow environmental conditions. Therefore in order to carry out a successful experiment, from cell culturing to DNA sequencing or assessing an enzyme’s activity, one has to pay utmost attention in calculating correct reaction components.

A solution is defined as a homogenous mixture of solute and solvent. In this case the amount solute is less as compared to the solvent. It is not always necessary that solvent is liquid and solute is solid. There are several examples where solute and solvents are found in other physical states such as gas or solids. For example, carbonated water CO₂ is solute and water is solvent; and smoke is solution of different smoke particles (solute) in air (solvent). The concentration of solution is described as amount of solute in a given volume of solvent. Several ways are there to express the concentration of a solution, such as molarity, normality, molality, and ppm. In this chapter we would discuss the general terms used in making solutions and a brief introduction about buffer solutions.

Nucleic acid extraction from clinical samples is the first step to be followed in molecular detection of cancer. Nucleic acids are the starting material for all downstream molecular diagnostic tools. Quality of test results largely depends upon the quality of starting material. Thus utmost importance should be given to the extraction protocols to isolate good quantity and good quality nucleic acids from patient samples.

Basic principle: The basic principle of nucleic acid extraction is to disrupt cell membrane and nuclear membrane to release and precipitate nucleic acid in the solution. Following precipitation,removal of other cellular contaminates such as protein, lipids, polysaccharides etc is performed to obtain a good quantity and quality of nucleic acid.

7.1 Basic concept

What is DNA sequencing?

The genome of any biological organism is a sequence of nucleotides arranged in a specific manner. The nucleotides are chemically a base which usually exists in 4 different forms; Purine – Adenine (A) and Guanine (G) and Pyramidine – Cytosine (C) and Thymine (T). These four nucleotides make up an entire sequence of Deoxyribose Nucleic Acid (DNA) which is encodes the information of any cell in the form of a gene. The genome, therefore, is the complete set of all genes that is required to build and maintain that organism. DNA sequencing is the process wherein the precise order of these nucleotides along the gene is detected and analysed which includes any method or technology that assists in determining the sequence.

An organic acid is an organic compound with acidic properties. The most common organic acids are the carboxylic acids, whose acidity is associated with their carboxyl group –COOH. However, sulphuric acids, containing the group –SO₂OH, are relatively stronger acids. Alcohols, with –OH, may also act as weak acids. Some other groups may also act as weak acids viz. thiol group and phenol group. In biological systems, organic compounds containing these groups are generally classified under organic acids. Some common organic acids are lactic acid, acetic acid, formic acid, citric acid, oxalic acid, uric acid, propionic acid and butyric acid. Organic acids have been used for decades in commercial compound feeds, mostly for feed preservation, for which formic and propionic acids are particularly effective. Since the ban of antibiotic growth promoters in many countries, organic acids have been used increasingly, not only because of their preservative qualities, but also for their nutritive properties. Organic acids and their salts are generally regarded as safe and have been approved by most member states of EU to be used as the feed additives in animal production.