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Carnation (Dianthus caryophyllus L.) belonging to the family Caryophyllaceae is very remarkable as a cut flower crop all over the world. It is well known for its attractive flowers with fragrance and varying colors. In India, the area under its cultivation is 2190 ha with a production of 8940 tones. But its production is affected by a number of insect pests and spider mite.
It is the two spotted spider mite or red spider mite which is considered as one of the most serious pests of carnation. High temperatures and low relative humidity favor the multiplication of these mites. These mites are found to be persisting throughout the year under polyhouse conditions.
Host range
Citrus, grapes, apples, pears, tomatoes, okra, potatoes, eggplant, beans, cucumbers, peppers, roses, jasmine, tea, coffee etc.

Lily (Lilium spp.) is a high-value bulbous ornamental plant prized for its large, attractive, and often fragrant flowers. Belonging to the family Liliaceae, lilies are widely grown for cut flowers, landscape decoration, and potted plant markets. Despite its beauty, lily cultivation is often challenged by several insect and mite pests that reduce aesthetic quality, market value, and bulb health.
Biology: Eggs are laid in clusters or singly on the undersides of lily leaves. They are yellowish to pale white, round, and smooth. Incubation lasts for about 3–6 days, depending on temperature and humidity. There are typically 5–6 larval instars. Newly hatched larvae are small and yellowish, later turning bright orange-red with black bands and spots. Caterpillars feed gregariously at first, skeletonizing leaves, then disperse to bore into stems or bulbs in later stages. The larval stage lasts for about 12–20 days. Pupation occurs in the soil, where larvae form a silken cocoon often mixed with soil particles. The pupal stage lasts 7–14 days. Pupae are reddish-brown and cylindrical. The adult is a medium-sized, metallic blue-black moth with bright yellow bands on the forewings and orange markings on the thorax. Adults are nocturnal and feed on nectar. Adult lifespan is around 4–6 days, during which female mate and lay eggs (Sathe, 2015).

Turfgrass refers to a group of grass species cultivated to form a dense, uniform ground cover, primarily used in lawns, golf courses, sports fields, parks, and landscape areas. Turfgrass serves both aesthetic and functional purposes, including erosion control, temperature regulation, and dust suppression. Turfgrass ecosystems provide a favorable habitat for various insect pests and mites that affect roots, stems, and leaves, compromising turf quality, appearance, and playability.
Distribution: Andhra Pradesh, Tamil Nadu, Maharashtra, Gujarat, Punjab, Uttar Pradesh, Odisha, and other regions, Mythimna separata (oriental armyworm), is more prevalent in the northern and eastern states.
Biology: Female moths lay eggs in clusters, often between leaf sheaths and blades, especially on dry grass. The eggs are initially pale yellow and darken before hatching. The larvae, also known as armyworms, go through several instars, feeding on grass blades.

Location and Layout
• As the laboratory will be handling materials hazardous to human and animal health, it must be isolated as much as possible from livestock yards, poultry farms and quarantine stations and should not function in or near establishments handling healthy animals, food products for human or animal consumption or near vaccine production laboratories.
• Animal houses, garages, workshops, stores, water tanks, residential accommodation, administrative blocks have to be constructed as per the need of ideal microbiological laboratory. Expansion possibilities should
also be kept in mind. The building can be arranged in 5 main groups:
• Administrative / laboratory rooms
• Decontamination and disposal area
• Animal house facilities
• Workshops / stores/garages and
• Residential accommodation for the staff, hospital, schools, playgrounds, parks, shops etc. • The laboratory building may be rectangular in blocks, arranged in L, T or U shapes and may be of one or several stories. The temperature, wind velocity, rainfall,

On the face of an outbreak of a disease, collection of right clinical specimens in proper preservative, their transportation to the laboratory under ideal conditions and quick processing of specimens in the laboratory to identify the causative agent are crucial to successful control of the disease. This attains more importance when a highly contagious disease appears.

Collection of specimens from a virus-infected host is the first step for demonstration or isolation of a virus. Specimens for virus isolation should be collected at the time when the maximum amount of virus can be expected to be present. i.e. collect the specimens as early as possible after the onset of clinical symptoms. In many viral diseases maximum viral production occurs before the patient becomes ill so that it is ideal to collect the specimen at an even early time.
The nature of the specimen taken in any particular instance is determined by the patient’s clinical syndrome and previous history. They are best considered under three main headings.

1. Specimen for virus isolation/antigen detection/ nucleic acid detection
2. Specimen for serological investigation
3. Specimen for direct examination

The chief functions of Clinical Microbiological laboratory are to examine and culture specimens for microorganisms, to make accurate species identification of important isolates and to perform antibiotic susceptibility tests when indicated. These tasks will assist physicians in the diagnosis and treatment of infectious diseases.

Once the specimen is received in the laboratory, the specimens are examined visually depending on the physicians order and the nature of the specimen. Wet mounts and smears may be prepared and stained for microscopic examination. Observations may or may not be immediately reported to the physicians depending on the definitiveness of the results. Timely information may often be used to establish a presumptive diagnosis and institute a specific course of therapy. Specimens that require definitive identification of potentially pathogenic microbes are processed further. All agar plates are streaked for colony isolation; then plates and broths. All cultures are placed in an incubator with appropriate temperature and environmental conditions to maximize the growth and replication of microbes. Often a presumptive microbial identificationcan be made. A final report should be delayed while subcultures and additional test procedures are performed to identify the organisms definitively.

Preliminary Identification of Unknown Bacteria
A number of techniques may be used in the direct microscopic examination of clinical specimens to demonstrate the presence of microbes The examination of wet mounts of unstained materials by phase contrast or
dark field microscopy is useful for demonstrating motility, spirochaetes and endospores. Giemsa, Wright or acridine orange stains may be helpful in observing bacterial forms that stain poorly or that have little contrast from
back ground material.

Special Staining
A. Spore stain
Spores may be spherical or oval and may be central subterminal or terminal in position within the cell. Shape, size and position of the spore in the cell are useful for the identification of sporulated organisms. Spores are difficult to stain and when once stained, they are difficult to decolourize. With Gram’s stain, mature spores remain unstained bodies. Modified Ziehl-Neelsen’s method of staining will be used for the staining of spores.
Composition
a. Concentrated carbol fuchsin
b. Sulphuric acid 0.5 % solution.
c. Loeffler’s alkaline methylene blue
Materials
Old cultures of B. subtilis and Cl. Chauvoei and staining solutions.
Procedure
1. Make smears and fix over flame.
2. Stain with carbol fuchsin and steam for 3 to 4 minutes.
3. Wash with tap water.
4. Decolourize with 0.5% sulphuric acid.
5. Wash with water.
6. Counter stain with Loeffler’s methylene blue for 2 minutes.

Antimicrobial Susceptibility Test
The routine use of the modified Kirby-Bauer method is recommended.
Material
• Sterile cotton swabs
• Soyabean – Casein digest broth or Tryptone soya broth
• Non selective agar such as nutrient agar.
• 0.5 Mc Farland turbidity standard:
Solution A
Bacl2, 2H2O. 1.75G
Make upto 100 ml with distilled water.
Solution B
H2SO4 1.0 ml
Make upto 100 ml with distilled water.
Stock Solution
0.5 ml of solution A + 99.5 ml of solution B. Dispense in 5 ml amounts.
Seal and store in dark.
• Glass vials or tubes 10 ml capacity
• Muller – Hinton agar
• Antimicrobial discs
• Sterile forceps
• Ruler or calipers

Viruses can be cultivated only in living cells. Laboratory animals, embryonated avian eggs and cell cultures are three systems available for this purpose. These three systems are used for the basic procedures of isolation and identification of viruses, maintenance of stock cultures and production of vaccines.
Cultivation of Viruses in Laboratory Animals
Animal inoculation helps to confirm the infectious nature of the disease. Laboratory animals used for many kinds of virological works such as clinical studies, pathogenicity of viruses, epizootiology, potency tests on vaccines and production of diagnostic and prophylactic antisera.

An ideal experimental animal is one that is
1. Uniformly and fully susceptible to the virus under study
2. Free from intercurrent infection and adventitious lesions and
3. Inexpensive purchase and maintain
If possible it is better to use germ free, specific pathogen free or colostrums deprived animals for experimental purpose. The same species of animals or atleast closely related species of the natural host is selected for inoculation, wherever possible. For example in case of swine fever, which shows a high degree of host specificity, the same species should be used for inoculation. in others such as in case of rabies with low host specificity, the causative virus will grow in a wide range of animals.

Molecular biology involves the study of DNA, RNA and proteins in cells. It emphasizes the biomolecules that carry out biological processes. One should understand the biomolecules participating in the process to understand the cellular function. In diagnostic microbiology, biomolecules are the analytes for testing and characterization. The analytes for molecular testing are the genomes (DNA), transcriptomes (RNA) and proteomes (Proteins) of the microorganism. Traditional methods of microorganism characterization depend on the culturing of the organisms, which is time-consuming and sometimes challenging to perform. Molecular techniques involve the characterization of DNA, RNA and protein to identify microorganisms. Nucleic acid sequence details are used to classify bacterial species, and mass spectrometry is used to identify microorganisms based on peptide profiles.

DNA (Deoxy ribonucleic acid)
DNA contains the genetic information required for the development and functioning of the living organism. It is the central informational molecule comprising a sequence of A, T, G and C nucelotides linked together by a phosphodiester bond. DNA is double helical in structure, composed of two complementary strands of nucleotides held together by hydrogen bonds between nitrogenous bases.

Precipitation and agglutination reactions are similar except in that for precipitation reaction antigen must be in soluble form. The precipitation reaction is highly specific and very sensitive for the detection of antigen being capable of demonstrating antigens in dilutions of 1:100,000 to 1: 1,000,000 or more. It is important to bear in mind that antigens used in precipitation reactions are of molecular size not cellular. A soluble antigen reacted with its homologous antibody at the optimum proportions produce an insoluble precipitate. The essential ingredients for precipitation test are soluble antigen (precipitinogen), antibody (precipitin) and an electrolyte (normal saline).
a) Precipitation ring test
The antigen is carefully layered over the antiserum, without mixing so that an interface is formed. Diffusion of each reagent will then occur into the other. If the system is homologous, precipitation will occur at the point in the tube, where the proper ratio of antigen to antibody is reached. Eg: Ascoli’s test for anthrax diagnosis.
In advanced decomposition of animals when cultures would be almost impossible, the precipitation method of Ascoli is used for diagnosis of Anthrax. This method is a mixing of immunized rabbit serum with an extract of organs under examination for the production of a precipitate.
Procedure
1. Grind up organs or blood of suspected animals.
2. Suspend in saline.
3. Boil for five minutes
4. Filter through filter paper and allow to cool
5. Carefully stratify 0.5 ml of this extract on 0.5 ml immune serum in small test tubes.

Among various immunodiagnostic tools enzyme immunoassay becomes increasingly popular for the detection of humoral immune response (seroconversion) or microbial antigens for the purpose of diagnosis. Enzyme Linked Immunosorbent Assay (ELISA) is widely used in studies of infectious diseases.Despite several merits of ELISA, it has some limitations such as requirement of ELISA reader to obtain accurate measurements. To overcome this, the dot immunobinding assay was developed .In Dot Immunoassay nitrocellulose membrane is used as an adsorbent matrix instead of plastic surface.The antigen is dotted onto the nitrocellulose membrane and the membrane then incubated with test antibody, and secondly with enzyme conjugated antibody directed against the first antibody.In case of positve
reaction, (specific antigen –antibody reaction) color dot is developed against the white background at the site of antigen deposition.In case of negative reaction, no color development takes place at the site of antigen deposition .
Principle
• In direct ELISA antibody is directly attached to solid phase and targeted by added antigen (detecting antigen).
• These added antigens are targeted by antispecies antibodies linked to enzyme termed as conjugates.
• The bound enzyme is developed by the addition of substrate /chromogen, then stopped and finally read using a spectrophotometer

Principle
The principle of the test is that antibodies possess two antigen-binding sites that will attach specifically with the antigenic determinants on the surfaces of the bacteria, cells or particles. Under suitable conditions one antibody molecule will combine with the determinant group on the surfaces of two bacteria and in this way a lattice is formed. These lattices are generally visible with the naked eye as clumps and they sediment readily due to the large size of the clump.
Pre-requisites for Agglutination Reaction
a) The antigen (agglutinogen) should be in particulate form, which will remain in suspension for sufficiently long time.
b) The antibody (agglutinin) should be directed to the target surface epitope.
c) Antigen – antibody should be present in optimal proportions.
d) An electrolyte is necessary for the reaction to take place and usually the reaction is carried out in physiological saline solutions (0.85% NaCl). Because of the overall negative charge on red cells or bacteria at neutral pH it is difficult for them to come close enough, so that for molecule such as IgG antibodies are able to form a bridge between epitope of two different cells or bacteria. The presence of salt tends to neutralize the charge effects to allow agglutination.
Plate Agglutination Test
This is employed to screen sera within a short period for preliminary testing.
Materials
• Brucella abortus coloured antigen
• suspected serum
• glass plate.

Principle
Some viruses like Orthomyxo, Paramyxoviruses etc. have the property to clump the erythrocytes of certain species of birds or animals. This is not an antigen-antibody reaction but a network of erythrocytes formed by the bridging virions. These virions on their envelop have peplomers that binds with the receptors on the erythrocytes. If large quantities of virus are used for HA and allowed to act on red blood cells
for a long period of time, the agglutinated red cells shed the virus particles which then float freely in the suspending fluid and neither attach to the same red cells nor agglutinate them. This process of dissociation of virus from the red cells is known as elution, and is due to the destruction of receptors on red cells by the virion associated receptor destroying enzyme - neuraminidase. The red cells freed from the virus particles are known as stabilized red cells. The elution will occur most rapidly if the temperature is raised to 37°C as the enzyme neuraminidase will be more active at this temperature.

Practical Application of Haemagglutination
a) Spot test: For rapid detection of the presence of a haemagglutinating virus. This can be done by mixing two or three drops of suspected sample (infected chick embryo fluids or cell culture fluids) with equal quantity of 5% chicken red cells in normal saline. The appearance of the clumps of agglutinated red cells, indicate the presence of a haemagglutinating
virus in the sample.
b) Titration of the virus using the haemagglutinating property.

Microbiology is the study of microorganisms or microbes. They are very small in size and hence cannot be seen with naked eyes. Microorganisms are excellent models for understanding cell function in higher organisms, including humans. Hence the science of microbiology is the foundation of all the biological sciences and microorganisms consist of bacteria, fungi, algae, protozoa and viruses. The fields according to the organisms studied are henceforth termed as:
Bacteriology - Bacteria (singular: bacterium)
Phycology (phyco, seaweed) - Algae (singular: alga)
Mycology (myco, a fungus) - Fungi (singular: fungus)
Protozoology (proto, first; zoo, animal) - Protozoa (singular: protozoan)
Virology - Viruses
Microbiology began when people learned to make lenses from glass and combine them to produce magnifications great enough to see microbes. The importance of bacteria in the lives of people, animals and the environment has been unfolded in the past 100 years. It was also believed that the incidence of the disease was due to divine reasons since man had no idea of microbes at this point of time. In the earlier days, leprosy was considered as a dangerous infectious disease and spread of the disease was attributed to direct contact with the affected persons. Hence, certain procedures were specified for
segregation of the affected persons. At this point of time various theories were also attributed to the genesis of disease. Some of the important theories are as follows:
1. Theurgical theory – Disease was attributed to the wrath of divine spirits as a punishment of individual sins.
2. Miasmatic theory – This theory was proposed by Hippocrates and further developed by his student Galen. It states that the disease was dueto the emanations from the

Classification of Organisms
Over the years, scientists have developed several systems for the classification of organisms. Greek phlosopher Aristotle (384-322 BC) grouped life forms as either plants or animals. Microscopic organisms were unknown at that time. Fungi were included in plants. Aristotle’s system distinguished only between plants and animals on the basis of movement, feeding mechanism, and growth patterns. In 1735, Carolus Linnaeus formalized the use of two Latin names to identify each organism, a system called Binomial nomenclature. He grouped closely related organisms and introduced the modern classification groups: kingdom, phylum, class, order, family, genus, and species. At that time singlecelled organisms were observed but not classified. This classification had two kingdoms.
Plantae: containing Plants and Fungi
Animalia: containing Animals
In 1866, Ernst Haeckel proposed a third kingdom- Protista, to include all single-celled organisms like amoebas and diatoms. Some taxonomists also placed simple multicellular organisms, such as seaweeds, in Kingdom Protista.
In 1938, Herbert Copeland proposed a fourth kingdom, Monera, to include only bacteria. This was the first classification proposal to separate prokaryotes from eukaryotes, at the kingdom level.
In 1957, Robert H. Whittaker proposed a fifth kingdom –Fungi.Fungi do not ingest food as animals do, nor do they make their own food, as plants do. They secrete digestive enzymes around their food, breaking it down before absorbing it into their cells.

1. Morphology of Bacteria
Structure of Bacteria
Bacteria are very small with a diameter of 0.5 – 1 μm in size. Due to this small size the surface area / volume ratio is very high compared to that of large organisms of similar shape. The large surface area compared to a small volume facilitates easy nourishment of all areas of the cell. So there is no circulatory mechanism to distribute the nutrients and there is little or no cytoplasmic movement within the cell.
i) Shape
Bacteria exhibit three types of morphology;
• Spherical - cocci (coccus) - Greek word kokkos, meaning a berry
• Straight rods - bacilli (bacillus) - Latin word bacillus, meaning a stick
• Helically curved rods -spirilla (spirillum)
Variations in these basic forms are also seen. Eg: cocobacillary, ovoid and filamentous.

First discovered by Scottish surgeon sir Alexander ogston (1880) in infected tissues. He named it as staphylococcus” (Greek staphyle, bunch of grapes;
KOKKAS, berry).
Systematics
Domain : Bacteria
Phylum : Firmicutes
Class : Bacilli
Order : Bacillales
Family : Staphylococcaceae
Genus : Staphylococcus
Species : Staph. aureus
Staph. Intermedius
Staph. hyicus
Habitat and Ecology
Staphylococcus occurs worldwide in mammals although the spread of staphylococcal strains between different animal species is limited. They colonise the nasal cavity, skin and mucous membranes and can be transient in the intestinal tract. It is an opportunist type organism.

Systematics
Domain : Bacteria
Phylum : Firmicutes
Class : Bacilli
Order : Lactobacillales
Family : Streptococcaceae
Genus : Streptococcus
Species : Str. agalactiae, Str.dysgalactiae, Str. equi subsp. zooepidemicus, Str.
uberis, Str. equi subsp equi, Str.suis, Str. canis, Str. pyogenes (human)
History
Rivolta (1873) described chain forming organisms in pus from a case of strangles in horses. In 1878-79, Pasteur recognized this organism as a pusforming agent. In 1903, Hugo Schottmuller introduced blood to differentiate various types of hemolysis. In 1928, Rebecca Lancefield reported a serological method of grouping Streptococci.
Habitat
Streptococci are worldwide in distribution. Most of the Streptococci of Veterinary interest live as commensals in the mucosa of the upper respiratory and lower urogenital tracts. They do not survive for long away from the animalhosts.

B.anthracis causes anthrax in animals and Wool sorter’s disease, hide porter’s disease, Malignant Pustule in humans. B.cerus causes food poisoning in humans. Other bacillus in this group is non-pathogenic and they are called as anthracoids. B.licheniformis is an emerging pathogen and it is implicated in sporadic abortions in cattle and sheep.
Systematics
Domain : Bacteria
Phylum : Firmicutes
Class : Bacilli
Order : Bacillales
Family : Bacillaceae
Genus : Bacillus
Species : B. anthracis, B. cereus, B. subtilis, B. mycoides,
B. megaterium, B.mesentricus
Family Characters
They are gram +ve large rods, aerobic (facultative anaerobic), endospore forming, capsulated, mostly catalase positive and fermentative organisms. They are motile by peritrichous flagella.
Bacillus Anthracis
History
• Discovery of the anthrax bacillus is credited to Davaine and Rayer (1863 –1868).
• Considerable historic interest attached to anthrax bacilli;