eChapters

A laboratory is essential for research work in science and development. Most of the research institutes have dedicated laboratories for researchers and students to conduct experiments, perform research work, as well as to provide hands-on learning/training to students. There are certain rules and regulations laid down by the laboratory in-charge which need to be followed strictly for safety purposes. Some of the laboratories keep ‘safety manual’ for researchers to read before entering into the laboratory to conduct experiments. Whenever safety is compromised in a laboratory, there is increase in risk of unwanted incidents in the laboratory. Storing food/beverages in the same refrigerator where laboratory chemicals are kept may lead to disastrous effects on health. This chapter aims to provide instructions in brief on the things to be performed (Do’s) before and after entering onto a laboratory as well as the things never to be done (Don’ts) in a laboratory for the safety reasons.

Good Laboratory Practices (Do’s)

• P Wear apron/labcoat while working in the laboratory.
• P Use gloves/respiratory mask/fumehood while handling toxic/volatile/inflammable chemicals/reagents.
• P Use pippet or pippetor for safety in transfer of solutions.
• P Protect eyes, face and naked parts of the body from UV radiations. Use safety goggles or face mask/apron.
• P Keep your eyes away from Liquid Nitrogen and use cryogloves while handling it.
• P Arrange all the necessary materials/solutions before starting the experiment.
• P Periodically fumigate/ozonize culture and inoculation rooms.
• P Autoclave all contaminated materials before discarding.
• P Pre-treat gel or solution containing Ethidium Bromide (EtBr) before discarding into laboratory waste-disposal bin, and use gloves while handling them.
• P Burn all the transgenic plant materials in an incinerator after taking necessary observations.
• P Make entry in the designated log-book and clean the instrument after using it properly.
• P Switch-off the instrument after use or when not in use.
• P Wash hands with soap and remove the labcoat before leaving/going out of the laboratory7.
• P Ensure that switches for fans/lights, and water taps are off while leaving the laboratory.
• P Follow the biosafety guidelines suggested for handling and disposal of the hazardous chemical.

Introduction
Haemoprotozoan diseases are highly prevalent in tropical and sub-tropical countries owing to the ubiquitous distribution of ticks and blood-sucking flies which are natural vectors for the pathogens. In India, theileriosis, babesiosis and trypanosomosis are major haemoprotozoan diseases and cause substantial economic losses to the livestock industry. Though vaccination of the susceptible stock by attenuated schizont vaccine is available for the control of tropical theileriosis in cattle, chemotherapy is the mainstay for the control of haemoprotozoan diseases. However, the limited available antihaemoprotozoan drugs is being depleted by a combination of two factors viz. emergence of drug resistance and the failure to replace old drugs (de Koning, 2017). Drug resistance against parasites is one of the emerging problems affecting veterinary health sector. Since last few decades, development of drug resistance in haemoprotozoan parasites to several drugs has been reported (Tuvshintulga et al., 2019; Ali et al., 2022). Though, understanding of drug resistance in parasitic protozoa is progressing steadily but possibly not as rapidly as parasites are becoming resistant to drugs. The genetic changes in the target sites, the reduced uptake and increased efflux of drugs, and metabolic regulations are the possible mechanism for the cause of drug resistance (Fairlamb et al., 2016). Anti-protozoan drug resistance has been reported in many economically important protozoa to isometamedium, diminazene aceturate, quinapyramine, suramin, melarsomine dihydrochloride, buparvaquone. The resistance initially evolves at genetic level of parasite with genetic traits that allow them to survive exposure to a drug, which is transferred to the subsequent generation thereby potentially increasing the percentage of resistant parasite that can survive subsequent exposure to the chemical. The emergence of drugresistant parasites has serious implications for treating clinical cases, leading to increased morbidity and mortality among the animals and control of the diseases.

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

Introduction

While experimenting with chemicals, sometimes we need to dry the chemical/ solution (solid, liquid or gas) using a process like evaporation or distillation, mainly to remove water or different solvents. Drying is the process of evaporation or distillation to remove water/solvent from a solution, solid or even from gas. Generally, a drying agent (usually an anhydrous inorganic salt) is used for this process, which reacts with the water (present in the chemical/ solvent to be dried) to form a hydrate. For example, anhydrous MgSO4 is one of the drying agents. The drying agents are recognized by their capacity (the rate at which they absorb water) and their intensity (the amount of water left behind in the solvent at equilibrium). No chemical reaction or change in phase occurs during the drying process.

Type of Solvents

Apart from water (a universal solvent), there are three families of organic solvents used in laboratories.

1) Oxygenated solvents: Alcohols, Ketones, Glycol ethers, Glycol esters, etc.

2) Hydrocarbon solvents: Aliphatic and Aromatic hydrocarbons.

3) Halogenated solvents: Chlorinated hydrocarbons.

Mould growth in raw materials or finished feeds result in production of mycotoxins. Mycotoxins are a large group of toxic chemicals causing wide range of pathological and physiological effects in animals. Symptoms depend on the amount of toxin present in the feed, the period for which the feed is ingested, the nutritional status of the feed and the susceptibility of the animal. Feed efficiency, growth rate and production are affected in animals consuming sub-acute doses.

Ruminants are generally considered less susceptible to mycotoxins than monogastric animals because of degradation of mycotoxin in rumen by rumen microorganism. However, certain mycotoxins resist rumen degradation and cause clinical mycotoxicosis. In high yielding dairy animals, during transition period, animals who are in negative energy balance (NEB), low body condition score and decreased resistance to infectious agents cause adverse effects in animal health with exposure of mycotoxins present in animal diet.

Effect of mycotoxin in ruminants

In cattle during metabolism, mycotoxin present in stored or ensiled feeds produce microbial volatile organic compounds (MVOCs) which are responsible for mouldy odour, reduced feed intake and increased feeding times. This insufficient feed intake further produces negative energy balance (NEB).

The ingestion of mycotoxin-contaminated feedstuffs by animals leads to adverse effect on animal health and the effects are more serious in monogastric animals depending on the species and the susceptibility to toxins within the species. Ruminants are less susceptible to mycotoxins than monogastric animals because of biotransformation of mycotoxins to non-toxic metabolites in rumen by rumen microbiota (Fink-Gremmels, 2008).

Introduction
India with a population of 1.30 billion people is highly focusing on “Development”i.e. good food, better health & living conditions for everyone. With the increase in the incomes, people can now afford better nutrition in the form of poultry egg and meat, now poultry industry has transformed from a mere backyard activity into a major commercial activity in just four decades. India is now the world’s 3rd largest egg producer and the 5thlargest producer of broilers. The Indian poultry market witnessed an increasing trend over the past five years, growing at a Compound Annual Growth Rate (CAGR) of 8.6% (DAHD, 2023). Poultry sector in India is valued at about Rs. 1,50,000 crore (2022-23) broadly divided into two sub-sectors – one with a highly organized commercial sector with about 80% of the total market share (say, Rs. 1,20,000 crore) and the other being unorganized with about 20% of the total market share of Rs. 30,000 Crore. (DAHD, 2023). Poultry production in India has become a profitable and most popular income generating sector for the educated unemployed youth. Most of the poultry farmers are interested in broiler production due to its quick returns, less space requirement and higher weight gains. The productive potential of poultry in India has not been fully exploited due to deficit feed resources and unutilization of available improved technologies for getting high productivity from the poultry at economical rate. Hence, it is essential to further enhance the feeding value of available feed resources so as to improve the efficiency of feed utilization and minimize the cost of feed per kilogram live weight gain. The use of antibioticas growth promoters in poultry has been banned due to concern about their residues in tissue and induction of bacterial resistance. Due to these concerns, recently many feed additives has been investigated for alternatives tofeed antibiotics. The international feed industry is facing the challenge of the awareness among the consumers of meat on the risk of bringing about antibiotic resistance in pathogenic microbiota through antibiotics used in animal and poultry feeds. It has directed them towards the non-antibiotic feed additives. Among them, the feed additives of plant origin, called as Phyto-additives or Phytogenic Feed Additives (PFA) or Phytobiotics are considered to be a better alternative as non-antibiotic growth promoters, even though there are well established non antibiotic growth promoters such as organic acids and probiotics. The Phytogenic feed additives also vary widely in their botanical origin, processing and composition. They have been used in solid, dried and ground forms or as extracts or essential oils.It is reported that aromatic plants may increase feed intake and may improve secretion of endogenous digestive enzymes. It has been shown thatthe dietary incorporation of herbs may provide beneficial effect on poultry performance and health due to the antimicrobial activity of their phytochemical components. Phytobiotics have gained increasing interest as natural growth promoting feed additives in broiler production in recent years. These have wide range of medicinal properties with no residual side effects and are best alternatives to antibiotic growth promoters (Chaudhary et al., 2018, 2019, Rahman et al., 2014). Beneficial effects of these substances in poultry nutrition are due to their high content of pharmacologically active compounds stimulating appetite and feed intake, improving endogenous digestive secretion and activating immune responses (Nouzarian et al., 2011 and Toghyani et al., 2010).

Introduction
A microorganism or microbe is a tiny living thing that can exist as a single cell or in groups of cells. People have suspected the existence of these unseen microbes since ancient times. These single-celled creatures have caused serious infections that often led to deaths and illness. These microorganisms can spread quickly and cause big outbreaks of disease. In 1928, Alexander Fleming discovered the first antibiotic, penicillin. This discovery started the development of one of the most important groups of medicines in medical history. In the next three decades, many different types of antimicrobial drugs were developed. In his Nobel Prize speech, Fleming warned that using penicillin too much might one day lead to bacteria becoming resistant to it, which would be a major problem. Antimicrobial resistance (AMR) is one of the biggest public health challenges today. Many of the microbes that cause diseases, like bacteria, viruses, and protozoa, no longer respond to common antimicrobial drugs such as antibiotics, antivirals, and antiprotozoals. Antimicrobial resistance is when a microbe becomes able to survive and grow even when treated with medications that used to work on it. Antibiotic resistance is a type of AMR, but it only applies to bacteria becoming resistant to antibiotics. Resistant microbes are harder to treat, and doctors may need to use other medicines or higher doses. These options can be more expensive and have more side effects. Microbes that resist multiple antimicrobials are called multidrug resistant (MDR). Those that are resistant to almost all drugs are sometimes called ‘superbugs’. Antimicrobial prophylaxis is used by doctors to prevent infections, but it should only be used when it’s really needed to avoid unnecessary costs, side effects, and the development of resistance. Antimicrobial prophylaxis can be used to prevent a first infection or to stop an infection from coming back or reactivating.

Aetiology
Classification of the causative agent • Ehrlichia ruminantium (formerly Cowdria ruminantium) Order Rickettsiales, Family Anaplasmataceae
• Small, Gram negative, pleomorphic coccus, and obligate intracellular parasite.
• Strains of E. ruminantium are very diverse and vary in virulence: while some strains are highly virulent, others appear to be less-pathogenic.
• E. ruminantium has a high level of genomic plasticity. Several different genotypes can co-exist in a geographical area, and may recombine to form new strains.
• E. ruminantium multiplies in vascular endothelial cells throughout the body to cause severe vascular compromise.
• It usually occurs in clumps of from less than five to several thousand organisms within the cytoplasm of infected capillary endothelial cells, and can be detected in brain smears by light microscopy.
Resistance to Physical and Chemical Action
Temperature
Heat labile and loses its viability within 12–38 hours at room temperature. Infective stabilities can be cryopreserved in DMSO (dimethyl sulphide) or better yet in sucrosepotassium phosphate-glutamate medium (SPG). Infective half-life of thawed stabilate kept on ice is only 20–30 minutes.
pH: Not applicable.
Disinfectants: Not applicable.
Survival:

Introduction

Proper electrical wiring and earthing are crucial for laboratory safety and functionality. A laboratory houses several installations/equipment that demand stable/continuous power supply. Inappropriate or faulty wiring and earthing may lead to significant risks, including electrical shocks, damage to equipment, and even incidence of fire. Therefore, understanding the requirements of electrical wiring and earthing is essential for designing, construction, and maintenance of laboratory. This chapter aims to provide a basic knowledge of electrical wiring and earthing necessary for a laboratory. Importance of earthing in mitigating electrical hazards and ensuring integrity of electrical installations are also described.

Basics of Electrical Wiring

Basic understanding of electrical wiring is necessary for laboratory safety and efficient electrical functioning. The potential difference between two electrical points (negative and positive) is known as Voltage, which drives the flow of electric current. This is measured as volt (V). The rate of flow of electricity in a circuit is known as Current (I), which is measured as ampere (A). Current can be either direct (DC) or alternating (AC). Opposition/resistance experienced in the flow of current in circuit is known as Resistance (R) and it is measured in ohms (Ω). Higher the resistance means lesser the current flows at a given voltage. The rate at which electrical energy is transferred/utilized in an electric circuit is known as Power (P), which is measured in watt (W). The Power can be calculated as the product of voltage and current (P = V × I).

A. Answer the following.

Q.1.     What are the different phagocytic cells ?
Ans.     The phagocytic cells of mammals belong to two complementary systems (1) The myeloid system consists of cells that are rapidly phagocytic but incapable of sustained effect. (2) Mononuclear phagocytic system act more slowly but capable of repeated phagocytosis.

Q.2.     How 3 types of granulocytes are differentiated ?
Ans.    Three types of granulocytes are distinguished by the staining property of their granules. Cells whose granules take up basic dyes such as haematoxylin (a blue dye) are called basophils, whose granules take up acidic dyes such as eosin (a red dye) are called eosinophils and those that take up neither basic nor acidic dyes are called neutrophils.

Emerging Infectious Diseases
Emerging infectious diseases are those due to newly identified and previously unknown infections which cause public health problems either locally or internationally.
Re-emerging Infectious Diseases
Re-emerging infectious diseases are those due to the reappearance and increase of infections which are known, but had formerly fallen to levels so low that they were no longer considered a public health problem.
What Causes Emergence or Re-emergence of Infectious Diseases?
Several factors contribute to the emergence and re-emergence of infectious diseases, but most can be linked with the increasing number of people living and moving on earth: rapid and intense international travel; overcrowding in cities with poor sanitation; changes in handling and processing of large quantities of food; and increased exposure of humans to disease vectors and reservoirs in nature. Other factors include a deteriorating public health infrastructure which is unable to cope with population demands, and the emergence of resistance to antibiotics linked to their increased misuse.
Avian Influenza
Avian influenza is a zoonotic, globally important disease of birds that can be categorised as either low pathogenic (LPAI) or highly pathogenic (HPAI) according to the virulence of the virus in animals. Outbreaks of LPAI are common around the world but LPAI typically causes no clinical signs or only minor illness in infected birds. LPAI strains are generally less of a threat to human health; in addition, LPAI can have significant economic repercussions as a result of export restrictions and culling of birds, particularly in developing countries where the use of vaccines and other veterinary medicines is difficult
due to weak veterinarian services and small farming units. Compared with LPAI, HPAI is more readily detected in poultry due to very high levels of mortality in infected birds, and can cause potentially catastrophic economic consequences, from significantly reduced livestock populations to lost export markets. Though relatively rare, sporadic human infections of HPAI have occurred in cases of close contact with infected birds and caused serious illness
and even death.

Emerging Infectious Diseases
Emerging infectious diseases are those due to newly identified and previously unknown infections which cause public health problems either locally or internationally.
Re-emerging Infectious Diseases
Re-emerging infectious diseases are those due to the reappearance and increase of infections which are known, but had formerly fallen to levels so low that they were no longer considered a public health problem.
What Causes Emergence or Re-emergence of Infectious Diseases?
Several factors contribute to the emergence and re-emergence of infectious diseases, but most can be linked with the increasing number of people living and moving on earth: rapid and intense international travel; overcrowding in cities with poor sanitation; changes in handling and processing of large quantities of food; and increased exposure of humans to disease vectors and reservoirs in nature. Other factors include a deteriorating public health infrastructure which is unable to cope with population demands, and the emergence of resistance to antibiotics linked to their increased misuse.

Introduction

The antibiotics are low molecular weight natural, semi-synthetic, or synthetic substance that kills or slows the growth of germs while causing little or no harm to the host is considered an antimicrobial (Walsh, 2000). Antibiotics are the type of medicine produced by some microorganisms that kill or slow the growth of other microorganisms. It includes any of a large group of chemical substances, as penicillin or streptomycin, produced by various microorganisms and fungi, having the capacity in dilute solutions to inhibit the growth of or to destroy bacteria and other microorganisms, used chiefly in the treatment of infectious diseases (De Vetten et al., 2003). However today, a large number of bacteria are resistant to various antibiotics. Antibiotic resistance occurs when a bacterium becomes able to survive and multiply despite the presence of an antibiotic that would normally control or kill it. In other words, the bacteria become “resistant” and continue to grow even at therapeutic levels of the antibiotic. While antibiotic resistance is a natural phenomenon, it is accelerated by the selective pressure exerted when an antibiotic is used. Bacteria that are naturally resistant to the antibiotic have a better chance of survival compared to those that are susceptible. As the susceptible bacteria are killed or inhibited, resistant strains are favored and continue to thrive (Holme et al., 2006). Some resistance arises without human influence, as bacteria can naturally produce antibiotics against other microorganisms, resulting in a low-level, natural selection for antibiotic resistance. However, the current surge in antibiotic resistant bacteria is largely attributed to the overuse and misuse of antibiotics. One of the main issues facing public health is antimicrobial resistance, particularly in developing nations where access to and use of medications are easier than in developed nations, leading to a disproportionately higher rate of inappropriate antibiotic use and higher resistance levels (Ganguly et al., 2011). Antimicrobial resistance will make it more difficult to control infections in the community and make health care services less effective (Bhatia et al., 2010). It is well known that India has one of the highest rates of infectious disease burdens in the world, and that the country’s high rates of malnutrition and inadequate sanitation make these circumstances worse (http://www. resistancestrategies.org/wpcontent/uploads/2011/03/Indiareport).

Introduction
Antimicrobial resistance (AMR) is a critical public health problem globally, which can shake the foundation of modern healthcare. Infections caused by drugresistant organisms could lead to increased mortality and prolonged duration of hospitalization, causing a huge financial burden to the affected persons, healthcare systems and hinder the goals of sustainable development. With rising AMR, antibiotics are increasingly becoming ineffective for treating diseases.The post antibiotic era is not a dreadful imagination but a potential reality unless effective global actions are initiated now (1). AMR is not the only problem of human health, it widely affects the animal husbandry as well as environment. In India in 2019, there were 297,000 deaths attributable to AMR and 1,042,500 deaths associated with AMR. India has the 145th highest age-standardized mortality rate per 100,000 population associated with AMR across 204 countries.The number of AMR deaths in India is higher than deaths from neoplasms, respiratory infections and tuberculosis, enteric infections, diabetes and kidney diseases and maternal and neonatal disorders.Out of 10 million projected human deaths in 2050, one-fifth may be from India (2).Annually, more than 50 000 new-born are estimated to die from sepsis due to pathogens resistant to first-line antibiotics. The median cost of treatment of a resistant bacterial infection in India is estimated to be more than a year’s wages of a rural worker. There are five pathogens to be aware of in India (number of deaths associated with AMR in parenthesis): Escherichia coli (152,700), Klebsiella pneumoniae (123,200), Staphylococcus aureus (111,400), Acinetobacter baumannii (103,500), and Mycobacterium tuberculosis (98,600).If there is no timely containment, AMR is likely to cause nearly 10 million deaths by 2050 and resulting in significant economic losses. It would also impact nutrition security, livelihood and hinder the attainment of the Sustainable Development Goals. In 1928, penicillin was developed and socalled magic drug was subsequently commercialised and used globally. When antibiotic resistance ?rst emerged, novel antibiotics to treat these bacteria were developed. However, it is dif?cult to develop effective new antibiotics every time as it is time consuming and costly and if developed a new resistance mechanism emerges in bacteria. There is a demand for new strategies to treat bacterial infections, such as endophytes therapy, phage therapy, antimicrobial peptides, CRISPER cas and so on.

Our animal companions are our family members. We want the best for them and want them to be healthy and happy. However, just as it is with everyone else, our companion animals can also fall ill and suffer from emotions such as grief, fear, and anxiety. And while we follow all expert medical advice, sometimes we may wish for something more – something that will make life easier for them as well as us. This is where the concept of energy healing for animals comes in – to comfort and support our animal companions. With joy, we watch them relax with a sigh; we see our nervous dogs calm down, and those recovering from an acute illness get back on their paws in no time. Energy healing for animals involves a holistic approach to ensure the physical, emotional, and spiritual well-being of our animal friends. It is based on the fundamental concept that just like us, our animal companions too have a subtle energy layer, and they too have vital life-force energies or Prana or Qi. And just like us, they, too, have chakras and meridians through which the Qi flows. Energy healing is a set of powerful techniques practiced to calm and restore the balance of the body. This system is largely based on the removal of energetic blocks and helps restore the body’s energy levels. It is crucial for those living with companion animals to understand the importance of energy healing for their animal companions, as it encompasses a broad range of alternative and complementary therapies designed to support the natural healing process of animals. Here are some proven, popular, non-invasive energy healing techniques that bring relief and comfort to companion animals.
1. Animal Reiki: The word Reiki means “mystical energy.” The system of Reiki for healing was revealed by Japanese Buddhist practitioner Mikao Usui in the early 1900s. And while it was initially meant for humans, practitioners soon discovered that animals responded much more swiftly and effectively to this gentle, non-invasive energy work.
2. EFT Tapping: This involves tapping on specific points on the body to free up emotional blocks. EFT Tapping for animals brings powerful transformations when an animal is struggling with emotional distress, such as grief over the loss of a loved human or animal.
3. Scalar Wave Healing: Scalar Wave was discovered in the 1900s by James Maxwell and later Nicolas Tesla. Scalar Wave harnesses the healing power of high vibrational frequencies meeting to create a standing wave, and practitioners use this to bring about healing at a cellular level.
4. Shamanic Healing: In this ancient technique, practitioners work with the elements of nature, such as plants, trees, and stones, as well as beings in other dimensions, to bring about healing. Shamanic healing can reach beyond space and time and has been very effective in releasing old trauma or emotions that are often hidden beneath layers of survival mechanisms.
5. Healing Mantra: Ayurveda, the Indian system of medicine, advocates about different types of Chikitsa. Daivavyapashraya Chikitsa is one of the treatment approaches concerned with Spiritual way of treatment. It includes so many approaches, amongst all Mantra Chikitsa is described in Ayurveda at various places as a potent approach. It is discussed for both the healthy and unhealthy conditions. Mantra comes from Sanskrit word which means sacred message or text, spell. The word Mantra is derived from Man+Tra, man—mananaat---just by chanting and tra—trayate---- we can protect ourselves. Means, just by chanting one can save himself in the universe. i.e. it is saved from onset of disturbances. Mantras are the words loaded with power and sounds capable of penetrating our body into deep levels. Mantra chikitsa is the ancient science originated from Vedas, this is a parallel science to Ayurveda also called alternative medicine system.Mantra Chikitsa is one among the Daivavyapashraya Chikitsa highlights the importance of concept of Mantra. In Atharva Veda and Kaushikasutra, the two most ancient and authentic sources of Daivavyapashraya. Mantra Chikitsa is mainly used for both preventive and curative aspect. It is also used for the enhancement of the Gunas of Aushadhi. Effect of Mantra is described as Prabhavajanya action. Prabhava means the specific and characteristic action of substances, it is called as Acintya. Mantra Chikitsa cure the Karmaja Vyadhi and Agantuja Vyadhi. In the Vedic period, Daivavyapashraya Chikitsa was followed in various rituals.

ALL MATTER IS IN MOTION

Everything moves:

In the atom electrons rotate around the nucleus

In gases, liquids and even solids molecules move away from other molecules

An accumulation of matter (i.e., a baseball) can move as a unit

The average velocity of movement is the distance travelled divided by the time

Bodies in motion have energy; the faster the velocity the more energy

Introduction
This method is used to determine the ‘total bacterial count’ (living and dead  organisms) by direct microscopic examination in samples of broth culture, cell suspension and milk. Simple and Gram’s staining techniques are used to stain the cells. This method is simple and a rapid. A known volume (0.01 ml) of the sample is spread over a known area (1cm2) on a glass slide. The sample is allowed to dry; then stained and examined microscopically. The average number of cells per field is determined and is then calculated by applying microscopic factor.
Materials
1. Microscopic slide with one sq. cm. area marked
2. Breed’s pipette (0.01 ml)
3. Methylene blue or Newman’s stain
4. Culture of E.coli or Bacillus subtilis
Procedure
1. Measure the diameter of microscopic field with a stage micrometer using oil immersion objective and 10X eye piece.
2. Area of field πr2.
Determination of microscopic factor (MF)

(A) Enumeration of Microbial Growth by Turbidity Measurement
Method and Estimation of Generation Time
Introduction
Bacterial growth usually refers to reproduction-increase in population sizerather than to enlargement of mycelia cells. “Reproduction in bacteria occurs mainly through binary (binary means one cell becomes two) or transverse fission that results in the formation of two daughter cells. This is repeated at intervals by each new daughter cell in turn, or with each successive round of division. The population increases (Fig. 1). The time required for a complete fission cycle i.e. parent cell to two daughter cells is called the generation or doubling time. In bacteria, it is a doubling process in which each new fission cycle or generation increases the population by a factor of 2. So the parent stage consists of 2 cells, the second of 4, then 8, 16, 32, 64, 128, 256 and so on (Fig. 2). Thus the population will double, over and over again as long as the environment remains favorable for growth. The length of generation time is a measure of the growth rate of an organism. The average generation time in bacteria is 30-60 minutes under optimum conditions. Some species have very rapid growth rate and short generation time (5-10 minutes) and others have very slow growth rates and long generation times (measured in days). Staphylococcus aureus and Salmonella enteritidis, both causes of food poisoning, are examples of pathogens having relatively short doubling times of 20-30 minutes, which result in a few million cells from a few cells in a few hours in the infested food. The longest generation time occurs in species of Mycobacterium, particularly Mycobacterium leprae (the cause of leprosy) of 10-30 days as long as in some animals.