1.Video Lesson

2.Objective

At the end of this lesson you will be able to:-

• define microbiota
• describe the use of microorganisms
• explain about controlling of microorganisms
• explain the process of microorganism isolation.

Brainstorming questions

• How does the normal microbiota contribute to human health beyond just pathogen exclusion, and what mechanisms can be explored to enhance these protective roles?
• What are the potential consequences of disrupting the normal microbiota (e.g., through antibiotics or lifestyle changes) on both individual health and broader ecological systems, and how can we mitigate these effects?

key words

• Resident Microbiota: Microorganisms that are consistently present in a specific location on the body for a long duration.
• Transient Microbiota: Microorganisms that are temporarily present in a specific location on the body.
• Normal Human Microbiota: The community of microorganisms that typically inhabit various parts of the human body without causing disease.
• Germ Theory of Disease: The theory that diseases are caused by microorganisms.
• Koch’s Postulates: Criteria established by Robert Koch to identify the causative agent of a specific disease
• Modes of Disease Transmission: The routes through which diseases spread, including contact, droplet, airborne, and common vehicle transmission.
• Uses of Microorganisms: Various applications of microorganisms in fields like agriculture, industry, food production, energy generation, and environmental cleanup.
• Bioremediation: The use of microorganisms to break down or neutralize pollutants and contaminants in the environment.
• Carbon Cycle: The process by which carbon is cycled through the environment, involving primary producers, consumers, and decomposers.
• Nitrogen Cycle: The process by which nitrogen is converted between its various chemical forms, facilitated by microorganisms such as nitrogen-fixing bacteria.
• Sulfur Cycle: The process of sulfur movement through the environment, involving its conversion into different chemical forms by microorganisms.
• Phosphorus Cycle: The process by which phosphorus moves through the environment, including its transformation by microorganisms from organic to inorganic forms.
• Sterilization: The process of eliminating all forms of life, including spores, from an object or environment.
• Germicide: A chemical agent that kills pathogenic microorganisms.
• Disinfection: The process of eliminating pathogenic microorganisms from inanimate objects, but not necessarily spores.
• Antiseptics: Chemical agents applied to living tissues to destroy or inhibit pathogens.
• Sanitization: The mechanical removal of microorganisms to reduce contamination levels to a safe standard.
• Pasteurization: A heat treatment process that kills pathogens in food and beverages, commonly used for milk.
• Bacterial Isolation Techniques: Methods used to isolate and identify bacteria from samples, including serial dilution, morphological examination, biochemical characterization, and molecular techniques.

Normal microbiota

The normal microbiota consists of microorganisms commonly found growing on the bodies of healthy individuals. Microbes that inhabit body sites for long durations are known as resident microbiota, while those present temporarily are called transient microbiota. Understanding normal human microbiota is important for several reasons: identifying the microorganisms in specific locations helps predict potential infections that might occur due to injuries at those sites; knowledge of normal microbiota aids physicians in understanding the causes and effects of colonization and growth by microbes that are usually not present in certain areas; and it enhances awareness of how these normal microbiota stimulate the host’s immune response, which is crucial for protecting against potential pathogens.

Germ theory of disease and Koch’s postulates

To determine if diseases are caused by microorganisms, Koch conducted experiments using mice. With proper controls, he showed that injecting a small amount of blood from a diseased mouse into a healthy mouse caused the healthy mouse to quickly develop anthrax. He then took blood from this second mouse and injected it into another. Koch established a set of stringent criteria, now known as Koch’s postulates, to definitively associate a specific microorganism with a specific disease.

Koch’s postulates state the following:

1. The disease-causing orgamsm must always be present in animals suffering from the disease but not in healthy animals.

2. The organism must be cultivated in a pure culture  away  from  the  animal  body.

3. The isolated organism must cause the disease when inoculated into healthy susceptible animals.

4. The organism must be isolated from the newly infected animals and cultured again in the laboratory, after which it should be seen to be the same as the original organism

Modes of disease transmission and ways of prevention

Microorganisms are transmitted in health care settings by four main routes

• Contact
• droplet
• air borne
• common vehicle

Uses of microorganisms

Microorganisms are crucial in agriculture, industry, food production, energy generation, and environmental cleanup. These microorganisms, which include bacteria, fungi, algae, protozoa, and viruses, aid in the decomposition of organic matter and the formation of humus.

Agriculture

Microorganisms play a crucial role in breaking down organic matter and forming humus. Their key functions include nitrogen fixation, phosphate solubilization, potassium mobilization, and providing antagonism against pathogens and pests, making their role in agriculture essential.

While the chemical elements carbon, nitrogen, oxygen, sulfur, and phosphorus are vital for life and are abundant, they are not always in forms that organisms can utilize. Microorganisms are primarily responsible for transforming these elements into usable forms for plants and animals.

Sewage treatment

Anaerobic bacteria are used in wastewater treatment on a normal basis. The main role of these bacteria in sewage treatment is to reduce the volume of sludge and produce methane gas from it. Methane gas can be used as an alternative energy source. This is a huge benefit considering the already high wastewater treatment energy consumption levels. Phosphorus removal from wastewater is another benefit of anaerobic microbes used in sewage treatment.

Bioremediation

Bioremediation is a natural process that uses microorganisms and plants—or their derivatives like enzymes or spent biomass—to break down or modify environmental contaminants as part of their normal life activities. Specifically, microbial bioremediation involves utilizing these microorganisms and/or their by-products to clean up pollutants. Microorganisms are ubiquitous, and their presence enhances the range of chemical pollutants that can be degraded or detoxified.

Although bioremediation is a natural process and takes some time, it is an effective waste treatment method for contaminated materials such as soil. Microbes that can degrade the contaminant proliferate in its presence, and their numbers decrease once the contaminant is broken down. The by-products of this treatment are typically benign, including water, carbon dioxide, and cellular biomass.

Food production and processing

The tart taste of yogurt, pickles, sharp cheeses, and some sausages is due to the production of lactic acid by one or more members of a group of bacteria known as the lactic acid bacteria. These bacteria includes Lactobacillus, Lactococcus, Streptococcus,   Leuconostoc, etc that used to ferment milks into different food staffs (figure 15)

The role of bacteria in recycling of minerals through ecosystem

Almost 90% of all living thing are made up of C,O,N and H, these substance are limited in their availability. For the continuity of life these substance should be recycled. This is done by decomposer (bacteria and fungi major one).

The carbon cycle

All organisms are composed of organic molecules such as protein, carbohydrate, lipid and others. The carbon travels through the food chain as primary producers are eaten by primary consumers, which are then eaten by secondary consumers. Decomposers then use the remains of primary producers and consumers.

Carbon Fixation

A fundamental steps of carbon cycle is carbon fixation, the defining characteristic of primary producers. Without primary producers, no other organisms, including humans, could exist. During respiration and fermentation organic materials break down to energy and carbon dioxide. Organisms use sugars, amino acids, and proteins as energy sources, but rapidly multiplying bacteria often the O₂ supply has a strong influence on the carbon cycle.  Not only do O₂ allow degradation of certain compounds such as lignin, it also helps determine the types of carbon­ containing gases produced. When organic matter is degraded aerobically, a great deal of CO₂ is produced showed in figure 16.  . When the O₂ level is low, in the case in marshes, swamps, and manure piles, the degradation is incomplete, generating some CO₂ and a variety of other products .

Methanogenesis and Methane Oxidation

In anaerobic environments, CO₂ is used by methanogens. These archaea obtain energy by oxidizing hydrogen gas, using CO2 as a terminal electron acceptor, generating methane (CH4). Methane that enters the atmosphere is oxidized by ultraviolet light and chemical ions, forming carbon monoxide (CO) and CO2. A group of microorganisms called methylotrophs can use methane as an energy source, oxidizing it to produce CO2 showed in figure 16.

The Nitrogen cycle

Root nodules are found on the roots of plants, primarily legumes, which form a symbiosis with nitrogen-fixing (Figure 2.38) bacteria. Under nitrogen-limiting conditions, capable plants form a symbiotic relationship with a host-specific strain of bacteria known as rhizobia. Nitrogen fixation in the nodule is very oxygen sensitive

The sulfur cycle

Sulfur is found in fewer types of organic molecules than nitrogen, but it is found in many proteins. Figure 17 shows the bacteria involved in the sulfur cycle and the roles they play.

The phosphorus cycle

Phosphorus (P) occurs in soils as both organic and inorganic forms (Figure 18). Phosphorus can be found dissolved in the soil solution in very low amounts or associated with soil minerals  or organic materials. The relative amounts of each form of phosphorus vary greatly among soils, with the total amount of P in a clayey-textured soil being up to ten times greater than in a sandy soil.

Organic P in soils

Large number of compounds makes up the organic P in soils, with the majority being of microbial origin. Organic P is held very tightly and is generally not available for plant uptake until the organic materials are decomposed and the            phosphorus release via the mineralization process. Mineralization is carried out by microbes, and as with nitrogen, the rate of P release is affected by factors such as soil moisture, composition of the organic material, oxygen concentration and pH.

Inorganic P in soils

The concentration of inorganic P (orthophosphates) in the soil solution at any given time is very small, amounting to less than 1 lb. /A. Phosphorus in the inorganic form occurs mostly as aluminum, iron or calcium compounds.

Controlling microorganisms

Sterilization is defined as a process by which an article, surface, or medium is freed of all living. either  the vegetative or in the spore state. Any material that has been subjected to this process is said to be sterile. These terms should be used only in the absolute sense. An object cannot be slightly sterile or almost sterile; it is either sterile or not sterile. Sterilization is performed with a physical agent, such as heat, a few chemicals called sterilants can be classified as sterilizing agents because of their ability to destroy spores.

A germicide, also called a microbicide, is any chemical agent that kills pathogenic microorganisms. A germicide can be used on inanimate (nonliving) materials or on living tissue, but it ordinarily cannot kill resistant microbial cells. Any physical or chemical agent that kills “germs” is said to have germicidal properties.

Disinfection refers to the use of a chemical agent that destroys or removes all pathogenic organisms or organisms capable of giving rise to infection. This process destroys vegetative pathogens but not bacterial endospores. It is important to note that disinfectants are normally used only on inanimate objects because they can be toxic to human and other animal tissue, when used m higher concentrations.

Disinfection:- processes also remove the harmful products of microorganisms (toxins) from materials. Examples

A). Applying a solution of 5% bleach to examining table

B). Boiling food utensils used by a sick person

C). Immersing thermometers in an isopropyl alcohol solution between uses

Antiseptics:-are chemicals that applied directly to the exposed body surfaces (e.g., skin and mucous membranes), wounds, and surgical incisions to destroy or inhibit vegetative pathogens.

e.g.

• Preparing    the    skin    before    surgical incisions with iodine compounds,
• Swabbing an open root canal with hydrogen peroxide, and
• Ordinary hand washing with a germicidal soap.

Sanitization is any cleansing technique that mechanically removes microorganisms (along with food debris) to reduce the level of contaminants. A sanitizer is a compound (e.g., soap or detergent) that is used to perform this task. Cooking utensils, dishes, bottles, cans, and used clothing that have been washed and dried may not be completely free of microbes, but they are considered safe for normal use. Air sanitization with ultraviolet lamps reduces airborne microbes in hospital rooms veterinary clinics, installations. and laboratory.

Preservation is a general term for measures taken to prevent microbe caused spoilage of susceptible products (pharmaceuticals, foods).

Decontamination is the removal or count reduction of microorganisms contaminating an object. The objective of aseptic measures and techniques is to prevent microbial contamination of materials or wounds. In antiseptic measures, chemical agents are used to fight pathogens in or on living tissue, for example in a wound.

Physical methods sterilization and disinfection

Heat

The application of heat is a simple, cheap and effective method of killing pathogens. Methods of heat application vary according to the specific application.

• Pasteurization.
• Disinfection
• Dry heat sterilization
• Moist heat sterilization
• Intermittent sterilization

Pasteurization: – is the antimicrobial treatment used for foods in liquid form (milk).

• Low-temperature pasteurization:-61.5°c for 30minutes  or 71°c, for 15 seconds.
• High-temperature pasteurization:-brief (seconds) of exposure to 80-85°C in continuous operation.
• Uperization:- heating to 150°C for 2.5 seconds in a pressurized container using steam injection

Disinfection:- using heat to sterilized materials and Important: boiling medical instruments, needles, syringes, etc. does not constitute sterilization! Many bacterial spores are not killed by this method

Dry heat sterilization:-. it includes sterilization by flaming, incineration and hot air oven.

 Flaming : Sterilization of inoculating loop or wire, the tip of forceps, searing spatulas, etc., is carried out by holding them in the flame of the Bunsen burner till they become red hot.

Incineration: Incineration is an excellent method for safely destroying infective materials by burning them to ashes. It has many uses:

Incinerators are used to carry out this process and are regularly employed in hospitals and research labs to destroy hospital and laboratory wastes. like  syringes, needles, culture material, dressings, bandages, bedding, animal carcasses, and pathology samples. It is fast and effective for most hospital wastes, but not for metals and heat­ resistant glass materials.

Hot air oven: Sterilization by hot-air oven requires exposure to l 60- l 80°C for 2 hours and 30 minutes, which ensures thorough heating of the objects and destruction of spores.

Moist heat sterilization: – Autoclaves charged with saturated, pressurized steam are used for this purpose:-

• 121°c, for 15 minutes, one atmosphere of pressure (total: 202 kPa).
• 134°C,for  minutes, two atmospheres of pressure (total: 303 kPa).

Intermittent sterilization

Certain substance have heat-labile substances (e.g., serum, sugar, egg, etc.) that cannot withstand the high temperature of the autoclave can be sterilized by a process of intermittent sterilization, known as tyndallization.

Tyndallization is carried out over a period of 3 days and requires a chamber to hold the materials and a reservoir for boiling water. Items to be sterilized are kept in the chamber and are exposed to free-flowing steam at 100°C for 20 minutes, for each of the three consecutive days

Bacterial isolation techniques

Microorganisms (bateria or fungi) can be isolated from food, soil, water or from other materials. For bacterial/fungal isolation, the soil (food) samples are collected from the desired sites (Figure 18). Microorganism are separated on artificial media by serial dilution method. Each of the isolates are purified on new media and experimented for the morphological characteristic like shape, gram nature and arrangement of cells, motility etc. Enzymatic activities were tested by biochemical characterization. Finally, molecular techniques are used for further identifications.