Microbial growth requirements: Chemical growth factors: What are the chemical growth factors required for isolation of microbes in vitro? 1-Carbon: organic: Glucose. Inorganic: CO2 2- Hydrogen occurs in organic hydrocarbon molecules and in inorganic molecules such as water. 3-Oxygen or gaseous requirements of microbes. 4-Nitrogen is used in protein/ amino acid synthesis and nucleic acid polymerization. 5- Phosphorus is essential for nucleic acid synthesis and formation of phospholipids. 6- Sulfur present in certain amino acids such as cysteine and methionine .

Physical growth factors: 1-Acidity (pH requirements): Pathogenic bacteria grow best at neutral or biological pH which is typically between pH 6.8 to pH 7.4. Fungi such as yeasts and molds prefer slightly more acidic conditions and grow best between pH 5 to pH 6. The pathogen Helicobacter pylori is able to survive with pH of the stomach by producing urease. So it is acidophilic bacterium. On the other extreme, bacteria that prefer alkaline (basic) conditions are known as alkaliphiles. Example: Vibrio cholerae (prefers pH 9).

2- Acids, Bases, Salts concentration: An ACID can be described as a hydrogen donor. A BASE can be described as a hydrogen acceptor. A SALT is an ionic compound that dissociates in water into anions and cations. 3- Temperature requirements: Mesophiles: Grow at optima between 25 C and 40 C. Normal microbiota and most pathogens grow at or near human body temp of 37 C. Thermophiles: heat-loving microbes of Optima between 50 and 60 C . a

4- Gaseous requirements and Humidity : According to oxygen requirements, microbes can be classified into: 1- Strict ,Obligate Aerobe: O2 must be present during cultivation of microbe. Example: Pseudomonas aeruginosa. 2- Strict or obligate Anaerobes: Molecular oxygen is toxic to these organisms. Lack enzymes catalase and superoxide dismutase (SOD) to neutralize hydrogen peroxide and free radicals, respectively. Example: Bacteriodes and Clostridium. 3- Facultative anaerobes: Adaptable organisms that use oxygen when present but can switch to anaerobic pathways in its absence. Example: Escherichia coli. 4- Microaerophilic: Also known as "Capnophilic" or carbon-dioxide loving. Only use low concentrations of oxygen. Example : Helicobacter pylori. Humidity: The percentage of water vapor in the cultivation atmosphere.

The Bacterial Growth Curve: Bacterial growth is the division of one bacterium into two daughter cells in a process called binary fission. During lag phase, bacteria adapt themselves to growth conditions. Exponential phase (sometimes called the log phase): is a period characterized by cell doubling. For this type of exponential growth, plotting the natural logarithm of cell number against time produces a straight line.

At death phase, bacteria run out of nutrients and die. During stationary phase, the growth rate slows as a result of nutrient depletion and accumulation of toxic products. This phase is a constant value as the rate of bacterial growth is equal to the rate of bacterial death. At death phase, bacteria run out of nutrients and die. Bacterial culture media: An undefined medium (also known as a basal or complex medium). It is a medium that contains: 1-A carbon source such as glucose for bacterial growth 2-water 3-various salts needed for bacterial growth. Defined media (also known as chemically defined media or synthetic media). a

Classification of media: Media can be classified into: 1-Minimal media ( simple medium): nutrient broths and agar media 2- Selective media: Selective media are used for the growth of only selective microbes. It contains antibiotics, dye, or specific chemicals that inhibits the growth of most types of microbe and stimulate the isolation of one type. Mannitol salt agar (MSA) which is selective for Gram-positive bacteria. Blood-free, charcoal-based selective medium agar (CSM) for isolation of Campylobacter.

3-Differential media: Differential media or indicator media distinguish one microorganism type from another growing on the same media. Indicators (such as neutral red, phenol red, eosin y, or methylene blue) could be used. Examples of differential media include: Eosin methylene blue (EMB), which is differential for lactose and sucrose fermentation. MacConkey (MCK), which is differential for lactose fermentation.

4- Enriched media: Enriched media contain the nutrients required to support the growth of a wide variety of organisms, including some of the more fastidious ones. Blood agar is an enriched medium in which nutritionally rich whole blood supplements the basic nutrients. Chocolate agar is enriched with heat-treated blood (40-45°C).

5- Transport media: Transport medium is a simple organic medium that Maintain the viability of all organisms in the specimen without altering their concentration. This type of medium mainly used for Temporary storage of specimens being transported to the laboratory for cultivation. Examples of transport media include: Thioglycolate broth for strict anaerobes.

Antibiotics: An antibacterial agent is a compound or substance that kills or slows down the growth of bacteria. Antibiotic(s) has come to include a broader range of antimicrobial compounds, including anti-fungal and other compounds. These include, for example, the beta-lactam antibacterials, which include the penicillins (produced by fungi in the genus Penicillium),and the cephalosporins. Compounds that are still isolated from living organisms are the aminoglycosides, whereas other antibacterial agents—for example, the sulfonamides,and the quinolones, are produced by chemical synthesis.

Classification of Antibiotics: According to agent action: In this classification antibacterial agents are divided into two broad groups according to their biological effect on microorganisms: bactericidal agents kill bacteria, and bacteriostatic agents slow down or stall bacterial growth. Bactericidal antibiotics: Antibiotics that inhibit cell wall synthesis: the Beta-lactam antibiotics (penicillin derivatives, and cephalosporins). Aminoglycosidic antibiotics are usually considered bactericidal, although they may be bacteriostatic with some organisms.

Tetracyclines, sulphonamides , trimethoprim ,chloramphenicol, Bacteriostatic antibiotics limit the growth of bacteria by interfering with bacterial protein production, DNA replication, or other aspects of bacterial cellular metabolism. This group includes: Tetracyclines, sulphonamides , trimethoprim ,chloramphenicol, and macrolides. The Beta-lactam: Penicillin. The Tetracycline.

The Erythromycin. The chloramphenicol The Trimethoprim

Antibiotic sensitivity test: Antibiotic sensitivity is a term used to describe the susceptibility of bacteria to antibiotics. Antibiotic susceptibility testing (AST) is usually carried out to determine which antibiotic will be most successful in treating a bacterial infection in vivo. Testing for antibiotic sensitivity is often done by the Kirby-Bauer method ( Disc-diffusion method). Other methods to test antimicrobial susceptibility include the  E-test (also based on antibiotic diffusion). Agar and Broth dilution methods for Minimum Inhibitory Concentration determination.

Antibiotic sensitivity test: Antibiotic sensitivity Test : antibiotic have been placed on an agar plate growing bacteria. Bacteria are not able to grow around antibiotics to which they are sensitive.