Microbial Growth

1. Microbial Growth Microbial growth: Growth involves an increase in size and number of organisms. Growth Requirements: (I) Chemical growth factors: What are the chemical growth factors required for isolation of microbes in vitro? 1- Carbon: organic: Glucose. Inorganic: CO2 2- Hydrogenoccurs in organic hydrocarbon molecules and in inorganic molecules such as water. 3- Oxygenor 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 .

2. Bacterial Nutrition • (II) Bacterial Nutrition: • Autotrophic Bacteria: • Most of saprophytic bacteria belong to this category. • Can utilize simple inorganic materials (CO2 as the source of carbon, and ammonium salts as the source of nitrogen). • Can form complex organic metabolites from these inorganic simple materials. • They derive their energy from light (e.g. photosynthetic bacteria) or from chemical reactions by oxidation of inorganic materials (e.g. chemolithotrophicbacteria). • Heterotrophic Bacteria: • Most of pathogenic bacteria belong to this category. • Require for their nutrition complex organic materials derived from animal and plant sources. • Can not synthetize complex organic substances from the simple inorganic materials . • They derive their energy by the oxidation or fermentation of organic compounds(e.g. glucose).

3. Physical Growth Factors (III) 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. Fungisuch 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).

4. Physical Growth Factors 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 anionsand cations. 3- Temperature requirements: Mesophiles:Grow at optima between 25ᵒC and 40ᵒC. Normal microbiota and most pathogens grow at or near humanbody temp of 37ᵒC. Thermophiles: Heat-loving microbes of optima between 50ᵒ and 60ᵒ C .

5. Physical Growth Factors 4- Gaseous Requirements: According to oxygen requirements, microbes can be classified into: 1- Strict ,Obligate Aerobe: O2must be present during cultivation of microbe. Example: Pseudomonas aeruginosa. 2-Strict or obligate Anaerobes: Require complete absence of O2. 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.

6. Physical Growth Factors 3- Facultative anaerobes: Adaptable organisms that use oxygen when present but can grow and multiply and switch to anaerobic pathways in its absence. Example: Escherichia coli. 4- Microaerophilic: Only use low concentrations of oxygen. Example : Helicobacter pylori. (II) According to CO2 requirements: The amount normally present in air is sufficient but some organisms require a higher percentage. They are called "Capnophilic" or carbon-dioxide loving. Example: Brucellaabortus(10% CO2) and Neisseria gonorrhoeae(5%CO2).

7. Bacterial Growth Curve The Bacterial Growth Curve: Bacterial growth is the division of one bacterium into two daughter cells in a process called binary fission. When bacteria are transferred to a suitable medium and the number of viable bacteria /ml is calculated at regular time intervals, a growth curve will be obtained and divided into 4 phases: 1- Lag phase. 2- Exponential phase. 3- Stationary phase. 4- Decline phase

8. Bacterial Growth Curve

9. Bacterial Growth Curve 1- The lag Phase: During this phase, there is no or little bacterial multiplication. It is the period between inoculation of the medium with the organism till the beginning of active multiplication. In this phase, the organisms adapts itself to the new environment by synthesizing new enzymes and macromolecules required for metabolism. The length of lag phase depends on: The nature of organism and its generation time (e.g. Escherichia coli has a short lag phase while Mycobacterium tuberculosis has a long lag phase). The nutritive value of the medium. The actual phase of the organism at the time of inoculation into the medium.

10. Bacterial Growth Curve 2- The logarithmic (exponential) phase: It is the most active phase of bacterial growth during which the number of bacteria increases steadily by time. This phase continues until: The available nutrients are exhausted, and/or The toxic waste products are accumulated. 3- TheStationary 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. 4- Death phase:Bacteria run out of nutrients and die. When exhaustion of nutrients, accumulation of waste products and change of the cultural conditions (e.g. pH) continue, the number of dying bacteria exceeds the number of newly formed bacteria. The number of viable living bacteria is decreasing.

11. Bacterial Growth Curve Clinical Significance of Growth Curve: The 4 phases of bacterial growth curve correlate with the different stages of natural infections and diseases. Stages of growth curve Stages of infections and diseases The lag phase: The incubation period of the disease The logarithmic & stationary The clinical signs and symptoms of phase: disease The phase of decline: The recovery & convalescence stages

12. Identification Of Bacteria Laboratory Methods For Identification of Bacteria: Bacteria are either identified in: A pathological specimen obtained from the patient (e.g. pus, sputum, urine, blood, stools, etc.) depending on the site of infection. After been grown on artificial nutrient media. Bacteria are then identified by: Microscopic Examination: Examination of fresh samples used for demonstration of bacterial motility; using hanging drop method. Morphology and staining reactions of bacteria.

13. Identification Of Bacteria Commonly used stains are: Simple stains: e.g. methylene blue stain. Differential stains: e.g. Gram stain: Primary stain: Methyl violet- Iodine mixture. Decolourization with Alcohol. Counter stain: Diluted carbolfuchin. Results: Gram (+)Purple Gram (-)Red Difference due to structure of cell wall • Gram (+) Thick cell wall • Gram (-) Thin cell wall

14. Identification Of Bacteria

15. Identification Of Bacteria Acid fast stain: (Ziehl-Neelsen stain) Differential Stain - divides bacteria into 2 groups Acid - Fast Non Acid - Fast Used to identify organisms in the Genera Mycobacterium (high lipid and wax content in cell wall) 1. Carbolfuchsin (Red) 2. Decolourization by sulphuric acid 20%. 3. Counter stain with Methylene blue. Acid - Fast organism: Red as Mycobacterium tuberculosis Non Acid – Fast organism: Blue

16. Identification Of Bacteria Special stains: e.g. Capsule stain, flagella stain, …..

17. Cultural Characters (II) Cultural Characters: Bacteria need nutritive culture media to multiply in vitro. An undefined medium(also known as a basalor 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). Classification of Media: Media can be classified into: 1-Minimal media ( simple medium): It contains the basic nutritive requirements, e.g.nutrient broths and agar media.

18. Cultural Characters 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. Mannitolsalt agar(MSA) which is selective for Gram +vebacteria. Blood-free, charcoal-based selective medium agar (CSM) for isolation of Campylobacter. Lowenstein- Jensen medium: enriched selective media for T.B. T.C.B.S (Thiosulphate- Citrate- Bile- Sucrose agar): selctive for Vibrio cholera due to alkalinepH.

19. Cultural Characters 3-Differential media: Differential media or indicator mediadistinguish one microorganism type from another growing on the same media. Indicators (such as neutral red, phenol red, eosin y, or methylene blue) 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.

20. Cultural Characters 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. It contains 5-10% human or animal blood. It shows the type of haemolytic activity of bacteria (complete, partial or non- haemolytic). Complete Partial Haemolysis of Haemolysis RBCs of RBCs (Beta (Alpha HaemolyticHaemolytic Streptococci) Streptococci)

21. Cultural Characters Chocolate agar (heated blood agar): is enriched with heat-treated blood (40-45°C). Loffler's serum media: Horse serum + glucose in a ratio 3:1 It is used for cultivation of Corynbacterium diphtheria.

22. Cultural Characters 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.

23. Antibiotics Antibiotics: Anantibacterial 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.

24. Antibiotics 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

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

26. Antibiotics The Erythromycin. The chloramphenicol The Trimethoprim

27. Antibiotics 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.

28. Antibiotics 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.