Bacterial Growth Requirements PDF

BACTERIAL GROWTH REQUIREMENTS TOPIC 4 : BACTERIAL GROWTH REQUIREMENTS At the end of this topic, you must be able to: ∙ Define microbial growth; ∙ Discuss the various nutritional and physical growth requirements; and ∙ Describe the different phases of microbial growth  MICROBIAL GROWTH...

BACTERIAL GROWTH REQUIREMENTS TOPIC 4 : BACTERIAL GROWTH REQUIREMENTS At the end of this topic, you must be able to: ∙ Define microbial growth; ∙ Discuss the various nutritional and physical growth requirements; and ∙ Describe the different phases of microbial growth  MICROBIAL GROWTH Growth involves an orderly and organized increase in the sum of all components of the organism which may entail replication of all cellular structures, organelles, and components. Microbial growth – concerned with the increase in number of cells and not increase in the size of the organism.  BINARY FISSION The process by which organisms divide and reproduce.  GENERATION TIME Time required for a cell to divide and its population to double. GENERATION TIME  GENERATION TIME Time required for a cell to divide and its population to double.  BACTERIAL GROWTH CURVE Illustrates the phases in the growth of the population of bacteria when they are grown in a culture of fixed volume. It reflects the different stages in the growth of a bacterium.  BACTERIAL GROWTH PHASES OF GROWTH LAG PHASE LOG PHASE Bacteria are still adjusting to their Exponential or logarithmic phase environment Rapid binary fission Bacteria absorb nutrients, Number of cells grow exponentially synthesize enzymes, and prepare Very susceptible to Gram Staining and for cell division. cell wall inhibitors Cells grow in size but not in number STATIONARY PHASE DEATH PHASE culture is at its greatest population aka logarithmic decline phase density. Accumulation of toxic metabolites Number of cells produced= number and autolytic enzymes of cells dying culture may die completely, or a few nutrients in the liquid medium are microorganisms or may continue to used up survive for months concentration of toxic waste formation of spores products from the metabolizing Morphologic changes occur (Bacteria bacteria build up assume different shapes)  BACTERIAL GROWTH PHYSICAL REQUIREMENTS 1. Water/Moisture 2. Temperature 3. pH 4. Osmotic pressure 5. Oxygen  BACTERIAL GROWTH PHYSICAL REQUIREMENTS 1. Water/Moisture The bacterial cell is composed mainly of water. All living organisms require water to carry out their normal metabolic processes, and most will die in environments containing too little moisture.  BACTERIAL GROWTH PHYSICAL REQUIREMENTS 2. Temperature a.Psychrophiles (0-20 oC) ✔live in the refrigerator a.Mesophiles (21-45 oC) ✔ very pathogenic b.Thermophiles (46 oC and higher) ✔ freeze at body temperature  BACTERIAL GROWTH PHYSICAL REQUIREMENTS 3. pH a.Alkalophiles – pH 8.4 – 9.0 b.Neutrophiles – pH 6.5 – 7.5 c.Acidophiles – pH less than 6  BACTERIAL GROWTH PHYSICAL REQUIREMENTS 4. Osmotic pressure a.Extreme halophiles – require very high salt concentration for growth. b.Obligate halophiles – require enough salt concentration for growth c.Facultative halophiles – do not require high salt concentration but able to grow at salt concentrations up to 2%. BACTERIAL GROWTH REQUIREMENTS  BACTERIAL GROWTH REQUIREMENTS PHYSICAL REQUIREMENTS 5. Oxygen  BACTERIAL GROWTH PHYSICAL REQUIREMENTS 5. Oxygen  BACTERIAL GROWTH CHEMICAL REQUIREMENTS 1. Carbon 2. Nitrogen, Sulfur, Phosphorus 3. Inorganic Ions 4. Growth Factors  BACTERIAL GROWTH CHEMICAL REQUIREMENTS 1. Carbon  BACTERIAL GROWTH CHEMICAL REQUIREMENTS 2. Nitrogen, Sulfur, Phosphorus - necessary for synthesis of proteins and nucleic acids  BACTERIAL GROWTH CHEMICAL REQUIREMENTS 3. Inorganic Ions a. Magnesium – stabilizes ribosomes, cell membranes, and nucleic acids; also serves as co-factor in the activity of enzymes b. Potassium – for normal functioning and integrity of ribosomes and enzyme activities c. Calcium – important component of gram-positive bacterial cell wall and contributes to the resistance of bacterial endospores d. Iron – component of cytochrome, a component of the electron transport chain and co-factor for enzymatic activities  BACTERIAL GROWTH CHEMICAL REQUIREMENTS 4. Growth Factors Essential to promote the growth and development of the bacterial cell. These include Vitamin B Complex and amino acids. TOPIC 5 : STAINING AND CULTURE MEDIA At the end of this topic, you must be able to: ∙ Compare the various staining methods used to visualize microorganisms; and ∙ Classify the different types of culture media based on their constituents, physical state, chemical composition, and functional type.  CULTURE MEDIA A nutrient material prepared for the growth of microorganisms. Can be classified into three primary levels: – Constituents – Physical state – Functional type  ACCORDING TO CONSTITUENTS Synthetic media – Contain chemically-defined substances, which are pure organic or inorganic compounds. Non-synthetic media – Complex media that contain at least one ingredient that is not chemically defined. Most are extracts of animals, plants, or yeasts.  ACCORDING TO CONSTITUENTS Synthetic media – Contain chemically-defined substances All components and constituents and concentrations are known Nutrients: C, H, O, N, S, P, K, Mg  ACCORDING TO CONSTITUENTS Non-synthetic media – Complex media that contain at least one ingredient that is not chemically defined. ACCORDING TO PHYSICAL STATE Liquid media – Commonly called broths, milks, or infusions. – Water-based solutions that do not solidify at temperatures above freezing point. Semi-solid media – Exhibit a clot-like consistency at ordinary room temperature and contains some amount of solidifying agent (agar or gelatin) that thickens the media but does not produce a firm substance. ACCORDING TO PHYSICAL STATE Solid media – Contain sufficient amounts of solidifying agent, giving them a firm surface on which cells can form discrete colonies. – Two forms: Liquefiable/Reversible solid media Non-liquefiable/Non-reversible solid media  ACCORDING TO FUNCTIONAL TYPE 1. General Purpose Media – Designed to grow a broad spectrum of microbes – Contain a mixture of nutrients that can support the growth of both pathogens and non- pathogens. – Ex: peptone water, nutrient broth, nutrient agar 2. Enrichment Media – Contain complex organic substances (blood, serum, special growth factors) – Increases the number of desired microbes without stimulating the rest of bacterial population.  ACCORDING TO FUNCTIONAL TYPE a. Blood Agar Certain Gram + bacteria produce Exotoxins which causes hemolysis of red blood cells contained in the blood agar Hemolytic reaction is categorized into three: 1. Beta-hemolysis – complete lysis of RBC 2. Alpha-hemolysis – incomplete lysis of RBC (greenish discoloration) 3. Gamma-hemolysis – no hemolysis, no change in the medium.  ACCORDING TO FUNCTIONAL TYPE a. Blood Agar Certain Gram + bacteria produce Exotoxins which causes hemolysis of red blood cells contained in the blood agar Hemolytic reaction is categorized into three: 1. Beta-hemolysis – complete lysis of RBC 2. Alpha-hemolysis – incomplete lysis of RBC (greenish discoloration) 3. Gamma-hemolysis – no hemolysis, no change in the medium.  ACCORDING TO FUNCTIONAL TYPE b. Chocolate Agar Used for culturing fastidious organisms such as Haemophilus sp. and Neisseria sp. Heat is applied to lyse the red blood cells, causing the medium to turn brown.  ACCORDING TO FUNCTIONAL TYPE 3. Selective Media – Contain one or more substances that encourage the growth of only a specific target microorganism and inhibit the growth of others. – Designed to prevent the growth of unwanted contaminating bacteria or commensals so only the target bacteria will grow. – Agar-based solid media that allow isolation of individual bacterial colonies.  ACCORDING TO FUNCTIONAL TYPE 3. Selective Media a. Thayer Martin Agar – contains antibiotics trimethoprim, nystatin, vancomycin, and colistin. Used for isolation of Neisseria b. Mannitol Salt Agar – contains 10% NaCl and used for the isolation of Staphylococcus aureus. c. MacConkey’s Agar – promotes the growth of gram- negative bacteria, and inhibits the growth of gram-positive bacteria. d. Lowenstein-Jensen Medium – for isolation of Mycobacterium tuberculosis. Made by the incorporation of malachite green. e. Sabouraud’s Dextrose Agar – for isolation of fungi SELECTIVE MEDIA SELECTIVE MEDIA Sabouraud’s Dextrose Agar SELECTIVE MEDIA Lowenstein-Jensen Medium  ACCORDING TO FUNCTIONAL TYPE 4. Differential Media – Allows the growth of several types of microorganisms. – Designed to show visible differences among certain groups of microorganisms. – Differences may be seen as variations in colony size or color, changes in media color, formation of precipitates or gas bubbles. – Ex: MacConkey agar, Triple Sugar Iron Agar  ACCORDING TO FUNCTIONAL TYPE 5. Transport Media – Used for clinical specimens that need to be transported to the laboratory immediately after collection. – This will prevent the drying of specimen and inhibit overgrowth of commensals and contaminating organisms. Ex: Cary Blair Transport Medium (feces of suspected cholera patients), Pike’s medium (throat specimens of patients with streptococcal infection)  STAINING Most microorganisms besides being very tiny are also devoid of any color and are thus difficult to see, even with the use of the microscope. To facilitate visualization, staining procedures have been developed by various scientists Staining procedures are meant to give color to the organisms, making them easier to see under the microscope. Staining can be simple, differential, or special  SIMPLE STAINING Make use of a single dye which can either be aqueous or alcohol-based. A quick and easy way to visualize cell shape, size, and arrangement of bacteria. Uses basic dyes such as safranin, methylene blue, or crystal violet.  DIFFERENTIAL STAINING Used to differentiate one group of bacteria from another. Two types of differential staining procedures: – Gram stain – Acid-fast stain  DIFFERENTIAL STAINING Gram stain developed in 1884 by the Danish bacteriologist, Hans Christian Gram. one of the most useful staining procedures because it classifies bacteria into two large groups: – gram-positive – gram-negative Exceptions: - Mycoplasma - Spirochetes - Mycobacteria  DIFFERENTIAL STAINING Gram stain  DIFFERENTIAL STAINING Gram Stain Result REAGENT FUNCTION GRAM + GRAM - Crystal violet Primary stain Purple or blue Purple or blue Gram’s iodine Mordant Purple or blue Purple or blue Acetone or 95% Decolorizer Purple or blue Colorless alcohol Safranin Counterstain/S Purple or blue Red or pink econdary stain  DIFFERENTIAL STAINING Gram Stain Result  DIFFERENTIAL STAINING Gram Stain Result  DIFFERENTIAL STAINING Acid Fast Stain Used for bacteria with high lipid content in their cell wall, hence cannot be stained using Gram stain. Microbiologists use this stain to identify all bacteria in the genus Mycobacterium and pathogenic strains of the genus Nocardia.  DIFFERENTIAL STAINING Acid Fast Stain : Two Methods Ziehl-Neelsen stain – Also known as the “hot method” because it requires steam-bathing the organism with aqueous dye – Developed by Franz Ziehl and Friedrich Neelsen Kinyoun stain – Also known as the “cold method”, as it does not utilize heat for staining.  DIFFERENTIAL STAINING Acid Fast Stain Result REAGENT FUNCTION RESULT Ziehl-Neelsen Kinyoun Acid-fast Non Acid-fast Carbolfuchsin Carbolfuchsin Primary stain Red or pink Red Acid alcohol Acid alcohol Decolorizer Red Colorless Methylene blue Malachite green Counterstain Ziehl Neelsen: Ziehl Neelsen: or Secondary Red organism/ Blue organism/ stain Blue Blue background background Kinyoun: Kinyoun: Red organism/ Green Green organism/ background Green background  DIFFERENTIAL STAINING Acid Fast Stain Result  SPECIAL STAINING Used to demonstrate specific structures in a bacterial cell. – Loeffler Alkaline Methylene Blue (LAMB) Stain – metachromatic granules – Hiss stain – capsule or slime layer – Dyer stain – cell wall – Fischer-Conn stain – flagella – Dorner and Schaeffer Fulton stains – spores – India ink or Nigrosine – capsule of the fungus Cryptococcus neoformans SPECIAL STAINING END