1. Classification of Bacteria and nutritional requirements,

classification of bacteria,
nutritional requirements, raw materials used for culture media and physical
parameters for growth, growth curve, isolation and preservation methods
for pure cultures, cultivation of anaerobes, quantitative measurement of
bacterial growth (total & viable count).

1. Classification of bacteria

2. Classification of Bacteria based on Mode of Nutrition Phototrophs Those bacteria which gain energy from light. Phototrophs are further divided into two groups on the basis of source of electron. Photolithotrophs:  these bacteria gain energy from light and uses reduced inorganic compounds such as H2S as electron source. E.g.  Chromatium okenii. Photo organotrophs:  these bacteria gain energy from light and uses organic compounds such as succinate as electron source. 2. Chemotrophs Those bacteria gain energy from chemical compounds. They cannot carry out photosynthesis. Chemotrophs are further divided into two groups on the basis of source of electron. Chemolithotrophs:  they gain energy from oxidation of chemical compound and reduces inorganic compounds such as NH 3 as electron source. E.g.  Nitrosomonas. Chemoorganotrophs:  they gain energy from chemical compounds and uses organic compound such as glucose and amino acids as source of electron. e.g.  Pseudomonas pseudoflava 

3. 3. Autotrophs Those bacteria which uses carbondioxide as sole source of carbon to prepare its own food. Autotrophs are divided into two types on the basis of energy utilized to assimilate carbondioxide. ie. Photoautotrophs and C hemoautotrophs. Photoautotrophs:  they utilized light to assimilate CO 2 . They are further divided into two group on the basis of electron sources. Ie.  Photolithotropic autotrophs  and  Photoorganotropic autotrophs Chemoautotrophs:  They utilize chemical energy for assimilation of CO 2. 4. Heterotrophs  Those bacteria which uses organic compound as carbon source. They lack the ability to fix CO 2 Most of the human pathogenic bacteria are heterotropic in nature. Some heterotrops are simple, because they have simple nutritional requirement. However there are some bacteria that require special nutrients for their growth; known as fastidious heterotrophs.

4. The requirements for microbial growth can be divided into two main categories  Physical and chemical. Physical - Temperature, pH and Osmotic pressure. Temperature:         Most microorganisms grow well at the temperature that humans favor.   Certain bacteria are capable of growing at extreme of temperature that would certainly hinder the survival of almost all eukaryotic organism. Microorganisms are classified into three primary groups on the basis of their preferred range of temperature Classification of Bacteria based on Temperature

5. Psychrophiles   (cold-loving microbes) Bacteria that can grow at 0°C or below but the optimum temperature of growth is 15 °C or below and maximum temperature is 20°C are called psychrophiles Psychrophiles have polyunsaturated fatty acids in their cell membrane which gives fluid nature to the cell membrane even at lower temperature. Examples:  Vibrio psychroerythrus, Vibrio marinus, Polaromonas vaculata, Psychroflexus

6. 2. Mesophiles: Those bacteria that can grow best between (25-40) o C but optimum temperature for growth is 37 0  C Most of the human pathogens are mesophilic in nature Examples:  E. coli, Salmonella, Klebsiella, Staphylococci. 3. Thermophiles: Those bacteria that can best grow above 45 o C . Thermophiles capable of growing in mesophilic range are called facultative thermophiles. Thermophiles contains saturated fatty acids in their cell membrane, so their cell membrane does not become too fluid even at higher temperature. Examples:  Streptococcus thermophiles, Bacillus stearothermophilus, Thermus aquaticus

7. Hyperthermophiles: Those bacteria that have optimum temperature of growth above 80 o  C. Mostly Archeobacteria are hyperthermophiles. Monolayer cell membrane of Archeobacteria is more resistant to heat and they adopt to grow in higher temperature. Examples:  Thermodesulfobacterium, Thiolava veneris The bacteria formed a massive white mat, around the summit of the newly formed Tagoro volcanic cone at depths of about 430 feet.  Scientists reporting the new species concluded that the unique metabolic characteristics of the bacteria allow them to colonize this newly formed seabed

8. Classification of bacteria based on optimum pH of growth 1. Acidophiles Those bacteria that grow best at acidic pH The cytoplasm of these bacteria are acidic in nature. Some acidophiles are thermophilic in nature, such bacteria are called   thermoacidophiles.          Examples:  Thiobacillus thioxidans, 2. Alkaliphiles Those bacteria that grow best at alkaline pH        Example: V ibrio cholerae  optimum pH of growth is 8.2 3. Neutrophiles  Those bacteria that grow best at neutral pH (6.5-7.5) Most of the bacteria grow at neutral pH       Example:  E. coli

9. Classification of bacteria based on gaseous requirement 1. Obligate aerobes: Those bacteria that require oxygen and cannot grow in the absence of O 2 . These bacteria carryout only oxidative type of metabolism.          Examples;  Mycobacterium, Bacillus 2. Facultative anaerobes: Those bacteria that do not require O 2 but can use it if available. Growth of these bacteria become better in presence of O 2 These bacteria carryout both oxidative and fermentative type of metabolism          Examples: E coli, Klebsiella, Salmonella 3. Aerotolerant anaerobes; Those bacteria do not require O 2 for growth but can tolerate the presence of O 2 . Growth of these bacteria is not affected by the presence of O 2 . These bacteria have only fermentative type of metabolism.               Example : Lactobacillus

10. 4. Microaerophiles  Those bacteria that grow on low concentration of Oxygen but at atmospheric level of Oxygen growth of these bacteria is inhibited. These bacteria only have oxidative type of metabolism           Example:  Campylo bacter 5. Obligate anaerobes  Those bacteria that can grow only in absence of Oxygen. Oxygen is harmful to obligate anaerobes These bacteria have only fermentative type of metabolism         Examples : Pepto coccus, Pepto streptococcus, Clostridium 6. Capnophiles  Those bacteria that require carbon dioxide for growth. They are CO 2 loving organism Most of the microaerophiles are capnophilic in nature.           Example : Campylobacter, Helicobacter pylori, Brucella abortus

11. N utritional requirements Raw materials used for culture media Physical parameters for growth  Unit 1.2

12. N utritional requirements Nutrients are materials that are acquired from the environment and are used for growth and metabolism .  Microorganisms (or microbes) vary significantly in terms of the source, chemical form, and amount of essential elements they need.  Some examples of these essential nutrients are carbon, oxygen, hydrogen, phosphorus, and sulfur. There are two categories of essential nutrients: Macro-nutrients (which are needed in large amounts)  Micro-nutrients (which are needed in trace or small amounts ).  Macro-nutrients usually help maintain the cell structure and metabolism.  Macromolecules found within bacterial cytoplasm include the nucleoid region, ribosomes, proteins, and enzymes Micro-nutrients help enzyme function and maintain protein structure.

13. The cytosol is a major component of the cytoplasm; it is the liquid portion of the cytoplasm   The cytosol is typically composed of water, salts, and organic molecules. Elements found within the cytosol include carbon, hydrogen, nitrogen, oxygen, phosphorous, and sulfur .  These elements are critical to metabolic processes used by microbes. Nutrients are necessary for microbial growth and play a vital role in the proper cultivation of microorganisms in the laboratory .  The types of nutrients that are required include those that supply energy, carbon and additional necessary materials. The nutrients used to propagate growth are organism -specific, based on their cellular and metabolic processes. The common nutrients which are found to be required in all living things include carbon, nitrogen, sulfur, phosphorus, potassium, magnesium, calcium, oxygen, iron and additional trace elements.

14. Raw materials used for culture media A microbial culture, is a method of multiplying microorganism by letting them reproduce in predetermined culture media under controlled laboratory conditions. Microbial cultures are used to determine the type of organism, its abundance in the sample being tested, or both. Culture Media: An artificial culture media must provide similar environmental and nutritional conditions that exist in the natural habitat of a bacterium. A culture medium contains water, a source of carbon & energy, source of nitrogen, trace elements and some growth factors. The pH of the medium must be set accordingly.  Uses:  • Enrich the number of bacteria.  •Select for certain bacteria and suppress others.  •Differentiate among different kinds of bacteria.

15. Basic requirements of culture media •Nutrients  •Energy source  •Carbon source  •Nitrogen source  •Mineral salts – Sulphate, phosphates, chlorides and carbonates of K, Mg and Ca  •A suitable pH about 7.2 – 7.6  •Accessory growth factors  •Water 

16. Many of the ingredients used in culture media are of natural origin, the most important are: Beef extract  Peptones  Yeast extract  Gelatin: Used in concentrations of 10 to 20 % in order to solidify media that tests the ability of an organism to produce an exoenzyme, called gelatinase, that hydrolyzes gelatin for growing different species of microorganisms. Agar – Agar: Complex polysaccharide : Used as solidifying agent for culture media in Petri plates, slants, and deeps. Generally not metabolized by microbes; Liquefies at 98°C; Solidifies at 42°C  Added to culture media in a concentration of 1.2% to 2% to render it solid • Peptone : Water soluble protein (milk, meat, soya)spray dried product obtained from hydrolysis(acid, enzyme e.g. Papain, pepsin, trypsin)  • Meat extract : Concentrated aqueous infusion of fresh beef 

17. Mineral salts:  Sulphate, phosphate, iron ​ • Carbohydrate: provides the microorganisms with carbon which is the source of energy ​ • Water deionized (or) Distilled water:  Free from chemicals that can suppress the growth of microorganisms​ Media Preparation •Accurately weigh each of the dry ingredients in your culture media. •Add each dry culture medium ingredient into a flask. •Add distilled water to make the correct volume. Heat and stir (agar will burn if it is not stirred) until all of the ingredients go into solution. When the media boils, it is ready for sterilization. Media Sterilization Sterilize by using the wet cycle (autoclave). 

18. Classification based on consistency 1. Liquid media.  2. Solid media.  3. Semi solid media

19. A. Liquid media:  These are available for use in test-tubes, bottles or flasks. Liquid media are sometimes referred as “broths” (e.g. nutrient broth).  In liquid medium, bacteria grow uniformly producing general turbidity. No agar is added.  Mostly used for inoculums preparation. (absence of agar) e.g. fluid thioglycolate broth. B. Solid media:  An agar plate is a Petri dish that contains a growth medium (typically agar plus nutrients) used to culture microorganisms.  2% of agar is added. Agar is the most commonly used solidifying agent.  Colony morphology, pigmentation, hemolysis can be appreciated. Examples include Nutrient agar and Blood agar. 

20. C. Semi-solid media: Such media are fairly soft and are useful in demonstrating bacterial motility and separating motile from non-motile strains. Examples of Semi-solid media Stuart's and Amies media 0.5% agar is added. 

21. Depending on functional type media  are classified into six types: (1) Basal media (2) Enriched media (3) Selective media  (4) Indicator media (5) Transport media (6) Storage media                     BASAL MEDIA - Basal media are those that may be used for growth (culture) of bacteria that do not need enrichment of the media .        Examples: Nutrient broth, nutrient agar and peptone water.        Staphylococcus and Enterobacteriaceae grow in these media.           ENRICHED MEDIA - The media are enriched usually by adding blood, serum or egg . Examples: Enriched media are blood agar and Lowenstein-Jensen media. Streptococci grow in blood agar media.

22. SELECTIVE MEDIA - These media favor the growth of a particular bacterium by inhibiting the growth of undesired bacteria and allowing growth of desirable bacteria. Examples: MacConkey agar, Lowenstein-Jensen media, tellurite media (Tellurite inhibits the growth of most of the throat organisms except diphtheria bacilli). Antibiotic may be added to a medium for inhibition. INDICATOR (DIFFERENTIAL) MEDIA -  An indicator is included in the medium. A particular organism causes change in the indicator, e.g. blood, neutral red. Examples: Blood agar and MacConkey agar are indicator media. TRANSPORT MEDIA -These media are used when specimen cannot be cultured soon after collection. Examples: Cary-Blair medium, Amies medium, Stuart medium. STORAGE MEDIA - Media used for storing the bacteria for a long period of time. Examples: Egg saline medium, chalk cooked meat broth

23. Physical parameters for growth  Temperature : Most microorganisms have a specific temperature range in which they grow best. This can vary widely: pH : The acidity or alkalinity of the environment affects microbial growth. Oxygen : Aerobes : Require oxygen to grow. Anaerobes : Do not require oxygen and may even be harmed by it. Facultative Anaerobes : Can grow with or without oxygen. Microaerophiles : Require low levels of oxygen. Aerotolerant Anaerobes : Can tolerate oxygen but do not use it for growth. Nutrient Availability : Microorganisms require various nutrients for growth, including: Carbon Sources : Such as glucose, acetate, or organic compounds. Nitrogen Sources : Such as ammonia, nitrates, or organic nitrogen. Minerals and Trace Elements : Such as calcium, magnesium, potassium, iron, and zinc. Vitamins and Growth Factors : Certain microorganisms require specific vitamins or growth factors not synthesized by themselves.

24. Physical parameters for growth  5. Osmotic Pressure : The concentration of solutes in the growth medium can affect microbial growth. Halophiles : Require high salt concentrations. Osmophiles : Adapted to high sugar concentrations. 6. Water Activity : The amount of free water available in the environment. Xerophiles : Can grow in very dry conditions. Hygrophiles : Require high moisture content. 7. Pressure : Some microorganisms, especially extremophiles, can thrive under high pressure. Barophiles : Grow best under high-pressure conditions, such as in deep-sea environments. 8. Light : Phototrophs : Require light for growth (e.g., certain algae and cyanobacteria). Non-phototrophs : Do not require light and may be harmed by it.