Nutritional , Physical Parameters, and Bacteriological Media Required for Optimal Bacterial Growth

Explore the critical nutrients and Physical conditions necessary for bacterial growth. This presentation covers the roles of carbon, nitrogen, sulfur, phosphorus, vitamins, and minerals and the effects of pH, temperature, osmotic pressure, and light. Learn about various bacteriological media and their importance in cultivating bacteria for research and industry.

1. Nutrition of Bacteria By Ms. Priyanka B. Rathod Assistant Professor G H Raisoni Institute of Life Sciences (Department of Pharmacy) 1

2. Nutritional Requirements of Bacteria General Requirements Bacteria require adequate nutrition, optimum pH, temperature, and oxygen for growth and multiplication. The rate at which bacteria grow and divide depends on the nutritional status of the environment. The growth of a single E. coli cell into new daughter cells may occur within 20-30 minutes in rich laboratory media. In contrast, the same process is much slower in nutritionally depleted media.   1. Source of Energy Bacteria that derive their energy from sunlight are called phototrophs (e.g. Rhodospirillum rubrum). Those that obtain energy from chemical reactions are chemotrophs (e.g. Escherichia coli). 2

3. 2. Source of Electrons All microorganisms require a source of electrons for their metabolism. Bacteria which use reduced inorganic compounds as electron donors are called lithotrophs (e.g., Pseudomonas pseudoflavo ). Some species which use organic compounds as electron donors are called organotrophs (e.g., Escherichia coli). Among phototrophic bacteria, some species use inorganic compounds (H2S) as a source of electrons and are called photolithotrophs (e.g. Chromatium okenii). Others which use organic compounds such as fatty acids and alcohols as electron donors are called photoorganotrophs (e.g. Rhodospirillum rubrum). Among chemotrophic bacteria, some species use inorganic compounds as the source of electrons and are called chemolithotrophs (e.g. Nitrosomonas europaea). Others use organic compounds such as sugars and amino acids as electron donors and are called chemoorganotrophs (e.g., Escherichia coli). 3

4. 3. Source of Carbon Microorganisms require carbon for synthesizing cell components. Some species use CO2 as the major source of carbon and are called autotrophs (e.g., Chromatium okenii). Other species require organic compounds as a source of carbon and are called heterotrophs (e.g., Escherichia coli).  4. Nitrogen Bacteria can use nitrogen from the atmosphere or from inorganic compounds such as nitrates, nitrites, ammonium salts, or organic compounds such as amino acids. Nitrogen is a major component of protein and nucleic acids. 5. Sulphur Many bacterial species use sulphur from organic sulphur compounds, inorganic sulphur compounds, and elemental sulphur . Sulphur is needed for the synthesis of amino acids ( e.g., cystine, methionine).   4

5. NUTRITION CLASSIFICATION OF BACTERIA Fig. 1 Nutritional Classification of Microorganisms Microorganism Energy Source Chemical reaction Chemotrophs (e.g. Escherichia Coli) Electron Sources Inorganic Compounds Chemolithotrophs (e.g. Nitrosomonas europaea Organic Compounds Chemoorganotrophs (e.g. Escherichia coli) Carbon Source Organic Compounds Chemoheterotrophs ( e.g Streptococcus sp.) CO2 Chemoautotrophs (e.g. Hydrogen, Sulfur, oxidizing bacteria) Sunlight Phototrophs (e.g. Rhodospirillum rubrum Electron Source Inorganic Compounds Photolithotrophs (e.g. Chromalium, okenii) Organic Compounds Photoorganotrophs (e.g. Rhodospirillum rubrum) Carbon Source Organic Compounds Photoheterotrophs (e.g. Purple non- sulphur bacteria) CO2 Photoautotrophs (e.g. Plants, algae Electron Source Inorganic Compounds Lithotrophs(e.g. Pseudomonas pseudoflava ) Organic Compounds Organotrophs(e.g. Escherichia coli) 5

6. 6. Phosphorus Phosphorus, usually supplied in the form of phosphate, is an essential component of nucleotides , nucleic acids, phospholipids , etc.  7. Mineral Salts Bacteria require salts, particularly the anions such as phosphate and sulphate, and the cations such as sodium, potassium, magnesium, iron, and calcium. These are normally present in natural environments or may be added to culture media. 8. Growth Factors or Bacterial Vitamins Some bacterial species require organic compounds in minute quantities for growth, known as growth factors or bacterial vitamins. Some bacteria are capable of synthesizing their entire requirement of vitamins from culture media. Other species cannot synthesize the vitamins from media and do not show growth in the absence of vitamins. Hence, vitamins are added to the culture media for the growth of these species. 6

7. 9. Water Water is the major essential nutrient as it accounts for about 80 to 90% of the total weight of cells. Water is a highly polar compound and contains micro-nutrients and trace elements which are required for the growth of bacteria. Species Required vitamin and Substances for Growth or Growth stimulation Staphylococcus aureus Thiamine, Nicotinic acid Bacillus anthracis Thiamine(Vitamin B1) Clostridium tetani Riboflavin Lactobacillus Species Pyridoxine (vitamin B6), Cobalamin (Vitamin B12) Bacteroides melaninogenicus Vitamin K Neisseria gonorrhoeae Glutathione Corynebacterium diphtheriae Β - Alanine Hemophilus influenza Hematin Table 1. Bacterial vitamins or growth factor: 7

8. BACTERIOLOGICAL MEDIA Nutrient agar is a common laboratory medium used for growth of many bacterial species. It is important to note that not a single media is suitable for growth of all microorganisms. Compound Amount Function or Use of Component Yeast/ Meat/ Beef Extract 10 gm Sources of carbohydrates, amino acid, and other growth factor Peptone 10 gm Sources of amino acids, N, S, P, and growth factors Sodium Chloride 5gm Electrolyte Agar 20gm Solidifying agent Distilled Water To make 100ml Vehicle Table 2. Composition of Nutrient agar: 8

9. 1. Water Types: Tap, pure, or distilled Role: Dissolves organic and inorganic compounds; serves as a vehicle for nutrient flow Cell Protoplasm: Contains 70 to 80% water; essential for enzymatic reactions Note: Copper distilled water inhibits bacterial growth   2. Peptone Source: Lean meat, heart muscle, casein, fibrin, soya meal, etc. Constituents: Proteases, amino acids, inorganic salts (phosphates, potassium, magnesium), growth factors (nicotinic acid, riboflavin) Function: Supplies nitrogenous material, acts as a buffer Storage: Tightly closed container (hygroscopic, becomes sticky when exposed to air) 9

10. 3. Yeast Extract Source: Cells of baker's yeast (Saccharomyces) Contents: Carbohydrates, amino acids, growth factors (vitamin B group), inorganic salts Function: Provides vitamins; can substitute meat extract   4. Meat Extract Source: Fresh lean meat, prepared by hot water extraction Contents: Gelatin, peptones, proteoses, amino acids, creatine, creatinine, purines, mineral salts, growth factors (thiamine, nicotinic acid, riboflavin, pyridoxine, pantothenic acid) 10

11. 5. Agar Source: Seaweeds algae (class: Rhodophyta), such as Gelidium , Gracilaria , Hyphea , Gelidiella , Ceramium Composition: Agarose (70%) and agaropectin (30%); contains calcium, chloride, magnesium, sulphate, iron Properties: Solidifying agent (2%) No nutritional value Bacteriologically inert Resistant to the action of all microorganisms Stable at incubation temperatures Melts at 95 to 98°C, remains liquid up to 40 to 42°C Solidifies below 40°C Economical and easily available 11

12. TYPES OF CULTURE MEDIA 1. Classification Based on Physical State: Solid Media Agar Concentration: 1.5% 2.5% Example: Nutrient Agar Semisolid Media Agar Concentration: 0.2% 0.5% Example: Nutrient Broth with 0.5% Agar   Liquid Media Agar Concentration: None Example: Fluid Thioglycollate Broth   12

13. 2. Classification Based on Oxygen Requirement : Aerobic Media Example: MacConkey's Broth Anaerobic Media Example: Robertson's Cooked Meat Medium   3. Classification Based on Chemical Composition: Simple or Basal Media Forms the basis of most media for studying common bacteria; e.g., Nutrient Agar. Example: Peptone Water, Nutrient Broth Synthetic or Defined Media Description: Prepared from pure chemical substances with known exact composition. Uses: Ideal for research and metabolic studies of microorganisms. Example: Media with precisely known chemical composition Non-Synthetic or Undefined (Complex) Media Contains biological materials like blood or yeast extract; used for unknown bacteria cultivation. Example: Media containing blood, milk, yeast extract, or beef extract 13

14. 4. Classification Based on Functional Type: Enriched Media Enrichment Media Selective Media Indicator Media Differential Media Sugar Media Transport Media Assay Media Storage Media A. Enriched Media: Description: Enhanced with additional substances to support the growth of fastidious microorganisms. Examples: Blood Agar: Used for Streptococcus Chocolate Agar: Supports Neisseria, Haemophilus Loeffler Serum Slope: Used for Corynebacterium diphtheriae 14

15. B. Enrichment Media: Description: Liquid media with specific substances that inhibit unwanted bacteria while promoting the growth of desired bacteria. Examples: Tetrathionate Broth Selenite F Broth Application: Effective for isolating pathogenic bacteria (e.g., Salmonella, Shigella) from fecal samples by suppressing coliforms (e.g., Escherichia coli). C. Selective Media: Description: Solid media with added substances that inhibit unwanted bacteria but allow desired bacteria to grow in colonies. Examples: MacConkey's Media: Contains sodium taurocholate, selects for Gram-negative bacteria. Deoxycholate Citrate Agar (DCA): Selects for Salmonella and Shigella species. Lowenstein Jensen Medium: Used for Mycobacterium tuberculosis. Additional Features: Physical conditions (temperature, pH, gaseous) can be adjusted to enhance selectivity. 15

16. D. Indicator Media: Description: Media containing indicators that change color in response to bacterial growth. Examples: Wilson and Blair Medium: Salmonella typhi reduces sulfite to sulfide, resulting in colonies with a black metallic shine. E . Differential Media: Description: Used to differentiate between bacterial types based on observable characteristics. Examples: MacConkey's Medium: (contains Peptone, Lactose neutral red & Taurocholate) Differentiates lactose fermenters (red colonies) from non-lactose fermenters (white or pale colonies). Blood Agar Medium: Enriched medium; bacteria causing hemolysis show clear zones around colonies, indicating differential growth patterns 16

17. F. Sugar Media: Description: Contains 1% sugar added to peptone water with a suitable indicator. Features: Includes a Durham's tube (small inverted tube) to detect gas production. Purpose: Used to test the fermentation of sugars by microorganisms.   G. Transport Media: Description: Designed to preserve delicate microorganisms during transport to the laboratory. Examples: Stuart's Transport Medium Amies Transport Medium 17

18. H. Assay Media: Description: Formulated with specific compositions for assaying substances. Uses: Antibiotics Amino Acids Vitamins Disinfectants Purpose: Provides a controlled environment for evaluating the effectiveness of various agents.   I. Storage Media: Description: Used for long term preservation and storage of bacteria. Examples: Dorset's Egg Medium Nutrient Agar Stabs Blood Agar Slants Robertson's Cooked Meat Medium Purpose: Ensures the viability of bacterial cultures over extended periods. 18

19. PHYSICAL PARAMETER FOR GROWTH In microbiology, understanding the physical parameters for bacterial growth is essential for cultivating and studying different microorganisms. Here's a summary of the temperature related classifications of bacteria based on their growth preferences:   1. Temperature: Optimum Growth Temperature: The temperature that supports the fastest growth rate for a microorganism over a short period. Maximum Growth Temperature : The highest temperature at which a microorganism can grow. Minimum Growth Temperature: The lowest temperature at which a microorganism can grow. 19

20. Based on temperature tolerance, bacteria are categorized into: Psychrophiles: Grow at temperatures as low as 0 ℃ . Obligate Psychrophiles: Optimal growth at 15 ℃ or lower. Psychrotrophs (Facultative Psychrophiles): Can grow at 0 ℃ but have optimal growth at 20 to 30 ℃ .   Mesophiles: Grow within a temperature range of 20 to 40 ℃ . Include bacteria that are pathogenic to humans and warmblooded animals. Thermophiles: Prefer high temperatures ranging from 45 to 70 ℃ for growth. Some can grow in the mesophilic range as well. Facultative Thermophiles : Optimal at 50 to 55 ℃ . Obligate or Steno: Optimal at 65 to 70 ℃ . Thermoduric: Tolerate high temperatures but do not grow above 60 ℃ . 20

21. Table 3. Temperature range for bacterial growth: 2. pH and Bacterial Growth  Classification by pH Acidophiles: Optimum pH 1 to 6.5 Examples: Thiobacillus thiooxidans (2-3.5), Lactobacillus acidophilus (5.8-6.6) Neutrophiles: Optimum pH 6.5 to 7.5 Examples: Escherichia coli, Salmonella typhi Alkalophiles : Optimum pH 7.5 to 14 Examples: Vibrio cholerae (9.0), Agrobacterium species (12.0) Group Minimum ( °C) Optimum (°C) Maximum( °C) Examples Obligate Psychrophiles 0 to 5 10 to 20 20 Vibrio psychroerythrus Facultative Psychrophiles 0 to 5 25 to30 35 Pseudomonas fluorescens Mesophiles 15 to 20 35 to 40 45 Corynebacterium diphtheria Facultative Thermophiles 35 to 40 50 to55 65 Streptococcus thermophilus Obligate Thermophiles 50 to 55 65 to 70 80 Thermus aquaticus 21

22. 3. Gaseous Requirements for Bacterial Growth Principle Gases Oxygen and carbon dioxide are crucial for bacterial growth. Oxygen requirement reflects the energy mechanism. Aerobic Bacteria Require oxygen for growth. Grow in an air atmosphere (21% oxygen). Examples: Escherichia coli, Staphylococcus aureus.   Anaerobic Bacteria Do not use oxygen for energy. Oxygen is toxic; cannot grow in an air atmosphere. Tolerant or Non-stringent Anaerobes: Can tolerate low levels of oxygen. Strict or Stringent Anaerobes: Cannot tolerate even low level oxygen; may die after brief exposure. Examples Clostridium species. 22

23. Facultatively Anaerobic Bacteria Do not require oxygen but use it if available for energy production. Grow in both aerobic and anaerobic conditions. Examples: Pseudomonas species.   Microaerophilic Bacteria Require low levels of oxygen for growth. Cannot tolerate full atmospheric oxygen levels. Examples: Campylobacter jejuni, Lactobacillus plantarum. 23

24. 4. Osmotic Pressure and Bacterial Growth   Osmotic Tolerance Bacteria are tolerant of osmotic variations due to the mechanical strength of their cell walls. Capable of growing in media with varying salt, sugar, and solute concentrations.  Plasmolysis Sudden exposure to high salt concentrations can cause plasmolysis. Culture media typically include 0.5% NaCl to maintain isotonic conditions. 24

25. 5.Light and Bacterial Growth Darkness generally favors the growth and viability of microorganisms. Microorganisms are sensitive to: Ultraviolet (UV) radiation Direct light Other forms of radiation   Key Considerations Exposure to light can inhibit growth or damage microorganisms. Culturing and storage often require dark conditions to ensure optimal growth. 25

26. Reference Kokare, Chandrakant.(2023) Pharmaceutical Microbiology, Eleventh Edition, Nirali Prakashan. 26

27. Thank you 27