Microbial cultivation

cultivation of microorganisms

1. MICROBIAL CULTIVATION PRESENTATION BY G. Mohana Priya S. Nandhini G. Nantha kumar 7 April 2020 Biochemical Engineering 1

2. What is cultivation? Cultivation is the process of propagating organisms by providing the proper environmental conditions . An understanding of these conditions enables us to characterize isolates and differentiate between different types of bacteria. Parasites, bacteria and viruses all generally require cultivation for detailed study. i.e., the process of growing microorganisms in culture by taking bacteria from the infection site (in vivo or environment) and grow them in artificial environment in the laboratory (in vitro). 7 April 2020 Biochemical Engineering 2

3. What is microbial cultivation? Bacteria may require adequate nutrition, temperature, oxygen, optimum pH for growth and multiplication. Suitable artificial media containing sources of carbon, oxygen, nitrogen, hydrogen, phosphorous, and other elements such as sodium, potassium, magnesium, iron and growth factor (vitamins) in very small amounts have been used for cultivation of microorganisms. 7 April 2020 Biochemical Engineering 3

4. Growth requirements for microorganisms A characteristic of microorganisms is their ability to grow and form a population of organisms . One of the results of microbial metabolism is an increase in the size of the cell. The many requirements for successful growth include those both chemical and physical. 7 April 2020 Biochemical Engineering 4

5. Chemical requirements: In order to grow successfully, microorganisms must have a supply of water as well as numerous other substances including mineral elements, growth factors, and gas, such as oxygen. Virtually all chemical substances in microorganisms contain carbon in some form, whether they be proteins, fats, carbohydrates, or lipids. 7 April 2020 Biochemical Engineering 5

6. Both chemoautotrophic and photoautotrophic microorganisms obtain their energy and produce their nutrients from simple inorganic compounds such as carbon dioxide. Chemoautotrophs do so through chemical reactions , while photoautotrophs use photosynthesis. 7 April 2020 Biochemical Engineering 6

7. Oxygen : The presence or absence of molecular oxygen (O2) is a significant factor for microbial growth and survival. Aerobe: Obligate aerobe - oxygen is an absolute requirement for their energy-yielding properties E.g. Mycobacterium tuberculosis. Microaerophile - bacteria species are microaerophilic, meaning that they grow in low concentrations of oxygen E.g. Helicobacter pylori. 7 April 2020 Biochemical Engineering 7

8. Anaerobe: Certain microorganisms grow in oxygen-free environments and are described as anaerobic. Facultative anaerobe - These species grow in either the presence or absence of oxygen E.g. Staphylococcus aureus. Aerotolerant anaerobe - E.g. Lactobacillus acidophilus. Obligate anaerobe - E.g. Clostridium difficile 7 April 2020 Biochemical Engineering 8

9. Other chemical requirements for microbial growth include such trace elements as iron, copper, and zinc . These elements often are used for the synthesis of enzymes. Organic growth factors such as vitamins may also be required by certain bacteria. Amino acids, purines, and pyrimidines should also be available. 7 April 2020 Biochemical Engineering 9

10. Physical requirements: Certain physical conditions affect the type and amount of microbial growth. Temperature: For many bacteria both extremely high and extremely low temperatures can be quite harmful, the former due to protein denaturation, the latter due to intracellular ice crystal formation upon freezing. 7 April 2020 Biochemical Engineering 10

11. Organisms can be divided into groups on the basis of their preferred temperature range Psychrophile: optimum less than 15℃ Mesophile: optimum 20 - 45℃ Thermophile: optimum 55 - 65℃ Hyperthermophile: optimum above 80℃ 7 April 2020 Biochemical Engineering 11

12. pH : Another physical requirement is the extent of acidity or alkalinity , referred to as the pH of a solution pH measures how acidic or basic a solution is, and microbes may grow in either acidic, basic, or neutral pH conditions. 7 April 2020 Biochemical Engineering 12

13. An organisms that grows in acidic (low pH of 2 or below) conditions is called an acidophile . E.g. Helicobacter pylori An organisms that grows in basic (high pH of usually 8.5 – 11) conditions is called an alkaliphile . E.g. Bacillus halodurans An organisms that grows in neutral pH (between 6.5 – 7.5) conditions is called a neutrophile . E.g. E.coli or streptococcus pyogenes and the protozoa Naegleria fowleri Most microbes grow in neutral pH conditions of around pH 7.0, but a number of bacteria can grow in acidic or basic conditions . 7 April 2020 Biochemical Engineering 13

14. Osmotic pressure: Osmotic pressure is the minimum pressure which needs to be applied to a solution to prevent the inward flow of water across a SPM. Types of solution: Hypotonic Isotonic Hypertonic 7 April 2020 Biochemical Engineering 14

15. Classification of bacteria according to osmotic pressure: Osmotolerant: Are those microorganisms which can grow at relatively high salt concentration. e.g. Staphylococcus sp , etc., Halophiles: Grow in the presence of salt at concentration above 0.2 to 0.6 e.g. Halobacterium halobium 7 April 2020 Biochemical Engineering 15

16. Classification of bacteria on the basis of hydrostatic pressure: Barotolerant: Does not get affected by increased pressure. Barophiles/ Piezophiles : Bacteria which grow at moderately high hydrostatic pressures. e.g. Halomonas salaria , Xenophyophores , etc., 7 April 2020 Biochemical Engineering 16

17. Methods of culturing microorganisms: 5 Basic techniques: to manipulate, grow, examine and characterize microorganisms. Inoculation Incubation Isolation Inspection Identification 7 April 2020 Biochemical Engineering 17

18. Types of culture: Pure culture: A container of medium that grows only a single known species or type of microorganisms. Most frequently used for laboratory study because it allows the systematic examination and control of one microorganism by itself. Mixed culture: A container that holds two or more identified, easily differentiated species of microorganisms. Contaminated culture: Was once pure or mixed but has since had contaminants introduced into it like weeds into a garden 7 April 2020 Biochemical Engineering 18

19. When microorganisms are cultivated in the laboratory, a growth environment called a medium is used. Microorganisms growing in or on such a medium form a culture. 7 April 2020 Biochemical Engineering 19

20. General microbial media: For the cultivation of bacteria, a commonly used medium is nutrient broth, a liquid containing proteins, salts, and growth enhancers that will support many bacteria. To solidify the medium, an agent such as agar is added. Agar: Agar is a polysaccharide that adds no nutrients to a medium, but merely solidifies it. The medium that results is nutrient agar. 7 April 2020 Biochemical Engineering 20

21. Many media for microorganisms are complex, reflecting the growth requirements of the microorganisms. For instance, most fungi require extra carbohydrate and an acidic environment for optimal growth. The medium employed for these organisms is potato dextrose agar, also known as Sabouraud dextrose agar. For protozoa, liquid media are generally required, And for rickettsiae and viruses, living tissue cells must be provided for best cultivation. 7 April 2020 Biochemical Engineering 21

22. For anaerobic microorganisms, the atmosphere must be oxygen free . To eliminate the oxygen, the culture media can be placed within containers where carbon dioxide and hydrogen gas are generated and oxygen is removed from the atmosphere. 7 April 2020 Biochemical Engineering 22

23. Media are classified based on Chemical constituents Physical nature Function 7 April 2020 Biochemical Engineering 23

24. 1. Chemical constituents: Defined media: all components and constituents are known. Complex media: contain some ingredients that are of unknown composition and / or concentration. 7 April 2020 Biochemical Engineering 24

25. 2. Physical nature: Based on the physical state Liquid medium e.g. Fluid Thioglycolate medium. Solid medium (1.5% (w/v) agar) e.g. Nutrient agar. Semi-solid medium (0.6-1.0%(w/v) agar) e.g. Nutrient broth. 7 April 2020 Biochemical Engineering 25

26. 3. Media function: Supportive – supports many organisms e.g. tryptone soy agar Enriched – general purpose media enriched with blood or other special nutrients to support the growth of fastidious bacteria e.g. Blood agar. Selection – allows growth for particular microorganisms while inhibiting the growth of others e.g. MacConkey agar – selects for enterics (i.e. bile salts) Differential – distinguished on different groups of microorganisms based on their biological characteristics e.g. blood agar: haemolytic versus non-haemolytic bacteria. MacConkey agar: lactose fermenter versus non fermenter 7 April 2020 Biochemical Engineering 26

27. Isolation methods: Streak plate method. Loop dilution or pour plate. 7 April 2020 Biochemical Engineering 27

28. Streak plate method: A sample of mixed bacteria is streaked several times along one edge of a Petri dish containing a medium such as nutrient agar. A loop is flamed and then touched to the first area to retrieve a sample of bacteria. This sample is then streaked several times in the second area of the medium. The loop is then reflamed, touched to the second area, and streaked once again in the third area. The process can be repeated in a fourth and fifth area if desired. During incubation, the bacteria will multiply rapidly and form colonies 7 April 2020 Biochemical Engineering 28

29. Pour plate method: In this method, a sample of bacteria is diluted in several tubes of melted medium such as nutrient agar. After dilution, the melted agar is poured into separate Petri dishes and allowed to harden. Since the bacteria have been diluted in the various tubes, the plates will contain various dilutions of bacteria, and where the bacteria are most diluted, they will form isolated colonies 7 April 2020 Biochemical Engineering 29

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31. Microbial reproduction and growth: Reproduction patterns: During their growth cycles, microorganisms undergo reproduction many times, causing the numbers in the population to increase dramatically. In fungi, unicellular algae, and protozoa, reproduction involves a duplication of the nucleus through the asexual process of mitosis and a splitting of the cell in cytokinesis. 7 April 2020 Biochemical Engineering 31

32. Bacteria reproduce by the asexual process of binary fission . In this process, the chromosomal DNA duplicates, after which the bacterial membrane and cell wall grow inward to meet one another and divide the cell in two. The two cells separate and the process is complete. One of the remarkable attributes of bacteria is the relatively short generation time , the time required for a microbial population to double in numbers. The generation time varies among bacteria and often ranges between 30 minutes and three hours . Certain bacteria have very brief generation times. Escherichia coli, for example, has a generation time of about 20 minutes when it is dividing under optimal conditions. 7 April 2020 Biochemical Engineering 32

33. Growth curve: The growth of a bacterial population can be expressed in various phases of a growth curve. Four phases of growth are recognized in the growth curve. 7 April 2020 Biochemical Engineering 33

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35. Lag phase: The population remains at the same number as the bacteria become accustomed to their new environment. Metabolic activity is taking place, and new cells are being produced to offset those that are dying. Log phase: Bacterial growth occurs at its optimal level and the population doubles rapidly . This phase is represented by a straight line, and the population is at its metabolic peak Stationary phase: The reproduction of bacterial cells is offset by their death, and the population reaches a plateau. The reasons for bacterial death include the accumulation of waste, the lack of nutrients, and the unfavorable environmental conditions that may have developed. Death phase: The bacteria die off rapidly , the curve turns downward, and the last cell in the population soon dies. 7 April 2020 Biochemical Engineering 35

36. Microbial measurements: In order to measure the number of bacteria in a population, various methods are available. Plate count method Direct microscopic counts Turbidity methods BOD( Biochemical oxygen demand) 7 April 2020 Biochemical Engineering 36

37. Plate count method: A sample of bacteria is diluted in saline solution, distilled water, or other holding fluid. Samples of the dilutions are then placed in Petri dishes with a growth medium and set aside to incubate. Following incubation, the count of colonies is taken and multiplied by the dilution factor represented by that plate. Generally, plates with between 30 and 300 colonies are selected for determining the final count, which is expressed as the number of bacteria per original ml of sample. 7 April 2020 Biochemical Engineering 37

38. Direct microscopic count: A specially designed counting chamber called a Petroff- Hausser counter is used. A measured sample of the bacterial suspension is placed on the counter, and the actual number of organisms is counted in one section of the chamber. Multiplying by an established reference figure gives a number of bacteria in the entire chamber and in the sample counted. The disadvantage of this method is that both live and dead bacteria are counted. 7 April 2020 Biochemical Engineering 38

39. Turbidity method: This can also be used to assess bacterial growth. As bacteria multiply in liquid media, they make the media cloudy. Placing the culture tube in a beam of light and noting the amount of light transmitted gives an idea of the turbidity of the culture and the relative number of bacteria it contains. 7 April 2020 Biochemical Engineering 39

40. BOD: The dry weight of a culture can also be used to determine microbial numbers. The liquid culture is dried out, and the amount of microbial mass is weighed on a scale. It is also possible to measure the oxygen uptake of a culture of bacteria. If more oxygen is used by culture A than by culture B and all other things are equal, then it may be deduced that more microorganisms are present in culture A. A variation of this method called the biochemical oxygen demand (BOD) is used to measure the extent of contamination in a water sample. 7 April 2020 Biochemical Engineering 40

41. Reference: https://www.cliffsnotes.com/study-guides/biology/microbiology/microbial-cultivation-and-growth/growth-requirements-for-microorganisms https://www.youtube.com/watch?v=2E4hNyHK3zM https://www.scribd.com/doc/17691787/Cultivation-of-Microorganisms 7 April 2020 Biochemical Engineering 41

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