Everything you need to know about cultivation and growth of microorganisms. Some of the frequently asked questions are as follows:-
Q.1. What is the optimum pH for most of the bacteria?
Ans. Near neutrality between 6.5 and 7.5.
Q.2. What are acidophiles?
Ans. Bacteria which can tolerate acidity.
Q.3. What are buffers?
Ans. Chemicals used to neutralize the acids and bases and maintain pH are called buffers. In other words the substance which tends to stabilize the pH of a solution.
Q.4. Why do plasmolysis or shrinkage of the plasma of cells occur?
Ans. Because of osmotic loss of water (also called exosmosis).
Q.5. A low molecular weight compound such as sodium chloride has a greater antimicrobial effect. Give a comparative example.
Ans. NaCl or Sodium chloride having low molecular weight in comparison to sugar possesses higher antimicrobial effect.
Q.6. What are extreme halophiles? Give one example.
Ans. The microorganisms adapted to high concentrations of salts which is needed for their growth, e.g., the bacteria isolated from saline waters of Dead Sea usually require 30 per cent salt.
Q.7. What are facultative halophiles?
Ans. The microbes that do not require high salt concentration but can grow at salt concentrations of up to 2 per cent.
Q.8. What is superoxide dismutase (SOD)?
Ans. In the presence of oxygen obligate anaerobes are known to form some superoxide free radicals. Therefore, all organisms attempting to grow in atmospheric oxygen produce an enzyme superoxide dismutase (SOD) to neutralize toxic free radicals.
Q.9. What is catalase? Give its function.
Ans. Catalase is an enzyme which converts hydrogen peroxide (H2O2) produced in aerobic microorganisms into water and oxygen to get rid of their toxic effects of H2O2 The presence of catalase can be detected easily when a drop of H2O2 is added to a colony and it effervesces (gives out bubbles).
Q.10. Why agar, which is a complex polysaccharide obtained from marine alga, and has been used to thicken foods like jellies, soups and ice-cream, is also used to solidify the media used in cultivation of microorganisms?
Ans. Agar melts at about 100°C and therefore, does not melt while incubating even thermopile microorganisms needing high temperature to grow (approaching 100°C). Once agar melts it remains in liquid form even while the temperatures come down to 40°C on cooling. It can therefore, be easily handled while pouring into petripates and culture tubes. In the laboratory agar is held in a water bath at 50°C, as at this temperature agar does not injure most of the microorganisms/bacteria when poured over the bacterial inoculum.
Q.11. What is a culture?
Ans. The microorganisms grown and multiplied in or on a culture medium are referred to as culture.
Q.12. What is a deep? How does it differ from a slant?
Ans. The agar solidified in a vertical tube is called a deep while the agar solidified in a tube in slanting position to increase the surface area for growth of the microorganisms is known as slant.
Q.13. Give a chemically defined medium for the growth of chemoautotrophic bacterium capable of using ammonium ions for energy.
Ans. It comprises ammonium sulphate [(NH4)2 SO)4] 0.5 g, potassium phosphate monobasic (KH2 PO4) 0.2 g, calcium chloride (CaCl2) 0.04 g, magnesium sulphate (MgSO4) 0.04 g, ferric citrate (FeC6H5 07) 0.0005 g and water 1 litre.
Q.14. Give the composition of a chemically defined medium for the growth of a typical chemoheterotroph (e.g., Escherichia coli).
Ans. It will comprise glucose-5.0 g, ammonium phosphate monobasic (NH4H2P04) -1.0 g, sodium chloride (NaCl)-5.0 g, magnesium sulphate MgS04 7H20 -0.2 g, potassium phosphate dibasic (K2HPO4) 1 g, and water 1 litre.
Q.15. Give the composition of a chemically defined medium to culture fastidious chemoheterotrophs (e.g., Neisseria gonorrhoeae).
Ans. The constituents are carbon source, salts, amino acids, organic growth factor, reducing agent and water and their composition is given below. Glucose 9.1 g, starch 9.1 g, sodium acetate 1.8 g, sodium citrate 1.4 g and oxalo acetate 0.3 g as sources of carbon and energy. The salts are potassium phosphate dibasic (K2HPO4) 12.7g, sodium chloride (NaCl) 6.4g, potassium phosphate monobasic (KH2 P04) 5.5g, sodium bicarbonate (NaHCO3) 1.2 g, potassium sulphate (K2S04) 1. 1 g, sodium sulphate (Na2S04) 0.9 91 magnesium chloride (MgCl2) 0.5g, ammonium chloride (NH4 CI) 0.4 g, potassium chloride (KC1) 0.4 g, calcium chloride (CaCl2) 0.006g, ferric nitrate [Fe (NO3)3] 0.006g.
The amino acids are cysteine 1.5 g, arginine 0.3 g, proline 0.3 g, glutamic acid 0.2g, Methionine 0.2 g, asparagine 0.2 g, isoleucine and serine (each) 0.2 g, cystine 0.06 g. Organic growth factors are calcium pantothenate 0.02 g, thiamine 0.02 g, Nicotinamide adenine dinucleotide 0.0lg, uracil 0.006 g, biotin 0.005 g, hypoxanthine 0.003 g. The reducing agent is sodium thioglycolate 0.00003 g and water 1 litre.
Q.16. Give the composition of nutrient agar a complex medium for the growth of heterotrophic bacteria.
Ans. A complex medium is a medium the exact chemical composition of which varies from batch to batch. They are made up of nutrients like extracts from yeast, meat or plants or digests of proteins from these and other sources. The generally used nutrient agar comprises peptone (partially digested protein) 5.0 g, beef extract (concentrated extract of beef which is a dark brown paste-like substance) 3.0 g, sodium chloride 8.0 g, agar 15.0 g and water 1 litre.
Q.17. Agar itself is not a nutrient. Why is then nutrient agar called so?
Ans. Because it contains nutrients.
Q.18. What is a nutrient broth?
Ans. The liquid medium of the aforesaid type without agar is called nutrient broth, i.e., nutrients without agar.
Q.19. Why are reducing media used for cultivation of anaerobes?
Ans. Media of a special type known as reducing media are used for the cultivation of anaerobic bacteria because they may be required on exposure to oxygen. For this an anaerobic container is used to cultivate anaerobic bacteria on Petriplates. Water is mixed with the chemical packets containing sodium bicarbonate and sodium borohydride which results in generation of hydrogen and carbon dioxide.
The palladium catalyst is placed in a screened reaction chamber, where the hydrogen and the atmospheric oxygen present in the jar combine to form water, freeing the environment in the jar from oxygen. Methylene blue is used as an anaerobic indicator which is blue when oxidized and turn colourless when the oxygen is depleted (removed) from the container.
Q.20. Why is Salmonella typhi (typhoid bacterium) cultured on bismuth sulphite agar?
Ans. Bismuth sulphite inhibits the growth of Gram positive bacteria and most Gram negative intestinal bacteria other than Salmonella typhi. It is a selective medium.
Q.21. Why is blood agar used as a differential medium to distinguish Streptococcus pyogenes?
Ans. The Streptococcus pyogenes causative agent of strep throat or sore throat show a clear ring around their colonies as the bacterium causes lysis of the surrounding blood cells around the colony.
Q.22. What is a pure culture? Why is pure culture needed? How are pure cultures obtained?
Ans. A population of one strain or species of bacteria or any other microorganisms is referred to as a pure culture. The need for pure cultures was first realized by the German physician Robert Koch to study some human pathogens. Infectious materials such as pus, sputum and urine contain many types of bacteria. In the same way samples of soil, water and food also contain many kinds of microorganisms.
Pure cultures are needed to identify and differentiate them on the basis of morphological and biochemical characteristics. Different organisms form unique colonies which are believed to have been formed from a single spore or individual vegetative cell on being spread on a medium.
The most common method to obtain pure culture of a bacterium or microorganism is the streak plate method. An inoculation loop is well sterilized by incinerating the loop (by heating over a flame). The loop is dipped in a mixed culture containing more than one type of microorganisms and is streaked on the surface of nutrient medium. While streaking, the bacteria are rubbed off the loop onto the medium in paths of fewer and fewer cells. In this way the last cells in the last part of the streak are sufficiently apart to grow and form isolated colonies.
Q.23. How are microorganisms preserved by deep-freezing and freeze-drying?
Ans. In deep-freezing pure cultures of microorganisms are placed in a suspending liquid and are frozen quickly at a temperature ranging from -50°C to -95°C. The cultures can be melted and used for several years. In freeze-drying or lyophilization a suspension of microorganisms is frozen rapidly ranging from -54°C to -72°C and water is removed by a high vacuum. Under vacuum the containers (vials) are sealed by a high temperature torch. The powder like residue which remains can be stored for many years and can be revived by hydration with a suitable liquid nutrient medium at an appropriate time.
Q.24. What is generation time?
Ans. It is the doubling time or the time required for a cell to divide into two.
Q.25. What is a typical bacterial growth curve? What are its four phases?
Ans. A typical bacterial growth curve is formed when a few bacteria are inoculated into a liquid growth medium and the population is counted at intervals recording growth of cells over time taken. It comes out to be like, an inverted cup. The phases that could be demarcated on this curve are:
1. Lag phase—the period in which there is little or no cell division.
2. Log phase or exponential growth phase-this is the phase of maximum growth.
3. Stationary phase-this is the period of equilibrium.
4. Death phase or logarithmic decline phase-the number of cells formed is less than the number of cells that die.
Q.26. List the methods for direct measurement of microbial growth?
Ans. These are:
(1) Plate counts,
(2) Serial dilution,
(3) Pour plate and spread plate,
(5) Most probable number or MPN, and
(6) Direct microscopic count.
Q.27. Give the indirect methods for the estimation of microbial numbers.
Ans. These are:
(2) Metabolic activity and
(3) Dry weight.