◄UNIT 18: MICROBIOLOGYUNIT 20: BIOTECHNOLOGY AND ITS APPLICATION►

UNIT 19: CULTURING MICRO-ORGANISMS

  • UNIT 19: CULTURING MICRO-ORGANISMS

    UNIT 19: CULTURING MICRO-ORGANISMS
    Key Unit Competence
    Explain the process of culturing microorganisms and the factors affecting their
    population growth.
    Learning objectives
    – List and describe the roles of microorganisms and their requirements for
    growth.
    – Explain the role of environmental variables in culturing microorganisms.
    – Describe the different types of culture media.
    – Draw and interpret the graph of the population growth of bacteria.
    – Carry out an experiment to stain bacteria for examination with a light
    microscope.
    – Observe and compare the numbers of bacteria present in fresh and stale milk.
    – Distinguish between gram negative and gram positive bacteria.
    – Describe the main features of aseptic techniques.
    – Explain how pure cultures of pure bacteria can be obtained.
    – Describe the methods of inoculation.
    – Use sterile techniques to prepare agar plates to culture bacteria and fungi
    – Carry out research on why microorganisms are particularly suitable for
    industrial use.
    – Appreciate the importance of culturing microorganisms.
    – Show perseverance when inoculating a solid and liquid medium.
    – Show concern for taking the basic precautions in the school laboratory when

    carrying out routine microbiological work.

    Introductory activity.
    Use different books and visit internet make a short summary about the culture of
    microorganisms and suggest why cultures are not incubated at 370 C in a school lab.

    19.1. Requirements for culturing of microorganisms
    Activity 19.1
    Use textbooks and other sources of information to discuss the requirements of
    growth of microorganisms.
    Many microorganisms can be grown in the laboratory. This allows scientists to learn
    a lot about them. We can find out which nutrients they need to survive and which
    chemicals will kill them. We can also discover which microorganisms can be useful
    to us and which cause deadly disease.
    To find out more about microorganisms, you need to culture them. Culturing
    microorganisms involves growing very large numbers of them so that you can see
    the colony as a whole.

    To culture microorganisms, you must provide them with everything they need. This
    usually involves providing a culture medium containing carbohydrates to act as an
    energy source. A long with this, various mineral ions some supplement of proteins
    and vitamins are included.

    The nutrients are often contained in an agar medium. Agar is a substance which
    dissolves in hot water and sets to form a jelly. You pour hot agar containing all the
    necessary nutrients into a Petri dish. Microorganisms are living organisms. Therefore,
    they have requirements for their growth, maintenance and multiplication. These
    include:
     Optimum temperature (30-40ᵒC) for enzymes to work better.
    – Source of energy such as glucose, maltose, juice.
    – Source of other nutrients (minerals such are as potassium, sodium, iron,
    magnesium and calcium, vitamins, proteins
    – Air for aerobic microbes or complete absence of air for anaerobic
    microorganisms.
    The medium for culture of microbes can be the dead organic matters (food,
    fruits, remaining of organism, juice, milk) or a prepared medium such as Agaragar
     (universal medium for any germ), Lowenstein medium (selective medium for
    tuberculosis bacillus). The medium can be wet or dry. Different types of media are

    used culture microorganisms. 

    19.1.1. Types of media
    There are many different types of media described by their components or
    ingredients.
    Universal media: this allow the growth of every type of bacteria e.g. agar-agar
    Differential/selective media: are specific to some types of bacteria for example
    Lowenstein for tuberculosis bacteria. Their ingredients will favour growth of certain
    types of bacteria.
    A pure culture: this contains only one kind of microorganism. The pure cultures are
    important for scientific method as they are free from other types of microorganisms.
    19.1.2. Principles of sterile culturing
    – Wash hands before touching a sterile Petri-dish
    – Open the Petri-dish as little as possible, and replace the lid quickly
    – Never cough or sneeze near the dish
    – Never touch the infected jiffy with fingers
    – When culturing is no longer required, they should be flooded with strong

    disinfectant

     After cleaning out the nutrient from Petri-dish, they should be washed and
    disinfected, and then if they are glass, heat sterilize.
    – Wash your hands thoroughly after all operation by using soap.
    – Never push hands near the mouth during experimental work.
    Safety precautions:
    Bacteria grow and reproduce more quickly when they are warm than when they
    are cold. It would be dangerous to incubate cultures at temperatures close to body
    temperature (37°C) because doing so might allow the growth of pathogens harmful
    to health. So the maximum temperature used in school and college labs is 25°C.
    However, higher temperatures can be used industrially, and these produce faster
    growth.
    Self-assessment 19.1
    1. What is meant by the term culturing bacteria?
    2. What do bacteria need to grow?
    3. Why do we culture microorganisms in the lab?
    4. Explain why cultures are not incubated at 370 c in a school laboratory.
    19.2. Culture media
    Activity: 19.2
    Describe different types of media used in culturing microorganisms.
    A medium is a solid or liquid preparation containing nutrients for the culture of
    microorganisms. A pure microbial culture undergoes the following steps namely:
    – Choice of the culture medium.
    – Sterilization of the culture medium.
    – A culture with a collection of microbial cells growing on or, in a medium.
    – Selection of a pure colony from a collection of microbial cells growing
    – Introduction of a microorganism into a suitable growth medium
    – Streaking to carrying out a pure culture.
    Microorganisms may be cultured in a solid medium or a liquid medium or broth.
    When there is not a culture with a collection of microbial cells growing on or,
    in a medium. A source of microorganisms is spread on the surface of an agar to
    produce individual colonies. Once individual colonies are obtained, this collection
    of microorganisms can then use to carry out a pure microbial culture. 

    a. Solid medium.

    Solid media are particularly suitable for bacteria and fungi and are prepared by mixing
    the liquid nutrient solution with a gelling agent, usually agar, at a concentration of

    about 1-5%, thus, producing nutrient agar that allows the growth of colonies. 

    b. Liquid media
    The liquid media are water – based solutions that are generally termed as broths,
    milks and infusions.
    Liquid media are often useful for measuring population growth. They may be placed
    in a test tube, stopped by a plug of cotton wool or a metal cap, or in a glass, screw crapped
    bottle such as a universal bottle which holds about 25cm2
     enough for one

    agar plate.

    The medium must be sterilized and after, adding a small quantity of cells to the
    medium is called inoculation.
    c. Enrichment media.
    An enrichment medium is a medium in which substances are added to meet the
    requirements of certain microorganisms in preference to others. As a result, certain
    microorganisms grow better than others.
    d. A selective medium
    It is a medium in which one or more substances are added to favor the grown of
    specific microorganisms and to inhibit the growth of others. Example, the addition
    of penicillin to a culture to select for those organisms resisting to it, or the selection

    of hybridizes cells during the production of monoclonal antibodies.

    Self-assessment 19.2
    1. How would you isolate from the soil an organism which could use
    atmospheric nitrogen as its only source of nitrogen (a nitrogen-fixing
    bacteria)?
    2. What is meant by nutrient agar?
    3. Distinguish between liquid media and solid media.

    4. Distinguish between enriched media and specific media.

    19.3. Aseptic technique.
    Activity 19.3.1
    Carry out a procedure of culturing fungi on a nutrient agar using sterile techniques.
    Aseptic technique is using sterilized equipment and solutions and preventing their
    contamination. Sterilization is the removal or destruction of all living microorganisms,
    including spores (inactive structures that enable some microorganisms to
    survive unfavorable periods). Bacterial and fungal spores are abundant in most
    environments including laboratories. A range of special techniques and apparatus
    are designed to prevent contamination of nutrients media. Autoclaves are used
    to sterilize equipment and culture media before experiments and also to sterilize
    equipment and specimens before disposal.
    In addition, after sterilization, a great care is taken during experiments to ensure that

    there is no infection.

    19.3.1. Spread plate technique
    This is one of the most basic and useful of microbiological techniques. Petri dishes
    are specially designed as shallow circular dish made of glass or plastic. The shape
    of the lid allows avoiding contamination, but gas molecules can diffuse between
    the inside of the dish and the environment through where the base meets the lid.

    Oxygen can therefore reach the culture and carbon dioxide can escape.

    The spread plate technique involves using a sterilized spreader with a smooth
    surface made of metal or glass to apply a small amount of bacteria suspended in
    a solution over a plate. The plate needs to be dried at room temperature so that
    the agar can absorb the bacteria more readily. A successful spread plate will have

    a countable number of isolated bacterial colonies evenly distributed on the plate.

    19.3.2. Methods of inoculation
    The introduction of a small number of microorganisms into a nutrient medium is
    called inoculation. Aseptic technique must be used to avoid contamination. The

    procedure differs for solid and liquid media.

    a. Inoculating a solid medium
    We use a wire loop. The loop is firstly flamed and after it is then used to lift a thin
    film of a liquid suspension or a small amount of solid material containing the
    microorganisms being investigated from the previous culture or any source of
    microorganisms. The loop is gently stroked across the surface of the medium in a
    series of sets of streaks.
    b. Inoculating a liquid medium
    If the cells to be inoculated are in a liquid, for example water or a broth, a sterile wire
    loop is used to transfer a sample to the medium, which is often a test medium. The
    wire loop is simply agitated gently inside the medium. If the cells to be inoculated
    are in or a solid medium such as soil nutrient agar, a wire loop may be used for
    transfer to the liquid medium. It can be rubbed on the inside surface of the vessel
    containing the liquid medium to ensure successful transfer.
    c. Carrying out a pure culture
    Pure culture technique is a method of culturing microorganisms in which all of the
    individuals in a culture have descended from a simple individual. The basis of pure
    technique is the isolation in colonies of individual cells. This is done so as to allow the

    characterization of specific types of microorganisms.

    d. Incubation on agar-agar.
    During incubation, the nutrients are contained in agar medium. Agar is a substance
    which dissolves in hot water and sets to form a jelly. You pour hot agar containing all
    necessary nutrients such as carbohydrates, proteins and vitamins into a Petri- dish.
    Then leave it to cool and set before you add any microorganism. The other way to
    provide nutrients to grow microorganisms is as a broth in a culture flask. The steps

    of culturing agar –agar are shown in the following activity.

    Activity: 19.3.2
    – Boil a mixture of 50 ml of water and 20g of agar-agar powder for 15 minutes
    as you are stirring
    – Pour the jelly mixture into four pre-sterilized glass Petri-dishes. Then allow
    the broth to coagulate at room temperature.
    – Number the dishes; 1, 2, 3 and 4 respectively; on the bases.
    – Place a nail scarping from between the teeth onto the jelly in dish 1 and 2,
    wave the dish 3 on latrine for 1minute and do not put anything on dish 4.
    – Warm the dishes 2 and 4 on the top of water vapour stream for 15 minutes
    and then cool them (do not open them)
    – Then fix the lids tightly to the bases of the four Petri-dishes with clear adhesive
    tape and place them upside down in an oven/incubator at 37 ˚C for 3 days.

    – Record and interpret your results. 




    19.3.3 Alcoholic fermentation
    Activity 19.3.3
    Describe how yeast would be used in alcoholic fermentation.
    Yeast releases digestive enzymes which allow the transformation of glucose into
    ethanol as result of anaerobic fermentation. The presence of bubbles is the evidence
    that carbon dioxide is released as waste product of the alcoholic fermentation.
    Making Beer depends on a process called malting. You soak and keep barley grains
    in water. As germination begins, enzymes break down the starch in the barley grains
    into a sugary solution. You then extract a solution produced by malting and use it
    as an energy source for the yeast. The mixture of yeast and sugar solution is then
    fermented to produce alcohol. Hops are added at this stage to give flavour. The beer
    is given time to clear and develops its flavour before putting it in bottles or to be
    sold. Interestingly, alcohol in large quantities is toxic to yeast as well as to people.
    This is why the alcohol content of wine is rarely more than 14%. Once it gets much
    higher, it kills all the yeast and stops fermentation.
    Self-assessment 19.3
    From questions 1-5, circle the letter corresponding to the right answer
    1. The method of culturing microorganisms in which all of the individuals in a
    culture have descended from a single individual is called:
    a. Pure culture technique
    b. Spread plate technique
    c. Aseptic technique
    d. Liquid media method
    2. Inoculating liquid medium, various instruments are used. Which one of the
    following is used to transfer the sample to the medium?
    a. Sterile wire loop
    b. Inoculating needle.
    c. Petri-dishes
    d. None of these.
    3. Large amounts of alcohol are dangerous to yeast during alcoholic fermentation.
    Which of the following explains the reason?
    a. It kills all the yeast and stops fermentation.
    b. Motivate yeasts
    c. It kills some few bacteria.
    d. Temperature affects fermentation.
    4. The technique of using sterilized equipment and solutions and preventing their
    contamination is referred to as:
    a. Pure culture technique
    b. Spread plate technique
    c. Aseptic technique
    d. Petri-dish technique.
    5. Petri dishes are specially designed as a shallow circular dish made of glass or
    plastic with a lid. Which one of the following best explains the function of the
    lid?
    a. Prevent contamination, but gas molecules can diffuse.
    b. Spread the bacteria on the plate.
    c. Allows contamination.

    d. None of the above.

    19.4. Population growth of bacteria
    Activity: 19.4.1
    Use text books and other sources of information to interpret the graphs of bacterial
    growth.
    When bacteria or any other microorganisms are incubated in a suitable culturing
    medium, they reproduce by binary fissions and the number of individuals increases.
    The ordinary growth of population is described as sigmoid curve or S-shaped curve
    made of 4 main phases:
    – The lag phase: period of adaptation of microorganisms to the new habitat
    (new environment)
    – The log or exponential phase: period of high rate of reproduction. Bacteria are
    sensitive to the limiting factors of the growth or anti-microbial agents
    – The stationary phase: Stationary phase of plateau-growth slows down. The
    population remains constant because the rate of dividing/growth is equal to
    the rate of death within the population. The maximum number that a habitat
    can accommodate for a long period is known as the carrying capacity.
    – The decline or death phase: period of high rate of death than the rate of 
    dividing/growth due to the scarcity of food, the abundance of metabolic waste
    products, presence of antibiotics or any other drugs killing the germs. Figure

    19.5 shows the phases explained above.

    19.4.1. Measuring population growth of bacteria 
    The typical growth curve of a population of bacteria is similar to the growth curve
    expected for yeast, a unicellular fungus or the growth of any population. When
    measuring the growth of a population of bacteria or yeast, we can carry out direct
    counting of the numbers of cells or indirectly by measuring some indication of the

    number of cells such as the coldness of a solution, or production of a gas

    It is usual to inoculate a small sample of the microorganisms in a sterilized nutrient
    medium and to place the culture in an incubator at the optimum temperature for
    growth. Other conditions are pH, oxygen concentration and ionic and osmotic
    balance. Growth can be measured from the time of inoculation. Two types of cell
    count are possible, namely viable count and total count. The viable count is the total
    of living cells only and total count is the total number of both living and dead cells

    and is easier to measure.

    Activity 19.4.2
    Investigating the bacterial content of fresh and stale milk.
    Materials required: Four sterile nutrient agar plates, inoculating loop, Bunsen
    burner, indelible marker or wax pencil, Fresh pasteurized milk, Stale milk (milk left
    at room temperature for 24hours) and Incubator set at 350C
    Procedure:
    – Place the inoculating loop in the Bunsen burner flame until the loop is red hot.
    – Allow the loop to cool and then dip into a sample of fresh, well shaken milk.
    – Lift the lid of the sterile agar plate slightly with the other hand and lightly
    spread the contents of the inoculating loop over the surface the agar.
    – Close the lid of the plate and return the loop to the Bunsen burner flame until
    red hot.
    – Label the base of the plate with an indelible marker or pencil.
    – Repeat the process with the second plate and another sample of fresh milk.
    – Flame the loop again and after cooling, place it in a sample of stale milk.
    – Spread the contents of the loop over the surface of a third plate and then
    close the lid.
    – Label the base of the plate with an indelible marker or pencil.
    – Repeat the process with the fourth plate and second sample of stale milk.
    – Put the four plates in an incubator at 350
    c for about 3 days. They should be
    placed upside down to prevent condensation falling onto the cultures. After
    incubation, the two halves of each plate should tape together for safety.
    – Record the appearance of the colonies

    Give the general comment based on your observations

    Self-assessment 19.4
    1. A culture of yeast, Saccharomyces cerevisiae, had been carried out in the banana
    juice for 7 days at 30°C.The table below shows the change in number of yeasts

    within that time:

    a. Draw a curve showing the growth of the yeast population
    b. What is the role of banana juice in that experiment?
    c. State any two conditions that should be maintained constant during that
    experiment.
    d. Describe the trend of the graph you have drawn in a
    2. Design an experiment to test the hypothesis that contact of an agar plate with a

    finger results in more bacterial growth than exposing the plate to classroom air.

    19.5. Staining of bacteria
    Activity 19. 5
    “Staining bacteria for practical purpose is important”. Discuss the validity of the
    statement.
    The microorganisms or parts of microorganisms that pick up the stain are clearly 
    distinctively observed from the rest of the background.
    In simple staining, all the cells and structures in general stain the same colour. In
    positive staining, cells structures take in the stain e.g. methylene blue while in
    negative staining the cells repel the stain and it is taken by the background e.g. Indian
    ink. Negative staining is mostly useful in viewing capsules and such structures that

    surround the bacteria.

    Differential staining on the other hand, multiple staining reactions are used to
    take advantage of the fact that particular types of microorganisms and/or specified
    structures of microorganism display varied staining reactions that are readily
    distinguishable by different colours. The stain must be fixed immediately and the
    dyed specimen is treated in some ways, e.g. by chemicals or heat to tightly bind the

    stain to the organism or its structures.

    19.5.1. The purpose of staining bacteria
    The purpose of staining bacteria is to see, for example, how thick of a layer of
    peptidoglycan their cell wall has. In the Gram stain, gram-negative bacteria will stain
    red or pink because the rinse took out the primary dye and the Safrinin (secondary
    dye) took over the coloring as the counterstain. In gram-positive bacteria, since it
    has a thick-layer of peptidoglycan, not all of the Crystal violet color will be rinsed out 
    of the cell wall, so it will be blue or purple. The following are reasons to explain why
    stained:
    – It’s for helping classifying and determining what the bacteria are composed of.
    – It’s very useful tool to help identify bacteria without necessarily killing the cell.
    – Gram staining is performed to distinguish between gram positive and negative
    bacteria.
    – To enable the person to visualize its physical features- shape, size, arrangement,
    etc the bacterial cells are stained with specific dyes or stains
    19.5.2. Procedure of staining and their corresponding stains.
    Activity 19.5.1:
    carry out an experiment to stain bacteria for examination under the light
    microscope
    In staining bacteria, we use various staining procedures each having specific set of
    stains or dyes. Some of them are:
    1. Gram’s Staining - Crystal violet, Iodine and Safrinin
    2. Capsule staining - Nigrosin, Safrinin or India Ink, Safrinin
    3. Spore staining - Malachite Green and Safrinin
    4. PHB staining - Sudan black.
    5. Using decolorizer – Alcohol wash
    Observe and identify some of the staining methods on figure19.6 as shown below:

    19.5.3 Growing viruses

    The culture of viruses is made more difficult than the culture of bacteria or fungi because

    viruses can only grow and multiply inside living cells. This can be done by infecting whole

    Figure 19.8: Gram positive and Gram negative bacteria.
    19.5.3. Growing viruses
    The culture of viruses is made more difficult than the culture of bacteria or fungi
    because viruses can only grow and multiply inside living cells. This can be done
    by infecting whole organisms such as plants or animals but, where possible, cell,
    tissue cultures are now used. An early technique was to grow certain viruses in chick

    embryos while the embryo was still growing inside the egg.

    19.5.4. Tissue Culture of Animal Viruses
    Viruses cannot be grown in standard microbiological broths or on agar plates;
    instead they have to be cultured inside suitable host cells. Note the following facts:
    – Tissue culture is a useful method for cultivating clinical samples suspected of
    harboring a virus. This method helps with the detection, identification, and
    characterization of viruses in the laboratory.
    – Tissue culture of animal viruses involves growing animal cells in flasks using
    various broth media and then infecting these cells with virus.
    – Transfect ion can be carried out using calcium phosphate, by electroporation,
    or by mixing a cationic lipid with the material to produce liposome’s, which
    fuse with the cell membrane and deposit their content inside.
    – Cytopathic effect is a non-lyrics damage that viruses cause to cells. These vary
    in their manifestation and damaging effect.
    – Cell culture is complex process by which cells are grown under controlled
    conditions, generally outside of their natural environment.
    Cell culture is the complex process by which cells are grown under controlled
    conditions, generally outside of their natural environment. The term “cell culture” is
    defined as the culturing of cells derived from multi-cellular eukaryotes, especially
    animal cells. However, there are also cultures of plants, fungi, and microbes, including

    viruses, bacteria, and protists.

    2. Explain why it is more difficult to culture viruses than culturing bacteria
    3. How/why are viruses specific to the cells they infect?
    4. Distinguish between vaccines and antibiotics
    End of unit assessment19
    1. What are different types of media used in the laboratories for culturing
    microorganisms?
    2. Define a pure culture.
    3. How do biologists differentiate between Gram –positive and Gram –negative
    bacteria?
    4. Describe the three methods of preventing bacterial growth in food.
    5. How does temperature affect the growth of bacteria in culture media?
    6. Assuming that you have a bacterial infection, would you ask for vaccination
    against the bacteria? Why or why not?
    7. How do bacteria maintain the balance in the environment?
    8. Explain why an infection by Gram–negative bacteria are more difficult to treat
    than Gram-positive bacteria.
    9. How would you investigate that temperature affect the bacterial growth?
    10.Write short notes on each of the following term related to the culture of
    microorganisms.
    a. Aseptic techniques.
    b. Staining bacteria
    c. Growing viruses

    11. Explain why microorganisms are particularly suitable for industrial use.

    ◄UNIT 18: MICROBIOLOGYUNIT 20: BIOTECHNOLOGY AND ITS APPLICATION►