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Microbiological Quality Assessment of Processed Fruit Juice

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Title: Microbiological Quality Assessment of Processed Fruit Juice


1

Microscope
2
Microscope

3
The Simple Stain
  • In a simple stain, the smear is stained with a
    solution of a single dye which stains all cells
    the same color. Differentiation of cell types or
    structures is not the objective of the simple
    stain. However, certain structures which are not
    stained by this method may be easily seen, for
    example, endospores and lipid inclusions.

4
Procedure
  • Prepare and heat-fix a smear of the organism to
    be studied.
  • Cover the smear with the staining solution. If
    crystal violet or safranin is used, allow one
    minute for staining. The use of methylene blue
    requires 3-5 minutes to achieve good staining.
  • Carefully wash off the dye with tap water and
    blot the slide dry with blotting paper, an
    absorbent paper pad or a paper towel.

5


Figure The Simple Stain
6
 Gram Stain
  • The Gram stain, performed properly,
    differentiates nearly all bacteria into two major
    groups. For example, one group, the gram-positive
    bacteria, include the causative agents of the
    diseases diphtheria, anthrax, tetanus, scarlet
    fever, and certain forms of pneumonia and
    tonsillitis. A second group, the gram-negative
    bacteria, includes organisms which cause typhoid
    fever, dysentery, gonorrhea and whooping cough.
    In Bacteria the reaction to Gram stain reagents
    is explained by different cell wall structures.
    Gram-positive microbes have a much thicker cell
    wall, while that found in Gram-negative microbes
    is thinner. Microbes from the Archaea domain
    contain different cell wall structures than that
    seen in microbes commonly found in the lab
    (Bacteria domain). However, they will still have
    a species specific Gram stain reaction, even
    though the underlying macromolecular structures
    are different.

7
  • The Gram stain is one of the most useful
    differential stains in bacteriology, including
    diagnostic medical bacteriology. The differential
    staining effect correlates to differences in the
    cell wall structure of microorganisms (at least
    Bacteria, but not Archaea as mentioned above). In
    order to obtain reliable results it is important
    to take the following precautions
  • The cultures to be stained should be young -
    incubated in broth or on a solid medium until
    growth is just visible (no more than 12 to 18
    hours old if possible). Old cultures of some
    gram-positive bacteria will appear Gram negative.
    This is especially true for endospore-forming
    bacteria, such as species from the genus
    Bacillus. In this class, many of the cultures
    will have grown for more than 2 days. For most
    bacteria this is not a problem, but be aware that
    some cultures staining characteristics may
    change!
  • When feasible, the cultures to be stained should
    be grown on a sugar-free medium. Many organisms
    produce substantial amounts of capsular or slime
    material in the presence of certain
    carbohydrates. This may interfere with
    decolorization, and certain Gram-negative
    organisms such as Klebsiella may appear as a
    mixture of pink and purple cells.

8
Gram stain procedure
  • Below is a procedure that works well in the
    teaching laboratories.
  • Cover the slide with crystal violet stain and
    wait one minute.
  • After one minute wash the stain off (gently!)
    with a minimum amount of tap water. Drain off
    most of the water and proceed to the next step.
    It may help to hold the slide vertically and
    touch a bottom corner to paper toweling or
    blotting paper.
  • Cover the slide with iodine solution for one
    minute. The iodine acts as a mordant (fixer) and
    will form a complex with the crystal violet,
    fixing it into the cell.
  • Rinse briefly with tap water.

9
  • Tilt the slide lengthwise over the sink and apply
    the alcohol-acetone decolorizing solution
    (dropwise) such that the solution washes over the
    entire slide from one end to the other. All
    smears on the slide are to be treated thoroughly
    and equally in this procedure. Process the sample
    in this manner for about 2-5 seconds and
    immediately rinse with tap water. This procedure
    will decolorize cells with a Gram negative type
    of cell wall but not those with a gram-positive
    type of cell wall, as a general rule. Drain off
    most of the water and proceed.
  • As the decolorized gram-negative cells need to be
    stained in order to be visible, cover the slide
    with the safranin counterstain for 30 seconds to
    one minute.
  • Rinse briefly and blot the slide dry. Record each
    culture as Gram positive (purple cells) or Gram
    negative (pink cells).

10
Gram Stain Procedure
11
Figure 3-11 The Gram Stain
  • A photomicrograph of gram-positive and
    gram-negative bacteria. Note that Gram reaction
    is dependent upon cell wall structure. A) E. coli
    a common gram-negative rod found in the colon. B)
    Staphylococcus epidermidis a gram-positive cocci
    found on the skin. C) Bacillus cereus a
    gram-positive rod found in the soil.

12
Microscopic view of E.coli Pseudomonas
13
Microscopic view of Staphylococcus B. anthracis
14
The Endospore Stain
Cells of Bacillus, Desulfotomaculum and
Clostridium (and several other, lesser-known
genera--see Bergey's Manual) may, as a response
to nutrient limitations, develop endospores that
possess remarkable resistance to heat, dryness,
irradiation and many chemical agents. Each cell
can produce only one endospore. It is therefore
not a reproductive spore as seen for some
organisms such as Streptomyces and most molds.
The endospore is essentially a specialized cell,
containing a full complement of DNA and many
proteins, but little water. This dehydration
contributes to the spores resistance and makes it
metabolically inert. The endospore develops in a
characteristic position (for its species) in the
vegetative cell. Eventually the cell lyses,
releasing a free endospore.
15
Endospore Stain Procedure
  • Endospore stains require heat to drive the stain
    into the cells. For a endospore stain to be
    successful, the temperature of the stain must be
    near boiling and the stain cannot dry out. Most
    failed endospore stains occur because the stain
    was allowed to completely evaporate during the
    procedure.

16
  • Place the heat-fixed slide over a steaming water
    bath and place a piece of blotting paper over the
    area of the smear. The blotting paper should
    completely cover the smear, but should not stick
    out past the edges of the slide. If it sticks out
    over the edges stain will flow over the edge of
    the slide by capillary action and make a mess.
  • Saturate the blotting paper with the 5-6
    solution of malachite green. Allow the steam to
    heat the slide for five minutes, and replenish
    the stain if it appears to be drying out.
  • Cool the slide to room temperature. Rinse
    thoroughly and carefully with tap water.

17
  • Apply safranin for one minute. Rinse thoroughly
    but briefly with tap water, blot dry and examine.
    Mature endospores stain green whether free or in
    the vegetative cell. Vegetative cells stain pink
    to red.

18
Figure The Endospore Stain
  • A photomicrograph of an enodspore stain. Spores
    present in the picture stain green, while the
    vegetative cells stain red. A) Staphylococcus
    epdiermidis which does not form endospores. B)
    The endospore-forming rod, Bacillus cereus.

19
The Acid fast Stain
  • Because of the waxy substance (mycolic acids)
    present on the cell walls, cells of species of
    Mycobacterium do not stain readily with ordinary
    dyes. However, treatment with cold carbol fuchsin
    for several hours or at high temperatures for
    five minutes will dye the cells. Once the cells
    have been stained, subsequent treatment with a
    dilute hydrochloric acid solution or ethyl
    alcohol containing 3 HCl (acid-alcohol) will not
    decolorize them. Such cells are thus termed
    acid-fast in that the cell will hold the stain
    fast in the presence of the acidic decolorizing
    agent. This property is possessed by few bacteria
    other than Mycobacterium.

20
  • This property is possessed by few bacteria other
    than Mycobacterium.
  • Microscopic examination of tissues or of sputum
    stained by the acid-fast staining procedure is an
    aid in the diagnosis of tuberculosis. If an
    individual has pulmonary tuberculosis, and if the
    tubercles in the lungs are open, the bacteria
    (Mycobacterium tuberculosis) will be present in
    the sputum. The bacteria which cause leprosy
    (Hansen's disease caused by M. leprae) can also
    be detected with this staining procedure. The
    finding of acid-fast cells in milk, on the skin,
    or in feces is of no great signifi-cance, because
    these bacteria may be commonly-found saprophytic
    species of Mycobacterium.

21
  • After preparation of the heat-fixed smear, place
    the slide over a steaming water bath.
  • Place a piece of paper towel or blotting paper
    over the smear. The paper should be about as wide
    as the slide and cover an area just slightly
    greater than the smear itself. Saturate the paper
    with carbol fuchsin and let the slide remain
    above the steaming water bath for five minutes.
    Add more carbol fuchsin to the paper if it
    appears the stain is drying out.
  • Allow the slide to cool to room temperature.
    Remove the paper and wash off the excess stain
    with water.

22
  • Decolorize the smear with acid-alcohol for 10-15
    seconds. Wash gently with tap water.
  • Counterstain with methylene blue for 3 minutes.
    Rinse the slide gently and dry.
  • Examine the smear first with the 10X and then the
    100X (oil-immersion) objective. Those cells which
    retained the primary stain (carbol fuchsin)
    through the acid-alcohol treatment are stained
    red these are the acid-fast organisms.
    Mycobacterium cells characteristically appear as
    clusters of long, red rods. All other cells are
    blue.

23
Figure The acid fast stain
  • A photomicrograph of Mycobacterium smegmatis
    (pink) and Micrococcus luteus (blue) at 1000x
    magnification. M. smegmatis is acid-fast,
    retaining the carbol fuchsin dye, thus appearing
    pink. M. luteus is not acid-fast, loses the
    carbol fuchsin during decolorizaiton, and is
    counter-stained with methylene blue.

24
Microscopic Observation of Stained Cell
Preparation
25
. Spirogyra sp.
  • Green in color
  • Filamentous in nature
  • Conjugation tube is present
  • One conjugating filament is empty

26
Volvox sp.
  • Spherical colony of green alga Volvox
  • Single celled flagellates embedded in a
    gelatinous matrix and organized into a hollow
    sphere.
  • The indivisual cells are joined by cytoplasmic
    threads.
  • Each parental colony has a number of developing
    projeny colonies,which are formed by repeated
    divison of a few specialized reproductive cells
  • Projeny colonies are released through
    disintegration of the parental colony.

27
Penicillium sp.
  • The mycelium is septed,long and branched
  • The conidiophores branched about two-thirds of
    the way to the tip in broom-like fashion
  • Single celled conidia developed at the end of
    sterigma in chains
  • The conidia are globose to ovoid and green in
    color

28
Aspergillus sp.
  • a.The hyphae were well developed,
    profusely branched and septed.
  • b.The conidiophore formed a bulbous
    head,the vesicle.
  • c.Conidia arose from sterigma,at
    their tips in a chain.
  • d.Conidia were typically
    globose,unicellular,enormous and black in color.

29
Mucor sp.
  • a.Sporese are oval
  • b. Nonseptate mycelium gives rise to single
    sporangium with globular
  • c. sporangium containing a columella.

30
Bacillus cereus
  • a.Gram positive cells (violet color)
  • b.Rod shaped cells
  • c.The cells are arranged in chains

31
Staphylococcus aureus
  • a.Gram positive cells (violet color)
  • b.Cocci in shape
  • c.The cells are arranged in clusters

32
Selective Differential Media
  • Selective Medium culture medium that allows the
    growth of certain types of organisms, while
    inhibiting the growth of other organisms
  •  dyes in the medium (e.g. methylene blue in EMB
    crystal violet in MacConkey's) or high salt
    concentration in the medium (e.g. 7 salt in
    MSA) inhibit the growth of unwanted
    microorganisms
  • Differential Medium culture medium that allows
    one to distinguish between or among different
    microorganisms based on a difference in colony
    appearance (color, shape, or growth pattern) on
    the medium.
  • dyes in the medium (e.g. eosin/methylene blue in
    EMB) or pH indicators change the color of the
    medium as sugars in the medium (e.g. lactose in
    EMB MacConkey's and mannitol in MSA) are
    fermented to produce acid products

33
EMB (Eosin Methylene Blue) Agar
  • selective for gram-negative bacteria
  • growth of gram-positive bacteria (e.g.
    Staphylococcus aureus in the image below) is
    inhibited by the eosin methylene blue dyes in
    the media
  • differential for lactose fermentation
  • gram-negative Enterobacteria Escherichia coli and
    Enterobacter aerogenes ferment lactose
  • E. coli produces colonies with a characteristic
    green metallic sheen on EMB agar
  • E. aerogenes produces pink colonies often with a
    central dark purple dot (fish eye colonies) on
    EMB agar
  • gram-negative bacteria Proteus vulgaris and
    Salmonella typhimurium grow on EMB agar, but do
    not ferment lactose

34
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36
MacConkey's Agar
  • selective for gram-negative bacteria
  • growth of gram-positive bacteria (e.g.
    Staphylococcus aureus in the image below) is
    inhibited by the crystal violet dye and bile
    salts in the media
  • differential for lactose fermentation
  • neutral red pH indicator turns red in the
    presence of acid by-products of lactose
    fermentation
  • gram-negative Enterobacteria Escherichia coli and
    Enterobacter aerogenes ferment lactose
  • E. coli produces pink to red colonies often with
    a reddish bile precipitate surrounding colonies
    on MacConkey's agar
  • E. aerogenes produces pink to red mucoid colonies
    on MacConkey's agar
  •  gram-negative bacteria Proteus vulgaris and
    Salmonella typhimurium grow on MacConkey's agar,
    but do not ferment lactose (media appears yellow
    to light pink in color colonies are colorless
    swarming of Proteus is inhibited)

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38
MSA (Mannitol Salt Agar)
  • selective for gram-positive Staphylococci
    bacteria
  • 7 salt in the medium inhibits the growth of most
    gram-positive and gram-negative bacteria
  • differential for mannitol fermentation
  • phenol red pH indicator turns yellow in the
    presence of acid by-products of mannitol
    fermentation
  • Staphylococcus aureus ferments mannitol
  • S. aureus changes the color of the medium from
    pink to yellow due to acid by-products of
    mannitol fermentation
  • Staphylococcus epidermidis grows on MSA, but does
    not ferment mannitol (media remains light pink in
    color colonies are colorless

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Hemolysis with Blood Agar
  • agar contains 5 sheep's blood
  • differential for hemolysis...particularly in
    streptococci
  •  based on the ability to break down hemoglobin or
    red blood cells, 3 groups of microorganisms can
    be described
  • alpha-hemolysis a green to light-brown halo is
    seen around the colonies bacteria partially
    break down hemoglobin leaving a green pigment
    (biliverdin)
  • beta-hemolysis a clearing is seen around the
    colonies bacteria produce a "beta-hemolysin"
    (streptolysin O or S), which lyses red blood
    cells in the medium
  • gamma-hemolysis (no hemolysis) no hemolysis is
    observed bacteria do not produce a hemolysin

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