Microbiological Quality Assessment of Processed Fruit Juice

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Microscope
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Microscope

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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.

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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.

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Figure The Simple Stain
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 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.

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• 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.

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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.

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• 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).

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Gram Stain Procedure
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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.

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Microscopic view of E.coli Pseudomonas
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Microscopic view of Staphylococcus B. anthracis
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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.
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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.

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• 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.

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• 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.

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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.

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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.

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• 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.

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• 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.

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• 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.

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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.

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Microscopic Observation of Stained Cell
Preparation
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. Spirogyra sp.

• Green in color
• Filamentous in nature
• Conjugation tube is present
• One conjugating filament is empty

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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.

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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

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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.

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Mucor sp.

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

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Bacillus cereus

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

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Staphylococcus aureus

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

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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

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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

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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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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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