Transcript Microbiological Quality Assessment of Processed Fruit Juice

Microscope
Microscope
The Simple Stain
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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.
Procedure
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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.
Figure :The Simple Stain
Gram Stain
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The Gram stain, performed properly, differentiates nearly all
bacteria into two major groups. For example, one group, the grampositive 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. Grampositive 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 sugarfree 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.
Gram stain procedure
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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 alcoholacetone 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).
Gram Stain Procedure
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.
Microscopic view of E.coli &
Pseudomonas
Microscopic view of Staphylococcus & B.
anthracis
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.
Endospore Stain Procedure
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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.
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.
The Acid fast Stain
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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 (acidalcohol) 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 significance, because these bacteria may be commonlyfound 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 1015 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 acidfast organisms. Mycobacterium cells
characteristically appear as clusters of long,
red rods. All other cells are blue.
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.
Microscopic Observation of
Stained Cell Preparation
. Spirogyra sp.
Green in color
Filamentous in nature
Conjugation tube is present
One conjugating filament is empty
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.
Penicillium sp.
The mycelium is septed,long and
branched
The conidiophores branched about twothirds 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
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.
Mucor sp.
a.Sporese are oval
b. Nonseptate mycelium gives rise to
single sporangium with globular
c. sporangium containing a
columella.
Bacillus cereus
a.Gram positive cells (violet
color)
b.Rod shaped cells
c.The cells are arranged in
chains
Staphylococcus aureus
a.Gram positive cells (violet
color)
b.Cocci in shape
c.The cells are arranged in
clusters
Selective & Differential Media
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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
EMB (Eosin Methylene Blue) Agar
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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
MacConkey's Agar
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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)
MSA (Mannitol Salt Agar)
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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 byproducts 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
Hemolysis with Blood Agar
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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