Morphology of bacteria. Morphological features of bacteria are very important in their...
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Transcript of Morphology of bacteria. Morphological features of bacteria are very important in their...
Morphology of bacteria
Morphological features of bacteria are
very important in their identification.
Bacteria are measured in terms of microns
(µ = 1/1000 0f millimeter).
Size
Bacteria can be broadly classified according to their
shape into:
Cocci: Spherical.
Bacilli: relatively straight rods.
Vibrios: definitely curved rods.
Shape
Spirilla: spiral non flexuous rods.
Spirochaetes: Thin spiral flexuous filaments.
Actinomycetes: long filamentous
branching rods.
I) Cocci may occur in :
Clusters grape like e.g. Staphylococci.
Arrangement
Pairs (diplococcic) e.g. Neisseria.
Chains e.g. Streptococci
Groups of 4 cells (Tetrads) e.g. Micrococci
II) Bacilli may be arranged as:
Separately arranged e.g: Salmonella.
Paired rods e.g: Klebsiella
Chains e.g: Anthrax.
Chinese letter arrangement and club shaped ends e.g: Corynebacterium diphtheria.
Staining reactions are of primary
importance in the identification and
differentiation of bacteria. The most
important stains commonly used are:
Staining properties
1. Gram’s stain:
With Gram stain bacteria can be divided into
two categories;
Gram positive bacteria that retain the
methyl violet-iodine dye complex and
appear purple blue.
Gram negative bacteria that destain with
95% alcohol and appear pink due to the
counter-staining with basic fuchsin.
2. Ziehl-Neelsen’s acid-fast stain:
The stain is used for detection of group of bacteria
described as “acid fast bacteria”. These organisms
are not readily stainable with ordinary stains but
they need exposure to a strong stain e.g. strong
carbol- fuchsin. Once stained they resist
decolorization with mineral acids e.g. H2SO4.
This property of acid fastness may be due to the
large amount of lipids and fatty acids particularly
the mycolic acid wax which these organisms
contain.
Used for the staining
of:
1. Mycobacterium
bacteria:
Mycobacterium
tuberculosis,
Mycobacterium lepra.
2. Bacterial spores. Mixture of acid fast and non acid fast bacteria
Examination of pathogens in wet preparationsAimTo examine specimens and cultures for motile bacteria
e.g. Vibrio cholera is an actively motile bacteria..2 ways of examining a bacterial suspension for
motile bacteria:
A) Place a small drop of suspension on a slide and cover
with a cover glass. Avoid making the preparation too
thick.
Seal the preparation with nail varnish or molten
petroleum jelly to prevent it drying out.Make sure the iris diaphragm of the condenser is
sufficiently closed to give good contrast.
B) Hanging drop preparation:
Use a depression or a cavity slide.
Place a drop of suspension on a cover glass
and inverting this over a cavity slide.
Smear preparation
Smear Preparation for Staining:
For the broth culture, shake the culture tube and,
with an inoculating loop, aseptically transfer 1 to 2
loopful of bacteria to the center of the slide.
Spread this out to about a 1/2-inch area.
When preparing a smear from a agar slant or agar
plate, place a loopful of distilled water in the center
of the slide.
With the inoculating needle, aseptically pick up a very
small amount of culture and mix into the drop of
water and spread it out.
Then for both
1. Allow the slide to air dry.
2. Fix it over a gentle flame, while moving the slide
in a circular fashion to avoid localized
overheating. As a result of heat fixation bacteria
gets firmly attached to the slide and is not lost
during staining and rinsing steps.
Procedure for making a bacterial smear
Precautions to take when staining smears
1. Use a staining rack.
2. Do not attempt to stain a smear that is too thick.
3. When you stain the slide, do not stain the whole
surface of the slide. Just staining the area
containing the smear is enough.
4. Follow exactly the staining technique.
5. To dispense stains, alcoholic and acetone
reagents, use dropper or dropper bottle.
6. While washing the slide after staining, the
water stream must flow slowly along the
surface as fall of water directly on the
smear may result in loss of the smear.
7. After staining, place the slides at an angle
in a draining rack for the smears to air-dry.
8. To check staining results, use quality
control smears of organisms, particularly
when a new batch of stain is used.
Staining techniques
1) Gram stain: bacteria can be classified
according to Gram reaction into Gram
positive or Gram negative.
2) Ziehl-Neelsen technique to detect AFB:
The stain binds to the mycolic acid in the
mycobacterial cell wall.
3) Auramine-phenol technique to detect
AFB: Auramine binds to the mycolic
acid in the mycobacterial cell wall &
fluoresces (Yellow) when illuminated
(excited) by ultra-violet (UV) light.
4) Methylene blue technique: The
methylene blue technique is a rapid
method which can be used to show
the basic morphology of bacteria.
Cells appear blue in color.
5) Wayson’s bipolar staining of bacteria:
is a rapid method which shows clearly
the bipolar staining morphology of
bacteria such as Yersinia pestis
(Safety pin like shape).
Yersenia stained by wayson
6) Albert staining of volutin granules: is used to
stain the volutin, or metachromatic, granules
of Corynebacterium diphtheriae. which is a
rod shaped bacteria with a swelling at one
end (Club shaped).
8) Acridine orange fluorochrome staining: is a
fluorochrome that causes deoxyribonucleic
acid (DNA) to fluoresce green and ribonucleic
acid (RNA) to fluoresce orange-red. It has
been recommended for the rapid
identification of Trichomonas vaginalis, yeast
cells, and clue cells in vaginal smears.
9) Toludine blue for staining of P. jiroveci cysts.
10)Polychrome Loeffler methylene blue staining
of anthrax bacilli.
7) Giemsa technique: widely used in
parasitology to stain malaria and other
blood parasites.