Download 2-Morphology-of-bacteria

Morphology of bacteria
Morphological features of bacteria are very important in
their identification.
Size
Bacteria are measured in terms of microns (µ = 1/1000 0f
millimeter).
Shape
Bacteria can be broadly classified according to their shape into:
 Cocci: Spherical.
 Bacilli: relatively straight rods.
 Vibrios: definitely curved rods.
 Spirilla: spiral non flexuous rods.
 Spirochaetes: Thin spiral flexuous filaments.
 Actinomycetes: long filamentous
branching rods.
Arrangement
I) Cocci may occur in :
 Clusters grape like e.g. Staphylococci.
 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 properties
Staining reactions are of primary importance in the
identification and differentiation of bacteria. The most
important stains commonly used are:
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 preparations
Aim
To 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.