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.