Morphological features of bacteria are very important in their identification.
Bacteria are measured in terms of microns (µ = 1/1000 0f millimeter).

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.

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 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. H
2
SO
4
.
 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

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 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.


1.
2.
Then for both
Allow the slide to air dry.
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

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.

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.