This document discusses bacterial shapes, arrangements, staining techniques, and colonial morphology. It begins by describing the three basic bacterial shapes as coccus (round), bacillus (rod-shaped), and spiral. Common arrangements of coccus and bacillus cells are then outlined. The document also explains smear preparation and different staining techniques, including simple staining, Gram staining, and Ziehl-Neelsen staining. Gram staining allows differentiation of bacteria as Gram-positive or Gram-negative. Ziehl-Neelsen staining is used to identify acid-fast organisms like Mycobacterium tuberculosis. Finally, colonial morphology characteristics that can help in bacterial identification are presented.

1. Medical Microbiology Laboratory
(Bacterial Shapes, Arrangements And
Staining Techniques)
Hussein A. Abid
Medical Laboratory Scientist
Member at American Society of Microbiology
Chairman of Iraqi Medical Laboratory Association
Teacher at Middle Technical University

2. BACTERIAL SHAPES AND
ARRANGEMENTS

3. 2
INTRODUCTION
 Bacteria (single bacterium) are single-celled, prokaryotic
organisms.
 They are microscopic in size and lack membrane-bound
organelles as do eukaryotic cells, such as animal cells
and plant cells.
 Bacteria are able to live and thrive in various types of
environments including extreme habitats such as
hydrothermal vents, hot springs, and in your
digestive tract.
 Most bacteria are reproduced by binary fission.

4. 3
INTRODUCTION
 A single bacterium can replicate very quickly, producing
large numbers of identical cells that form a colony.
 Not all bacteria look the same.
 Some are round, some are rod-shaped bacteria, and
some have very unusual shapes.
 Bacteria can be classified according to three basic
shapes into:
1. Coccus: spherical or round
2. Bacillus: rod shaped
3. Spiral: curve, spiral, or twisted

5. 4

6. 5
COMMON BACTERIAL CELL ARRANGEMENTS
 Diplo – cells remain in pairs after dividing.
 Strepto – cells remain in chains after dividing.
 Tetrad – cells remain in groups of four and divide in two
planes.
 Sarcinae – cells remain in groups of eight and divide in
three planes.
 Staphylo – cells remain in clusters and divide in multiple
planes.

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COCCI ARRANGEMENTS
 Diplococci: cells remain in pairs after dividing.
 Streptococci: cells remain in chains after dividing.
 Tetrad: cells remain in groups of four and divide in two
planes.
 Sarcinae: cells remain in groups of eight and divide in
three planes.
 Staphylococci: cells remain in clusters and divide in
multiple planes.

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BACILLI ARRANGEMENTS
 Monobacillus: remains single rod-shaped cell after
dividing.
 Diplobacilli: cells remain in pairs after dividing.
 Streptobacilli: cells remain in chains after dividing.
 Palisades: cells in a chain are arranged side-by-side
instead of end-to-end and are partially attached.
 Coccobacillus: cells are short with a slight oval shape,
resembling both coccus and bacillus bacteria.

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12. COLONIAL MORPHOLOGY

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COLONIAL MORPHOLOGY
 Bacteria grow on solid media as colonies.
 A colony is defined as a visible mass of microorganisms
all originating from a single mother cell, therefore a
colony constitutes a clone of bacteria all genetically
alike.
 In the identification of bacteria and fungi much weight is
placed on how the organism grows in or on media.
 Colony morphology is how a colony of bacteria appears!.
 Mostly, eight characteristics for bacterial colony
morphology can help in primary diagnosis includes:

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COLONIAL MORPHOLOGY DESCRIPTION
1.
2.
3.
4. Size Punctiform Small Moderate Large

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COLONIAL MORPHOLOGY DESCRIPTION
5. Texture: Smooth, granular or rough.
6. Appearance: glistening (shiny) in mucoid colonies or dull.
7. Pigmentation (Chromogenesis):
A) Pigmented: e.g. red, purple, yellow, ..etc.
B) Non-pigmented: e.g. cream, white, ..etc.
8. Optical property: Opaque, translucent, transparent

16. SMEAR PREPARATION AND
STAINING

17. BACTERIAL SMEAR TOOLS
14Burner
Distilled water
Wire loop
Marker pen
Glass slide

18. 15
BACTERIAL SMEAR PREPARATION
1. Place one needle of solid
bacterial growth or two loops
of liquid bacterial growth in
the center of a clean slide.
2. If working from a solid
medium, add one drop (and
only one drop) of distilled
water to your specimen. If
using a broth medium, do not
add the water.

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BACTERIAL SMEAR PREPARATION
3. Now, with your inoculating
loop, mix the specimen with
the water completely and
spread the mixture out to
cover about half of the total
slide area.
4. Place the slide on a slide
warmer and wait for it to dry.
The smear is now ready for
the staining procedure.

20. SMEAR PREPARATION (broth)

21. 18
STAINING
 Microbes are colorless and highly transparent structures.
 They should be stained, to be visible under the light
microscope.
 Staining: the process in which microbes are stained.
 Microbial staining, giving colour to microbes.
 Stains (dyes): are organic compounds which carries
either positive charges or negative charges or both.
 Stains could be classified according to:
o Charges (basic, acidic and neutral stains).
o Function (simple, differential and special stains).

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PRINCIPLE OF STAINING
 Each staining method have its own principle, but the
following may be common:
1. Basic stain (+ve charge):
 To stain negatively (-ve) charged molecules of bacteria.
 Mostly used because cell surface is -ve charge.
2. Acidic stain(-ve charge):
 To stain positively (+ve) charged molecules of bacteria.
 Used to stain the bacterial capsules.
 As cell surface is -ve charged, basic dyes mostly used.

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STAINS ACCORDING TO FUNCTION
 Simple stains: only one dye used to describe bacterial
shapes and arrangements, the differentiation is
impossible (e.g. Crystal violet, methylene blue).
 Differential stains: more than one dye used, the
differentiation is possible (e.g. Gram’s stain, Ziehl-
Neelsen stain).
 Special stains: more than one dye used, to detect
special structures (e.g. capsule stain, flagellar stain,
spore stain, silver nitrate stain)

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SIMPLE STAIN
 Properties: rapid & effective way of preparing smear for
viewing.
 Basic stains includes (crystal violet, methylene blue
and safranin).
 Procedure:
1. Stain heat-fixed slide for 1 min with basic stain.
2. Wash, dry and view.
 Purpose: to determine size, shape and arrangement of
bacterial cells.

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SIMPLE STAIN
Methylene blue alone, as simple stain to describe bacterial
shape and arrangement as staphylococci

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DIFFERENTIAL STAIN (GRAM STAIN)
 Introduced by Danish bacteriologist Hans Christian Gram
(1880).
 Based on this reaction, bacteria classified Into Gram
positive and Gram negative bacteria.
 The cell wall composition differences makes difference.
 Four reagents (including stains) are used:
1. Crystal violet (primary stain)
2. Gram’s iodine (mordant/fixative)
3. 3. Acetone (95%) (decolorizer)
4. 4. Safranin/dilute carbol fuchsin (counter stain)

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GRAM STAIN CHEMICALS
Beaker with water for washing
Crystal violet Iodine Alcohol Safranin

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DIFFERENTIAL STAIN (GRAM STAIN)
1. Flood air-dried, heat-fixed smear of cells for 1 minute
with crystal violet staining reagent. Please note that
the quality of the smear (too heavy or too light cell
concentration) will affect the Gram Stain results.
2. Wash slide in a gentle and indirect stream of tap water
for 2 seconds.
3. Flood slide with the mordant: Gram’s (Lugol's) iodine.
Wait 1 minute.
4. Wash slide in a gentle and indirect stream of tap water
for 2 seconds.

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DIFFERENTIAL STAIN (GRAM STAIN)
5. Flood slide with decolorizing agent (Acetone-alcohol
decolorizer). Wait 10-15 seconds or add drop by
drop to slide until decolorizing agent running from
the slide runs clear.
6. Flood slide with counterstain, safranin. Wait 30
seconds to 1 minute.
7. Wash slide in a gentile and indirect stream of tap water
until no color appears in the effluent and then blot dry
with absorbent paper.
8. Observe the results of the staining procedure under oil
immersion (100X) using a Bright field microscope.

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DIFFERENTIAL STAIN (GRAM STAIN)
Results interpretation:
1. Gram-negative bacteria will stain pink/red
2. Gram-positive bacteria will stain blue/purple.

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Gram +ve Staphylococci
Gram -ve Diplococci Gram -ve Bacilli
Gram +ve Streptobacilli

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ZIEHL-NEELSEN STAIN
 Also known as the acid-fast stain, was first described by two
German doctors (bacteriologist Franz Ziehl and pathologist Friedrich
Neelsen).
 Used to identify acid-fast organisms, mainly Mycobacterium
tuberculosis is the most important of this group because it is
responsible for tuberculosis (TB).
 Acid-fast organisms like Mycobacterium contain large amounts of
lipid substances within their cell walls called mycolic acids.
 These acids resist staining by ordinary methods such as a Gram
stain.
 It can also be used to stain a few other bacteria, such as Nocardia.
 The reagents used are Ziehl–Neelsen carbol fuchsin, acid alcohol,
and methylene blue.

33. 30
ZIEHL-NEELSEN STAIN (tools & chemicals)
Methylene blue Carbol fuchsin Acid alcohol
(3% HCl in 70% alcohol)
Bunsen burner
Glass slide
Loop

34. 31
ZIEHL-NEELSEN STAIN PROCEDURE
1. Make a thin smear of the material for study and heat fix
by passing the slide 3-4 times through the flame of a
Bunsen burner or use a slide warmer at 65-75 ºC. Do
not overheat.
2. Place the slide on staining rack and pour carbol fuschin
over smear and heat gently underside of the slide by
passing a flame under the rack until fumes appear
(without boiling!). Do not overheat and allow it to stand
for 5 minutes.
3. Rinse smears with water until no color appears in the
effluent.
4. Pour 20% sulphuric acid, wait for one minute and keep
on repeating this step until the slide appears light pink in
color (15-20 sec).

35. 32
ZIEHL-NEELSEN STAIN PROCEDURE
5. Wash well with clean water.
6. Cover the smear with methylene blue or malachite
green stain for 1–2 minutes.
7. Wash off the stain with clean water.
8. Wipe the back of the slide clean, and place it in a
draining rack for the smear to air-dry (do not blot dry).
9. Examine the smear microscopically, using the 100x oil
immersion objective.

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ZIEHL-NEELSEN STAIN TECHNIQUE

37. 34
ZIEHL-NEELSEN STAIN RESULTS
AFB (acid fast bacilli) or Mycobacterium tuberculosis = red
Other cells = blue