Bacteria are unicellular prokaryotic organisms that are studied in medical microbiology. They have distinct cell structures including a cell wall, cell membrane, cytoplasm, and nucleic material. Bacteria can be visualized under light, phase contrast, or electron microscopes and stained using simple, differential, or acid-fast staining techniques. Gram staining divides bacteria into Gram-positive and Gram-negative categories based on cell wall structure. Bacteria exhibit a variety of external structures such as flagella, pili, capsules, and endospores, and follow a defined growth curve with lag, log, stationary, and death phases.

1. Introduction to bacteriology, morphology & staining
DR ADILRAZA
DEPARTMENTOF MICROBIOLOGY,
JNMC.AMU

2. Medical Microbiology:
• It is the study of microbes that infect humans,
the disease they cause, their diagnosis,
prevention and treatment. It also deals with
the response of the human host to microbial
and their antigens.

3. • Branches of Microbiology:
– Bacteriology
– Virology
– Mycology
– Immunology
– Parasitology
– Mycobacteriology

4. Bacteria:
• Bacteria are prokaryotic microorganisms that
do not contain chlorophyll.
• They are unicellular.

5. Differences between prokaryotic and
eukaryotic cells
CHARACTER PROKARYOTES EUKARYOTES
NUCLEUS
Nuclear membrane Absent Present
Nucleolus Absent Present
Deoxyribonucleoprotein Absent Present
Chromosome One circular More than one ( linear)
Mitotic division Absent Present
Cytoplasm
Cytoplasmic streaming Absent Present
Pinocytosis Absent Present
Mitocho, golgi App, ER Absent Present
Chemical composition
Sterols Absent Present
Muramic acid Present Absent

6. Size of Bacteria
• The unit of measurement used in bacteriology is the
MICRON (micrometre, µM).
• 1 micron (µ), or micrometre (µM)= 10-6 M or one
thousands of a millimetre.
• 1 millimicron (mµ ) or nanometre (nm)= 10-9M= one
thousands of a micron or one millionth of a
millimetre.
• 1 Angstrom unit (Å)= one tenth of a nanometre.

7. Cocci: sphere, 1μm
Bacilli: rods , 0.5-1 μm in width -3 μm in length
Spiral bacteria: 1~3 μm in length and 0.3-0.6 μm in width

8. Microscopy
1. Optical or light
microscope:
 Bacteria may
be examined
under the
compound
microscope,
either in the
living state or
after fixation
and staining.
Ocular lens
Specimen
Objective lens
Light source
Condenser lens

9. 2. Phase contrast
microscope:
 Different cell orgenelles
have different refractive
indices.
 Retardation by a fraction
of a wave length of the
rays of light that pass
through the different cell
orgenelles results phase
difference between the
two types of the rays.
 In the phase contrast
microscope phase
differences are converted
into differences in
intensity of light,
producing light and dark
contrast of the image.

2
Light source

4

10. • produces a bright image of
the object against a dark
background
• used to observe living,
unstained preparations.
• Used to observe
spirochetes.
Dark field microscope/dark ground microscopy

11. The Fluorescence Microscope
• exposes specimen to
ultraviolet, violet, or
blue light
• specimens usually
stained with
fluorochromes
• shows a bright image of
the object resulting
from the fluorescent
light emitted by the
specimen

12. Electron microscope

13. STAINING
• Increases visibility of specimen
• Accentuates specific morphological features
• Preserves specimens

14. Fixation
• Process by which internal and external
structures are preserved and fixed in position
• Process by which organism is killed and firmly
attached to microscope slide
– heat fixing
• preserves overall morphology but not internal
structures
– chemical fixing
• protects fine cellular substructure and morphology of
larger, more delicate organisms

15. Simple Staining
• Simple staining
– a single staining agent is used
– basic dyes are frequently used
• dyes with positive charges
• e.g., crystal violet

16. • Negative staining
– often used to
visualize capsules
surrounding bacteria
– capsules are colorless
against a stained
background

17. Differential Staining
• divides microorganisms into groups based on
their staining properties
– e.g., Gram stain
– e.g., acid-fast stain

18. 18
Gram staining
• most widely used differential staining
procedure
• divides Bacteria into two groups based on
differences in cell wall structure

19. Crystal
violet
Gram's
iodine
Decolorise with
acetone
Counterstain with
e.g. methyl red
appear purple
Gram-negatives
appear pink
The Gram Stain

20. Escherichia coli – a gram-negative rod

21. Acid-fast staining
• particularly useful for staining members of the
genus Mycobacterium
e.g., Mycobacterium tuberculosis – causes tuberculosis
e.g., Mycobacterium leprae – causes leprosy
– high lipid content in cell walls is responsible for
their staining characteristics

22. Structure of Bacteria
Particular structures
capsule
flagella
pili
spore
Essential structures
cell wall
cell membrane
Cytoplasm
nuclear material

23. Gram +
Gram -
Cell wall
Cell (inner) membrane Outer membrane
Ribosomes
Granule
Cell wall
NucleoidCell membrane
Capsule
Flagellum
Pili

24. Cell wall
• Situation:
outmost portion.
15-30nm in
thickness, 10%-
25% of dry
weight.

25. Cell wall :Common peptidoglycan layer
• A backbone of N-acetyl glucosamine and N-
acetylmuramic acid: Both discovered in Gram
positive and Gram negative bacteria.
• A set of identical tetrapeptide side chain attached
to N-acetyl-muramic acid: different components
and binding modes in Gram positive and Gram
negative bacteria.
• A set of identical peptide cross bridges: only in
Gram positive bacteria

28. Special components of Gram positive
cell wall
Teichoic acid

29. Special components of Gram
negative cell wall

30. Functions of Cell Wall
• Maintaining the cell's characteristic shape- the rigid wall
compensates for the flexibility of the phospholipid
membrane and keeps the cell from assuming a
spherical shape
• Countering the effects of osmotic pressure
• Providing attachment sites for bacteriophages
• Providing a rigid platform for surface appendages-
flagella, fimbriae, and pili all emanate from the wall
and extend beyond it
• Play an essential role in cell division
• Be the sites of major antigenic determinants of the cell
surface。
• Resistance of Antibiotics

31. Cell membrane
• Bearing the
enzymes & carrier
molecules that
functions in the
biosynthesis of DNA,
cell wall polymers &
membrane lipids
• Electron transport
and oxidative
phosphorylation
• Excretion of
hydrolytic
exoenzymes

32. Mesosomes
• Mesosomes are specialized structures formed
by convoluted invaginations of cytoplasm
membrane, and divided into septal and lateral
mesosome.

33. Cytoplasm
• Composed largely of water, together with proteins, nucleic
acid, lipids and small amount of sugars and salts
• Ribosomes: numerous, 15-20 nm in diameter with 70S;
distributed throughout the cytoplasm; sensitive to
streptomycin and erythromycin site of protein synthesis
 Plasmids: extrachromosomal
genetic elements
 Inclusions: sources of stored
energy, e,g volutin

34. Nucleus
• Lacking nuclear
membrane, absence
of nucleoli, hence
known as nucleic
material or nucleoid,
one to several per
bacterium.

35. Capsules and slime layers
• Many bacteria secrete a viscid material
around the cell surface, when this is organized
into a shapely defined structure, as in
pneumococcus, (Capsule).
• When is loosely attached (slime layer).
• Capsule is antigenic in nature.

36. Flagella
Monotrichate/Amphitrichate/Lophotrichate/Peritrichate
 Identification of Bacteria
 Pathogenesis
 Motility of bacteria
 Made up of flagellin

38. Pili
• Pili are hair-like projections of the
cell , They are known to be
receptors for certain bacterial
viruses. Chemical nature is pilin
• Classification and Function
a. Common pili or fimbriae: fine ,
rigid numerous, related to
bacterial adhesion
b. Sex pili: longer and coarser,
only 1-4, related to bacterial
conjugation

39. Endospores (spores)
• Dormant cell
• Resistant to adverse
conditions
- high temperatures
- organic solvents
 Identification of Bacteria
 Pathogenesis
 Resistance
• Produced when starved
• Contain calcium dipicolinate
DPA, Dipicolinic acid
• Bacillus and Clostridium

40. Wall-less forms of Bacteria/L forms
• When bacteria are treated with
• 1) enzymes that are lytic for the cell
wall e.g. lysozyme or
• 2) antibiotics that interfere with
biosynthesis of peptidoglycan, wall-less
bacteria are often produced.
• Usually these treatments generate
non-viable organisms.

41. Bacterial growth curve
• Lag phase
• Log (logarithmic) or exponential phase
• Stationary phase
• Phase of decline

43. Phases of Growth
• Lag
– Adapt to nutrients
• Log
– Active growth
• Stationary
– Death = Growth rate
• Death
– Nutrients consumed
– pH too low (why?)
• Optimize curves in production

44. Points to remember:
• Staining
• Fixation
• CW of bacteria
– Techoic acid
– LPS
• Classification of flagella
• Spores
• Pili
• Bacterial growth curve