Bacteria are microscopic, single-celled organisms that thrive in diverse environments. These organisms can live in soil, the ocean and inside the human gut. Humans' relationship with bacteria is complex. Sometimes bacteria lend us a helping hand, such as by curdling milk into yogurt or helping with our digestion

1. BACTERIA
Structure of Bacterial Cells
Md. Saiful Islam
BPharm, MPharm (PCP)
North South University
Fb Group: Pharmacy Universe

2. • Staphylococcus sp. is used when referring to the genus
as a whole when the species is not identified.
• “sp.” – singular (Staphylococcus sp.)
• “spp.” – plural (Staphylococcus spp.)
# When in print, genus and species are italicized.
(Staphylococcus aureus)
# When written, genus and species are underlined.
(Staphylococcus aureus)

3. Introduction
• Bacterium (pl. bacteria) refers to a single-celled organism
without having a true nucleus or functionally specific
components of metabolism that belongs to the kingdom
Prokaryotae .(a name which means primitive nucleus.
• All other living organisms are called eukaryotes, a name
implying a true or proper nucleus.
• Bacteria share with the blue-green algae this unique place
in the world of living organisms.
• Recently, a different class bacteria and blue-green algae
was identified, termed as the Archaebacteria; which has
different wall and membrane structure and pattern of
metabolism. They are thought by many to be the first
living organisms to have appeared on earth.

4. The main features distinguishing
prokaryotic and eukaryotic cells
Feature Prokaryotes Eukaryotes
• Nucleus No enclosing membrane Enclosed by a membrane
• Cell wall Peptidoglycan Cellulose
• Mitochondria Absent Present
• Mesosomes Present Absent
• Chloroplasts Absent Present

5. Eukaryotic Cell

6. • Bacteria can synthesize nucleic acids (DNA, RNA), other
important proteins and can reproduce independently, but
may essentially need a host to supply food and also a
supportive environment.
• In reality, millions of non-pathogenic bacteria live on the
skin and mucous membranes of the human gastrointestinal
tract (GIT) ; these are termed as normal flora.
• Importantly, bacteria that cause disease are usually known
as pathogens. Example- ?????

7. Size
• An average rod-shaped bacterium measures
approximately 1 μm in diameter and 4 μm in
length.
• They usually vary in size considerably from
< 0.5 to 1.0 μm in diameter to 10–20 μm in
length in some of the longer spiral forms

8. SIZE OF BACTERIA
• Unit for measurement :
Micron or micrometer,μm:
1μm=10-3mm
• Size:
Varies with kinds of bacteria, and
also related to their age and
external environment.
 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

9. There are 3 principal forms of bacteria-
(a) Spherical or Ovoid: Bacteria occur as single cells
(micrococci), or in pairs (diplococci), clusters
(staphylococci), chains (streptococci) or cubical
groups (sarcinae).
(b) Rod-shaped: Bacteria are termed as bacilli, more
oval ones are known as coccobacilli and those
forming a chain are called as streptobacilli.
(c) Spiral: Bacteria are rigid (spirilla), flexible
(spirochaetes) or curved (vibrios).
Shape

11. Food and Oxygen Requirements
Bacteria are of different types based upon their food
and oxygen requirements as given below :
(a) Heterotrophic : require organic material as food,
(b) Parasites : feed on living organisms,
(c) Saprophytes : feed on non-living organic material,
(d)Autotrophic : i.e., self-nourishing–obtain their
energy from inorganic substances, including most of
the soil bacteria,
(e) Aerobes : essentially require oxygen for their very
existence and growth, and
(f) Anaerobes : do not require oxygen for their
existence and growth. e.g., most bacteria found in the
gastrointestinal tract.

12. • Three fundamental divisions of the bacterial
cell occur in all species:
1. Cell wall
2. Cytoplasmic membrane
3. Cytoplasm
Bacterial Cell Structure

14. 1. Cell wall
The bacterial cell wall has two major roles to play :
(a) to protect the cell against osmotic rupture
particularly in diluted media, and also against
certain possible mechanical damage(s).
(b) to assign bacterial shapes, their subsequent major
division into Gram positive and Gram negative
microorganisms and their antigenic attributes.

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

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

17. 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

18. At a Glance
• Cell Wall Structure
– Bacterial cell walls are composed of peptidoglycan
• the glycan portion of peptidoglycan is made of a huge
polymer of carbohydrates containing:
• N-acetylmuramic acid (NAM) and
• N-acetylglucosamine (NAG)
• These long chains of alternating NAM and NAG are
held together by short peptide cross-bridges

20. A, peptidoglycan of Escherichia coli. B, repeating unit of
peptidoglycan of E. coli. L-ala, L-alanine; D-glu, D-glutamine; DAP,
diaminopimelic acid: D-ala, D-alanine.

21. Special components of Gram positive cell wall
Teichoic acid

22. Special components of Gram
negative cell wall

23. 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
( additions) - flagella, fimbriae, and pili all originate 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

24. Wall-less forms of Bacteria
• 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.
• Wall-less bacteria that can not replicate are referred to
as spheroplasts (when an outer membrane is present)
or protoplasts (if an outer membrane is not present).

26. Cell membrane
• Site of biosynthesis of DNA, cell wall polymers and
membrane lipids.
• Selective permeability and transport of solutes into cells.
• Electron transport and oxidative phosphorylation.
• Excretion of hydrolytic exoenzymes.

27. Hans Christian Joachim Gram
Danish Bacteriologist

28. Dyes become Stains
• With interest in the effects of dyes on living
tissue. In 1884 the Danish microbiologist Hans
Christian Gram discovered that crystal violet
irreversibly stains certain bacteria but can be
washed from others.
• The dye has been widely used ever since for
the Gram stain technique, which identifies
bacteria as gram-positive (the stain is
retained) or gram-negative (the stain is
washed).

29. Gram Staining Technique
• Staining procedures that make visible the differences between
bacterial cells or parts of a bacterial cells are termed differential
staining.
• Gram staining is one of the most important and widely used
differential staining technique.
• This technique was introduced by Christian Gram in 1884.
• In this process the fixed bacterial smear is subjected to the
following reagents in the order listed: crystal violet, iodine
solution, alcohol ( decolorizing agent) and safranin.
• Bacteria stained by the Gram method fall into two groups :
Gram positive bacteria, which retain the crystal violet and
hance appear deep violet in color.
• And Gram negative bacteria, which lose the crystal violet, are
counterstained by the safranin and hence apper red in color.

30. Based on the ability to retain crystal violet during decolorization
with alcohol
Gram-positive cell wall Gram-negative cell wall

31. Organizing the Staining Bottles
Dr.T.V.Rao MD 31

32. Making a Smear
• First prepare your slide. You
do this by placing bacteria
on a slide in a drop of water,
allowing them to dry and
then heat fixing them.
Heating the slide kills the
bacteria and makes sure
that the bacteria a stuck to
the slide and wont wash
away during the staining
procedure .

33. Choosing a Right Smear
• Before choosing a field for
microscopic examination,
it is important to look at
the smear macroscopically
• Note that the smear is
easily visible in ordinary
light

34. Dr.T.V.Rao MD

35. Steps in Gram Staining Procedure- Follow
the Clock
1. On a rack, flood with filtered crystal violet 10 sec
2. Wash briefly in water to remove excess crystal violet
3. Flood with Gram’s iodine 10 sec
4. Wash briefly in water, do not let the section dry out.
5. Decolourise with acetone until the moving dye front
has passed the lower edge of the section
6. Wash immediately in tap water
7. Note : If the section appears too blue repeat steps 6
and 7
8. Counterstain with safranin 15 seconds

36. Step 1

37. Dr.T.V.Rao MD
Step 2

38. Dr.T.V.Rao MD
Step 3

39. Step 4

40. Step 5

41. Step 6

42. Step 7

43. Colors makes the Difference in Gram
staining
• Bacteria that manage to
keep the original purple dye
have only got a cell wall -
they are called Gram
positive.
• Bacteria that lose the
original purple dye and can
therefore take up the
second red dye have got
both a cell wall and a cell
membrane - they are called
Gram negative.

44. Observe with Caution to differentiate

45. Gram-positive bacteria
• Steptococcus
• Staphylococcus
• Lactobacillus
• Bacillus
• Clostridium

46. Gram-negative bacteria
• Escherichia
• Salmonella
• Vibrio
• Treponema

47. Common errors in Staining procedure
• Excessive heat during
fixation
• Low concentration of crystal
violet
• Excessive washing between
steps
• Insufficient iodine exposure
• Prolonged decolourization
• Excessive counterstaining

48. Trouble shooting in Gram Staining
method
• It is important to note that gram-positivity (the
ability to retain the purple crystal violet-iodine
complex) is not an all-or-nothing phenomenon
but a matter of degree. There are several
factors which could result in a gram-positive
organism staining gram-negatively
1.The method and techniques used.
Overheating during heat fixation, over
decolourization with alcohol, and even too much
washing with water between steps may result in
gram-positive bacteria losing the crystal violet-
iodine complex.

49. Trouble shooting in Gram Staining method
2. The age of the culture. Cultures more than 24
hours old may lose their ability to retain the
crystal violet-iodine complex.
3. The organism itself. Some gram-positive
bacteria are more able to retain the crystal
violet-iodine complex than others.
** One must use very precise techniques in
gram staining and interpret the results with
discretion

50. Gram+ vs. Gram- Cell Walls
• Gram+ cells have a very thick, multilayered cell wall
– they also contain teichoic acids and lipoteichoic acids
– Lysozyme
• Gram- cells have a very thin layer of peptidoglycan
– they also have an outer membrane in addition to the
cytoplasmic membrane
– the space between these two membranes is called the
periplasmic space or periplasm

51. Insert figure 4.12
Comparative cell envelopes

52. – the outer membrane is an asymmetric bilayer:
• Phospholipids on the inside
• lipopolysaccharides (LPS) on the outside
• LPS structure:
–Lipid A - also called endotoxin because it
damages cells and tissues (also causes fever and
shock)
–Core Polysaccharide
–O antigen – distinguishes serotypes of a species
**Porins allow non-specific transport across the
membrane
Gram+ vs. Gram- Cell Walls

54. Gram+ vs. Gram- Cell Walls
In the walls of Gram-positive bacteria, molecules of a
polyribitol or polyglycerolphosphate are attached by covalent
links to the oligosaccharide backbone; these entities
are teichoic acids.
The glycerol teichoic acid may contain an alanine residue.
Teichoic acids do not confer additional rigidity on the cell wall,
but as they are acidic in nature they may function by
sequestering essential metal cations from the media on which
the cells are growing.
** This could be of value in situations where cation
concentration in the environment is low.

55. Gram+ vs. Gram- Cell Walls
# The Gram-negative cell envelope is even more complicated;
essentially, it contains lipoprotein molecules attached
covalently to the oligosaccharide backbone.
# In addition, on its outer side, a layer of lipopolysaccharide
(LPS) and protein attached by hydrophobic interactions and
divalent metal cations, Ca2+ and Mg2+.
# On the innerside is a layer of phospholipid (PL). The LPS
molecule consists of three regions, called lipid A, core
polysaccharide and O-specific side chain .
# The O-specific side chain comprises an array of sugars that
are responsible for specific serological reactions of organisms,
which are used in identification.
# The lipid A region is responsible for the toxic and pyrogenic
(fever-producing) properties of this group .

56. The complex outer layers beyond the peptidoglycan in the
Gram-negative species, the outer membrane, protect the
organism to a certain extent from the action of toxic
Chemicals .
Thus, disinfectants are often effective only at concentrations
higher than those affecting Gram-positive cells and these
layers provide unique protection to the cells from the action of
benzylpenicillin and lysozyme.