Introduction to Bacteriology: Gram Stain and Bacterial Structures

Leading Causes of Death Globally

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Global
Both sexes, All ages, 1990, Deaths
i
IHD
Stroke
Lung C
Stomach C
Neonatal
LRI
Annual % change
1990 Y to 2019
Deaths/100,000
±
+
-3%
TB
Breast C
Liver C
Esophag C
Maternal
-1%
Diarrhea
HIV Meningitis
Whooping
Leukemia
Brain C
Ovary C
19
HTN HD
RHD
CMP
A Fib
Lymphoma
Cervix C
Aort An
PAD
Lip Oral C
1%
Diabetes
CKD
Cirrhosis
Lels
COPD
Asthma
Falls
Drown
Road Inj
2%
Upper Digest Ileus
Mech
Fre
F Body
Med Treat
Self Harm
Violence
Oth Unint
3%-
Global
Both sexes, All ages, 2019, Deaths
IHD
Stroke
Lung C
Colorect C |Stomach C
Neonatal
LRI
Annual % change
1990 ¥ to 2019 Y
Deaths/100,000
H
+
-3%-
-2%
Breast C
Other MN Liver C Esophag C
TB
Maternal
Diarrhea
HIV
-1%
Leukemia
Brain C
Ovary C
Kidney C
HTN HD
RHD
CMP
A Fib
Valvular
Lymphoma
Myeloma
PAD Cervix C
Lip Oral C
Uterus C
Diabetes
CKD
Cirrhosis
Falls
Road Inj
COPD
Asthma
2%
Poison
ILD
F Body
Parkinson's
Drugs
Alcohol
Animal
3% -
IHME
Masosring what matters
https://vizhub.healthdata.org
2019
Prostate C
Pancreas C
INTS
Whooping Hep
0%
Malaria
PEM
Oth Cardio
Aort An
1%
Drown
Mech Fire
Self Harm Violence
Alzheimer's
Colorect C
Congenital
-2%
Typh + Paratyph
Measles
Hep
0%-
Pancreas C
Tetanus
STI
1990
Malaria
PEM
Prostate C
Urinary
Hemog
Oth Cardio
Alzheimer's
i
Congenital
Urinary
Meningitis
Upper Digest Ileus
Other MN

Mortality and Morbidity due to Selected Infectious Diseases

Number of Deaths Caused by Selected Communicable Diseases Annually Worldwide as of 2019

Number of deaths caused by selected communicable
diseases annually worldwide as of 2019 (in 1,000)
Tuberculosis
1,208
HIV/AIDS]
675
Malaria
411
Measles
166
Cholera
143
Hepatitis
100
0
200
400
600
800
1,000
1,200
1
https://www.statista.com/statistics/282715/deaths-from-communicable-diseases-worldwide/

Major Infectious Diseases in Recent Years

TB deaths in 2022: 1.3 x 106
New cases in 2022: 10 x 106
Living with TB: 1.8 billion
HIV deaths in 2022: 630,000
New cases in 2022: 1.3 x 106
Cases since start: 80 x 106
Deaths from AIDS since start: 40 x 106
Living with HIV: 39 x 106
Malaria deaths 2022: 600,000
Living with malaria: 239 x 106
Swine flu deaths: 51,700 to 575,400
Total cases: 700 x 106 to 1.4 billion
Total COVID-19 deaths: 7 x 106
Total cases: 774,300,000 (as of 3-2024)

Lecture 17: Introduction to Bacteriology

Learning Objectives for Bacteriology

Learning Objectives:
LO1: Describe the differences between the gram-positive and the gram-
negative bacteria.
. LO1a: Describe various bacterial structures and detail the function of
these structures
· LO1b:Describe the differences in transcription mechanism of eukaryotes
vs. prokaryotes (Describe lac operon)
· LO1c: Summarize bacterial virulence factors and specifically describe the
differences between exotoxins and endotoxins
LO2: Describe the difference between aerobic and anaerobic bacteria in terms of
the enzymes they encode and growth conditions they prefer.
. LO2a: Detail the role of oxygen in the process of glycolysis, anaerobic
fermentation, aerobic fermentation
· LO2b: Describe the mechanisms by which bacteria are able to disarm
oxygen radicals
· LO2c: Identify organisms as strict anaerobes, strict aerobes, facultative
anaerobes or air-tolerant
LO3: Describe techniques used to grow bacteria
LO4: Describe the various stages of the growth curve of bacteria in liquid media
and calculate the number of bacteria, generation times etc. based on information
given

Bacterial Structure and Morphology

Bacteria are the smallest living things and lack mitochondria, nucleus, Golgi, ER,
lysosomes. This structure is distinguished from eukaryotic cells and is called
prokaryotic.

1. There are several common shapes of bacteria. They are referred to as: coccus
   (spherical), coccobacillus, vibrio (comma shaped), bacillus (rods), spirillum, and
   spirochete.
   Coccus
   Coccobacillus
   Vibrio
   Bacillus
   Spirillum
   Spirochete
2. In addition to shape, bacteria can arrange in aggregates (due to their "stickiness").
   For cocci, these include diplococci (pairs, figure right) streptococci (chains), and
   staphylococci (irregular or grape-like).

The basic structure of bacteria can be divided into envelope (containing many complex
and unique molecules) and the interior (nuclear body and cytosol).

Bacterial Envelope Components

Envelope (Cell Membrane, Cell Wall, Capsule, Flagella, Pili)

Envelope (cell membrane, cell wall, capsule, flagella, pili)
· Protects bacteria against chemical and biological threats
· Carries out many bacterial processes
· Contains appendages that make it possible for bacteria to colonize surfaces

Cell Membrane Structure and Function

Cell membrane
. Electron transport system of the cell, vital for
cell growth
. Similar to eukaryotic cells except that it has
many more proteins and no sterols (except
Mycoplasma)
. The bacterial chromosome is attached to cell
membrane
· Has proteins controlling the entrance and exit
of solutes and proteins
Outside cell
Cell
Membrane
Cytoplasm
Proteins

Cell Wall - Essential for Bacterial Life

Envelope (cell membrane, cell wall, capsule, flagella, pili)
Cell wall - essential for bacterial life
· Almost all bacteria have a rigid peptidoglycan (murein) layers that surround the
cytoplasmic membrane.
. This structure is unique to prokaryotes.
· Cell wall consists of alternating sugars: N-acetylglucosamine (NAG) and N-
acetylmuramic acid (NAM).
. These are cross-linked into sheets by amino acids.
· Size of cell wall is the main feature that distinguishes gram-positive and gram-
negative bacteria.
. The stain used to differentiate G(+) and G(-) bacteria is called the gram stain.

Gram-Positive Bacteria Characteristics

Gram-positive bacteria
G(+) bacteria have a thick multilayered cell
wall (20-80 nm) with embedded teichoic
acid.
· Gram-positive bacteria have a thick multilayered
cell wall with embedded teichoic acid.
. The teichoic acids consist of polymers of glycol
phosphate or ribotol phosphate with extra
moieties such as amino acids.
. A type of teichoic acid called lipoteichioc acid
also has a fatty acid that anchors it into the
cytosolic membrane.
. Both of these elements promote bacterial
adhesion to the host.
Peptidoglycan Monomer
NAM
NAG
CH3
CH2 OH
H
NH-C=O
H
O
H
M
H
OH
H
H
-0-
H
H
YLO-
H
NH-C=O
CH2 OH
H3C-CH-C=O
CH3
L-Alanine
D-Glutamine
I
- pentapeptide
L-Lysine
I
D-Alanine
D-Alanine
surface proteins
teichoic acid
- lipoteichoic acid
peptidoglycan
NAM NAG
peptide
cytoplasmic
membrane
phospholipid
Gram-positive
http://faculty.ccbcmd.edu/courses/bio141/labmanua/lab14/diseases/enterococcus/u1fig9b.html

Antibiotics Targeting Bacterial Cell Wall

Antibiotics can target bacterial cell wall
Beta-lactams
· Beta-lactams prevent bacteria from the final stages (cross-linking of
the peptide subunits) of constructing a cell wall, creating an
osmotically imbalanced bacteria
. Mostly used against Gram-positive bacteria
. Amoxicillin is a synthetic derivative of penicillin, and has a greater
activity against Gram-negative microorganisms due to the added free
amino group
· Side-chains may contribute to allergic reaction

Gram-Negative Bacteria Characteristics

Gram-negative bacteria
G (-) bacteria have more complex outer structure.
· They have a thin (5 - 10 nm) peptidoglycan layer, no teichoic
acid, BUT they have an additional outer membrane (OM)
external to the peptidoglycan.
· The space between cytoplasmic (or inner) membrane (IM) and
OM is called the periplasmic space and contains
peptidoglycan wall (not extensively cross-linked), many
metabolic enzymes and virulence factors.
. Outer leaflet of OM contains lipopolysaccharide (LPS), also
called endotoxin. OM serves as a barrier to organic molecules
and hydrophobic molecules. LPS consists of:
o
Lipid A (lipid component of an endotoxin responsible for
the toxicity of gram-negative bacteria)
o Core polysaccharide
o
O antigen.
· Active transport and diffusion of nutrients is facilitated by
proteins called porins that traverse the entire OM.
protein
protein
porin
LPS
O polysaccharide
lipid A
outer
membrane
lipoprotein
.
peptidoglycan
cytoplasmic
membrane
phospholipid
https://bio.libretexts.org/Bookshelves/Microbiology/
Gram-negative
LPS
man
rha
abe
O-antigen
repeat 40 units
gal
Core polysaccharide
P
gin
O HN
gln
O HN
P
Lipid A
https://microbeonline.com/lipopolysaccharide

Gram Stain Procedure

Gram stain
LIPO POLYSACCHARIDE
PORIN
S-layer
Gram-negative
OUTER MEMBRANE
PERIPLASMICSPACE
PEPTIDOGLYCAN
CYTOPLASMIC
MEMBRANE
surface proteins
teichoic acid
lipoteichoic acid
Gram-positive
peptidoglycan
NAM NAG
peptide
cytoplasmic
membrane
phospholipid
https://schaechter.asmblog.org/schaechter/2013/02/the-gram-stain-its-persistence-and-
GRAM +
GRAM -
its-quirks.html
O
Crystal
Violet
L
1
lodine
treatment
I
Decolorization
0
1
Counter stain
(safranin)
red
Fixation

Bacterial Envelope Appendages

Capsule - Virulence Factor

Envelope - continued
Capsule
· Capsules are found on both G(+) and G(-) bacteria.
· Polysaccharide layer (slime layer) is a major
virulence factor, but NOT essential for survival.
· Capsules protect against phagocytosis and resist
complement binding.

Flagella - Locomotion

Flagella
· Responsible for locomotion
· Found on both G (+) and G (-) bacteria
. Bacteria may have none, one or many
. Made of helically coiled proteins that are anchored into
bacterial IM
· Flagella consist of proteins, called flagellin that differ between
strains and therefore can be used for differentiation of bacteria.
infolding of
plasma
membrane
capsule
cell wall
DNA coiled
into nucleoid
basal body
888888
8888
flagellum
Q
888
70S
ribosomes
cytoplasmic
inclusion
pili
plasmid
cytoplasm
plasma
membrane
@ Elsevier. Goering et al: Mims' Medical Microbiology 4e - www.studentconsult.com.

Pili - Attachment and Genetic Exchange

Pili
. Made of proteins called pilin that surround a hollow core
. Much smaller than flagella in diameter, and are all over the
surface of the bacteria
. Main function is to promote attachment to host cells often in a
specific manner (pili that do this are called fimbriae)
. In bacterial mating - can also function in genetic exchange of
DNA between bacteria (these are called F pili or sex pili).
These are usually found one per cell.
· Found on both G (+) and G (-) bacteria
infolding of
plasma
membrane
capsule
cell wall
DNA coiled
into nucleoid
basal body
888888
8888
flagellum
0
888
70S
ribosomes
cytoplasmic
inclusion
pili
plasmid
cytoplasm
plasma
membrane
C) Elsevier. Goering et al: Mims' Medical Microbiology 4e - www.studentconsult.com.

Spores/Endospores in Gram-Positive Bacteria

Spores/Endospores (Some G+ bacteria only)
· Dormant state of bacteria
. Usually dehydrated structures with calcium + dipicolinate
· Spores have an inner membrane, two peptidoglycan
layers and a keratin-like protein coat.
· Spores resist chemicals, boiling, and radiation.
. The presence of water and alanine cause the
germination for the spore.
Coat
Outer Membrane
Core
Cortex
Germ Cell Wall
Inner Membrane
free endospore
vegetative cell
spore coat
L
sporulation
mother
cell
developing
spore coat
ASM Digital Image Collection. Merkel
https://micro.cornell.edu/research/epulopiscium/bacterial-endospores/