Comments from background information sheets last

week

Differing levels of microbiology- range of marks on the

quiz- formative

Worried about lab

Allergies etc

Appointment
Syllabus Outline till Christmas
Week Date /Time Event Reading
9 26/9 Lecture 1 Unit Introduction/History of Chapter 1
Microbiology
10 3/10 Lecture 2 Microscopes and Microbial Cell Chapter 1 and 2
Structure
All dates 11 10/10 Lecture 3 Cell structure and function Chapter 2

and times 12 14/10 Lecture 4 Microbial nutrition Chapter 3 (15

further reading)
are 12 17/10 (9-10, 11-12, Laboratory introduction and training Online Lab
2-3, 4-5) Manual
subject to 13 24/10 (9-11, 12-2, Laboratory 1 Lab Manual
3-5, 6-8)
change- 13 25/10 Lecture 5 Microbial Growth and Control Chapter 5

please 15 7/11 (9-11, 12-2, 3- Laboratory 2 Lab Manual

check your 5, 6-8)
14/11 (9-11, 12-2,
16 Laboratory 3 Lab Manual
portal! 3-5, 6-8)

18 28/11 (9-11, 12-2, LAB EXAM Lab Manual

3-5, 6-8)

20 9/12 Lecture 6 Viruses Chapter 8

Formative MCQ assessment online
Microscopy and
Microbial cells
Figure 2.20
Light Microscopy
• Compound light microscope uses visible light to illuminate
• Many different types of light microscopy:
– Bright-field, Phase-contrast, Dark-field, Fluorescence

• Magnification= the ability to make an object larger

• Resolution= the ability to distinguish two adjacent objects as
separate and distinct
• Limit of resolution for light microscope is about 0.2 μm
Fig. 2-1 Magnification Light path
100, 400, Visualized
1000 image
Eye
Ocular
Specimen on
glass slide
Eyepiece
10 (ocular) lens
Intermediate image
(inverted from that
Objective
of the specimen)

Stage

Condenser
10, 40, or Objective lens
100 (oil)
Focusing knobs Specimen
None Condenser lens
Light source
Field diaphragm
[“Light source” in (a)]
Figure 2.5
Brightfield, Phase Contrast and Darkfield Illumination
• Brightfield
• Dark objects are visible against a bright background.
• Light reflected does not enter the objective lens.
• Phase Contrast
• Increases contrast without stain
• Light reaches specimen from sides only
• Darkfield
• Light objects are visible against a dark background.
• Light reflected enters the objective lens.
Fluorescence Microscopy
• Uses UV light.
• Fluorescent
substances absorb
UV light and emit
visible light.
• Cells may be
stained with
fluorescent dyes
(fluorochromes).
 Confocal Microscopy
• Uses fluorochromes
and a laser light.
• Laser illuminates each
plane in a specimen

http://www.microbeworld.org/component/jlibrary/?view=article&id=12553
 FISH-CLSM analysis of the bacteria in the
sponge sample 6b. Volume–renderings of a
confocal stack showing sponge
autofluorescence (A, cyan); (B) Gam42a–
stained bacteria (B, blue) and EUB338MIX–
stained bacteria (C, red); (D) overlap of (A–C),
where Gammaproteobacteria appear purple
for the overlap of red and blue, while other
bacteria remain only red; (E) three–
dimensional model of (C), where bacteria are
converted into spheres and the sponge tissue
into semi–transparent iso–
surfaces; Gammaproteobacteria are indicated
by arrowheads, while arrows point to other
bacteria. Scale bars: 10 µm.

https://www.nature.com/articles/s41598-017-06055-9
 Electron Microscopy
• Uses electrons instead of light.
• The shorter wavelength of electrons gives greater
resolution.
• TEM
• SEM
Transmission Electron Microscopy (TEM)
• 10,000-100,000; resolution 2.5 nm.

Figure 3.9a (2 of 2)
 Scanning Electron Microscopy (SEM)
• 1,000-10,000; resolution 20 nm.

Figure 3.9b (2 of 2)
Archaeal and Bacterial
Cell Structure
 Prokaryotic Cells
Cytoplasm Nucleoid Ribosomes
• Comparing Plasmid

prokaryotic and Cytoplasmic

eukaryotic cells Cell wall
Prokaryote
membrane

– Prokaryote comes Cytoplasmic

membrane
Endoplasmic
reticulum

from the Greek Ribosomes

Golgi
Nucleus
words for Nucleolus Mitochondrion
prenucleus. Nuclear
membrane Chloroplast

– Eukaryote comes Cytoplasm

from the Greek Eukaryote

words for true

nucleus.
 Bacteria and Eukaryote
Archaea
• One circular chromosome, • Paired chromosomes,
not in a membrane in nuclear membrane
• Plasmids • No plasmids
• No histones • Histones
• No organelles • Organelles
• Peptidoglycan cell walls • Polysaccharide cell walls
• Binary fission • Mitotic spindle
Cell Morphology
• Average size: 0.1 -1.0 µm  2 - 8 µm
• Basic shapes:
Fig. 4-1

Coccus

Rod

Spirillum

Spirochete
Hypha
Stalk
Budding and
appendaged bacteria

Filamentous bacteria
• Unusual shapes
– Star-shaped Stella
– Square Haloarcula
• Most bacteria are monomorphic
• A few are pleomorphic
Bacterial cell arrangements
Some bacteria cells separate completely after ÷, others stay
attached

Attached cells have a characteristic arrangement that differs

depending on the bacteria, the shape, and the plane.
Diplo- = cells remain attached in pairs (e.g.,
diplococcus)

Strepto- = cells remain attached in chains

Tetrads = cells arranged in squares

Sarcinae = cells arranged in cubes

Staphylo- = random planes of division

resulting in sheets and clumps
Fig. 4-2

r = 1 µm
Surface
=3
Volume

r = 2 µm
Surface
= 1.5
Volume
The Small World
• Lower limits of cell size
• Cellular organisms <0.15 µm in diameter are unlikely.
• need volume to house proteins, nucleic acids, ribosomes, and
so on
• Open oceans tend to contain small cells (0.2–0.4 µm in
diameter) known as “ultramicrobacteria.”
• Genomes are highly streamlined, missing functions that must
be supplied by other microbes or hosts (plants and animals).
Bacterial and Archaeal cell anatomy

www.cellsalive.com
 Cell Wall
• Prevents osmotic lysis
• Pressure inside cell
• Rigidity and cell structure
• Bacteria= peptidoglycan
• Archaea = pseudomurein
and S-layers
• Disease and other specific interactions
 Bacteria
Gram-Negative Gram-Positive
Cell Walls Cell Walls
• Thin peptidoglycan • Thick peptidoglycan
• No teichoic acids • Teichoic acids
• Outer membrane • In acid-fast cells, contains
• LPS mycolic acid

Exception!
Prokaryotes that lack cell walls -Mycoplasmas
-Thermoplasma (Archaea)
Figure 2.24
 Peptidoglycan (murein)
• Monosaccharides N-acetylglucosamine (NAG)
and N-acetylmuramic acid (NAM)
• Polymer of disaccharide
• Linked by polypeptides
Peptidoglycan structure. The tetrapeptides linked to NAM
are cross-linked by a pentaglycine peptide, shown as red
lines linking the D-glutamine (L) to the D-alanine (A).
Fig. 2.25
N-Acetylglucosamine (G) N-Acetylmuramic acid (M)

N-Acetyl
group

Lysozyme-
sensitive
bond
Peptide
cross-links
L-Alanine

D-Glutamic acid

Meso-diamino-
pimelic acid

D-Alanine
Figure 2.11
Gram-Positive Cell Walls
• Up to 90% peptidoglycan
• Teichoic acids – common (e.g glycerol and P)
– Lipoteichoic acid links to plasma membrane
– Wall teichoic acid links to peptidoglycan
• May regulate movement of cations.
• Polysaccharides provide antigenic variation.
Gram-Negative Outer Membrane

Figure 4.13c
 Gram-Negative Outer Membrane
• Lipopolysaccharides, lipoproteins, phospholipids
• Periplasm / outer memb and the plasma memb.
• LPS= 2 components- core and the O-polysaccharide
• O polysaccharide antigen, e.g., E. coli O157:H7
• Lipid A is an ENDOTOXIN
• Porins (proteins) form channels (non-specific).

O-specific polysaccharide Core polysaccharide Lipid A

n
 Lysozyme-insensitive N-Acetyl

Cell Walls of Archaea (1,3)

group

•No peptidoglycan NAG

N-Acetyltalosaminuronic acid

•Typically no outer membrane Peptide

cross-links

•Pseudomurein -methanogenic Archaea

•NAG and N-acetylalosaminuronic acid (NAT)

•Some Archaea lack Psedomurein.

•S-Layers- most common cell wall type among Archaea

• Paracrystalline protein layer- lysozyme and penicillin- no effect.

Damage to Cell Walls
• Lysozyme digests disaccharide in peptidoglycan.
• Penicillin inhibits peptide bridges in
peptidoglycan.
• Protoplast is a wall-less cell.
Wall
Membrane
Lysozyme
digests wall
H2O enters Lysis

• Spheroplast is a wall-less
H2O enters H2O enters Low solute solution

Gram-negative cell.
• Protoplasts and spheroplasts Lysozyme
digests wall

are susceptible to osmotic lysis. Protoplast

Isotonic solute solution

Preparation of Specimens for Light Microscopy

• Smear: A thin film of a solution of microbes on a slide.

• A smear is usually fixed to attach the microbes to the
slide and to kill the microbes.

 Live or unstained cells- little contrast

 Cell behavior -live specimens.
 Preparing Smears for Staining
• Stains consist of a positive and negative ion.
• Basic dye, the chromophore is a cation (+ve).
• Acidic dye, the chromophore is an anion (-ve).
• Staining the background instead of the cell is
called negative staining.
 Simple Stains
• Simple stain: Use of a single basic dye.
• A mordant may be used to hold the stain or coat
the specimen to enlarge it.
• Crystal Violet
• Methylene Blue
• Aqueous Safranin

• Iodine
Differential Stains
 Differential Stains: Gram Stain
• Gram stain classifies bacteria into gram-positive
or gram-negative.
• Gram-positive bacteria tend to be killed by
penicillin and detergents.
• Gram-negative bacteria are more resistant to
antibiotics.
• 1884 Hans Gram

http://www.ncl.ac.uk/dental/oralbiol/oralenv/tutorials/gramstain.htm
 Gram Stain Mechanism
• Crystal violet-iodine crystals form in cell.
• Gram-positive
– Alcohol dehydrates peptidoglycan
– CV-I crystals do not leave
• Gram-negative
– Alcohol dissolves outer membrane and leaves holes
in peptidoglycan.
– CV-I washes out
 Atypical Cell Walls
• Mycoplasmas
• Such as?
– Lack cell walls
– Sterols in plasma membrane- mycolic acid
– ACID FAST- retain a basic stain in the presence of
acid-alcohol are called acid-fast (Ziehl Neelsen)
• Stain is more soluble in mycolic acid than acid
alcohol- does not dissolve away.
 Special Stains
• Negative staining is useful for
capsules.
• Heat is required to drive a
stain into endospores.
• Flagella staining requires a
mordant to make the flagella
wide enough to see.

Figure 3.13a–c
• Finish and annotate notes using Chapter 1/2
• Cell size webpage
• Laboratory pages