MAKERERE UNIVERSITY

P.O. Box 7072 Kampala, Uganda Phone:256 414 533098

Email: principal@chs.mak.ac.ug Fax:256 414 533098

COLLEGE OF HEALTH SCIENCES

DEPARTMENT OF MICROBIOLOGY

ASSIGNMENTS ON GENERAL MICROBIOLOGY AND BACTERIOLOGY (YR II)

Author,

Name Registration Number Course

Timugibwa Persis 21/U/19951/p/s BNUR

Assignment One:

History and taxonomy

1. Who is the Father of Microbiology?

Anthony van Leeuwenhoek

2. Name five giants of the early days of microbiology and their major contributions
Louis Pasteur: Introduced germ theory and pasteurization of milk in 1864.
Alexander Fleming: Discovered penicillin in 1929.
Hans Christian Gram: Introduced the gram staining technique in 1884.
Joseph Lister: Developed a system of antiseptic surgery in 1864.
Robert Koch: Discovered the use of methylene blue to stain bacteria in 1877.

3. What is the concept of spontaneous generation and how did Louis Pasteur defeat
it?
The theory of spontaneous generation suggested that living organisms could develop
from non-living matter.
Louis Pasteur disproved this theory by conducting the swan-neck experiment.
-He used a special flask whose neck was shaped like an S or the neck of a swan, hence
the name “Swan Neck Flask.” He put a nutrient rich broth in the flask, which he called
the “infusion.” He then boiled the infusion killing any microorganisms which were
already present. Then he allowed the infusion to sit.
- Because of the shape of the flask, the infusion was exposed to air. However, dust
particles and other things in the air never made it into the infusion. Because they were
trapped in the curve of the Swan Neck Flask. No matter how long he allowed the flask to
sit, microorganisms never appeared in the infusion. However, if he tipped the flask and
allowed the things trapped in the neck to get into the infusion, then micro-organisms
began to appear in the infusion and multiply rapidly. This demonstrates that
microorganisms do not appear as a result of Spontaneous Generation. Instead, they are
introduced into food through dust particles and other things that happen to land on the
food.

4. Proof that some microorganisms cause disease provided the greatest impetus for
the development of microbiology as an independent biological science. Even as early
as the sixteenth century it was thought that something that induced disease could be
transmitted from a diseased person to a healthy person. It was not until the work of a
German physician, Robert Koch (1843-1910) that the concept of infectious disease was
given experimental support. Koch formulated a set of rigorous criteria, known as
Koch’s postulates, for definitively linking a specific microorganism to a specific
disease. Using an example, state and explain Koch’s postulates.
Koch’s postulates are used to substantiate claims that a particular organism causes a
specific ailment. An example of an organism which readily meets the criteria of these
postulates is Mycobacterium tuberculosis. They require that:
-The organism is demonstrable in every case of the disease and is absent in healthy
subjects.
-The organism can be isolated and propagated in pure culture in vitro.
-Inoculation of the pure culture by a suitable route into a suitable host should reproduce
the disease.
-The organism can be re-isolated from the host with the disease.

5. Why is taxonomy important in microbiology?

Taxonomy is the classification, nomenclature and identification of microbes.
Classification is important because it allows for orderly grouping of micro-organisms.
Nomenclature is important for naming of the micro-organisms. Identification helps us to
isolate and distinguish desirable organisms from undesirable ones.

6. Using an example, list the taxonomical hierarchies of bacteria.

 Kingdom (Prokaryotae)
 Division (Gracilicutes)
 Class (Betaproteobacteria)
 Order (Burkholderiales)
 Family (Burkholderiaceae)
 Genus (Burkholderia)
 Species (Burkholderia cepacia)

Bacterial Morphology
A Typical Bacterial Cell
1. . What is the difference between a bacterial cell and an animal cell?
Bacterial Cell Animal Cell
They have a proteoglycan cell wall They lack a cell wall
70S ribosomes 80S ribosomes
Lack membrane bound organelles Have membrane bound organelles
Lack a true nucleus Has a true nucleus
Have circular DNA Have linear DNA
 Diameter is approximately 1.0µm Diameter is approximately 10µm

2. What characteristic shapes can bacteria assume?

There are two basic shapes of bacteria: spherical (cocci) and rod shaped (bacilli).
The rod shaped bacteria show different variations such as spirochete, vibrio,
spirilla (spiral/helical), filamentous and hyphae.

3. List some of the common bacterial structures and their functions

 Cytoplasmic membrane: responsible for protein secretion, processing and
packaging; ATP synthesis; signal transduction; transport.
 Cell wall: maintain the shape of the cell; support the cytoplasmic
membrane against high internal osmotic pressure.
 Fimbriae: adhesion
 Flagella (H antigen): motility (flagellin)
 Pilli: conjugation (reproduction)
 Capsule: antigenic (K antigen); resists phagocytosis,
 Bacteria nucleoid: stores genetic information;
 Plasmid: carries antibiotic resistant genes
 Ribosomes: protein synthesis

Bacteria Cell Envelope

4. Describe the bacterial cell envelope
The bacterial cell envelope consists of the cell wall and inner cell membrane, but in
gram-negative bacteria, an outer cell membrane is also included. It serves to protect
these organisms from their unpredictable and hostile environments.
The Plasma Membrane
5. Of all the layers of the cell envelope, the plasma membrane is the most
important because it encompasses the cytoplasm and defines the cell. If it is
removed the cell’s contents spill into the environment and the cell no longer
exists. The most widely accepted model for membrane structure is the fluid
mosaic model. Describe the fluid mosaic model for cell membranes.
The fluid mosaic model is composed of a phospholipid bilayer with cholesterol and
proteins randomly distributed across the membrane. Phospholipids are arranged in
such a way that their hydrophilic heads face outwards (towards the cytosol or
extracellular space), and their hydrophobic tails facing each other. They are able to
move freely between the layers and the randomly placed cholesterol increases their
fluidity. The proteins are globular masses, floating in the lipid bilayer which
constitute about 70% of the mass of the membrane and are of 2 types: integral and
peripheral.
6. All plasma membranes function as barriers. Yet they must also allow
movement of nutrients into the cell. If a microbe does not obtain nutrients
from its environment, it will quickly exhaust its supply of amino acids,
nucleotides and other molecules needed to survive. In addition, if a microbe is
to thrive and reproduce, it must have a source of energy. The energy source is
used to generate the cell’s major energy currency: the high energy molecule
ATP. Clearly, obtaining energy and nutrient sources is one of the most
important jobs an organism has, and it is primarily a function of the plasma
membrane. Concerning nutrient uptake, on what basis are elements divided
into macroelements (macronutrients) and trace elements (micronutrients)?
If they are taken up in large amounts for survival, they are termed
macronutrients. If they are taken up in relatively small amounts, then they are
termed micronutrients.

7. Describe facilitated diffusion, primary active transport (ABC transport),

secondary active transport (ion-coupled transport) and group translocation in
terms of their distinctive characteristics and mechanisms. What advantage
does a bacterium gain by using active transport rather than facilitated
diffusion?
Facilitated diffusion is the carrier mediated movement of molecules down a
concentration gradient. It uses SLC transporters and usually transports large
molecules and charged molecules that cannot diffuse freely across the cell
membrane. Facilitated diffusion is selective and rare in eukaryotes in which it is
only used for transport of glycerol.

Primary active transport uses energy directly from the hydrolysis of ATP to
transport molecules against their concentration gradient. The transports involved
in primary active transport are of the ABC superfamily. In secondary active
transport, the energy required to move a molecules against its concentration
 gradient is facilitated by energy obtained by the downhill movement of sodium
ions. It also uses SLC transporters.

Group translocation is primarily for glucose and mannose transport which are
phosphorylated during the course of transport by carrier proteins. No
concentration gradient is required and it uses energy from energy rich organic
compounds such as Phosphoenolpyruvate (PEP).

With active transport, the bacterium will be able to transport nutrients against
their concentration gradient. Even thought there is a high concentration of
nutrients within the bacterium, it can still take up more of its required nutrients
to ensure its survival.

8. What are uniport, symport and antiport?

 Uniport = catalyze transport of a substrate independent of any coupled ion
e.g. Calcium channel
 Symport = catalyze simultaneous transport of two substrates in the same
direction using a single carrier. e.g. sodium-glucose symporter
 Antiport = catalyze simultaneous transport of two like charged compounds
in opposite directions by a common carrier. e.g. Sodium-Hydrogen pump

9. What are siderophores? Why are they important?

They are low molecular weight iron chelators, produced by microbes, that have a
high affinity for iron (Fe3+) which enables them to accumulate it. It is produced
by E. coli, Klebsiella pneumoniae and Listeria monocytogen. Examples include
hydroxamic acid and yersiniabactin in pathogenic yersiniae.

10. The feature common to nearly all bacterial cell walls is the presence of the
peptidoglycan. Describe the peptidoglycan structure.
Peptidoglycan is a structural polymer of glycan chains which consists of repeating
N -acetylglucosamine and N -acetylmuramic acid residues.
N-acetylmuramic acid consists of L-alanine, D-glutamic acid and D-alanyl-D-
alanine. In gram-negative bacteria, N-acetylmuramic acid also contains meso-
diaminopimelic acid, while in gram-positive bacteria, it also contains L-lysine.
 Cross-links are formed either directly between meso-diaminopimelic acid or L-
lysine of one strand and the penultimate D-alanine of the next, or through an
interpeptide bridge composed of up to five amino acids. In both cases, the D-
alanine is lost in the cross-linking reaction.
In gram-positive bacteria, there are as many as 40 sheets of peptidoglycan,
comprising up to 50% of the cell wall material. In gram-negative bacteria, there
are only one or two sheets, comprising of 5-10% of the cell wall material.

11. What are the differences between Gram-positive cell wall and Gram-negative
cell wall?
Gram-Positive Bacteria Gram-Negative Bacteria
Thick peptidoglycan layer Thin peptidoglycan layer
Teichoic acid present Teichoic acid absent
Outer membrane absent Outer membrane present
Endotoxin absent Endotoxin present
Porin protein absent Porin protein present
Periplasmic space absent Periplasmic space present

12. Describe the lipopolysaccharide structure of the Gram-negative bacteria. What

is its function?
The lipopolysaccharide (LPS) has three components: Lipid A, O-polysaccharide
and core polysaccharide. Lipid A is the most intrinsic and is responsible for the
pyrogenic activity of the endotoxin. The O-polysaccharide extends outward from
the core polysaccharide and functions as an antigen. The core polysaccharide
attaches to lipid A and provides stability.

13. What are the functions of the other parts of the bacterial cell wall?
 Porin proteins: allow for passive diffusion of molecules through the outer
membrane of gram-negative bacteria
 Teichoic acid: form surface antigens of gram-positive bacteria
 Teichuronic acid: same function as teichoic acid but produced when there
is limited phosphate
 Periplasmic space: has beta-lactonase that provides resistance to
penicillin
 14. Bacterial cell wall synthesis is targeted by several antibiotics. List the different
antibiotics that target cell wall synthesis and the points of target on the cell
wall
 Penicillin: limits peptidyl cross-linkages
 Cephalosporin: binds and blocks enzymes involved in peptidoglycan
synthesis
 Chloramphenicol: blocks growth of peptide chain
 Vancomycin: blocks incorporation of NAG-NAM-PEP into the growing
peptidoglycan chain
 Cycloserine: analogue of D-alanine
 Carbopenems: same action as Penicillin

15. What are protoplasts, spheroplasts and L forms? What is the significance of L
forms in chronic infections?
 Protoplasts: viable spherical osmotically sensitive bacterial bodies lacking
a cell wall which are typically gram-positive bacteria
 Spheroplasts: viable spherical osmotically sensitive bacterial bodies with a
weakened cell walls. They are typically gram-negative bacteria and
maintain their outer membrane.
 L-forms: when a spheroplast of protoplast grow and divide, they are
called L-forms. They are resistant and difficult to demonstrate because
they do not stain with Gram or Acid Fast methods. They may not
propagate in vitro.
Cell Envelope Layers Outside the Glycocalyx
16. What is the difference between a capsule and a slime layer?
A capsule has a well-organized glycocalyx while a slime layer has a loosely
associated disorganized glycocalyx. The slime layer is soluble in liquid medium.

17. What does the term glycocalyx refer to?

The polysaccharide containing material lying outside the cell. It can be made of
homopolymers ( one polysaccharide) or heteropolymers (more than one
polysaccharide) and it is negatively charged.

Bacterial Cytoplasm
 18. Describe the nature and the function of the cytoplasm, and the regions and
structures within it.
The cytoplasm is a soft aqueous gel containing a variety of organic and inorganic
solutes, ribosomes and polysomes.
There are 3 regions of the cytoplasm: the nucleoid zone (nucleic acids and
protein complexes); the structural zone (cytoskeletal proteins) and the metabolic
zone (the space between structural and nucleoid zones).
It lacks membrane bound organelles and shows signs of internal morbidity. It
also contains inclusions like starch and glycogen for storage, gas vesicles and
parasporal bodies. It contains actin and non-actin filaments in its cytoskeleton.

19. How do plasmids differ from chromosomes? What is an episome?

Plasmid
Non genomic, circular, small DNA
molecule, present in bacteria and
archaea
Shows self replication
Centromeres and chromatids are
absent
Lacks introns and exons
Always double stranded
Chromosome
Thread-like structure composed of
DNA and proteins
Replicates during cell division
Centromeres and chromatids are
present
Contains introns and exons
Double or single stranded

And episome is extrachromosomal DNA

External Structures
20. What is the difference between pili, fimbrae and flagella?
 Pili: filamentous appendages involved in exchange of genetic material
between 2 cells (conjugation)
 Fimbrae: filamentous appendages involved in adhesion between bacterial
cells and their host cells (shorter, more numerous and thicker than pili;
straighter than flagella)
 Flagella: long twisted thin filamentous appendages involved in
locomotion
21. Describe the way many flagella operate to move the bacterium
 Flagella contain flagellin and their movement is powered directly by the proton
gradient from the electron transport chain. Bacteria located in alkaline
environments use energy from the sodium ion gradient instead. They are
chemotactic and move either in a clockwise (tumbling) or anticlockwise (linear)
motion.

22. Explain in a general way how bacteria move toward substances such as
nutrients and away from toxic materials
Attractants and repellents bind to specific receptors in the bacterial membrane.
If the cell is moving towards the chemotactic source, it moves linearly and so the
flagella move in an anticlockwise motion.
A cell moving away from the chemotactic source (e.g. toxic materials) tumbles
away by reversing the direction of rotation at intervals.

Bacterial Endospores
23. Describe the structure of the bacterial endospore using a labeled diagram?
 Core: spore protoplast which contains normal cell structures which are
metabolically inactive
 Cortex: thickest layer of the spore envelope. It has cortex peptidoglycan
which is extremely sensitive to lysozyme.
 Spore wall: innermost layer surrounding the inner spore membrane that
contains normal peptidoglycan and becomes the cell wall of the
germinating vegetative cell.
 Spore coat: enclosing the cortex and is relatively thick
 Exosporium: consists of glycoprotein.
24. Briefly describe endospore formation and germination. What is the importance
of the endospore?
1. Axial filament Formation
The adjustment of the genetic material of the bacterial cell in the accurate
center plane
2. Septa Formation
A septum known as forespore septum is formed as a result of the infolding of the
plasma membrane into the cell lumen. Its creation causes a small segment of
DNA to detach from the remaining genetic material.
3. Forespore Engulfment
The newly formed immature spore is entirely engulfed by the membrane of the
mother cell as it proceeds to grow. The engulfment causes the forespore to be
enveloped by an intermembrane space and two plasma membrane.
4. Cortex Formation
The inner membrane space between the two membranes gives rise to the
formation of the cortex. At this stage, the accumulation of dipicolinic acid and
calcium in larger quantities is observed.
5. Protein coat Formation
The cortex of the hence formed spore is covered with a protein coating
6. Spore Maturation
At this stage, the cell turns inactive metabolically and the core turns increasingly
dehydrated.

7. Enzymatic destruction
Endospores are released as a result of the destruction of the enzymes of the
spore mother cell.

25. What features of the endospore contribute to its resistance to harsh conditions
 They have a high concentration of calcium and dipiclonic acid for
stabilization of DNA
 Low water content
 Their cortex and cell wall are impermeable
 They have low metabolic and enzymatic activity
  They are surrounded by an outer covering which is high in protein that
provides enzymatic and chemical resistance
 They contain SASPs (Small, Acid Soluble Proteins) which are responsible
for binding and condensing DNA. It is also responsible for the UV-light
resistance property of endospores and protects them from DNA-
damaging chemicals.

Assignment Two: Microscopy and staining

1. Define and compare the terms magnification and resolution.

-Magnification is the ability of a microscope to produce an image of an object at a scale
larger than its actual size.
-Resolution is the ability to reveal closely adjacent structural details as separate and
distinct.
2. With the 100X objective, an optical-grade oil is placed between the specimen and
the
objective. Lenses on which oil is used are called oil-immersion lenses. What is the
purpose of this immersion oil?
-It enhances resolution by preventing light rays from dispersing and changing
wavelength after passing through the specimen.

3. A veterinary doctor investigating the massive death of buffaloes at Queen Elizabeth

National Park presents with an ulcer on his left arm, and the arm is markedly swollen.
The ulcer is healing to produce a black scab. A Gram stain on the exudates from the
lesion shows large Gram-positive rods.

a. How was the Gram stain procedure carried out?

1. Make a smear.
2. Allow it to dry.
3. Fix it with gentle heat by passing it through a flame 3 times.
4. Add a triphenyl methane dye, for example crystal violet for 30-60 seconds and
then wash the slide with water.
5. Add Lugol’s iodine for 30-60 seconds and then wash the slide.
6. Decolourize using acetone alcohol for not more than 5 seconds and wash the
slide.
7. Add a red dye e.g. Safranine or dilute carbol fuschin for 60 seconds and wash the
slide.
8. Allow the slide to dry.
9. Add a drop of oil then observe under the oil immersion objective (x100).

b. How did the microbiologist come to the conclusion that the organisms seen on the
slide were Gram-positive rods (and not Gram-negative rods)?
Gram-positive rods don’t decolourize and thus appear purple when observed under a
microscope whereas Gram-negative rods do decolourize and take up the colour of the
dye, thus appear pink or red when observed under a microscope.

c. One clue that would help to make a diagnosis is to determine if the organism seen
on the slide produces spores. What microscopy staining technique could the
microbiologist use?
The microbiologist could use the Schaeffer Fulton method.

4. A patient presenting with a prolonged history of cough and fever is suspected to be

having tuberculosis. How can this be proved or disproved in the lab?
Tuberculosis is caused by Mycobacterium tuberculosis bacteria. It is detected using the
Ziehl Neelsen acid fast stain which is carried out following the procedure below.
1. Make a smear.
2. Allow it to dry.
3. Fix it with gentle heat by passing it through a flame 3 times.
4. Apply carbol fuschin and heat until steam rises.
5. Wait for 5 minutes and heat again until steam rises; this procedure is repeated 2
more times.
6. Wash the slide with water.
7. Decolourize with 3% acid alcohol or 20% H2SO4 and then wash the slide with
water.
8. Counter-stain with methylene blue or malachite green and then wash the slide.
9. Allow the slide to dry and examine using oil immersion objective.

When observed under a microscope, acid fast bacteria such as Mycobacterium

tuberculosis appear red, while non-acid fast bacteria appear blue because they are
decolourized by the acid alcohol and take up the colour of the dye.
Assignment Three: Pathogenesis
1. What does it mean when it says, “Shigella are Gram-negative bacteria”?
This means that they appear reddish-pink under a microscope after Gram staining. This
is because their cell wall is very thin (with only one to two sheet of peptidoglycan) and is
unable to retain the crystal violet stain so are colored only by the safranin counterstain.

2. What does facultative anaerobic mean?

This is a microorganism that can grow in both oxygen-deficient and oxygen-rich
environments.

Using the figure below, explain the pathogenesis of Shigellae

Reservoir: Human Colony
Route of transmission: fecal-oral route or person-to-person
Virulence: ingestion of only about 1 to 10 organisms is required to start infection
because they are extremely resistant to acid, enabling them to travel through the
gastro-intestinal tract to the colon and rectum without being destroyed. Within the
Peyer’s patches, the bacteria invade the M cells by membrane ruffling and endocytosis.
Once within the phagosome in the M cells, it produces proteins that prevent
phagolysosome formation and so the bacteria a released into the cytoplasm where they
replicate and increase in number. The then move from cell to cell by rapid
depolarization of the actin filaments from the M cell cytoskeleton forming actin
comet/jet cells. These allow form bacteria-containing protrusions at the cell plasma
membrane that invade adjacent cells laterally without going out into the extracellular
milieu. This will cause the colon epithelium to become ulcerated resulting in fluid loss,
release of cytokines and white blood cells traveling to the injured site. This is what
causes inflammatory diarrhea/dysentery which is a combination of fecal blood, food
waste and leukocytes (pus). There are four species of Shigella capable of causing this
dysentery: S. dysenteriae, S. flexneri, S. boydii, and S. sonnei which is most common in
the United States.
Shigella dysenteriae is unique because it causes bacillary dysentery and it produces
Shiga Toxin (an exotoxin). The Shiga Toxin binds to glycosphingolipids on the endothelial
cells of the glomerulus to enter the cell where it binds to the 60S ribosome and prevents
translation thus damaging the cells. Damage to endothelial cells will activate platelets
causing them to aggregate and to reduce in circulation (thrombocytopenia). The platelet
aggregates will then lyse red blood cells as they try to pass through the blood vessel
forming schistocytes and thus causing Hemolytic Uremic Syndrome (HUS) which could
progress and cause acute renal failure, especially in children.
Assignment Four: Bacterial Growth
Influences of environmental factors on growth
1. Define pH, acidophile, neutrophile, and alkaliphile. Give examples of bacteria that
fall under each category.
-pH is a quantitative measure of the acidity or basicity of aqueous or other liquid
solutions.
-Acidophiles are organisms which grow at a pH lower than 5; such as Acidithiobacillus
ferroxidans and Acidihalobacter prosperus.
-Neutrophiles are organisms which grow at neutral pH; from about 6.0-8.0 e.g.
Escherichia coli and Staphylococcus species.
-Alkaliphiles are organisms which grow at pH greater than 9; examples include
Natronomonas pharaonis and Vibrio cholerea.

b. Explain how extreme pH values might harm microbes.

At extreme pH levels, misfolded proteins are formed because of change in ionization of
the amino acid functional groups and disruption of hydrogen bonding which, in turn,
promotes changes in the folding of the molecule.
At very high pH, DNA undergoes alkaline denaturation because of abundance of
hydroxide ions which combine with hydrogen ions from the base pairs of DNA thus
breaking hydrogen bonds and causing denaturation of DNA strands.
Normally, H+ ions are responsible for maintaining the proton gradient across the
membrane for ATP production. At high pH, they are neutralized by the hydroxide ions,
leading to collapse of the concentration gradient and impairment of ATP production.
2. Define psychrophile, pyschrotroph, mesophile, thermophile, and hyperthermophile.
Give examples of bacteria that fall under each category.
Psychrophiles are organisms which grow best at low temperatures (less than 15°C), for
example Flavobacterium spp. and Listeria monocytogenes.
Psychrotrophs are organisms which grow at temperatures between 20 and 30°C e.g.
Staphylococcus aureus and Pseudomonas fluorescens.
Mesophiles are organisms which grow within a temperature range of 30-37°C e.g.
Escherichia coli and Streptococcus pyogenes.
Thermophiles are organisms which grow within a temperature range of 50-80°C, for
example Bacillus stearothermophilus and Alicyclobacillus tolerans.
Hyperthermophiles are organisms which grow at temperatures above that of boiling
water, such as Aquifex aeolicus.
b. What metabolic and structural adaptations for extreme temperatures do
psychrophiles and thermophiles have? How might these protect the organisms from
their environment?
Psychrophiles have antifreeze proteins and solutes that decrease the freezing
temperature of the cytoplasm.
They also have unsaturated lipids in their plasma membranes which increase the
membrane fluidity.
The enzymes in psychrophiles have more accessible active sites to accommodate for the
decreased rate of diffusion at lower temperatures.
In thermophiles, the DNA sequences have a higher proportion of guanine–cytosine
nitrogenous bases, which are held together by three hydrogen bonds in contrast to
adenine and thymine, which are connected in the double helix by two hydrogen bonds
and these require higher temperatures to be broken.
They have a higher proportion of saturated lipids in the plasma membrane which
decrease fluidity of the membrane.

3. Describe the five types of oxygen relationships seen in microoganisms.

Obligate aerobes: These microorganisms strictly require oxygen as a hydrogen acceptor
for them to survive for example Pseudomonas aeruginosa, Mycobacterium tuberculosis.
Obligates anaerobes: These microorganisms cannot tolerate oxygen and require another
substance rather than oxygen as a hydrogen acceptor for them to survive for example
Clostridium tetani, Bacteroids fragilis
Facultative anaerobes: These microorganisms can grow in both oxygen-deficient and
oxygen-rich environments for example Staphylococcus aureus.
Microaerophilic organisms: These microorganisms require small amounts of oxygen (2 –
10%) for example Helicobacter pylori, Campylobacter.
Aero tolerant anaerobes: These microorganisms are indifferent to oxygen that is they
can grow in the presence of oxygen but do not require it for example Streptococcus
pneumonia , Clostridium perfrigens.
b. What are the toxic effects of oxygen? How do aerobes and other oxygen-tolerant
microbes protect themselves from these effects?

During aerobic metabolism, which requires oxygen, reactive compounds such as

hydrogen peroxide and superoxide are produced as by-products. In the presence of
iron , hydrogen peroxide and superoxide generate a hydroxyl radical which can damage
any biologic macromolecule.

Aerobes and other oxygen-tolerant species have superoxide dismutase, an enzyme that
catalyzes a reaction that protects them from these reactive compounds.

Aerobes and other oxygen – tolerant species also have catalase, an enzyme that
catalyzes a reaction that protects them from these reactive compounds.

4. Below is a figure showing the relationships between oxygen and bacterial growth.
Each dot represents an individual bacterial colony within the agar or on its surface.
The surface which is directly exposed to atmospheric oxygen is oxic. The oxygen
content of the medium decreases with depth until the medium becomes anoxic
toward the bottom of the tube. What are the different types of oxygen relationships
for each of the tubes? What is the enzyme content in each of the tubes? What
enzymes am I thinking about? What is the purpose of the enzymes?

Oxygen relationship Enzyme content

-Catalase
1 Obligate aerobes
-Superoxide dismutase

2 Obligate anaerobes None

-Catalase
3 Facultative anaerobes
-Superoxide dismutase
Small amounts of;
4 Microaerophiles -Catalase
-Superoxide dismutase

5 Aerotolerant -Superoxide dismutase

Catalase: Catalyzes conversion of hydrogen peroxide to oxygen and water.

Superoxide dismutase: Catalyzes conversion of superoxide ion to oxygen and hydrogen
peroxide, in presence of hydrogen ions.

Microbial growth in natural environments.

5. What is a biofilm? List two ways life in a biofilm is advantageous for microbes.
A biofilm is an assemblage of surface-associated microbial cells enclosed in an
extracellular polymeric substance matrix.
A biofilm is advantageous for microbes in that;
-It makes the initial bacteria less susceptible to immune clearance since it forms a layer
upon layer of bacterial growth.
-The external growth shields penetration by antibiotics.

b. What medical challenges do biofilms present?

Biofilms are significant in human infections which are persistent and difficult to treat
such as Staphylococcus epidermidis and S. aureus infections of central venous catheters,
eye infections in people with contact lenses and intraocular lenses, in dental plaque, and
in prosthetic joint infections. Also, cystic fibrosis patients are prone to P. aeruginosa
airway infections.

Laboratory culture of cellular microbes

6. What is a culture medium?
A culture medium is a solid, liquid, or semi-solid designed to support the growth of a
population of microorganisms by attempting to replicate the conditions of their natural
environment.
b. Culture media may be liquid, semisolid or solid. Give an example of each.
Liquid - Nutrient broth
Semisolid - Amies media
Solid - Blood agar
c. What is the difference between selective media and differential media?
Selective media inhibit growth of other bacteria while having little/no effect on the
organisms they are isolating while differential media differentiates bacteria based on
their colonial appearance, allowing closely related organisms to be distinguished.
d. How is blood agar an enriched and differential medium?
Blood agar is an enriched medium because extra growth nutrients (5% sheep blood) are
added. It is also a differential medium because the 5% sheep blood acts an indicator for
hemolysis since the red blood cells are highly sensitive to hemolytic toxins released by
bacterial cells.
e. How is MacConkey agar a selective and differential medium?
MacConkey agar is selective because it has bile salts and crystal violet which favour
growth of Gram negative enteric bacteria and inhibit growth of Gram positive and
Gram-negative mucosal cocci. It is a differential medium that differentiates lactose
fermenters from non-lactose fermenters because it contains lactose monohydrate, a
fermentable source of carbohydrate, which is only broken down by lactose fermenters.

f. What are some of the characteristics of agar that make it a particularly useful
solidifying agent?
-It is resistant to microbial action.
-It is a solid at 37°C.
-It has no nutritive value for most bacteria.
g. Why are peptones, yeast extract and beef extract used in culture media?
They provide essential nutrients, vitamins and nitrogenous factors required for growth
of microorganisms.
7. What are some of the terms used by microbiologists to describe colony
morphology?
The shape of colony can be circular, irregular, filamentous or rhizoid.
Elevation of the colony describes how much the colony rises above the agar e.g. raised,
convex, flat, crateriform.
Surface of the colony, can be smooth, glistening, rough, rugose or dull.

The Growth curve

8. Describe the four phases of the growth curve.
Lag phase: Cells are adapting to the new environment they have been introduced to, so
number of cells is initially constant. Cells increase in size because they are preparing for
growth. There is formation and accumulation of enzymes and metabolic intermediates.
Exponential phase: Cells start dividing by binary fission. There is exponential increase in
the number of cells. The cells have a constant growth rate and uniform metabolic
activity. This is the stage in which bacteria are most susceptible to disinfectants and
antibiotics.
Stationary phase: Bacterial growth slows down because of exhaustion of nutrients,
oxygen depletion, etc. At this stage, number of new cells being formed is equal to the
number of cells dying. The total number of cells reaches a maximum and stabilizes.
Decline phase: The number of dying cells exceeds the number of cells being formed due
to nutrient exhaustion, accumulation of toxic metabolites. Many cells lyse and release
their nutrients into the medium, allowing survival cells to maintain viability and form
endospores. Persister cells, which are characterized by a slow metabolic rate remain and
these are important clinically in chronic infections e.g. TB.
Bacterial pathogenesis
1. What are the steps involved in pathogenesis of bacteria?
Exposure: For an organism to be able to cause infection, it has to be able to gain access
to the host tissue. Most pathogens gain exposure via portals of entry which are in direct
contact with the external environment, for example skin, mucous membranes,
parenteral routes, breaches in the skin. In addition, the respiratory and GIT mucosal
surfaces are very susceptible points of entry because many particles and inhaled and
ingested.
Adhesion: This refers to the capacity of pathogens to attach to cells using adhesion
factors after being exposed to the host tissue. Some bacteria use adhesins that bind to
specific receptors. Others produce slime layers and capsules allowing certain bacteria to
attach to cells. Some bacterial cells form biofilms that attach on the surface of host
tissues such as in Staphylococcus epidermis.
Colonization: Is the establishment of a stable population of bacteria in the host. It
requires adhesion to the mucosal surface. Successful colonization requires bacteria to
acquire essential nutrients such as iron for growth.
Invasion: The pathogen then disseminates throughout the body. This may occur by
production of toxins serving as virulence factors that allow them to colonize and
damage host tissues.

2. What are the characteristics of bacteria that are pathogens?

 Transmissibility
 Adherence to host cells
 Persistence
 Invasion of host tissues
 Toxigenicity
 Ability to evade host’s immune system

3. What are the roles of mobile genetic elements and pathogenicity islands in bacterial
pathogenesis?
Mobile genetic elements include transposons, phages and plasmids. Transfer of these
between members of one species can result in transfer of virulence factors including
antimicrobial resistance genes. Pathogenicity Islands (PAIs) are large organized groups
of genes that increase the pathogenicity of an organism when expressed. They have
more than one virulence gene and are only present in the pathogenic members of large
species. An example of this is SPI-1 in Salmonella serotype Tyhimurium which increases
its invasion and damage of host cells causing diarrhea.
4. What does the term virulence mean?
This is the ability of an cause infection and spread disease.
b. Give examples of virulent factors that different bacteria have
 Staphylococcus aureus: protein A binds to the Fc portion of antibodies that
prevent complement from binding
 Adherence Factors: many pathogenic bacteria colonize mucosal sites by using pili
(fimbriae) to adhere to cells.
 Invasion Factors: surface components that allow the bacterium to invade host
cells can be encoded on plasmids, but more often are on the chromosome.
 Capsules: many bacteria are surrounded by capsules that protect them from
opsonization and phagocytosis
 Endotoxins: the lipopolysaccharide endotoxins on Gram-negative bacteria cause
fever, changes in blood pressure, inflammation, lethal shock and many more
toxic events
 Exotoxins: these include several types of protein toxins and enzymes produced
and/or secreted from pathogenic bacteria. Major categories include cytotoxins,
neurotoxins and enterotoxins
 Siderophores: these are iron-binding factors that allow some bacteria to compete
with the host for iron, which is bound to hemoglobin, transferrin and lactoferrin.

5. Toxins produced by bacteria are generally classified into two groups: exotoxins and
endotoxins. What are the differences between exotoxins and endotoxins?
Exotoxins Endotoxins
Present in both Gram positive and Gram Present only in Gram negative
positive
Polypeptide Lipopolysaccharide
Highly antigenic and toxic Weakly antigenic and toxic
Released by living bacteria Released upon bacterial cell death
Action is specific Action is general
Highly immunogenic Pyrogenic
6. Some pathogenic bacteria are able to evade or survive the host’s immune system.
Using examples, what are some of the mechanisms bacteria use to evade the host’s
immune system?
 Presence of a capsule: Streptococcus pyogenes, Streptococcus agalactiae
 Antigenic variation: Neisseria gonorrhoeae
 Proteases: (IgA1) Neisseria gonorrhoeae, Neisseria meningitidis, Hemophilus
influenzae
 Serum resistance: Escherichia coli
 Iron Acquisition: Neisseria meningitidis, Corynebacterium diphtheriae
 Resistance to killing by phagocytic cells: Shigella sonnei and Listeria
monocytogenes secrete proteins that destroy the phagosome before it fuses with
the lysosome

7. Bacterial secretion systems are important in the pathogenesis of infection and are
essential for the interaction of bacteria with the eukaryotic cells of the host. Using
examples, describe the different bacterial secretion systems and their functions.
The major mechanism of protein secretion in Gram negative and positive bacteria is the
general secretion pathway (Sec): it is involved in insertion of bacterial membrane
proteins and is the main pathway by which proteins cross the cytoplasmic membrane
Gram negative bacteria have 6 types of secretion systems (1 to 6) while Gram positive
bacteria only have 2 types of secretion systems (type 1 and 4).
The Sec dependent systems are types 2 and 5.
Sec independent systems ae types 1, 3, 4 and 6.
 Type 1: protein secretion is a continuous process where the protein secreted
traverses the inner and outer membrane in one step. It requires 3 secretory
proteins: an inner membrane ABC transporter; an outer membrane protein; and
a membrane fusion protein. Examples include alpha hemolysis in E. coli.
 Type 2 and 5: these require the Sec pathway. Proteins produced from the
ribosomes are pre-proteins with an extra leader sequence of 15 to 40 amino
acids. The Sec pathway comprises of inner membrane proteins, a cell membrane
associated ATPase that provides energy for protein export, a chaperone that
binds to the pre-protein and a periplasmic signal peptidase that cleaves the
leader sequence. Examples of these are the Cholera toxin in V. cholera and IgA1
protease and adhesins in H. influenzae.
  Type 3: this is activated by contact with a host cell for example when a toxin
protein is injected into a host cell. The proteins are approximately 20 and they
are mostly in the inner membrane. An example of this is the cytotoxin in P.
aeruginosa.
 Type 4: these secrete either polypeptide toxins or protein-DNA complexes
between two bacterial cells or between a bacterial cell and a eukaryotic cell.
Examples of this are the cytotoxin and DNA uptake and release systems in H.
pylori and the DNA export system in N. gonorrhoeae.
 Type 6: this is a sec independent pathway which consists of 15 to 20 proteins.
These include the pore-forming toxin Hcp 1 in P. aeruginosa and virulence
proteins in V. cholerae.

8. What is the role of bacterial biofilms in pathogenesis of bacteria?

A biofilm is able to create a barrier between the pathogenic bacteria and the immune
cells of the host, thus preventing the destruction of the bacteria. The biofilm can also
establish an environment where the waste product of one organism becomes the
nutrient for another. For example, facultative bacteria consume oxygen, creating
anaerobic regions that promote the growth of anaerobes. This occurs in many
polymicrobial infections that involve both aerobic and anaerobic pathogens.