BIO602 Applied Microbiology Semester II, 2020 Tutorial 3

1.) List the various factors that affect the antimicrobial activity and describe the
different physical methods to control the microbes.
- The concentration and the kind of chemical used: is often thought that the
more conc the chemical reagent, the more the reaction to destroy the micro
bacterial pathogen.
Example: Ethanol: 70% to sterilize tables for work.95%: used to sterilize the
scalpel, and other lab equipment's.Therefore, 70% is more effective because it is
enhanced with water.
-The Population also matters.
Effectiveness is very much dependent on their Nature.

-Length of Exposure: The longer the exposure , the more organism are removed.

-Temperature: lower conc of disinfectant should be used in a higher temperature.

Heat kills more readily at a certain PH.

Physical Control Method:

They are 2 ways to physically control microbes:
1. Heat
2. Radiation.
Heat:
We have moist heat ,Dry heat Sterilisation, Steam sterilisation and Pasteurisation
-Moist Heat:
Moist heat destroys microorganisms by the irreversible denaturation of enzymes and
structural proteins. The temperature at which denaturation occurs varies inversely with
the amount of water present. Sterilization in saturated steam thus requires precise control
of time, temperature, and pressure
-Steam destroys all vegetative spores.
-Destroys viruses, fungi, and bacteria.
-Pressure serves as a means to obtain the high temperatures necessary to quickly
kill microorganisms. Specific temperatures must be obtained to ensure the microbicidal
activity. Minimum sterilization time should be measured from the moment when all the
materials to be sterilized have reached the required temperature throughout.
-Destroys viruses, fungi, and bacteria.

Pasteurisation:
Controlled heating at temperatures well below boiling
Used for milk, beer, and other beverages
• Process does not sterilize but does kill pathogens present and slow spoilage by
reducing the total load of organisms present

Steam Sterilisation:
– They are often carried out above 100oC which requires saturated steam under
pressure.
– -Uses an Autoclave
Autoclave: is a heated chamber used to sterilize various types of media, by
means of dry saturated steam under pressure. In order to achieve dry saturated
steam, air has to be removed from both the material inside the autoclave, and the
autoclave chamber itself

Dry Heat Sterilisation:

They are less effective than moist heat sterilization, requiring higher temperatures
and longer exposure times.
Several Processes includes:
-Direct Flaming
-Incineration
-Hot Air Sterilization

2.) Radiation:
The 2 types of radiations include:
-Ionising Radiation
-Non-Ionising Radiation

2.) Differentiate between thermal death point and thermal death rate?

Thermal Death Point (TDP): Lowest temperature at which all of the microbes in a
liquid suspension will be killed in ten minutes.Thermal Death Time (TDT): Minimal
length of time in which all bacteria will be killed at a given temperature.

3.) Discuss the mechanism of thymine – thymine dimer formation with the
effect of ultraviolet radiation and discuss photo reactivation to make normal
DNA.
When cells are exposed to sunlight, radiant energy can damage the DNA . For e.g
Ultra violet Radiation causes covalent bond formation between adjacent thymines
on the same strand of DNA. When DNA is damaged in this way, it cannot be
replicated or tanscribed. Most cells are able to repair damage to DNA for example,
an enzyme called photolyase can break the covalent between the thymine dimer.
This enzyme requires visisble light which is used for energy source for bond
cleavage. Therefore, if this happen to the DNA of Bacteria, it will be hard for them
to recover which will cause death to this bacterias.

4.) What are the different chemical methods to control the growth of
microbes?
There are several chemical methods to control the growth of microbes but 3 of the
main methods includes:
STERILISATION: destruction or removal of all viable organisms.
DISINFECTION: Killing, inhibition, or removal of disease causing
(pathogenic)organism
-disinfectants: agents, usually chemical, used for disinfectants.
-usually used on inanimate.
SANITIZATION:reduction of microbial population to levels deemed safe (based on
public health standards)
ANTISEPSIS: prevention of infection of living tissue by microorganisms.
-Antiseptics: chemical agents that kill or inhibit the growth of microorganisms when
applied to tissue.
Soaps and Detergents contains alcohol and the following chemicals that helps In
preventing the growth of Microbes
1.) Alcohol:
Among the most widely used disinfectants and antiseptics
• Two most common are ethanol and isopropanol
• Bactericidal, fungicidal, but not sporicidal
• Inactivate some viruses
• Denature proteins and possibly dissolve membrane lipids
2.) Halogens
• Any of five elements: fluorine, chlorine, bromine, iodine, and astatine
• Important antimicrobial agents.
-Halogens - Iodine
• Skin antiseptic
• Oxidizes cell constituents and iodinates proteins
• At high concentrations may kill spores
• Skin damage, staining, and allergies can be a problem
• Iodophore – iodine complexed with organic carrier – released slowly to
minimize skin burns

3.) Quaternary Ammonium Compounds

-detergents that have antimicrobial activity and are effective disinfectants –
amphipathic organic cleansing agents
-cationic detergents are effective disinfectants – kill most bacteria, but not M.
tuberculosis or endospores – safe and easy to use, inactivated by hard water and
soap

4.) Aldehydes
-Commonly used agents are formaldehyde and glutaraldehyde
• Highly reactive molecules
• Sporicidal and can be used as chemical sterilants
• Combine with and inactivate nucleic acids and proteins

5.) Halogens – Chlorine

Important in disinfection of water supplies and swimming pools, used in dairy and
food industries, effective household disinfectant
• Destroys vegetative bacteria and fungi
• Chlorine gas is sporicidal
• Can react with organic matter to form carcinogenic compounds

5.) Discuss the properties and different modes of action of antimicrobial agent
The properties of Antimicrobial agents are as follows:
1.) Solubility in Body fluids.
2.) Selective toxicity.
3.) Spectrum of Activity.
-Broad-spectrum Activity- should be highly effective even in low concentrations is
applied.
-Narrow-Spectrum: Should not be necessarily the temperature.
4.) Non-Allergic.
5.) Stability.
How does this antimicrobial agents work????
-Inhibition of Cell wall synthesis: This antimicrobial agents prevents the peptidoglycan
from undergoing synthesis in the cell wall of bacteria.
Human cells are not affected. (because we do not have cell wall or peptidoglycan in their
cell membrane.)
Examples of Antimicrobial agents: Cephalosporin, Vancomycin and penicillin.

-Destruction of Plasma Membrane: Important substances used in metabolic

activities leaves the bacterial cell.
-Lysis of cell is going to occur.(cell tends to burst)
Examples include: Polymyxin B, Nystatin, Amphotericin B and Miconazole
-Inhibition of Protein synthesis: They affect the ribosome structure (Ribosomal
large and small Sub unit is affected) therefore they will be no protein synthesis
because the ribosomes are being affected which affects the growth of micro
bacteria.
Examples include: Chloramphenicol, erythromycin, Streptomycin, Neomycin,
Kanamycin and Gentamicin

Inhibition of Nucleic acid: Transcription and translation process is stopped.

-Inhibition of Synthesis of Essential metabolites: The synthesis of folic acid (B

Vitamin) which is needed to make nitrogenous bases of DNA is Inhibited

- Inhibition of nucleic acid synthesis: DNA replication and the copying of the
information from DNA by mRNA is affected (transcription)

6.)What is metabolism? Differentiate between anabolism and catabolism.?

Metabolism: the sum of the chemical reactions that take place within each cell of a
living organism and that provide energy for vital processes and for synthesizing new
organic material.

Anabolism: is the set of metabolic pathways that construct molecules from smaller
units. These reactions require energy, known also as an endergonic process. Anabolism
is the building-up aspect of metabolism.

Catabolism: also known as destructive metabolism; the breaking down in living

organisms of more complex substances into simpler ones, with the release of energy
(opposed to anabolism).

7.) What are enzymes? How do they affect the reactants and which factors
influence the enzyme activity?
Enzyme: a catalyst that regulates the rate at which chemical reactions proceed in living
organisms without itself being altered in the process
How do they affect the reactants and which factors influence the enzyme activity?
They increase the rate of reaction by decreasing the activation energy, but they are not
going to be used up.
Factors that influence the enzyme activtiy?
Enzyme activity can be affected by a variety of factors, such as temperature, pH, and
concentration
Temperature: Higher temperatures tend to speed up the effect of enzyme activity,
while lower temperatures decrease the rate of an enzyme reaction. However, if the
temperature is too high, an enzyme will denature, which causes the shape of the
enzyme to change. If the enzyme's shape changes, it cannot bind to the substrate.

Concentration: Increasing enzyme concentration will speed up the reaction, as long as

there is substrate available to bind to. Once all of the substrate is bound, the reaction will
no longer speed up, since there will be nothing for additional enzymes to bind to.

PH: Each enzyme has an optimum pH range. Changing the pH outside of this range
will slow enzyme activity. Extreme pH values can cause enzymes to denature.

8.) Define respiration and discuss the different types of respiration. Which type
yields more ATP (energy)?
Respiration is the biochemical process in which the cells of an organism obtain energy
by combining oxygen and glucose, resulting in the release of carbon dioxide, water, and
ATP (the currency of energy in cells).

-External respiration is the breathing process. It involves inhalation and exhalation of

gases.

-Internal respiration involves gas exchange between the blood and body cells.

-Cellular respiration involves the conversion of food to energy. Aerobic respiration is

a cellular respiration that requires oxygen while anaerobic respiration does not.

Aaerobic Respiration: consists of three stages: glycolysis, citric acid cycle (Krebs Cycle),
and electron transport with oxidative phosphorylation.

Anaerobic Respiration: This type of respiration occurs without oxygen and involves
the consumption of another molecule (nitrate, sulfur, iron, carbon dioxide, etc.) instead
of oxygen.
Which types yields more ATP?

Aerobic respiration produces much more ATP than anaerobic respiration. Anaerobic
respiration occurs more quickly than aerobic respiration.

9.) Explain the oxidation-reduction reaction during aerobic respiration. Give the
detail of total ATP yield per glucose molecule in aerobic cellular respiration

During aerobic respiration, oxygen is reduced, donating an electron to hydrogen to

form water. The entire process of cellular respiration oxidizes glucose. This produces
the majority of the energy released in cellular respiration
According to some of newer sources the ATP yield during aerobic respiration is not 36–
38, but only about 30–32 ATP molecules / 1 molecule of glucose , because:
ATP : NADH+H+ and ATP : FADH2 ratios during the oxidative phosphorylation appear
to be not 3 and 2, but 2.5 and 1.5 respectively. Unlike in the substrate-level
phosphorylation, the stoichiometry here is difficult to establish.

ATP synthase produces 1 ATP / 3 H+. However the exchange of matrix ATP for
cytosolic ADP and Pi (antiport with OH− or symport with H+) mediated by ATP–ADP
translocase and phosphate carrier consumes 1 H+ / 1 ATP as a result of regeneration of
the transmembrane potential changed during this transfer, so the net ratio is 1 ATP : 4
H+.

The mitochondrial electron transport chain proton pump transfers

acrosstheinnermembrane10H+ /1NADH+H+ (4+2+4)or6H+ /1FADH2 (2+4).

So the final stoichiometry is 1 NADH+H+ : 10 H+ : 10/4 ATP = 1 NADH+H+ : 2.5 ATP

1 FADH2 : 6 H+ : 6/4 ATP = 1 FADH2 : 1.5 ATP

ATP : NADH+H+ coming from glycolysis ratio during the oxidative phosphorylation is

1.5, as for FADH2, if hydrogen atoms (2H++2e−) are transferred from cytosolic
NADH+H+ to mitochondrial FAD by the glycerol phosphate shuttle located in the inner
mitochondrial membrane.

2.5 in case of malate-aspartate shuttle transferring hydrogen atoms from cytosolic

NADH+H+ to mitochondrial NAD+

So finally we have, per molecule of glucose

Substrate-level phosphorylation: 2 ATP from glycolysis + 2 ATP (directly GTP) from

Krebs cycle Oxidative phosphorylation

2 NADH+H+ from glycolysis: 2 × 1.5 ATP (if glycerol phosphate shuttle transfers
hydrogen atoms) or 2 × 2.5 ATP (malate-aspartate shuttle)
2 NADH+H+ from the oxidative decarboxylation of pyruvate and 6 from Krebs cycle: 8
× 2.5 ATP 2 FADH2 from the Krebs cycle: 2 × 1.5 ATP

Altogether this gives 4 + 3 (or 5) + 20 + 3 = 30 (or 32) ATP per molecule of glucose

The total ATP yield in ethanol or lactic acid fermentation is only 2 molecules coming
from glycolysis, because pyruvate is not transferred to the mitochondrion and finally
oxidized to the carbon dioxide (CO2), but reduced to ethanol or lactic acid in the
cytoplasm.

10.) Discuss the alcoholic and lactic acid fermentation in detail.

Alcohol fermentation is a chemical reaction that uses yeast and sugar to produce
energy, which you can see as the solution bubbles; it can be aerobic or anaerobic (work
in the presence or absence of oxygen). After the carbon dioxide is removed, the resultant
acetaldehyde is then reduced to form ethanol. Yeast cannot metabolize ethanol; as far as
the parent cells are concerned, it is a waste product.

lactic acid fermentation is the process that occurs after glycolysis in anaerobic
respiration. An enzyme called lactate dehydrogenase prompts a reaction to start
glycolysis, forming lactate in the process.

This lactate protonates into lactic acid and continues accumulating in muscle cells until

oxygen is reintroduced and aerobic respiration returns. ("Protonate" means to add a

proton to another atom or molecule, which creates a bond and transforms the lactate into

an acid.) Lactic fermentation occurs through anaerobic respiration, which occurs when

there is a lack of oxygen in an organism. This prevents muscles from getting energy

from cellular respiration.

Primarily, lactic acid fermentation differs from ethyl alcohol fermentation in that lactic

acid, rather than ethanol, is the resulting by-product. When exposed to oxygen, lactic

acid molecules break down into carbon dioxide and water. When used in food

production, this lactic acid breaks down sugars, preventing food from spoiling.
Alcohol fermentation can take place in environments both with and without oxygen,

with differing results.

11.) Describe the fat and protein respiration. Also discuss the inter – conversion of
fat, carbohydrates and proteins.

Fat Respiration:

Fats are stored as triglycerides in cells, primarily of adipose tissue. They have a high
energy content, and form a better fuel than the carbohydrates. They break up into fatty
acids and glycerol in the cytoplasm before use in respiration.

Under Fat respiration we have:

Fatty Acids: Fatty acids are broken by a series of reactions into 2-carbon acetyl
coenzyme A. The latter enters the Krebs cycle.

Glycerol: Glycerol combines with a phosphate group, forming phosphoglyceraldehyde.

The latter enters glycolysis.

Energy Output: A molecule of 18-carbon stearic acid on complete oxidation produces

147 high-energy phosphates. A 6-carbon glucose molecule yields 36 or 38 ATP. With
this rate, an 18-carbon molecule is expected to give 3 times more energy (36 or 38 x 3 =
108 or 114 ATP) but it provides about 4 times more energy (36 or 38 x 4 = 144 or 152
ATP) than 6-carbon glucose produces.

(ii) Protein Respiration:

The proteins split into amino acids in the cytoplasm for use in respiration. The amino
acids enter respiratory routes in two ways: deamination and transamination.

Deamination:

In deamination, an amino acid loses its amino group (- NH2) and changes into a keto
acid. The latter may further change into pyruvic acid or acetyl coenzyme A. Pyruvic acid
is oxidized to acetyl coezyme A. The latter enters the Krebs cycle.

Transamination:
In transamination, an amino group of an amino acid is transferred to an appropriate keto
acid, forming a new amino acid and a new keto acid. The keto acids so formed are
normal participants of glycolysis or Krebs cycle.

-inter – conversion of fat, carbohydrates and proteins.

Metabolism in the body converts carbohydrates, protein and fat into energy. Any
excess of these nutrients will be stored as fat in the body. In other words, regardless of
whether it is carbohydrates, protein or fat, it will be converted into fat storage in the
body if it is not used.