Download BSc in Applied Biotechnology 3 BO0045 ‑ MICROBIOLOGY

PROGRAM
BSc in Applied Biotechnology
SEMESTER
3
SUBJECT
BO0045 - MICROBIOLOGY
BOOK ID
B0762
SESSION
Winter 2015
No
Q 1
Question/Answer key
Marks Total Marks
10
Describe the procedure of gram staining and endospore staining.
( Unit 2 ; Section 2.5 )
A 1
Gram staining procedure:
1. Cover the slide with crystal violet stain and wait one minute.
• 2. After one minute wash the stain off gently with a minimum amount of tap
water. Drain off most of the water and proceed to the next step. It may help to
hold the slide vertically and touch a bottom corner to a blotting paper.
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• 3. Cover the slide with iodine solution for one minute. The iodine acts as a
mordant (fixer) and will form a complex with the crystal violet, fixing it into the cell.
• 4.
Rinse briefly with tap water.
• 5. Tilt the slide lengthwise over the sink and apply the alcohol-acetone
decolorizing solution (drop wise) such that the solution washes over the entire
slide from one end to the other.
• All smears on the slide are to be treated thoroughly and equally in this
procedure.
• Process the sample in this manner for about 2-5 seconds and immediately
rinse with tap water.
• This procedure will decolorize cells with a Gram negative type of cell wall but
not those with a Gram-positive type of cell wall, as a general rule.
• Drain off most of the water and proceed.
• 6. As the decolorized Gram-negative cells need to be stained in order to be
visible, cover the slide with the safranin counter stain for 30 seconds to one
minute.
• 7. Rinse briefly and blot the slide dry. Record each culture as Gram positive
(purple cells) or Gram negative (pink cells).
Endospore staining:
a) Place the heat-fixed slide over a steaming water bath and place a piece of
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blotting paper over the area of the smear.
• b) The blotting paper should completely cover the smear, but should not
stick out past the edges of the slide. If it sticks out over the edges, stain will flow
over the edge of the slide by capillary action and make a mess.
• c) Saturate the blotting paper with the 5-6% solution of malachite green.
Allow the steam to heat the slide for five minutes, and replenish the stain if it
appears to be drying out.
• d) Cool the slide to room temperature. Rinse thoroughly and carefully with
tap water.
• e) Apply safranin for one minute. Rinse thoroughly but briefly with tap water,
blot dry and examine. Mature endospores stain green, whether free or in the
vegetative cell. Vegetative cells stain pink to red.
Q 2
10
Explain the four glycolytic pathways found in different bacteria.
( Unit 3 ; Section 3.2 )
A 2
Explaining the four glycolytic pathways found in different bacteria.
There are 4 major glycolytic pathways found in different bacteria which are:
• 1. Embden-Meyerhoff-Parnas pathway (classic glycolysis):
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• In prokaryotes the glycolysis takes place within the cytosol of the cell.
• A phosphate from the hydrolysis of a molecule of ATP is added to glucose, a
6-carbon sugar, to form glucose 6-phosphate.
• The glucose 6-phosphate molecule is rearranged into an isomer called
fructose 6-phosphate.
• A second phosphate provided by the hydrolysis of a second molecule of ATP
is added to the fructose 6-phosphate to form fructose 1,6-diphosphate.
• The 6-carbon fructose 1, 6-biphosphate is split into two molecules of
glyceraldehyde 3-phosphate, a 3-carbon molecule.
• Oxidation and phosphorylation of each glyceraldehyde 3-phosphate produces
1,3-biphosphoglycerate with a high-energy phosphate bond (wavy red line) and
NADH.
• Through substrate-level phosphorylation, the high-energy phosphate is
removed from each 1, 3-biphosphoglycerate and transferred to ADP forming ATP
and 3-phosphoglycerate.
• Each 3-phosphoglycerate is oxidized to form a molecule of
phosphoenolpyruvate with a high-energy phosphate bond.
• Through substrate-level phosphorylation, the high-energy phosphate is
removed from each phosphoenolpyruvate and transferred to ADP forming ATP
and pyruvate.
• 2. Hexose monophosphate pathway:
• The hexose monophosphate pathway begins with the oxidation of
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glucose-6-phosphate to 6-phosphoglucono-d-lactone by glucose-6-phosphate
dehydrogenase, followed by the oxidation of 6-phosphoglucono-d-lactone to
pentose ribulose 5-phosphate and CO2.
• NADPH is produced during these oxidations. The capability of this oxidative
metabolic system to bypass glycolysis explains the term shunt.
• All cyanobacteria, Acetobacter suboxydans, and A. xylinum possess only the
hexose monophosphate shunt pathway.
• 3.Entner-Doudoroff pathway:
• The Entner-Doudoroff pathway describes a series of reactions that catabolize
glucose to pyruvate, using a different set of enzymes from those used in either
glycolysis or the pentose phosphate pathway.
• This pathway can occur only in prokaryotes.
• A distinct feature of Entner-Doudoroff pathway is that it uses
6-phosphogluconate dehydrase and 2-keto-3-deoxyglucosephophate aldolase to
create pyruvates from glucose.
• The Entner-Doudoroff Pathway has a net yield of 1 ATP for every glucose
molecule processed, as well as 1 NADH and 1 NADPH.
• 4.Phosphateketolase pathway:
• Glucose-6-phosphate is oxidized to 6-phosphogluconate, which becomes
oxidized and decarboxylated to form pentose phosphate.
• Unlike Embden-Meyerhof pathway, NAD-mediated oxidations take place
before cleavage of substrate being utilized. Pentose phosphate subsequently is
cleaved to glyceraldehyde-3-phosphate (GAP) and acetyl phosphate.
• Acetyl phosphate is reduced in two steps to ethanol, which balances the two
oxidations before cleavage but does not yield ATP.
• Overall reaction = Glucose®1 lactate + 1ethanol +1 CO2 with net gain® 1
ATP. Efficiency is about half that of the E-M pathway.
Q 3
10
Describe the origin and mechanism of drug resistance.
A 3
( Unit 5 ; Section 5.5 )
Describing the origin of drug resistance:
The origin of drug resistance can be Non-genetic or genetic:
• In non-genetic drug resistance, Active replication of bacteria is usually required
for most antibacterial drug action. Consequently, a microorganism that is
metabolically inactive may be phenotypically resistant to drugs.
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• Microorganisms may lose the specific target structure for a drug over several
generations and thus be resistant.
• In genetic Origin of drug resistance, Most drug resistant microbes emerge as a
result of genetic change and subsequent selection process by antimicrobial drugs
by chromosomal resistance and extra chromosomal
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resistance.
Describing the mechanism of drug resistance:
Following are the different mechanisms by which microorganisms might exhibit
resistance to drugs.
• 1) Microorganisms produce enzymes that destroy the active drug.
Ex. Staphylococci resistant to penicillin produce -lactamase that destroys the
drug.
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• Gram-negative bacteria resistant to aminoglycosides produce adenylating,
phosphorylating or acetylating enzymes that destroy the drug. Gram-negative
bacteria may be resistant to chloramphenicol if they produce chloramphenicol
transferase.
• 2) Microorganisms change their permeability to the drug.
Ex. Tetracyclines accumulate in the susceptible bacteria but not in resistant
bacteria. Streptococci have a natural permeability barrier to aminoglycosides.
Polymyxin resistant occurs due to change in permeability to the drug.
• 3) Microorganisms develop an altered structural target for the drug.
Ex. Erythromycin resistant organisms have an altered receptor on the 50s subunit
of the ribosome, resulting from the methylation of a 23s ribosomal RNA.
1. Microorganisms develop an altered enzyme that can still perform its metabolic
function, but is much less affected by the drug than the enzyme in the susceptible
organisms.
Ex. In some sulphanamide susceptible bacteria, the tetrahydropteric acid
synthase has a much higher affinity for sulphanomide than for PABA. In
sulphanomide resistant mutants, the opposite is the case.
Q 4
10
Discuss any 5 groups of chemical antimicrobial agents.
( Unit 4 ; Section 4.3 )
A 4
Discussing any 5 groups of antimicrobial agents among the following:
1) Phenol and phenolic compounds:
• Phenolic substances may be either bactericidal or bacteriostatic, depending
upon the concentration used. Bacterial spores and viruses are resistant.
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• The antimicrobial activity is reduced at an alkaline pH, by organic material,
presence of soap and low temperature.
• It causes disruption of cells, precipitation of cell protein, and inactivation of
enzymes and leakage of amino acids from the cells.
• Lethal effect is associated with physical damage to the membrane structures in
the cell surface.
• 2) Alcohols:
• Used to reduce the surface microflora of skin and for disinfection of clinical oral
thermometers.
• Alcohols denature proteins and damage lipid complexes in the cell
membranes. They are also dehydrating agents.
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• 3) Halogens:
• a) Iodine: Iodine is one of the most effective germicidal agents.
• Iodine is mainly used as the best skin disinfectant, used for disinfection of
water, air, (iodine vapors) and sanitization of food utensils.
• Iodine oxidizes essential metabolic compounds such as proteins with sulfhydryl
groups. The action, may also involve the halogenation of tyrosine units of
enzymes and other cellular proteins requiring tyrosine activity.
• b) Chlorine and Chlorine Compounds:
• Chorine compounds are also used to disinfect open wounds, to treat athlete’s
foot, to treat other infections and as a general disinfectant.
• The antimicrobial action is due to hypochlorous acid formed when free chlorine
is added to water.
• Hypochlorite and Chloramines undergo hydrolysis with the formation of
hypochlorous acid.
• It has killing effect which is partially due to the direct combination of Chlorine
with proteins of the cell membranes and enzymes.
• 4) Heavy metals and their compounds:
• Heavy metals like Mercury, Silver and Copper, either in combination or alone
in certain compounds bring about detrimental effects upon microorganisms.
• These compounds combine with cell proteins and inactivate them. Mercuric
chloride acts upon sulfhydryl group of enzyme.
• High concentrations of salt of heavy metals coagulate cytoplasmic proteins and
precipitate them causing death of the cells.
• 5) Dyes: There are two categories of dyes having antimicrobial property. They
are:
a) Triphenylmethane and b) Acridine Dyes
• Triphenylmethane dyes in low concentrations are included in certain culture
media to inhibit the growth of Gram-positive bacteria. Species of Brucella bacteria
can be identified based on their resistance pattern to several dyes. It interferes in
cellular oxidation process.
• Acridine Dye: There are two derivatives of acridine dye. It includes 1)
Acriflavine and 2) Tryptoflavine.
• 6) Detergents:
• These are surface tension depressants, or wetting agents used mainly for
cleaning purposes. Soap is a poor detergent in hard water. Synthetic detergents
used in laundry, dishwashing powders, shampoos etc. are superior to soaps as
they do not precipitate in alkaline or acidic water and do not produce deposits
with minerals in hard water.
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• They are classified as anionic and cationic detergent.
• 7) Quaternary Ammonium Compounds:
• These are cationic detergents having superior antimicrobial properties.
• They have chemically four carbon groups (R1, R2, R3 & R4) linked to the
nitrogen atom and R group may be any of the alkyl groups.
• It is used as skin disinfectants, as a preservative in ophthalmic solutions and in
cosmetic preparations.
• It is used widely in hospitals to disinfect surfaces, food processing plants and
as sanitizers of utensils in restaurants.
• 8) Aldehydes: These are low molecular weight compounds with general
formula RCHO, and have most effective antimicrobial; even sporicidal property.
Compounds include a) Formaldehyde and b) Glutaraldehyde.
• 9) Gaseous agents: Sterilization by means of gaseous agents is employed for
plastic syringes, blood transfusion apparatus and other laboratory materials which
cannot be sterilized by heat. There are two types of gaseous agents; a) Ethylene
oxide and b) β-Propiolactone. Formaldehyde fumes are also a gaseous agent.
Q 5
10
Define fermentation. List and explain the types of fermentations.
( Unit 3 ; Section 3.2 )
A 5
Defining fermentation
Fermentation is an anaerobic breakdown of carbohydrates in which an organic
molecule is the final electron acceptor.
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Listing the types of fermentations:
1) Mixed Acid Fermentation
2) Butanediol Fermentation
3) Butyric acid fermentations
4) Butanol-acetone fermentation
5) Propionic acid fermentation
2
Explaining the types of fermentations.
1) Mixed Acid Fermentations:This is mainly the pathway of the
Enterobacteriaceae. End products are a mixture of lactate, acetate, formate,
succinate and ethanol, with the possibility of gas formation (CO2 and H2) if the
bacterium possesses the enzyme formate dehydrogenase, which cleaves formate
to the gases.
• 2) Butanediol Fermentation: Forms mixed acids and gases, but, in addition, 2,
3 butanediol from the condensation of 2 pyruvate. The use of the pathway
decreases acid formation (butanediol is neutral) and causes the formation of a
distinctive intermediate, acetoin.
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• 3) Butyric acid fermentations: Butyric acid fermentations, as well as the
butanol-acetone fermentation, are run by the Clostridia, the masters of
fermentation. In addition to butyric acid, these Clostridia form acetate, CO2 and
H2 from the fermentation of sugars. Small amounts of ethanol and isopropanol
may also be formed.
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• 4) Butanol-acetone fermentation: Butanol and acetone were discovered as the
main end products of fermentation by Clostridium acetobutylicum during World
War I. This discovery solved a critical problem of explosives manufacture
(acetone is required in the manufacture of gunpowder).
• 5) Propionic acid fermentation: This is a bizarre fermentation carried out by the
propionic acid bacteria which include Corynebacteria, Propionibacterium and
Bifidobacterium. Although sugars can be fermented straight through to
propionate, propionic acid bacteria will ferment lactate (the end product of lactic
acid fermentation) to acetate, CO2 and propionate. The formation of propionate is
a complex and indirect process involving 5 or 6 reactions. Overall, 3 moles of
lactate are converted to 2 moles of propionate + 1 mole of acetate + 1mole of
CO2, and 1 mole of ATP is squeezed out in the process. The propionic acid
bacteria are used in the manufacture of Swiss cheese, which is distinguished by
the distinct flavor of propionate and acetate, and holes caused by entrapment of
CO2.
Q 6
10
List and explain the four main morphological virus types.
( Unit 2 ; Section 2.9 )
A 6
Listing the types of morphological virus types.
1) Helical viruses
2) Icosahedral viruses
3) Enveloped viruses
4) Complex viruses
2
Explaining the morphology of virus types.
1) Helical viruses:
• Helical capsids are composed of a single type of subunit stacked around a
central axis to form a helical structure, which may have a central cavity, or hollow
tube.
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• The genetic material - generally single-stranded RNA, but also ssDNA in the
case of certain phages - is bound into the protein helix, by charge interactions
between the negatively-charged nucleic acid and positive charges on the protein.
• Overall, the length of a helical capsid is related to the length of the nucleic acid
contained within it, while the diameter is dependent on the size and arrangement
of protomers.
• The well-studied Tobacco mosaic virus is an example of a helical virus.
• 2) Icosahedral viruses
• Icosahedral capsid symmetry results in a spherical appearance of viruses at
low magnification, but actually consists of capsomers arranged in a regular
geometrical pattern, similar to a soccer ball.
• Hence they are not truly "spherical". Capsomers are ring shaped structures
constructed from five to six copies of protomers.
• These associate via non-covalent bonding to enclose the viral nucleic acid,
though generally less intimately than helical capsids, and may involve one or
more protomers.
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• 3) Enveloped viruses
• In addition to a protein capsid many viruses are able to envelope themselves in
a modified form of one of the cell membranes - the outer membrane surrounding
an infected host cell, or from internal membranes such as nuclear membrane or
endoplasmic reticulum - thus gaining an outer lipid bilayer known as a viral
envelope.
• This membrane is studded with proteins coded for by the viral genome and
host genome; however the lipid membrane itself and any carbohydrates present
are entirely host-coded.
• The Influenza virus and HIV use this strategy.
• 4) Complex viruses
• These viruses possess a capsid which is neither purely helical, nor purely
icosahedral, and which may possess extra structures such as protein tails or a
complex outer wall.
• Some bacteriophages have a complex structure consisting of an icosahedral
head bound to a helical tail, the latter of which may have a hexagonal base plate
with many protruding protein tail fibres.
• The Poxviruses are large, complex viruses which have an unusual
morphology.
• The viral genome is associated with proteins within a central disk structure
known as a nucleoid.
• The nucleoid is surrounded by a membrane and two lateral bodies of unknown
function.
• The virus has an outer envelope with a thick layer of protein studded over its
surface.
• The whole particle is slightly pleiomorphic, ranging from ovoid to brick shape.
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