BACTERIAL ENZYMES

DNA polymerases are enzymes that synthesize DNA molecules from deoxyribonucleotides, the

building blocks of DNA. These enzymes are essential to DNA replication and usually work in
pairs to create two identical DNA strands from a single original DNA molecule. The DNA
copies are created by the pairing of nucleotides to bases present on each strand of the original
DNA molecule. This pairing always occurs in specific combinations, with cytosine along
with guanine, and thymine along with adenine. During this process, DNA polymerase “reads”
the existing DNA strands to create two new strands that match the existing ones. These
enzymes catalyze the following chemical reaction
deoxynucleoside triphosphate + DNAn   diphosphate + DNAn+1
The first evidence of the existence of an enzymatic activity capable of synthesizing DNA came
in 1958 with the discovery of E. coli Pol I by A. Kornberg and colleagues. Five DNA
polymerases are now recognized inEscherichia coli, 8 in Saccharomyces cerevisiae, and at least
15 in humans.

DNA ligase is a specific type of enzyme that facilitates the joining of DNA strands together by
catalyzing the formation of a phosphodiester bond. It plays a role in repairing single-strand
breaks in duplex DNA in living organisms. DNA ligase is used in both DNA repair and DNA
replication. 
In molecular biology, it is used in laboratories for recombinant DNA experiments. Purified DNA
ligase is used in gene cloning to join DNA molecules together to form recombinant DNA.
The mechanism of DNA ligase is to form two covalent phosphodiester bonds between 3'
hydroxyl ends of one nucleotide, ("acceptor") with the 5' phosphate end of another ("donor").
ATP is required for the ligase reaction, which proceeds in three steps:
1. Adenylation (addition of AMP) of a lysine residue in the active center of the
enzyme, pyrophosphate is released;
2. Transfer of the AMP to the 5' phosphate of the so-called donor, formation of a
pyrophosphate bond;
3. Formation of a phosphodiester bond between the 5' phosphate of the donor and the 3'
hydroxyl of the acceptor.
Helicases are a class of enzymes vital to all living organisms. Their main function is to
unpackage an organism's genes. They are motorproteins that move directionally along a nucleic
acid phosphodiester backbone, separating two annealed nucleic acid strands. Helicases are often
used to separate strands of a DNA double helix or a self-annealed RNA molecule using the
energy from ATPhydrolysis, a process characterized by the breaking of hydrogen
bonds between annealed nucleotide bases. They also function to remove nucleic acid-associated
proteins and catalyze homologous DNA recombination.[3] Metabolic processes of RNA such as
translation, transcription, ribosome biogenesis, RNA splicing, RNA transport, RNA editing, and
RNA degradation are all facilitated by helicases.
 The Clean Room classifications

Negative Staining Technique

In a negative staining technique, an acidic, anionic dye is mixed with a cell sample. The dye
changes the color of the background, not the cells. This process can be considered the opposite of
simple staining.
An anion is a negatively charged ion, therefore an anionic dye has a negative charge. When the
negatively charged dye is added to the negatively charged cells, the two repel each other,
meaning they push apart. When the mixture is placed on a slide and air dried, what results is a
darkly dyed background, surrounding clear, unstained cells. The transparent cells are now highly
visible but are unaffected by direct contact with the dye and distortion from heat fixing, which is
not needed in a negative stain.
India ink is the classic example of a negative stain. It will turn the background a dark brown to
black, leaving the clear, bright cells unstained and highly visible. These are cells of the fungal
pathogen Cryptococcus. The India ink has colored the background brown, leaving the cells their
natural color.
SPECIAL STAINS
􀁺 Stains for Metachromatic granules
􀁺 Stain for spores
􀁺 Stain for capsules
􀁺 Stain for spirochetes
􀁺 Stain for flagella
Albert’s Staining for C. diphtheriae
In all cases of suspected Diphtheria, stain one of the smears with Gram stain. If Gram stained
smear shows morphology suggestive of C.diphtheriae, proceed to do Albert staining which
demonstrates the presence or absence of metachromatic granules. Present in the body of the
bacillus (C. diphtheriae) are numerous metachromatic granules which give the bacillus beaded
or barred appearance. These granules are best demonstrated by Albert’s stain.
Capsule staining
The purpose of the capsule stain is to reveal the presence of the bacterial capsule. Capsule may
appear as clear halo when a fresh sample is stained by Grams or Leishman stain, Negative
staining- using - India ink, Nigrosin
Endospore Staining
Bacterial endospores are metabolically inactive, highly resistant structures produced by some
bacteria as a defensive strategy against unfavorable environmental conditions. Primary stain - is
malachite green, which stains both vegetative cells and endospores and heat is applied to help
the primary stain penetrate the endospore. Decolorized with water, which removes the malachite
green from the vegetative cell but not the endospore, Safranin – counterstain any cells which
have been decolorized, At the end of the staining process, vegetative cells will be pink, and
endospores will be dark green.
Flagella stain
􀁺 Flagella are fragile appendages
􀁺 Cannot be seen under ordinary microscope
􀁺 Hence the surface is coated with a precipitate to form a colloidal substance
􀁺 This precipitate serves as a layer of stainable material
Dye Components
1. 1% Osmic acid
2. Mordant
10% Tannic acid
Sat.potassium alum
10% Ferric chloride
3. Fontana’s silver solution
Use
This is used to demonstrate the flagella and the organisms stain black andflagella appear light
brown.

ELECTRON MICROSCOPE
The electron microscope is a type of microscope that uses a beam of electrons to create an
image of the specimen. It is capable of much higher magnifications and has a greater resolving
power than a light microscope, allowing it to see much smaller objects in finer detail.
Types of Electron Microscopes
1. Scanning Electron Microscope (SEM)
In SEM, a beam of electrons scans the surface of a sample (Figure 1). The electrons interact with
the material in a way that triggers the emission of secondary electrons. These secondary
electrons are captured by a detector, which forms an image of the surface of the sample. The
direction of the emission of the secondary electrons depends on the orientation of the features of
the surface. There, the image formed will reflect the characteristic feature of the region of the
surface that was exposed to the electron beam.

2. Transmission Electron Microscope (TEM)

In TEM, a beam of electrons hits a very thin sample (usually no more than 100 nm thick). The
electrons are transmitted through the sample (Figure 2). After the sample, the electrons hit a
fluorescence screen that forms an image with the electrons that were transmitted.

Figure 2. Transmission Electron Microscope. A

beam of electrons is focused on a sample. The
electrons pass through the sample to form an image
on a fluorescent screen.
3. Scanning Transmission Electron Microscope (STEM)
A scanning transmission electron microscope orSTEM combines the capabilities of both an
SEM and a TEM. The electron beam is transmitted across the sample to create an image (TEM)
while it also scans a small region on the sample (SEM). The ability to scan the electron beams
allows the user to analyze the sample with various techniques such as Electron Energy Loss
Spectroscopy (EELS) and Energy Dispersive X-ray (EDX) Spectroscopy which are useful
tools to understand the nature of the materials in the sample.
Electron microscopy application areas
Semiconductor and data storage Research
 Circuit edit  Electron beam induced
 Defect analysis deposition
 Failure analysis  Materials qualification
Biology and life sciences  Materials and sample
preparation
 Cryobiology
 Nanoprototyping
 Protein localization
 Nanometrology
 Electron tomography
 Device testing and
 Cellular tomography
characterization
 Cryo-electron microscopy Industry
 Toxicology
 Biological production and viral load  High-resolution imaging
monitoring  2D & 3D micro-
 Particle analysis characterization
 Pharmaceutical QC  Macro sample to nanometer
 3D tissue imaging metrology
 Virology  Particle detection and
 Vitrification characterization
 Direct beam-writing
fabrication
 Dynamic materials
experiments
 Sample preparation
 Forensics

Spirochaetes  belong to a phylum of distinctive diderm (double-membrane)bacteria, most of

which have long, helically coiled (corkscrew-shaped or spiraled, hence the name) cells.
[2]
 Spirochaetes arechemoheterotrophic in nature, with lengths between 5 and 250 µm and
diameters around 0.1–0.6 µm.
Spirochaetes are distinguished from other bacterial phyla by the location of their flagella,
sometimes called axial filaments, which run lengthwise between the bacterial inner membrane
and outer membrane in periplasmic space. These cause a twisting motion which allows the
spirochaete to move about. When reproducing, a spirochaete will undergo asexual transverse
binary fission. Most spirochaetes are free-living and anaerobic, gram-negative bacteria.
The spirochaetes are divided into three families (Brachyspiraceae, Leptospiraceae,
and Spirochaetaceae), all placed within a single order (Spirochaetales). Disease-causing
members of this phylum include the following:

 Leptospira species, which causes leptospirosis

 Borrelia burgdorferi, B. garinii, and B. afzelii, which cause Lyme disease
 Borrelia recurrentis, which causes relapsing fever
 Treponema pallidum subspecies which cause treponematoses such as syphilis and yaws.
 Brachyspira pilosicoli and Brachyspira aalborgi, which cause intestinal spirochaetosis[
Rickettsia is a genus of nonmotile, gram-negative, nonspore-forming,
highly pleomorphic bacteria that can present as cocci (0.1 μm in diameter), rods (1–4 μm long),
or thread-like (10 μm long). The term rickettsia, named after Howard Taylor Ricketts,
Rickettsia cannot live in artificial nutrient environments and is grown either
in tissue or embryo cultures; typically, chicken embryos are used. Rickettsia species are
transmitted by numerous types of arthropod, including chigger, ticks, fleas, and lice, and are
associated with both human and plant disease.
The classification of Rickettsia into three groups (spotted fever, typhus, and scrub typhus) was
based on serology. This grouping has since been confirmed by DNA sequencing. All three of
these contain human pathogens.