Question

1.Explain how polymerase chain reaction (PCR) can be used to test the identity of a species.

2. Use technical microbiology terms to describe the shapes and arrangements of bacteria

Answer 1

1.

PCR is an amplification tool, it will enhance the number of amplicons or desired genes in the reaction which u can analyze further as well in details. No matter the quantity of genome of any pathogen in your original sample, PCR will simply amplify this number high enough for diagnosis and for further assessments.

PCR is a technique that takes specificsequence of DNA of small amount andamplifies it to be used for further testing.

PCR allows for rapid and highly specific diagnosis of infectious diseases, including those caused by bacteria or viruses. The human immunodeficiency virus (or HIV), is a difficult target to find and eradicate. The earliest tests for infection relied on the presence of antibodies to the virus circulating in the bloodstream. However, antibodies don't appear until many weeks after infection, maternal antibodies mask the infection of a newborn, and therapeutic agents to fight the infection don't affect the antibodies. PCR tests have been developed that can detect as little as one viral genome among the DNA of over 50,000 host cells.[29] Infections can be detected earlier, donated blood can be screened directly for the virus, newborns can be immediately tested for infection, and the effects of antiviral treatments can be quantified.

steps of PCR

• Initialization: This step is only required for DNA polymerases that require heat activation by hot-start PCR. It consists of heating the reaction chamber to a temperature of 94–96 °C (201–205 °F). if extremely thermostable polymerases are used, which is then held for 1–10 minutes.
• Denaturation: This step is the first regular cycling event and consists of heating the reaction chamber to 94–98 °C (201–208 °F) for 20–30 seconds. This causes DNA melting, or denaturation, of the double-stranded DNA template by breaking the hydrogen bonds between complementary bases, yielding two single-stranded DNA molecules.
• Annealing: In the next step, the reaction temperature is lowered to 50–65 °C (122–149 °F) for 20–40 seconds, allowing annealing of the primers to each of the single-stranded DNA templates. Two different primers are typically included in the reaction mixture: one for each of the two single-stranded complements containing the target region. The primers are single-stranded sequences themselves, but are much shorter than the length of the target region, complementing only very short sequences at the 3' end of each strand.

It is critical to determine a proper temperature for the annealing step because efficiency and specificity are strongly affected by the annealing temperature. This temperature must be low enough to allow for hybridization of the primer to the strand, but high enough for the hybridization to be specific, i.e., the primer should bind only to a perfectly complementary part of the strand, and no where else. If the temperature is too low, the primer may bind imperfectly. If it is too high, the primer may not bind at all. During this step, the polymerase binds to the primer-template hybrid and begins DNA formation.

• Extension/elongation: The temperature at this step depends on the DNA polymerase used; the optimum activity temperature for the thermostable DNA polymerase of Taq polymerase is approximately 75–80 °C (167–176 °F), though a temperature of 72 °C (162 °F) is commonly used with this enzyme. In this step, the DNA polymerase synthesizes a new DNA strand complementary to the DNA template strand by adding free dNTPs from the reaction mixture that are complementary to the template in the 5'-to-3' direction, condensing the 5'-phosphate group of the dNTPs with the 3'-hydroxy group at the end of the nascent (elongating) DNA strand. The precise time required for elongation depends both on the DNA polymerase used and on the length of the DNA target region to amplify.

The processes of denaturation, annealing and elongation constitute a single cycle. Multiple cycles are required to amplify the DNA target to millions of copies.

• Final elongation: This single step is optional, but is performed at a temperature of 70–74 °C (158–165 °F) (the temperature range required for optimal activity of most polymerases used in PCR) for 5–15 minutes after the last PCR cycle to ensure that any remaining single-stranded DNA is fully elongated.
• Final hold: The final step cools the reaction chamber to 4–15 °C (39–59 °F) for an indefinite time, and may be employed for short-term storage of the PCR products.

2)  shapes and arrangements of bacteria

Morphologically, bacteria are microscopic single-celled organisms that are small in size and lack membrane bound organelles. A majority of these organisms also have a cell wall and capsule that protects the inner contents of the cell where the nucleoid, ribosome, plasmid, and cytoplasm are found.

While a majority of bacteria share these characteristics, they vary in shape which allows different types of bacteria to be classified based on their general shape.

Cocci : Cocci bacteria appear spherical or oval in shape. Cocci bacteria may exist as single cells or remain attached to each other.

Diplococci bacteria - Diplococci bacteria are the type of cocci bacteria that occur as a pair (two joined cells). examples:

• Streptococcus pneumoniae
• Moraxella catarrhalis
• Enterococcus spp
• Neisseria gonorrhea

Tetrad bacteria - Tetrad bacteria are arranged in groups of four cells. Examples

• Pediococcus
• Tetragenococcus

Sarcinae sarcina/ Bacteria - Sarcina bacteria occur in groups of 8 cells. Examples

• Sarcina aurantiaca
• Sarcina lutea
• Sarcina ventriculi

Streptococci Bacteria - Streptococci bacteria are a type of bacteria that arrang in a chain form (resembling chains). Examples

• Streptococcus pyogenes
• Streptococcus pneumoniae
• S. mutans

Staphylococci Bacteria- Staphylococci Bacteria are a type of bacteria that form grape-like clusters. This type of arrangement is the result of division that occurs in two planes.Examples

• Staphylococcus epidermidis
• Staphylococcus haemolyticus
• Staphylococcus aureus
• Staphylococcus capitis

Bacillus Bacteria (Rod-Shaped)

Bacillus bacteria have the following traits:

• They are all rod-shaped
• They form endospores
• They are facultative anaerobes

bacillus bacteria are arranged differently. While some exist as single, unattached cells (e.g. Salmonella enterica subsp, Bacillus cereus, and Salmonella choleraesuis), others are attached.

Diplobacilli bacteria :occur in pairs. Following cell division, the two cells do not separate and continue existing as a pair. Examples

• Klebsiella rhinoscleromatis
• Moraxella bovis

Streptobacilli - Streptobacilli bacteria occur as elongated chains. As such, they are the result of division on a single plane. Examples

• Streptobacillus moniliformis
• Streptobacillus Levaditi

Coccibacilli bacteria - Compared to other bacilli, Coccibacilli bacteria are shorter in length and thus appear stumpy. Examples

• Chlamydia trachomatis
• Haemophilus influenzae

Vibrio bacteria - Generally, vibrio bacteria are comma-shaped and thus not fully twisted (curved rods). Examples

• Vibrio parahaemolyticus
• Vibrio cholerae

Spirochete - Spirochetes are characterized by a helical shape. Examples

• Leptospira
• Spirochaeta
• Treponema

• Rectangular bacteria- They appear rectangular in shape e.g. Haloarcula marismortui
• Star-shaped bacteria- Look like stars (star-shaped) e.g. Stella humosa
• Haloarcula- Triangular in shape
• Pleomorphic bacteria- Bacteria with the ability to change their shape and size in different environments, e.g. M. pneumonia
• Stalked bacteria- These include such bacteria as C. crescentus that possess a stalk on one end of the cell.