Identification of Bacterial Species by 16S rRNA gene sequencing

1. What is the purpose of 16S ribosomal RNA PCR?

To identify species by sequencing the variable regions of the 16S rRNA gene.

2. What is the application of 16S rRNA gene sequencing?

It is used in clinical diagnosis, the NIH Human Microbiome Project, and metagenomics to study bacterial diversity in the environment.

3. How does heat sterilization work?

Steam sterilization is performed in an autoclave at 121°C for 15-30 minutes at 15 psi to denature proteins and enzymes.

4. What is the purpose of autoclave tape?

Autoclave tape is used to monitor the condition of sterilization. White tape indicates before sterilization, and black tape indicates after sterilization.

5. What is the pore size of a filter used in filtration?

The pore size of the filter is 0.22μm, which is smaller than most microorganisms, allowing retention of microbes on the membrane.

6. How does UV irradiation inhibit bacterial growth?

UV irradiation causes DNA or RNA damage, leading to the inactivation and inhibition of bacterial growth.

7. What is the purpose of Gram stain?

Gram stain is used to classify bacteria into Gram positive, Gram negative, and Gram variable bacteria, and to visualize the shape and arrangement of bacterial cells.

8. What are the differences between Gram positive and Gram negative bacteria?

Gram positive bacteria have a single membrane, a thick peptidoglycan layer, and lower lipid content in the cell wall, while Gram negative bacteria have a double membrane, a thin peptidoglycan layer, and higher lipid content in the cell wall.

9. What is peptidoglycan?

Peptidoglycan is a carbohydrate backbone of alternating units of NAG and NAM residues linked to peptides, forming a larger polymer network.

10. What is the principle of the Gram stain?

The Gram stain involves staining with crystal violet dye, forming a complex with Gram's iodine solution. Ethyl alcohol or acetone is then used to decolorize the sample, and a counter stain is applied using safranin.

11. How does the oil immersion lens in a light microscope improve image quality?

The oil immersion lens has the same refractive index as glass, allowing more light to be gathered, resulting in a brighter image and higher resolution.

12. What are the different shapes of bacteria?

Bacteria can be spherical (coccus), rod-shaped (bacillus), or spiral-shaped (vibrio, spirillum, spirochete).

13. What are the different arrangements of bacterial cells?

Bacterial cells can be arranged in pairs (diplo-), in a line (strepto-), in groups of four (tetrads), in packets of eight (sarcinae), or in clusters (staphyl-).

14. What is the Beer-Lambert Law used for?

The Beer-Lambert Law is used to determine the concentration of nucleic acids by measuring absorbance.

15. What are the formulas of the Beer-Lambert Law?

T = I/I0 and A = log(1/T) = log(I0/I) = ελcℓ, where T is transmittance, I0 is the intensity of incident light, I is the intensity of transmitted light, ελ is the molar absorbance coefficient, c is the concentration of the absorbing solution, and ℓ is the path length of the sample.

16. What does the A260/A280 ratio indicate about the purity of nucleic acid?

An A260/A280 ratio of approximately 1.8 for DNA and 2.0 for RNA indicates purity, while a lower ratio suggests protein contamination.

17. What does the A260/A230 ratio indicate about the purity of nucleic acid?

An A260/A230 ratio greater than 2.0 indicates pure DNA or RNA, while a lower ratio suggests possible contamination with phenol or guanidium salt.