Week 4

Today's Topics:
○ Viri

Announcements:
○ Today's practical - video on lab and microbial techniques, aseptic technique, etc.
○ Group F - benches 23/24

Lecture Topic:
During the lecture, take notes here.
○ Viral Infection of cell
§ Attachment
§ Penetration/ infection

○ HIV replication
§ Attachment - GP120:CD4
§ Fusion/ endocytosis
§ Uncoating
§ Reverse Transcription (cf Central Dogma)
§ RNA-->DNA (Reverse Transcriptase)
§ Integration of DNA into genome (integrase)
§ Transcription (viral RNA & proteins)
§ Assembly
§ Release (budding)
§ 1 T-lymphocyte can produce 10M HIV virions in 36 hours, before dying from exhaustion
○ Outcome of infection
§ HIV: 2-3 years
§ Karposi's Sarcoma - tumour of blood vessel lining
§ Long-term latency - eg Chicken Pox & Shingles - Herpes Zoster (Varicella zoster)
§ Possible effects:
□ Tumour (eg Rous sarcoma or papilloma)
□ Lysis (eg polio)
□ Persistent infection (eg HIV)
□ Latent infection (eg Epstein Barr virus or herpes simplex)

○ Subviral agents
§ Viroids:
□ Naked RNA
□ circular
□ 240-400 nucleotides
□ cause at least 20 plant diseases (eg potato spindle tuber or coconut kdang kdang)
□ Only infect plants.
§ Virusoids
□ Larger than viroids
□ 1600-2000 nucleotides
□ Need helper virus (need capsid, but don't have one)
□ Hepatitis D ("The Delta Agent") is a virusoid, helped by Hepatitus B.
□ A parasite of a parasite.
§ Prions
□ Cause slow neurological conditions (transmissible spongiform encephalopathies)
□ Eg: Kuru, scrapie, BSE, CJD
□ Misfolded brain protein.
□ Proteins form fibril plaques which are cytotoxic.
□ Cell death leaves holes in tissue.
§ Article - bacterium living in insect cells, has lost ability for independent reproduction.
○ Assessment of bacterial growth - Bacterial Enumeration.
○ Usually consider populations rather than individual cells
○ Number of cells, or biomass (weight of cells)
○ Bacterial numbers:
§ Total count (haemocytometer or coulter counter): every cell.
□ Haemocytometer - blood cell counter - thicker slide, with well. Graticule under well, to assist counting.
□ Coulter counter - automated particle counter.
□ Usually expressed, eg as cells per cm3.
□ Direct count
§ Viable count - only live cells. (eg colony count or most probable number)
□ Dilute to allow spread, then innoculate petri dish. Incubate, then count colonies. - known volume of known dilution. Ideal count is 30 - 300 colonies for best accuracy; more are hard to count, less may not be representative.
□ Viable count = colony count x 1/dilution x 1/volume (cm3)
□ Volume is usually 0.1 cm3 or 0.2 cm3 (100 or 200μl)
□ Incubation in nutrient broth in series of tubes - count proportion of tubes that have growth.
□ Usually expressed as colony forming units per cm3.
□ Indirect count
○ Biomass
§ Wet and dry weight measure
1) Weigh centrifuge tube
2) Add culture
3) Weigh
4) Centrifuge
5) Remove supernatant
6) Weigh pelleted cells (wet weight)
7) Dry pellet in oven/incubator at >100°C
8) Weigh dried cells (dry weight)

§ Spectrophotometric measurement
□ Measure turbidity of culture