Replication + Cultivation of Microorganisms

In viral replication, host cell metabolism switched to

synthesis of viral parts under influence of viral genome

Stages of viral replication

1.Attachment
2.Penetration
3.Uncoating
4.Eclipse period
5.Morphogenesis
6.Release

Attachment stage of viral replication

virion + receptors on cell surface interaction

presence of absence of receptor on cell surface in viral replication determines

cell tropism

What stage of viral replication is - virus is taken up inside cell

#2, penetration

Ways a virus can penetrate the cell (stage 2)

• endocytosis (endosomes)

• translocation (direct)

• membrane fusion

___________ involves separation of viral nucleic acid from outer components, and ________ is lost

uncoating (stage 3), infectivity

In the uncoating viral replication stage, RNA viruses enter _________ and DNA viruses enter ________

cytoplasm, nucleus (thru pores by fusion)

The eclipse period of viral replication allows for appearance of

new viral particles

Processes of eclipse period of viral replication

• mRNA transcribed + translated from viral nucleic acid

• proteins made (early for nucleic acid replication + late for structure)

What is assembled in the morphogenesis stage of viral replication (#5)

• genomes + capsid = nucleocapsid

• other viral parts (may build up as inclusions)

Fast vs slow release of virus from host cell (stage 6)

fast - after cell lysis
slow - exocytosis + budding (enveloped viruses)

Bacterial replication is a coordinated process in which 2 daughter cells made through ________ ___________

binary fission

Division in bacterial replication requires (#)

• cell wall extension

• chromosome replication

• septum formation

For bacterial replication there must be enough

• metabolites (for component synthesis)

• nucleotides (for DNA synthesis)

4 phases of multiplication of bacteria in vitro

▪Lag
▪Logarithmic phase of growth (exponential phase)
▪Stationary phase
▪Decline

Lag phase

adaptation period, # germs unchanged, 2hrs

Exponential phase

bacterial cells start dividing, 20-30min, high virulence

Stationary phase

nutrients from culture media depleted, # bacteria constant, sporogenesis starts, 2-3 days

Decline phase

high # toxic metabolites + sporulation

Diauxic vs continuous growth

diauxic - second lag (metabolism switches)
continuous - in open systems

Types of multiplication of yeasts

• budding + detaching of daughter cells

• fragmentation

• direct division

• pseudohyphae (yeast chain)

Asexuate multiplication of filamentous fungi

by spores, hyphae grows continuously

Sexuate multiplication of filamentous fungi

hyphae fuse, meiosis, zygo+basidiospores formed

Diauxic growth for fungi multiplication involves

reutilization of previously excreted metabolites

Rhizoids

roots of fungi, infiltrate into nutrient substrates and absorb

How do single cell parasites replicate?

binary fission (cystic forms don't replicate)

How do flat worms replicate?

produce embryonated eggs, egg releases parasite if ingested

How do round worms replicate?

females lay eggs

fatal attraction (parasites)

host is assumed to be under the genetic control of the parasite and to represent its "extended phenotype" (ex mice toxoplasma)

Wild-type vs recombinant viruses

wild-type - in world, can cross host barriers
recombinant - in lab, viruses can be combined/produced

Primary cell cultures for viruses

from organ that make cells, for research

Cell lines cultures vs established cell lines cultures - for viruses

cells repetitively subcultured 50-70 times, established allows subcultured 70+ times

Cell cultures for viruses are supplemented w/ and buffered for

supplemented w/ antibiotics + antimycotic + FCS
buffered for pH (w/ indicator)

Cell culture inoculation for viruses

specimens diluted in filtered transport medium, medium placed over cell layer (after 1h at 37°), 2 weeks incubation

cytopathic effect (CPE)

cell morpho alteration from virus infection, allows visualization of viral replication

in light microscopy, what is visible in detection of viral replication?

visible - nucleoproteins + virus inclusion
invisible - individual viruses

What can you cultivate on 1. culture media 2. animals 3. cell cultures

1. bacteria, fungi, some parasites

2. bacteria, viruses'3. viruses, some bacteria

Streak-plating method is used to obtain

isolated bacterial colonies

Quantification (physical inoculation method)

for # microbes in sample, successive dilutions in agar

Semiquantitative inoculation uses

urine and sputum

S(mooth) colonies

round edges, shiny, homogenous, for patho bacteria

R(ough) colonies

rugged + flat surface, uneven edges, dry, adheres to medium, for nonpatho bacteria (except TB, diphtheriae + anthracis)

M(ucous) colonies

big, shiny, smooth surface, flowing, for encapsulated bacteria

Creamy colonies

round, smooth surface, defined edges + creamy, for yeasts

Swarming colonies

continuous growth in waves, for Proteus spp.

In liquid media, no _______ are present

colonies

What bacteria has homogenous growth on liquid media

S germ types, S aureus

What bacteria has adherent ring on liquid media

e coli

What bacteria has pellice/veil on liquid media

vibrio cholerae, pseudomonas, TB

What bacteria has deposits on liquid media?

strep, anthracis

Incubation time (+ temp) for 3 diff fungi

24h for inf, 24-72h for sup, 3-7 days for dermatophytes (@ 30-32°)

What type of colony is typical for filamentous fungi?

fluffy

Physical inoculation methods for filamentous fungi

air (exposure to spores), homogenization in liquid, on agar blocks