L4: Microbial Cell Walls/Technique (incomplete)

Question 1

Describe the gram stain on microbial cell walls

Answer 1

• cytological characteristics for cytological types

Question 2

Name key G+ features, key examples

Answer 2

Have peptidoglycan (FAT C!)
* Firmicutes - some have no cell wall may stain (-), includes Baccilus

*Actinobacteria: many antibiotics

*Chloroflexi: have leaky cell wall, hard to culture, might stain -

*TM7 (Sacha…) only one cultured isolate

-Chloroflexi are likely to stain gram -
- rest are Gram-

Question 3

How did Woese think of Gram+

Answer 3

• Group 5 = Firmicutes plus Actinobacteria, clustered together as only G+
• these were the only Gram-positive organisms known at the time

Question 4

Describe the steps of gram stain

Answer 4

1. apply crystal violet
2. Apply iodine morfant
3. wash with alchohol for decoloration (will stain ppt purple if correct, if no ppt will leak)
4. apply safarin (counter stain for lip)

Question 5

Archea layer

Answer 5

slayers?

Question 6

What are 4 methods of studying microbial ecology

Answer 6

1. Pure Culture
2. Microcosm
3. Culture Independant
4. Genomics

Question 7

What are pure culture studies

Answer 7

• focus on one or few species in synthetic media
• can do growth curves, physiological, genetic, medical studies
• limited relvance to ecologial role study because most media cannot be grown on pure culutre
• can supply different carbon sournce (like glucose) see how mutants compare to wildtype

Question 8

What are microcosm studies, parameters to control,

Answer 8

• make natural habitat in the lab like winogradky coloumns
• not pure but mained in control conidtions where u manipulated parameters
• enrichement for specific organisms like N2 bacteria

*moniter response to manipulation

*use molecualr biology tools to moniter prescence of genes / gene products in poplaution (like gene products (DNA), gene expression (RNA)

*try isolate pure culture

Question 9

What are responses to moniter in micrcosm

Answer 9

nutrient consumption, pollutant biodegradation, waste products, N 2 fixation, CH 4 oxidation, CO 2 evolution, etc

Question 10

What are inputs/outpute of soil columns

Answer 10

input: irrigation, fresh air

output: exhaust air, percolate

Question 11

What are culture independant studies

Answer 11

• in situ = without manipulation, immeditaley sample so u have a snapshot
• direct study of complex communities in env sample but no culturing or enrichment (like time or depth)

Question 12

What do culture independant studies help to investifate

Answer 12

DIQ C
1. where the microbes are (Detect)
2. which species are present (identify)
3. population sizes/fluctuations (Quanity)
4. physiiological and ecological functions (characterise)

Question 13

List examples of culture independant methods

Answer 13

• measurement of gasses (CO 2 , H 2 S, N 2 O, CH 4 etc) and other metabolites, often using radioactive isotope tracers
• molecular biological methods (many)
• microscopy (many methods)
  
  • FISH (fluorescent in situ hybridization) microscopy to visualize specific cells, CARD-FISH
• stable isotope probing (SIP) studies i.e., DNA-SIP

Question 14

How do we know that the sequences detected using molecular
techniques represent real microbes

Answer 14

hey can be specifically visualized via FISH
– microbiologists can sometimes coax them to grow in culture
using their natural environment as a growth medium
– understand that some species cannot survive on their own
(obligate symbionts, organisms that depend on consortia of
different species, etc) and so will never be isolated as pure
cultures
– some organisms have been nearly isolated via enrichment culture
from environmental samples with thousands of species to mixed
cultures with few species
– promising technology currently being developed is the iChip