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Lecture 8 Introduction to the bacteria

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Often important in making vaccines-why? ... Obligate aerobe eg Pseudomonas aeruginosa. Obligate anaerobe eg Clostridium botulinum ... – PowerPoint PPT presentation

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Title: Lecture 8 Introduction to the bacteria


1
Lecture 8Introduction to the bacteria
  • Lecture aims
  • To understand the nature of bacteria
  • To understand how bacteria can be detected and
    isolated
  • To understand how Bacteria grow
  • Referenceseg Black Chp 4 p71-85 Chp 6p137-160

2
Basic external structuresOften important in
making vaccines-why?
  • Pili hair like structures used for attachment
    and mating (sex pili)
  • Flagella whip like structure for movement
  • Capsules sticky sugary base, can be used for
    attachment and to avoid immune system

3
Basic internal structures
NAG
  • The Cell wall
  • A major component is glucose based polymer called
    peptidoglycan
  • Consists of cross linked molecules of
  • N-acetylglucoseamine
  • N-acetylmuramic acid

NAM
4
Cell wall contin...
  • Petidoglycan content differs between Gve and
    G-e bacteria
  • Gram bacteria have a dominant thick
    peptidoglycan layer (with some linkage changes as
    well)

5
Cell wall contin...
  • While G-e bacteria have a more complex cell wall
    layer with inner and outer cell membranes (LPS)
    thinner layer of pepidoglycan

6
Seeing bacteria
  • Bacteria have a size in the range of 1x10-15µM
    (exceptions exist)
  • Consequently we need to view with oil immersion
  • Many have a cell wall
  • Nearly all are colourless when viewed
    microscopically
  • Need to use specialized viewing techniques for
    live specimens (Darkfield Phase contrast)
  • Fixed (killed can be observed by specialized
    stains
  • The most common is the Gram stain

7
Gram stain Divides bacteria on the basis of cell
wall thickness
  • Thick cell wall present
  • Gram positive
  • Dark purple colour
  • eg Staphylcoccus aureus
  • Thin cell wall present
  • Gram negative
  • red colour
  • eg Escherichia coli

8
So how does the Gram stain work?
  • Basic procedure following fixation of bacteria on
    slide
  • Stain all bacteria with crystal violet (30sec)
    wash
  • Add Grams iodine (30sec) wash
  • Decoulourize with acetone (3-5 sec) wash
  • Counterstain with safranin (30sec) wash, dry,
    observe x1000

9
How does the Gram stain work?
  • The crystal violet stains all the bacteria purple
  • The iodine complexes with the crystal violet
  • The acetone dehydrates the cell wall (shrinks)
    and washes out complex from thin CW
    bacteria-these become colourless
  • To see thin CW bacteria- we counterstain with a
    red dye

10
Bacterial growth1. Physical requirements
  • Temperature preferences
  • Note tends to be a range with growth most rapid
    at preferred temperature
  • Bacterial growth measured by numbers of
    bacteria/time

11
Bacterial growth contin...
  • Bacteria defined according to temp. preferences
  • Psychrophyles (0-20oC)
  • eg Listeria monocytogenes
  • Mesophyles (20-40oC)
  • eg E.coli (most endogenous bacteria including
    pathogens)
  • Thermophyles (gt45oC)
  • eg Thermophilus aquaticus 90o

12
Growth requirements2. Oxygen needs
  • Obligate aerobe eg Pseudomonas aeruginosa
  • Obligate anaerobe eg Clostridium botulinum
  • Facultative anaerobe eg E.coli
  • Microaerophilic eg Haemophilus influenzae
  • Aerotolerant eg Micrococcus sp

13
Oxygen preferences contin...
  • Figure illustrates growth of various bacteria in
    liquid culture according to their tolerance/need
    of oxygen

14
Bacterial isolation (labs)
  • Need to supply nutrients physical chemical
  • Some very finicky (fastidious)
  • Can take a long time (TB gt6 weeks)
  • Often need special media (Choc agar)
  • Supply correct temp and oxygen needs
  • Can be enhanced by using special media

15
Bacterial isolation contin...
  • Selective media
  • Selective differential (MSA, McConkey)
  • Mannitol Salt Agar (Staphylococcal)
  • High salt selects for staphylococci
  • Mannitol separates S.aureus (yellow) Others
    (pink)
  • McConkey (enteric bacteria)
  • Bile salts inhibit other than enteric(gut)
    bacteria)
  • Enrichment (selenite broth BHI)
  • Inhibits non target bacteria, allows small
    numbers of target to grow and be isolated

16
Bacterial growth in a closed cultureeg. in a
broth
  • Lag adaptive(slow)
  • Log fastest possible under conditions (newgtthan
    dying)
  • Stationary newdying
  • Deathdyinggtnew
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