Lecture 8 Introduction to the bacteria

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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

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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

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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
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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)

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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

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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

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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

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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

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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

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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

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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

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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

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Oxygen preferences contin...

• Figure illustrates growth of various bacteria in
  liquid culture according to their tolerance/need
  of oxygen

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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

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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

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Bacterial growth in a closed cultureeg. in a
broth

• Lag adaptive(slow)
• Log fastest possible under conditions (newgtthan
  dying)
• Stationary newdying
• Deathdyinggtnew