Bacterial Staining (Gram Staining)

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Bacterial Staining(Gram Staining)

• Sang-heum, Shin
• Hyoung-Joo, Lee
• Fermentation and Metabolic Lab.
• URL http//www.ajou.ac.kr/fereng/

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???? ??? ??? ??

• ????
• ???? ??, ??, ???
• ???? ?? ? ???? ??
• ??? ??? ??? ??? ??
• ????? ??? ??? ??
• ????? ???? ??? ???? ????

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???

• ??
• Magnification
• Contrast
• Resolution ( Resolving power)

• ??
• Bright-field microscopy
• Dark-field microscopy
• Phase-contrast microscopy
• Fluorescence microscopy
• Electron microscopy
• SEM (???????)
• TEM (???????)

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(No Transcript)
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???? ?? ??? ?? ??

• ?? ??????
• (ocular micrometer)
• ??
  ??????
• (stage
  micrometer)

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1. ?? ?????? 1 ??? ?? ?? ??
?? ??????? ?? ? 1?? ??????? ?? ? 10 ???
----- ?? ?????? 1 ??? ?? 1/10 x 10 1 µm
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2. ??? ??? ?? ???? ?? ?????? 1 ??? ???, ????
???? ??? ?(?, ??? ?? ???????? ??? ?? ???????? ??
?????? 1??? 10 ???.)
   ?? ?????? 1 ??? ?? 3/5 X 10 6 ?    ????
??? 28 X 6 168 ?
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Bacterial morphology
Rod
Spherical (cocci)
Herical and Curve
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???? ???

• Binomial nomenclature system
• - Latinized name.
• - ???? ?? ??
• - First word Genus name
• Second word Species name
• Escherichia coli

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Microscope Techniques Dyes Staining

• Simple (positive) staining
• ? basic dye (methyle blue, crystal violet,
  safranin O ?)? cell? ??
• Negative staining nigrosin, india ink ??? ???
  ??
• Differential staining uses combination of dyes.
• The Gram stain divides bacteria into TWO groups
  based upon their cell wall composition
• Gram-positive vs. Gram-negative
• 1?(?) ?? (crystal violet) ? ??? ? ?? ? 2?(?) ??
  (safranin)
• The acid-fast stain Mycobacterium
  tuberculosis(??) and leprosy (??)
• 1?(?)?? (carbol fucshin) ? heating ? ??
  (HCl-alcohol) ? 2?(?) ??(MB)
• Specific stains ?? ??? ??? ?? ??, capsule,
  spore, flagella staining,

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Cell structure

• Gram positive bacteria

• Gram negative bacteria

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Gram Stain
1884? Christian Gram ? ??? ??? ?? ??? 1921?
Hucker ? ?? ??? ???? ?? ??? ??? ??? ??
Hucker ?? Hucker ?? Hucker ??
???? ?? ??
1. ? ? ? Crystal violet 30?
2. ? ? ? Gram iodine 30?
3. ? ? ? Acetone-alcohol 5?
4. ???? Safranin O 30?
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Typical Gram stain
Gram positive Gram negative
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?? ? ?????

1. ?????? ?
2. ????? ???? ???? ??.
3. ??? ??? ??? ??.
4. Serial Dilution ?? ?

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???? ??? ????

• Sang-heum, Shin
• Hyoung-Joo, Lee
• Fermentation and Metabolic Lab.
• URL http//www.ajou.ac.kr/fereng/

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Purpose

• This section is designed to instruct you in
• ????? ??? ???? ??? ????
• ???? ????? ????.
• Culture transfer ??? ??? ????? ???.
• ??? ??? ??? ??? ?? ??? ???.

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Introduction

• Pure culture
• ??
• ??? ???? ?? ???? ????? ???? ???? ???? ?
• L.????? R.??? ??? ??? ???, ?? ?? ????
• ??? ?? ???? ???? ???? ?? ??? ???? ??
• ???? ??? ??? ??.
• ??
• solid media streak plate
• poured plate
• spread plate
• dilution shake culture
• Liquid media dilution method
• single cell isolation
• two membered culture
• ? ??? ??? ??

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Culture transfer techniques

• Broth

• Agar Slant

• Agar deep

• Agar plate

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Experimental Device
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Experimental Device
incubator
clean bench
Stirrer
Water bath
centrifuge
Deep freezeer
Shaking incubator
vortex
Anaerobic jar
pH meter
Fermenter

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Procedure for preparation of a pure culture
Streaking
Colony selection
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Medium

• ???? ????
• Heat
• Filtration
• Chemicals
• ??? ??
• Arar slants
• Agar deep tube
• Agar plate
• ????? ?? ??? ??
• Glucose, Peptone, Tryptone
• Yeast extract, Beef extract, NaCl
• K2HPO4, MgSO4, FeSO4, Agar

• ????? ??
• (a) Label
• (b) ??? ??? ??? ???
• V ??? ? ?? ??.
• (b) Selected colony
• (c) Flame the needle or loop
• (d) Flame the necks of the tubes
• (e) Transfer zigzag motion
• streaking
• (f) Flame the necks of the tubes
• (g) Recap the tubes
• (h) Reflame the loop or needle

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??????? ???? ????? ?? ? ????

• Nutrient plate
• 0.1 strach(???????)
• 0.1 skim milk
• (??? ?????)
• 0.1 tributyrin (???????)
• Balance? ???? ??
• ??? ??, ???? ???
• pH meter? pH? ???
• Agar ???, autoclave? 15? ??
• ??? ??? 48-50? ????? ?? ???
• ??
• ??? ??

• Nutrient broth
• ????? ?? ?? 1-3? ??? ??? ???? ??? pH? ???
• Dispenser? ???? 5ml? ??
• Autoclave? ??
• ???? ????? ?? ??

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??????? ???? ????? ?? ? ????

• ????
• ???? ?? ?? ???? ??? ??
• ??? ??? ????? ??
• ??? 1? ???? ??? ?, 10? ??? ? 0.1 strach ??
  nutrient agar plate? spreading
• Spreading ? ??? 55? ????? 1-2?? ??
• Colony ??? ????? ?? clear zone? ??? ??? nutrient
  broth? ?? ??
• Broth?? ??? ???? nutrient agar plate? ???.
• Colony? ????? ??
• ?????? glycerol(20) stock
• -20? ?? -70? ??? ??

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Serial Dilution
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?? ? ?????

• 1. ??? ?? ??? ??
• 2. ??? ?? ?? ??
• 3. ??? ??? ??? ??

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???? ?? ?? ? ?? ?? ??
Sang-Heum, Shin Hyoung-Joo, Lee URL
http//www.ajou.ac.kr/fereng/Department of
Molecular Science and TechnologyAjou University
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?? ??
???? ?? ?? ??? ??, ?? , ??
Spectrophotometer ??
Temperature, pH ,NaCl glucose concentration
Optimal condition
???? ???? ??
?? ?? ??
??????, ??????
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Introduction

• ??? ????? ??
• ??? ?? ?? ??
• ??? ??? ??? ??
• ?? ??

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??? ?? ??? ??

• ?? ?? ?? ???(lag phase), ?? ???(exponential
  phase),
• ???(stationary
  phase), ???(death phase)? 4 ?? ?? ??

• ???(lag phase)
• ???? ??? ??
• ?? ???? ??
• RNA ??, DNA ??
• ??? ???? ??
• ???(exponential phase)
• ?? ????? ???? ??
• ????, ??? ??? ??
• ???? ????, ???? ??? ??
• ??? ??? ??? ?? ?? ??
• ??? ??? ??? ?? ???? ?? ??
• RNA ??? ??, DNA ??

• ???(stationary phase)
• ???? ??, ???? ??
• ????? ??, ?????? ??
• pH? ??, ??????? ???? ??
• ??(spore)? ???? ??
• ???? ??? ???? ???
• ???(death phase)
• ???? ???? ???? ???? ??
• ????? ?? DNA, RNA??, ?????, ?????, ?? ??? ?? ?

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Growth curve
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??? ??? ??? ??

• ??? ??
• (1) ??
• ????? -1095? ???? ??
• Optimum, Miminum, Maximum temperature
• - ??? (Psychrophile)
• - ??? (Mesophile)
• - ??? (Thermophile)
• (2) ??
• ???? ????? 1???? ??
• (3) ???
• -????(???? ?)
• - ????, ?????, ?????, ?????
• (4) pH
• - ?? ?? (Acidophile)
• - ?? ?? (neutrophile)
• - ???? ?? (Alkalophile)

• ??? ??
• (1) ????(AW water activity)
• (2) ??
• ?? ???? (obligate aerobes)
• ?????? (facultative anaerobes)
• -?? ???? (aerophobic anaerobes)
• ????? (microaerophiles)
• (3) ????
• - ?? ?? ???
• ???? ??
• (1) Metabolism(??)
• ?? ????
• (2) Competition(??)
• ???? ?? ??, ????? ??? ??
• (3) Antagonism(??)
• ???? ???
• (4) Synergism(????)
• ?? ???? ???? ??? ?? ??

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??? ?? ?? ??

• ?? ???(Dry weight)
• - ??? ??, Desicator ??,????
• - ?? ???? ??
• - ????? ?? ??, ??? ?? ??
• - ???? ???? ??? ?? ???
• ?????
• ?? ??? ???? ???? ????
• ???? ??
• - ???? ???? ????? ??
• Packed volume ???
• - ???? Packed volume ???
• - ?????? ?? ?????,
• ???? ??? ??
• - ??? ?? ?? ??? ??? ??
• - ??????? ????? ?,
• ??? ???? ?? ??
• - ???? ????? ??? ????

• ??? ???(???)
• ?? ???? ???? ??(O.Doptical density) ???? ??
• 660nm ?? ??
• ?????
• 1.????(counting chamber)
• - heamatometer
• 2.??????(Coulter count)
• - ??? ??? ??? ??? ??,
• ????? ? ??? ????? pulse? ???
• 3.Membane filter?
• - ???? ??? ? ?? memebrane filter?
• ???? ???? ??? ???? ? ? ??
• ???? ?? ???? ??
• - 100ml/100? ??
• ?????
• plate counting, MPN method (??)
• ?? ??? ??

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Indirect Measurements of Microbial Growth
Turbidity

• ???? ??? ???? ???? ???? ?? ??? ????? ???? ?? ???
  ??? ???? ??? ?????? ????? ??? ? ?? ??? ??.
• ??? ??? ???? ??? ? ??? ??? ??? ????? ??? ??? ???
  ????? ??? ??? ?? ??.

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Spectrophotometer

• ??? 600nm ? ??.
• ???? ?? ??? ????.
• ???? ????.
• O.D(optical density) 0.2-0.8 ????? ??.
• ???? ??? ? ????? ???.

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• ??? ????? ??? Photometer(???) ??
  Spectrophotometer(????)? ?? ??.

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Generation time

• 1 ?? ??? 2?? ??? ???? ??
• ???????, ??? ???? X 2???? X b
• 
  a X 2n (a??? ??, n ???, b??)
• ?) 30??? ???? ?? 2??? 3?? ?? ? ??? ?????
• 128??
• ?) ????? 14?? 24?? 36?? ?? ???? ? ??? ??????
• 24?

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Materials and Methods

• Strains
• ? Escherichia coli
• Medium
• ? LB ??
• Culture condition
• A. pH pH4, pH7
• B. Glucose 0, 0.5, 1
• C. Temperature 37, 50OC
• D. NaCl 0, 1, 5
• 37OC Shaking incubator
• Spectrophotometer
• 50ml / 250ml flask

• Seed culture
• ???? ???? ??? ???
• clean bench ?? ??(2-3?)
• Main culture
• ?? ? 12 h ??
• Sampling
• 2 h? ? 12 h ??
• ??? ??
• 9ml ???? ??? 1ml ??
• Vortex

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?? ???

• ????? ??, ????
• ????? activity ????
• - Kirby-Bauer antibiotic sensitivity procrdure
  (paper disc method)
• - MIC (Minimum inhibitory concentration)
• ??? LOX

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????? ?? ??? ?? ??
Sang-heum, Shin Hyoung-Joo, Lee Fermentation and
Metabolic Lab. URL http//www.ajou.ac.kr/fereng
/
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• ???(Antibiotics)? ???(Antibacterials)

1928? Alexander Fleming? penicillin? ?? ????,
1943? ???????? ??? ??, ?? ??? ???? ???? ???? ??
?? ???? ???? ??. ??? 1997?? ??? ???? ??? ????
???? Vancomycin?? ???? ?? ??? ???? ??? ?? ?? ???
??? ???? ??? ?? ??? ???? ??.

• ??? ??(Antibiotic Resistance)

???? ??? ?? ?? ?? ?? ??? ?? ????? ?????. ? ??
???? ???? ? ??? ?? ???? ????, ????? ?? ???? ?????
???? ??. ?? ????? ??? ?? ??? ??? ???? ???? ???
???? ????? ? ???.
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??? ??
Bacteria are classified as gram positive or gram
negative depending on whether or not they take
up gram stain. (Hans C. J. Gram)

• Gram positive bacteria

• Gram negative bacteria

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Gram-positive bacteria
Gram positive bacteria retain the dye Gentian
violet taken to the thick cross-linked
polysaccharide. Staphylococcus aureus,
Streptococcus pneumoniae, Enterococci
Gram-negative bacteria
Have additional thick outer membrane, which
consists of lipopolysaccharides, proteins, and
phospholipids.
Neisseria meningitidis, Escherichia coli,
Haemophilus influenzae, Pseudomonas aeruginosa
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???? ??? ? ???

• - b-Lactam ?
• - Aminoglycoside ?
• - Quinolone ?
• - Tetracycline ?
• - Sulfonamide? trimethoprim ?
• - Glycopeptide ?

• ?? ? ??
• ????

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???? ??? ??? ????
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b-Lactam ? ???
ß-Lactam ????? Penicillin, Cephalosporin,
Monobactam, Carbapenem ? ?? ??.
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b-Lactam ? ???? ??? ?????

• ???(cell wall)? ??? ??? ????, ???? ?? ??? ???
  ????. ???
• ??? ???? ???? ???? ???? ???? ??.
• ???? polysaccharide? polypeptide chain ?
  covalent link? peptidoglycan?? ??.
• ???? ???? transcarboxylase? transpeptidase?
  ????, ?? ??? penicillin?
• ? ???? ??? ?? ??? penicillin binding protein
  (PBP)?? ???. ?? ß-lactam
• ???? ?? PBP? ???? ? ??? ????, ????? ??? ???
  ????
• ???? ????

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?????? b-Lactam ? ???
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Aminoglycoside ? ???
Aminoglycoside ????? Gentamicin , Micronomicin
Streptomycin , Kanamycin ?? ??.
Gentamicin
Kanamycin
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Aminoglycoside ? ???? ??? ?????

• Aminoglycoside? ?? ribosome? 30S subunit? ??????
  ???? ?????
• ???? ??? ?????.

• Aminoglycoside? ??? ribosome? ?????? mRNA? ??
  (translation)? ?
• ?? ??.

• ?? aminoglycoside? ribosome? ?? ?? ??
  (misreading)? ????
• ??? ???? ????? ??, ?? ??? ??? ???? ??.

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Quinolone ? ???
Quinolone ? ???? 1962? nalidixic acid? ??? ?? ??
????? ??????, ?? fluoroquinolone ? ????? ?? ????
???? ??. (Norfloxacin, Ofloxacin, Ciprofloxacin,
Enoxacin ?) ????? ???, ???, ?? ? ??? ???? ???
??.
Norfloxacin
Nalidixic acid
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Quinolone ? ???? ??? ?????

• Quinolone? ??? DNA ??? ???? ??? ?????.

• ? ??? DNA gyrase(topoisomerase IV)??.
• ??? DNA? ??? ?????, ? ??? ??? ??? ?? ??.

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• ???? ? DNA? ?? ?? ?? ???? ???? ? ?? ???,
• ???? ???? supercopiling??? ??.

• ?? ??? ???? ???? DNA? ???? ???,
• ?? ???? supercoiling? 2?????? ??? ??.
• DNA gyrase? ? ??? ???? ????,
• quinolone? ? ??? ?????? DNA ??? ????.

DNA gyrase
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Tetracycline ? ???

• Tetracycline? ????? ??? 3?? group?? ???.
• Short-acting tetracyclines Chlortetracycline,
  Oxytetracycline,
• Tetracycline
• Intermediate-acting tetracyclines
  Domeclocycline,
• Methacycline
• Long-acting tetracyclines Doxycycline,
  Minocycline

Tetracycline? ?? ??
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Tetracycline ? ???? ??? ?????

• Tetracycline? ??? ?? ??? ??? ???? ????.

• ? ???? ??? ??? ??? ??? ?? ?? ????,
• ribosome? 30S subunit? ????? ????.

• Tetracycline? ??? 30S subunit? aminoacyl tRNA?
  ???? ???,
• ?? ??? ??? ??? ?? ???.

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Sulfonamide? trimethoprim ? ???

• Sulfonamide? ??? folic acid ??? ????
  para-aminobenzoic
• acid (PABA)? ??? ??? ??? ??

• Trimethoprim (TMP)? pyrimidine ?????

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Sulfonamide? trimethoprim ? ???? ??? ?????

• Sulfonamide? ??? folic acid ????? ?? ???? PABA?
  dihydrofolic acid?
• ???? dihydropteroate synthetase ??? ????.

• ?? TMP? ??? ?? ???? dihydrofolic acid?
  tetrahydrofolic acid?
• ???? dihydrofolate reductase ??? ????.

???? ??? folic acid? ???? ????, ??? purine ???
sulfonamide ?? TMP? ??? ???? ???.
?? ??? ??? ?? purine ??? ????, ?? ??? DNA ??? ???
??.
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Glycopeptide ? ???
Glycopeptide ? ????? Vancomycin, Teicoplanin,
Ristocetin, Avoparcin ?? ??.
Vancomycin
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Glycopeptide ? ???? ??? ?????

• ??? ???? peptidoglycan? teichoic acid? ???? ??.

Peptidoglycan? ???? penicillin-binding protein
(PBP)? ?? (transcarboxylation ?
transpeptidation)? ??? ?? ??? ?? ?? ????.
Glycopeptide? peptidoglycan ????
D-alanyl-D-alanine ??? ???? PBP? ? ??? ???? ???
??.
? ??? peptidoglycan ???? transpeptidation? ??
???? ??? ??? ??, ??? ?? ?? ??? ??? ??? ??? ??.
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??? ? ???? ????? ?? ????!
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??? ???? ??? ??? ?????

• Case 1. ??? ?? ???? ?? ? ?? ??

• Case 2. ?? ??? ??

• Case 3. ???? ??? ??? ??

• Case 4. ?? ??? ???? ??(using efflux pumps)

Bacteria become resistant to antibiotics either
through genetic mutations or by acquiring
resistance genes from other bacteria. Resistance
is cyclical. Current emphasis is on gram-positive
infections.
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??? ?? ???? ????

• ß-Lactamase? ? ???? ???.

• ß-Lactamase? ß-lactam ??? ?????? ????
• ??????.

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?? ??? ??

• ???? ???? ??? ?? ???? ??????,
• ?? ???? ????? ??? ??? ???? ??? ??.

S. aureus? ??? PBP (PBP 2)? ??? ??? methicilin?
??? ???. PBP 2? ??? PBP? ??? b-lactam ???? ????
?? ??.
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???? ??? ??? ??
?? ??? porin ???? ???? ?? ?? ???? ????. ????
porin ???? ??? ??? ?? ??? ??? ? ????, porin ?
??, ???? ??? ???? ??? ???.
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?? ???? ??? ??
Gram-negative bacteria can have one- or
three-component efflux pumps that remove the
antibiotic from a cell. Related but different
pumps may work together to efflux the same
substrates
?? ??? ???? ??? ???? ????? ????.
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Kirby-Bauer antibiotic sensitivity procedure
Paper disc method.
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Clear zone ?? ??