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Mother plate First quadrant streak
colony characterization
First glycerol stock Second quadrant streak Second glycerol stock Third glycerol stock
Gram’s staining Oxidase test Catalase test Broth
Broth characterization
Motility check at 18th hour Antibiotic sensitivity test
Spore staining IMViC assay TSI test
Carbohydrate fermentation test:
Minimum of 3 carbohydrates Growth curve
Remember to
document the
results of
each and
every
experiment
Characterization of bacteria
Macro- & micro-
morphological analysis
Chemotaxonomy Molecular taxonomy
 Colony morphology
 Cell shape and size
 Mobility
 Total cellular protein fingerprinting
 16S rRNA gene sequencing
 Restriction digestion
 Extracellular polysaccharide profiling
 Cell wall composition
 KoH assay
 Oxidase
 Catalase
 IMViC
 TSI
 Carbohydrate fermentation
 Urease test
 Nitrate reduction test
 Antibiotic sensitivity assay
Pour plate Spread plate
Same soil sample: @10-5 dilution
Mother plate First quadrant streak
colony characterization
First glycerol stock Second quadrant streak Second glycerol stock Third glycerol stock
Gram’s staining Oxidase test Catalase test Broth
Broth characterization
Motility check at 18th hour Antibiotic sensitivity test
Spore staining IMViC assay TSI test
Carbohydrate fermentation test:
Minimum of 3 carbohydrates Growth curve
Remember to
document the
results of
each and
every
experiment
NCT45 = Tax. Score = 0.692
Tax. Score = sum of all score / no. of assay
Capsule and negative staining
 Butt should be 40% length of the tube.
 Slant should be in equal length to butt (up to 80% length of the tube).
 Way of inoculation: stab & streak
Triple sugar iron test
Glucose (0.1%)
Sucrose (1%)
Lactose (1%)
Observed within 15
seconds
68 KDa
1
1
2
3
4
0
0
0
1
2
3
4
NCT27 = BSA004
1
1
1
2
3
4
5
6
7
8 NCT70 = BSA118
Include student name at the bottom of the gel
Student who did the isolation, not the gel.
NCT45 = Tax. Score = 0.692
Tax. Score = sum of all score / no. of assay
Step 1 Step 2 Step 3
Visualization of the
experiment before
entering the lab
Execution of the
experiment
Cleaning before
leaving the lab
How to identify a bacterial species? Dos and Don'ts
How to identify a bacterial species? Dos and Don'ts
How to identify a bacterial species? Dos and Don'ts
How to identify a bacterial species? Dos and Don'ts

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How to identify a bacterial species? Dos and Don'ts

  • 1. Mother plate First quadrant streak colony characterization First glycerol stock Second quadrant streak Second glycerol stock Third glycerol stock Gram’s staining Oxidase test Catalase test Broth Broth characterization Motility check at 18th hour Antibiotic sensitivity test Spore staining IMViC assay TSI test Carbohydrate fermentation test: Minimum of 3 carbohydrates Growth curve Remember to document the results of each and every experiment
  • 2. Characterization of bacteria Macro- & micro- morphological analysis Chemotaxonomy Molecular taxonomy  Colony morphology  Cell shape and size  Mobility  Total cellular protein fingerprinting  16S rRNA gene sequencing  Restriction digestion  Extracellular polysaccharide profiling  Cell wall composition  KoH assay  Oxidase  Catalase  IMViC  TSI  Carbohydrate fermentation  Urease test  Nitrate reduction test  Antibiotic sensitivity assay
  • 3.
  • 4. Pour plate Spread plate Same soil sample: @10-5 dilution
  • 5.
  • 6. Mother plate First quadrant streak colony characterization First glycerol stock Second quadrant streak Second glycerol stock Third glycerol stock Gram’s staining Oxidase test Catalase test Broth Broth characterization Motility check at 18th hour Antibiotic sensitivity test Spore staining IMViC assay TSI test Carbohydrate fermentation test: Minimum of 3 carbohydrates Growth curve Remember to document the results of each and every experiment
  • 7. NCT45 = Tax. Score = 0.692 Tax. Score = sum of all score / no. of assay
  • 8.
  • 9.
  • 10.
  • 11.
  • 12.
  • 13.
  • 14.
  • 15.
  • 16.
  • 18.
  • 19.  Butt should be 40% length of the tube.  Slant should be in equal length to butt (up to 80% length of the tube).  Way of inoculation: stab & streak Triple sugar iron test Glucose (0.1%) Sucrose (1%) Lactose (1%)
  • 20.
  • 22.
  • 23.
  • 24.
  • 25.
  • 26.
  • 27.
  • 28. 68 KDa 1 1 2 3 4 0 0 0 1 2 3 4 NCT27 = BSA004 1 1 1 2 3 4 5 6 7 8 NCT70 = BSA118
  • 29. Include student name at the bottom of the gel Student who did the isolation, not the gel.
  • 30.
  • 31. NCT45 = Tax. Score = 0.692 Tax. Score = sum of all score / no. of assay
  • 32. Step 1 Step 2 Step 3 Visualization of the experiment before entering the lab Execution of the experiment Cleaning before leaving the lab