5. Determine cell biomass by observing the absorbance of cultures at 600 nm using a spectrophotometer.
6. Check the OD at every 30 min interval and record.
7. Plot a standardized growth curve of the organism by means of
this OD value (absorbance versus time).
8. Calculate generation time.
2.4 Gram Stain
Microscopic
Observation
Symbiotic bacterial isolates are additionally examined for their gram
reaction and shape. Typically all the isolates are Gram negative and
rod shaped.
2.4.1 Materials Required
l
Gram stain kit.
l
Culture.
l
Glass slide.
l
Absorbent paper.
l
Light microscope.
2.4.2 Procedure
1. Smear a complete loop of culture on a clean glass slide.
2. Air-dry the slide and later heat-fix for 1 min.
3. Add one or two drops of crystal violet staining reagent.
4. Rinse the slide gently using tap water.
5. Add one or two drops of Gram’s iodine and wait for 1 min.
6. Wash the slide gently with tap water.
7. Add decolorizing agent (acetone-alcohol decolorizer) gently,
one drop at a time to slide until decolorizing agent goes clear
from the slide.
8. Add counter stain, one or two drops of safranin and wait for
30 s to 1 min.
9. Rinse slide gently using tap water until no color is visible on the
slide.
10. Spot dry with absorbent paper.
11. Observe the results of the staining procedure under oil immersion (100Â) employing light microscope.
2.4.3 Expected Result
Gram-negative bacteria: stain pink.
Gram positive bacteria: stain purple.
2.5 Motility
There are a numerous methods to establish the motility of the
bacterium-biochemical experiments and microscopic examination.
Microscopy is the most precise way to assess bacterial motility, and
hanging drop method is a frequently used microscopic technique.
Morphological Characterization
121
6. Check the OD at every 30 min interval and record.
7. Plot a standardized growth curve of the organism by means of
this OD value (absorbance versus time).
8. Calculate generation time.
2.4 Gram Stain
Microscopic
Observation
Symbiotic bacterial isolates are additionally examined for their gram
reaction and shape. Typically all the isolates are Gram negative and
rod shaped.
2.4.1 Materials Required
l
Gram stain kit.
l
Culture.
l
Glass slide.
l
Absorbent paper.
l
Light microscope.
2.4.2 Procedure
1. Smear a complete loop of culture on a clean glass slide.
2. Air-dry the slide and later heat-fix for 1 min.
3. Add one or two drops of crystal violet staining reagent.
4. Rinse the slide gently using tap water.
5. Add one or two drops of Gram’s iodine and wait for 1 min.
6. Wash the slide gently with tap water.
7. Add decolorizing agent (acetone-alcohol decolorizer) gently,
one drop at a time to slide until decolorizing agent goes clear
from the slide.
8. Add counter stain, one or two drops of safranin and wait for
30 s to 1 min.
9. Rinse slide gently using tap water until no color is visible on the
slide.
10. Spot dry with absorbent paper.
11. Observe the results of the staining procedure under oil immersion (100Â) employing light microscope.
2.4.3 Expected Result
Gram-negative bacteria: stain pink.
Gram positive bacteria: stain purple.
2.5 Motility
There are a numerous methods to establish the motility of the
bacterium-biochemical experiments and microscopic examination.
Microscopy is the most precise way to assess bacterial motility, and
hanging drop method is a frequently used microscopic technique.
Morphological Characterization
121