Examination of Bacterial Plates

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Practical 9: Examination of Bacterial Plates
Objective:
1. To examine bacteria pigmentation, colony, margin characteristics, elevation properties, broth characteristics and agar stroke properties. 2. To examine bacteria growth characteristics on different culture media.

Introduction:
Bacterial species can sometimes be identified on the basis of how they appear on or in the different media. The pigmentation, size and shape of bacterial colonies as they grow on and in agar plates can provide identifying signs. Observing the growth characteristics of organisms in broth cultures can also be helpful. The major problems that may arise are contamination and species variations. These must be eliminated if good results are to be obtained.

Materials
1. Nutrient agar plates of
a) Sarcina lutea and Serratia marcescens
b) Bacillus subtilis and Escherichia coli
c) Streptococcus fecalis and Pseudomonas fluoroscens
2. 24 hour pour plate preparation of
a) Serratia marcescens
b) Sarcina lutea
c) Bacillus subtilis
d) Escherichia coli
3. 24 hour agar slant of the following cultures incubated at 25ºC a) Bacillus subtilis
b) Escherichia coli
c) Serratia marcescens
d) Pseudomonas fluoroscens
4. 48 hour nutrient broth cultures of the following cultures incubated at 25ºC a) Bacillus subtilis
b) Serratia marcescens
c) Escherichia coli

Procedures

1. The various cultures provided were examined and observation was recorded. 2. The broth was observed carefully without disturbed or shaken as this may dislodge the surface growth. 3. All results included the following observations

a) Pigmentation
b) Colony form
c) Margin characteristics
d) Elevation properties
e) Broth characteristics
f) Agar stroke properties

.

Results:
Indicator:
a) Pigmentation
b) Colony form
c) Margin characteristics
d) Elevation properties
e) Broth characteristics
f) Agar stroke properties

1. Nutrient agar plates of
Bacteria species| a| b| c| d| e| f|
Sarcina lutea and Serratia marcescens| | | | | | |
Bacillus subtilis and Escherichia coli| | | | | | |
Streptococcus fecalis and Pseudomonas fluoroscens| | | | | | |

Indicator:
a) Pigmentation
b) Colony form
c) Margin characteristics
d) Elevation properties
e) Broth characteristics
f) Agar stroke properties

2. 24 hour pour plate preparation of
Bacteria species| a| b| c| d| e| f|
Serratia marcescens| | | | | | |
Sarcina lutea | | | | | | |
Bacillus subtilis| | | | | | |
Escherichia coli| | | | | | |

Indicator:
a) Pigmentation
b) Colony form
c) Margin characteristics
d) Elevation properties
e) Broth characteristics
f) Agar stroke properties
3. 24 hour agar slant of the following cultures incubated at 25ºC Bacteria species| a| b| c| d| e| f|
Bacillus subtilis| | | | | | |
Escherichia coli| | | | | | |
Serratia marcescens| | | | | | |
Pseudomonas fluoroscens| | | | | | |

4. 48 hour nutrient broth cultures of the following cultures incubated at 25ºC Bacteria species| a| b| c| d| e| f|
Bacillus subtilis| | | | | | |
Serratia marcescens | | | | | | |
Escherichia coli| | | | | | |

Discussion
A differential staining technique (the Schaeffer-Fulton method) is used to distinguish between the vegetative cells and the endospores. A primary stain (malachite green) is used to stain the endospores. Because endospores have a keratin covering and resist staining, the malachite green will be forced into the endospores by heating. In this technique heating acts as a mordant. Water is used to decolorize the cells, as the endospores are resistant to staining, the endospores are equally resistant to de-staining and will retain the primary dye while the vegetative cells will lose the stain....
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