Examination of Bacterial Plates

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

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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. The addition of a counterstain or secondary stain (safranin) is used to stain the decolorized vegetative cells. When visualized under microscopy the cells should have three characteristics: the vegetative cells should appear pink, the vegetative cells that contain endospores should stain pink while the spores should be seen as green ellipses within the cells. Mature, free endospores should not be associated with the vegetative bacteria and should be seen as green ellipses.

Conclusion
The Schaeffer-Fulton stain is a technique designed to isolate endospores by staining any present endospores green, and any other bacterial bodies red.The green stain is malachite green, and the counterstain is safranin, which dyes any other bacterial bodies red. | | |

References 1. Tortora, Funke, Case (2010). Microbiology : An introduction,10th

Questions

1. From the procedure above, can you determine the intracellular location of the spore? Can this also used for taxanomic purposes?
When viewed unstained, endospores of living bacilli appear edged in black and are very bright and refractile. Endospores strongly resist application of simple stains or dyes and hence appear as nonstaining entities in Gram-stain preparations. However, once stained, endospores are quite resistant to decolorization. This is the basis of several spore stains such as the Schaeffer-Fulton staining method which also differentiates the spores from sporangia and vegetative cells. However this method is not suitable for taxanomic purposes.

References: 1. Tortora, Funke, Case (2010). Microbiology : An introduction,10th Questions 1. From the procedure above, can you determine the intracellular location of the spore? Can this also used for taxanomic purposes? When viewed unstained, endospores of living bacilli appear edged in black and are very bright and refractile. Endospores strongly resist application of simple stains or dyes and hence appear as nonstaining entities in Gram-stain preparations. However, once stained, endospores are quite resistant to decolorization. This is the basis of several spore stains such as the Schaeffer-Fulton staining method which also differentiates the spores from sporangia and vegetative cells. However this method is not suitable for taxanomic purposes.