Thursday, March 15, 2012


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Microorganisms are extremely small and can be isolated by different methods. The purpose of this lab was to take an unknown mixture containing two different types of bacteria and isolate a pure culture of each type and be able to identify what each bacteria was. This was done using the procedures we have learned thus far in lab.

More than one type of organism may show up in the selective environments that were used, that is why we kept isolating the bacteria from day to day. A pure culture is not present until there is one type of colony. The unknown bacteria was identified by streaking them on different petri plates, such as; DNAse, Pseudomonas Afar P (PAP), Pseudomonas Agar F (PAF), Nutrient Agar (NA), Blood Agar Plate (BAP), Colistin-Nalidxic Agar (CNA), MacConkey Agar (MAC) and Trypticase Soy Broth (TSB) plates. TTC deeps, TSI slants, tryptone broth, bile esculin broth, gram stains, catalase and oxidase tests were also used.

The first day the unknown suspension was streaked onto a CNA plate, MAC plate and a TSB plate. These plates were used because the CNA plate inhibits the growth of most gram negative rods. Streptococcus and Staphylococcus grow well on this type of a plate. Also some strains of Bacillus are able to grow. The MAC plate enables most gram negative bacteria to grow. Yeasts and gram positive bacteria are inhibited. When lactose utilizers are present the colonies appear pink to red, when absent they appear colorless to yellow. The TSB plate is an all purpose medium for isolating bacteria, to make sure there is actually bacteria present.

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During the second day gram stains were taken from the plates used in day one. This enabled me to determine what types of media and further tests to used to identify the bacteria based on the gram reaction. For gram negative bacteria a TSI slant, Tryptone broth, PAP plate, PAF plate, TTC deep and a DNAse plate were used. Colonies were taken from the MAC plates and streaked onto the plates, stabbed into the slants and mixed in with broths. The TSI slant was used to test for gas production and sugar utilization. The Tryptone broth detects the production of indole from tryptophan. Kovac’s reagent is added and it turns red if indole is produced. This is useful for the identification of E. coli. PAP plate and PAF plates are non-selective medias used for detection and differentiation between Pseudomonas. The pigments diffuse from the colonies of Pseudomonas into the agar. A PAP plate enhances the elaboration of pyocyanin and inhibits the formation of pyoverdin. Pyocyanin on Pseudomonas is a blue non-fluorescent color. On the PAF plate enhances the elaboration of pyoverdin and inhibits the formation of pyocyanin. Pyoverdin on Pseudomonas is a yellow-green pigment. The TTC deep tests for motility of the bacteria. A DNAse plate tests for production of DNAse, it differentiates between Serratia, E. coli, Enterobacter, S. aureus and S. epidermis.

For gram positive bacteria a DNAse plate, TTC deep, Mannitol salt plate, Bile esculin broth, NA plate and BAP plates were used. Colonies were taken from the TSB plates (because I had no growth on the CNA plate) and streaked onto the plates, stabbed into the deeps and mixed in with the broth. The Mannitol salt plates has a high salt concentration and inhibits most organisms except Staphylococci. It differentiates between S. epidermis and S. aureus. S. aureus have opaque colonies surrounded by a yellow zone and most S. epidermis fail to ferment mannitol, so the colonies appear red-purple. In the Bile esculin broth the bacteria grows on 4% bile and hydrolyzes esculin to esculetin and glucose, the media turns black. It is selective and differential for growth of Enterococcus. The Nutrient agar plate is an all purpose medium that allows the growth of Bacillus spores. A BAP plate is used to detect for hemolysis especially in Staphylococcus. Enterococcus won’t lyse sheep red blood cells. a᠃hemolysis is the partial lysis of red blood cells around the colony, resulting in a greenish discoloration around the colony. b hemolysis is the complete lysis of red blood cells around the colony, resulting in a transparent area around the colony. Non-hemolytic results in no lysis of red blood cells around the colony and no change in the agar.

Catalase and Oxidase tests were used for both gram negative and gram positive stains. In the Catalase test, the enzyme catalase decomposes hydrogen peroxide to water and oxygen, which results in vigorous bubbling if the test is positive. This is used to separate Staphylococci, which are positive from Streptococci that are negative. The oxidase test tests for cytochrome oxidase which is found in bacteria using oxidative phosphorylation. A positive test of a colony deposit turns purple within 10 seconds. This separates Pseudomonas and Azotobacter, which are positive from E. Coli, Staphylococcus, Streptococcus and Serratia marcescens, which are negative.


After a lot of observations of the plates, slants and deeps, gram stains, catalase and oxidase tests I was able to determine which unknowns I had. Please see attached tables of unknowns. From the tables, I have concluded that my unknowns are Escherichia coli and Bacillus subtilis.


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