Microorganism Cultivation Method in Liquid Media

							Exercise 1 : Methods of cultivating microorganisms

1.1 Inoculation of liquid media
      Microorganism                       E.coli               Pseduomonas aeruginosa
   Colour of the colonies           Pale yellowish                   Bluish green
     Amount of growth                  Moderate                        Moderate
      Surface growth             No pellicle formation             Pellicle formation
        Turbidity               Slightly turbid, uniform                 turbid
         Sediment                       granular                       flocculent

Discussion :
Broth media are usually used as enrichments, general cultivation and sterility testing. In this
experiment, Escherichia coli is a facultative anaerobe. It can grow with or without the oxygen
supply. Thus, there is no distinct layer of pellicle formation. However, Pseduomonas aeruginosa
is a strict aerobe which can grow only with oxygen supply. Thus, the colonies formed pellicle as
there was more oxygen supply at the top layer of the broth. The mild colorization of broth in
greenish-blue was due to the presence of a greenish-blue pigment called pyocyanin which was
produced by the bacteria Pseudomonas aeruginosa.

1.2 Inoculation on Solid Medium : Streak-plate Method
                                                                  Mixed culture
Characteristics         E.coli        P.aeruginosa           E.coli         epidermis
   Shape               Circular         Circular            Circular          Circular
    Size                1mm              1-2mm              1-2mm             1-2mm
Chromogenesis      Pale yellowish,    Bluish green      Pale yellowish, Pale yellowish,
                      insoluble         pigment,           insoluble         insoluble
                                         soluble
   Opacity             Opaque          Translucent          Opaque             Opaque
   Elevation           Convex            Raised             Convex             Raised
    Surface            Smooth            Smooth             Smooth             Smooth
     Edge               Entire            Entire             Entire             Entire

Discussion :
Streaking is a technique used in microbiology to isolate a pure strain from a single species of
microorganism. The inoculation loop is resterilized and dragged across the inoculated quadrant
of the streak plate. Each time the loop gathers fewer and fewer bacteria until it gathers just one
single bacterial cell that can grow into a colony. A bacteria can be grown by using this method so
that the it can be identified, studied, or tested. There are different type of agar medium that can
be used. For example, nutrient agar, minimum agar, blood agar, MacConkey and Thiosulfate
Citrate Bile Sucrose ( TCBS ). Different kinds of bacteria cultured in different media will show
different morphology and different results. TCBS and MacConkey are selective and differential
media which selects and limits the growth of certain types of bacteria. For example, there are red
colonies of E.coli grow on the MacConkey plate. This indicates that E.coli can utilize and
ferment lactose, forming acidic products which turn the indicator in the growth medium red, thus
causing the colonies to appear reddish as well. However, if other bacteria that cannot ferment
lactose were grown in this medium, no colours will be observed because they cannot form acidic
product that change the indicator into red colour. Besides, TCBS is suitable to be used in the
selective isolation of vibrios. Different type of vibrios will show different colour depending on
their ph because thymol blue is used as an indicator in this agar.
Exercise 2 : Microbial Flora Skin
          Organ                        Hand                                 Ear
          Shape                      Circular                             Circular
            Size             Very small, few punctiform               Relatively larger
                                     colonies                           ( 1–2mm )
          Colour                     Off white                           Off white
         Opacity                      Opaque                              Opaque
        Elevation                     Convex                              Convex
         Surface                      Smooth                             Glistering
           Edge                        Entire                              Entire
       Haemolysis                     Absent                              Present

Discussion :
For this exercise, bacteria were swab from ear and hand and streak on the blood agar. There are
very few bacteria colonies that swab from hand grow on the blood agar. It is believed that the
experiment conducter had sterilize or wash her hand before doing this exercise. However, for the
bacteria colonies that swab from ear, there is a big yellow circular colonies observed surrounding
one of the off-white colony. It is Haemolytic colonies which can break down red blood cells.

Exercise 3 : Characterization of Bacteria
3.1 Catalase Test
3.2 Oxidase Test

     Bacteria Type                     Catalase Test                   Oxidase Test
          E.coli                             +                              -
Pseuduomonas aeruginosa                      +                              +
Staphylococcus epidermis                     +                              -
  Staphylococcus aures                       +                              -
      Streptococcus                          -                              -

Discussion :
For catalase test, most aerobic or facultatively anaerobic that possesses catalase activity will give
positive result by production of bubble or effervescence. Since Streptococcus is an anaerobic
bacteria, it showed a negative result. For oxidase test, aerobic or facultatively anaerobic
orgamisms will show positive results. In this experiment, Pseuduomonas aeruginosa is the only
bacteria that shows positive result. Thus, it possesses oxidase activity and cytochrome oxidase
system. This test can be used to differentiate Pseudomonas family and enterobacteria family. E.
coli is one of the most important model organisms in enterobacteria family.

Exercise 4 : Microscopy

  Bacteria Type            Monochrome Staining                      Gram Staining
                      Cocci / Rod shape, singular in       Cocci / Rod shape, singular
                      arrangement and blue                 arrangement / arranged in
      Mixed                                                cluster, pink and purple



                      Cocci, arranged in cluster and       Cocci, arranged in cluster,
                      blue                                 purple
  Staphylococcus
     epidermis


                      Rod shape, singular in               Rod shaped, singular
                      arrangement and blue                 arrangement, pink
      E.coli




Discussion :
Monochrome staining is not sufficient to observe detailed morphological characteristics. Under
microscope observation, all the cells Escherichia coli and Staphylococcus epidermidis were
stained blue and only certain aspects ( colour, shape and arrangement ) were able to be observed.
However, gram staining is a better method to observe microorganism. In gram staining,
Staphylococcus epidermidis are stained purple while Escherichia coli are stained pink.
Hence, Staphylococcus epidermidis is gram positive whereas Escherichia coli is gram negative.
The bacteria are first stained with crystal violet and next treated with iodine to promote dye
retention. When gram positive bacteria are decolourized with ethanol, the alcohol is thougth to
shrink the pores of the thick peptidoglycan. Thus the dye-iodine complex is retained during the
short decolourization step and the bacteria remain purple. In contrast, gram negative
peptidoglycan is very thin, not as highly cross-linked, and has larger pores. Alcohol treatment
also may extract enough lipid from the gram negative wall to increase its porosity further. For
these reasons, alcohol more readily removes the purple crystal violet-iodine complex from gram
negative bacteria. The cell is stained pink with the counterstain carbol fushin.
         Some bacteria do not stain easily by staining procedures. Staining of these organisms is
facilitated by the application of heat during staining. Once stained, these organisms retain the dye,
even when treated with a decolourization agent such as acid alcohol; They are said to be acid-fast.
Another special stain method is spore staining. With the help of heat treatment, a primary stain
penetrates the coat and stains the spores whereas the cytoplasm will take up the secondary stain.
The spores are stained green while the cytoplasm is stained red.