Laboratory Report 10 and 11
VMICRO 2200 LAB
Name: Joan S. Estrella
Section and Laboratory: DVM 2-2 / LAB B
Laboratory Schedule: Wednesday (07:00 – 10:00 am)
QUESTIONS
1. Discuss the importance of evaluating microbial growth after a given period of
incubation. (5 pts)
Microbes can expand rapidly when given the right conditions such as food or nutritional
requirement, the appropriate temperature, radiation, osmotic pressure and oxygen. Which could
either be detrimental or advantageous for humans so knowing how they grow will help us forecast
or manage their development under specific circumstances.
The significance of monitoring microbial growth after a specific incubation period will help
us predict, monitor and even control their growth. Predicting the growth of microorganism based
on the calculated growth curve or by measuring the increase in population, monitor its increasing
or decreasing population and manage or control the fast proliferation process of the microbes.
Furthermore, evaluating the microbial growth of a cultured media after an incubation will aid us
in knowing if there is an increase or decrease in the colony’s population, whether they thrive or
not on a specific environment and set of conditions.
2.
Describe the different criteria or parameters that should be considered in evaluating
different species of bacteria. (5 pts)
There are different criteria or parameters that we should consider while evaluating different
species of bacteria which includes its morphological structure and attributes, and its biochemical
composition or characterization.
Bacterial colony morphology is a method employed by scientists and researchers to best
describe and identify bacteria by their species. A bacterial colony may differ in its morphological
features such as their form, size, elevation, margin, surface, opacity and color. Form is basically
the shape that forms in a bacterial colony of a cultured medium, it includes a circular, irregular,
filamentous and rhizoid shape. Size refers to the diameter of the colony formed, how big or small
the bacterial growth that has taken place. Small colonies are known as punctiform while elevation
describes the side view of a given colony. Its elevation can either be raised, convex, flat, umbonate,
crateriform or pulvinate. Margins or border represents the edge of the colony which includes the
following: entire, undulate, lobate, filamentous and curved. Other than that, the surface describes
the appearance of the colony. Its texture can either be smooth, rough, glistening, wrinkled or dull.
For opacity, it provides us with a better description if a cultured media is transparent (clear),
opaque, or translucent (like a frosted glass). The last physical attribute that should be considered
is their color or pigmentation.
Biochemical characterization of different bacteria based on different test to distinguish their
biochemical properties that sets them apart from other bacterial microbes. The following
biochemical and physiological traits are used to identify bacteria: motility, enzyme production
(catalase, coagulase, hemolysins, etc.), resistance to inhibitory substances (high salt, antibiotics,
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etc.), nutrient utilization (carbohydrate utilization, amino acid degradation, and lipid degradation),
and lipid degradation.
Indole test is used to determine if the cultured bacterial medium can metabolize tryptophan,
whereas VP test determines whether acetylmethylcarbinol is produced from glucose fermentation
from a given cultured organism. For lactose test, the result can be distinguish based on observation
as the medium changes its color into yellow then the culture is capable of neutralizing lactose and
otherwise if it turns red then it is not capable of neutralizing lactose. These are some of the
biochemical tests that are used to differentiate and evaluate species of microbes. Different reacting
agents are used for every biochemical test to determine its properties that make them distinct from
other bacteria.
3.
Describe a typical colony of fungus. (5 pts)
A fungal colony typically grows in a solid nutrient agar medium as molds and they’re made
up of fungal hyphae of a single cell spore. They flourish at a pH range of approximately 5 to 6 pH
level. This colony has a filamentous and rhizoid shape. Their appearance also has a hairy look and
their texture is powder-like. It often has fuzzy edges too. Other than that, a fungus in a colony may
be unicellular or multicellular organisms. Furthermore, it has a filamentous margin unlike bacterial
colonies that have a fixed margin.
Fungal colony thrives in a medium that is rich in nitrogen and carbohydrates sources, a pH
range of about 5 to 6, and a temperature range of 15 to 37⁰C. High moisture content and moderate
temperatures are ideal for fungal colony growth as it can affect the growth of fungus in a cultured
medium. It can also grow or be cultured not only in a solid mycological nutrient agar but also in
mycological broth.
4.
What is the Indole Test? Discuss how an indole test is done by giving emphasis on the
media and reagents used and the changes that take place in a positive indole reaction in
comparison with a negative one. (5 pts)
Indole test is a procedure used to determine if a microbial organism can metabolize tryptophan.
This test shows that some bacteria can break down the medium-accumulating amino acid
tryptophan to produce indole. Furthermore, the test for indole synthesis is significant in identifying
and distinguishing members of the Enterobacteriaceae family.
There are two methods employed in the indole test. This includes the traditional technique
in tube method, which after an overnight incubation will detect weak indole-producing microbes
and spot indole test that can detect if a bacterial microorganism produces indole faster in
comparison with tube method.
For a rapid spot test, a piece of filter paper is moistened and placed in a clean petri dish
until it is saturated. Then, with the use of an inoculating loop a portion of the incubated colony
was rub into the filter paper and was applied with a couple of drops of cinnamaldehyde reagent
afterwards to test the microbes. The resulting color of the sample will vary depending on the
outcome of the test. A positive result of the Indole rapid spot test will mark a change in color of
the bacterial smear, the original color of the solution will turn blue to blue-green color. Within a
few seconds or more upon the introduction of cinnamaldehyde agent (containing pdimethylaminocinnamaldehyde, HCl and deionized water) that is used in this test, a change in
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appearance (by means of color) is observed. A positive reaction will result to a blue to blue-green
appearance whereas a colorless or no change in color after the addition of the indole reagent on
the sample will indicate that the result of the test is negative.
On the other hand, a tube method is employed when a combination of broth culture and Kovacs
reagent (benzaldehyde reagent) or Ehrlich’s reagent (alternative) are used. Ehrlich's reagent is
utilized for anaerobes and weak indole makers, while Kovac's reagent is used for aerobic
organisms. A small portion of the inoculated broth is used in a separate test tube to be incubated
at 37⁰C for a day or 24 hours. Then Kovacs reagent or Ehrlich is added to the broth to test if the
culture medium is indole positive or not. The resulting color of the positive reaction will change
into pink to red color. Within seconds of introducing the reagent, the medium's top layer of the
reagent layer begins to take on a pink to red hue. It forms a "cherry-red ring" on the top layer of
the solution as it rises to the surface of the soup and leaves an oily, red film, whereas, a colorless
or no change in color to a slightly yellow after the addition of the reagent on the sample will
indicate that the result to the indole test is negative.
5.
What is the Voges-Proskauer test? Discuss how the VP test is done by giving emphasis
on the media and reagents used and the changes that take place in a positive VP reaction in
comparison with a negative one. (5 pts)
Voges-Proskauer test, also known as VP test is a procedure which is used to distinguish
between the two major types of facultative anaerobic enteric bacteria based on the generation of
neural products. VP test determines whether acetylmethylcarbinol is produced from glucose
fermentation from a given cultured organism. This test was named and created by German
microbiologist/scientist Daniel Wilhelm Otto Voges and Bernard Proskauer in 1898. They’re the
ones who first notices the red color reaction caused by an appropriate culture media after a
treatment procedure was done with potassium hydroxide.
Voges-Proskauer test used methyl-red Voges-Proskauer broth as its culture medium which is
made up of the following material or ingredients: buffered polypeptone 7 g, glucose 5 g,
dipotassium phosphate 5 g, and distilled water 1 L. The final pH is 6.9. Other than that, it also uses
agents such as 5% α- naphthol in 95% ethyl alcohol (5 g/100 mL) and Potassium hydroxide at 40%
(KOH).
First, methyl-red Voges-Proskauer broth should be inoculated with pure culture of the
organism and once it is done, incubate the medium for about 48 hours. In the next procedure, a
couple of droplets of reagents such as 5% α- naphthol in 95% ethyl alcohol (5 g/100 mL) and
Potassium hydroxide at 40% (KOH) must be added to a separate test tube with nutrient broth.
Lastly, allow the tube to stand undisturbed for 10 to 15 minutes after gently shaking it to expose
the medium to ambient oxygen. Observe the change in color to distinguish if it is positive with the
reacting agent. A positive reaction in a VP test will have a crimson to ruby pink or red hue of color
as its outcome, whereas, if there are no change in coloration then the result is negative.
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