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LABORATORY EXERCISE 12

ASEPTIC TRANSFER TECHNIQUES

INDIVIDUAL OUTPUT

STUDY QUESTIONS: Maximum of three short sentences per number. Cite your references.

1. Why is it important to sterilize the inoculating instrument before and after each inoculation?

The importance of flaming the inoculating instruments prior to and after each inoculation
is to assure sterility of the instruments so that there is no source of an external contaminant.

2. List the possible sources of contamination that you should be concerned about while transferring
bacteria from one culture to another.

a. Improper laboratory attire, contaminants from skin is exposed and is released into the air
b. Dust and other particles of hair gets near of the cultures
c. Unsterile work space and equipment (petri dishes & swabs)
d. Airborne contaminants

3. Why is it necessary to flame the mouth of the tube before and after performing an inoculation?

Passing the mouth of the test tube through a flame produces a convection current, which
forces air out of the tube. Doing this ensures that no microorganisms enter the mouth of the
vessel to contaminate the culture or the medium.

4. Differentiate the following terms:

a. Stock culture vs. Pure culture

A stock culture is a culture of a microorganism maintained solely for the purpose of


keeping the microorganism in a viable condition by subculture, as necessary, into fresh medium.
A pure culture is, in the ordinary bacteriologic sense, a culture consisting of a single species and
strain of a bacterium. (www.MediLexicon.com)

b. Aseptic vs. Sterile

Aseptic technique minimizes contamination by harmful virus and bacteria. A sterile


technique on the other hand, involves the elimination or absence of all microbial life.
(www.MediLexicon.com)
LABORATORY EXERCISE 13-A

SIMPLE TECHNIQUES TO COLLECT, CULTURE, AND VISUALIZE MICOROORGANISMS

INDIVIDUAL OUTPUT

STUDY QUESTIONS: Maximum of three short sentences per number. Cite your references.

1. What part of the bacterial cell (cell wall or protoplast) appears to play the most important role
in determining whether an organism is gram-positive?

Peptidoglycan, also called murein, is a polymer that makes up the cell wall of most
bacteria. Bacteria are classified as being either Gram-positive or Gram-negative based in
differences in the structure of their peptidoglycan cell wall. (biologydictionary.net)

2. What is the purpose of heat fixation? What happens when too much heat is applied?

Heat fixation kills bacteria without destroying it, causing it to stay fixed and adhered on
to the slide and makes microbes more permeable to stains. Applying too much heat, however,
destroys the cell, which makes it useless for viewing. There is also a possible chance that the glass
slide will break.

3. Which is more effective in staining, acidic or basic dyes? Why?

Basic dyes. They have a positively charge chromogen that forms an ionic bond with
negatively charged bacterial cell and thus colorize the bacterium.

4. What is negative staining? Describe the advantages and disadvantages of negative staining.

In contrast to direct stains that bind to bacteria directly, a negative stain colors the
background of a smear rather than the bacteria. The advantages of negative staining are; bacteria
are not heat fixed so they do not shrink, and some bacterial species resist basic stains and one
way they can be visualized is with the negative stain. However, negative staining does not
differentiate bacteria – one can only determine morphology. (Watson, 2010).

5. Describe the advantages of differential staining procedures compared with simple staining
techniques.

The simple stain is a very simple staining procedure involving only one stain in one type
of bacteria. It can be used to determine cell shape, size, and arrangement. Differential staining
can help identify a culture with more than one type of bacteria or other microorganism and its
physical characteristics, which can identify the makeup of the bacteria’s cell wall. (Bisen, 2014).
6. What are the three most common bacterial shapes? Give at least one example of bacterial
species for each shape.

a. Cocci (spherical) – Staphylococcus aureus


b. Bacilli (rod-shaped) – Escherichia coli
c. Spirilla (twisted) – spirochete Leptospira

7. What do you call bacteria that are isolated from hot springs? From glaciers? Why do these
organisms thrive where they do?

Thermophiles have cellular structures adapted for heat, including protein molecules that
are heat-resistant and enzymes that work better at high temperatures. Psychrophiles are
protected from freezing by ice-induced desiccation and vitrification (glass transition) and contain
unsaturated fatty acids on their cell membrane in order to overcome stiffening. (Gupta, 2014 et.
al)

(Gupta, G.N., et al. 2014. Extremophiles: An Overview of Microorganism from Extreme


Environment)

8. Give the purpose of each of the following reagents in a differential staining procedure:

a. Primary stain – Crystal Violet. This violet stain is used first and stains all cells purple.
b. Counter stain – Safranin. The final reagent. Used to stain pink those cells that have been
previously decolorized.
c. Decolorizing agent – Ethyl Alcohol, 95%. Serves a dual function as a protein-dehydrating agent
and as a lipid solvent. Removes the primary stain and decolorizes Gram-negative bacteria.
d. Mordant – Killing agent and increases the cells’ affinity for a stain. It binds to the primary stain
which forms an insoluble complex.

(Cappuccino, J.G and Sherman, N. 2014. Microbiology: A Laboratory Manual, Tenth Edition)
LABORATORY EXERCISE 13-C

SURVEY OF MICROORGANISMS: FUNGI

INDIVIDUAL OUTPUT

STUDY QUESTIONS: Maximum of three short sentences per number. Cite your references.

1. Define:
a. Budding – Budding is an asexual mode of reproducing new organisms. In this process, a new
organism is developed from a small part of the parent’s body. A bud which is formed detaches
to develop into a new organism or remains in the parent’s body to form colonies.
(“Reproduction from budding”, 2012).
b. Hyphae – Are long filamentous branches found in fungi and actinobacteria. Hyphae are
important structures required for growth in these species, and together, are referred to as
mycelium. (Steinberg et al., 2017).
c. Yeast – Yeasts are a group of eukaryotic fungi with a well‐defined cell wall whose growth is
either entirely unicellular or a combination of hyphal and unicellular reproduction. Yeasts
grow rapidly and have simple requirements, for which reason they have been used as model
systems in biochemistry, genetics and molecular biology. (Lachance, 2011).

2. Why do yeasts generally have to be cultured for longer periods than most bacteria?

Fungi are eukaryotic protists which are more complex and differ in many ways with
bacteria such as in the time it needs to culture fungi. Bacteria reproduce rapidly through binary
fission, while yeasts (with all its organelles and the much larger chromosome) would take more
time than the smaller bacterial cell. (Kumar, 2012).

3. What are some similarities and differences of the yeasts that were cultured from your mouth?

4. What is the difference between vegetative and aerial mycelia?

The portion of the mycelium that anchors the mold and absorbs nutrients is called the
vegetative mycelium while the portion that produces asexual reproductive spores is termed the
aerial mycelium. Vegetative mycelium does not produce spores. Aerial mycelia produces asexual
spores. (Kaiser, 2018).

5. Can bacteriological media be used for the cultivation of molds? Explain your answer.

Yes. Most fungi occur in nature and grow readily on simple sources of nitrogen and
carbohydrate, so bacteriological media that contain glucose and modified peptone with a neutral
pH may also be used to cultivate molds.

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