Republic of the Philippines

LEYTE NORMAL UNIVERSITY

Paterno, St., Tacloban City

Name: Ariane Mae D. Caberio Course: Microbiology (Laboratory)

Program, Year & Section: BSBIO AB-21 Instructor: Mr. Jefferson E. Flores

Module 3 (UPDATED)
Methods for the culturing and Microscopic Analysis of Microorganism

1. Explain the Five I’s and what each step entails.

 In culturing microorganisms there are these five I's basic techniques that are used
namely: inoculation, incubation, isolation, inspection, and identification.
Inoculation is the process where the samples are placed into a container of
nutrient medium which serves as the environment where they multiply. Next is
incubation where it is placed in a temperature controlled area known as incubator
for multiplication in a temperature ranging from freezing to boiling between 20°C
and 45° C. After, it is isolated in which a technique used for the reason that an
individual bacterial cell is separated from other cells to provide adequate space on
a nutrient surface then it will grow into discrete mound of cells. Then, the next
process is inspection, here the cultures are observed for growth characteristics
which can be useful in specimen's data analysation. After performing all the
processes, it can now be determined in to what type of microbe belongs upto its
species level, and this is the last technique known as the identification.

2. Discuss three physical states of media and when each is used.

 Physical state of media are classified into three parts namely the liquid media,
semisolid media and solid/reversible to liquid media. The water based solutions
where it does not solidify at temperature above freezing point and freely flow
when the container is tilted is known as the liquid media, this is used in showing
biochemical reaction. Next is semisolid media it have more body that liquid but
less body than solid media, it cannot flow freely, have a soft and clotlike
consisteni at room temperature, this is used in determining the motility if bacteria
and localizing a reaction at a specific site. Solid/reversible to liquid media these
are those that are reversible solid and can be liquefied with heat, poured into
various container and then resolidified. This contains 1%-5% agar that when tilted
or inverted remains solid enough in place, this is used in nutrient gelatin surface.
3. Compare and contrast selective and differential media, and give an example of each.
 Selective media contains one or more agents that inhibit the growth of a certain
microbes like it selects for the growth of the desired microorganism an example is
MacConkey agar is for Gram-negative bacteria. On the other hand differential
media allows multiple types of microorganisms to grow and it displays visible
variations in the way they grow, an example of this type is blood agar.

4. Provide brief definitions for defined media and complex media.

 Defined media also known as synthetic are those composition precisely
chemically defined while complex media are those components that are not
chemically definable since the medium came from complex category such as
extracts of animals, plants, or yeasts and others.

5. Why is aseptic technique important in all aspects of the 5 I’s?

 Aseptic technique is important in all aspects of the 5 I's for the reason that it is a
process and so it needs to be prevented from transferring pathogens that may lead
to the formation of infections. Also this technique will prevent the contamination
from a process to the next one. Since in the process of 5 I's microorganisms are
placed from a container to the other it really needs the aseptic technique and it
uses different medium to attain or identify the microorganisms well
contaminations are prohibited and are highly considered.

6. Rationalize the use of small metric units in microbiology.

 The usual subjects are so little that the need for a measurement of very little
organism is a must have

7. List and describe the three elements of good microscopy.

 Magnification – the ability to process of enlarging the apparent size, not physical
size.
 Resolution – the ability of the microscope to discern two different object and
distinguish details
 Contrast – the difference in light intensity between the image and the background,
also it is the bending of light. The more bending, the sharper the contrast

8. Differentiate between the principles of light microscopy and the principles of electron
microscopy.
 The mentioned microscope differ in light they are using to produce image of the
specimen, light microscope uses the natural light source while the electron
microscope uses beam of electrons rather than the beam of light.

9. Give examples of simple, differential, and special stains.

 Simple – crystal violet stain of Escherichia coli
 Differential – Acid-fast stain. Reddish-purple cells
  Special - Flagellar stain of Proteus vulgaris

Module Assessment

1. A mixed culture is
 One that contains two or more known species
2. Resolution is _______ with longer wavelength
 Worsened
3. A microscope has a total magnification of 1500x when using oil immersion objective has an?
 15x
4. A cell is 25um wide when viewed at a 1000x magnification this measurement can also be
written as
 0.025 mm
5. DNA fingerprinting and antibody based ELISA tests would be used during which step of
microbial analysis
 Identification
6. Motility is best done with
 Hanging drop preparation
7. Bacteria tend to stain more readily with cationic dyes because bacteria
 Contain large amounts of acidic substances on their surfaces.
8. A fastidious organism must be grown on what type of medium
 Defined or enriched medium
9. A microbiologist inoculates Staphylococcus aureus into a culture medium. Following
incubation both Staphylococcus aureus and Staphylococcus epidermidis are determined to be
growing in this culture. What is the most likely explanation?
 The culture was contaminated by Staphylococcus epidermidis
10. Magnification is achieved in a compound microscope through the initial magnification of the
specimen by the ocular lens. This image is then projected to the objective lens that will
further magnify the specimen to form a virtual image received by the eye.
 a. ocular, objective
 Thoughts Questions and Task
1. Research situation in which an infectious disease was, at first, incorrectly identified.
What issues may have contributed to the misidentification.
 Factors that affect to misidentification of patient’s disease are as follow: incorrect record
of patients identification at the registration, the time pressure of the one recording during
the treatment, lack of trainings of the staffs, duplication of records, human error,
inefficient information being pass on, and the patients behavior of giving wrong
information.

2. Research countries in the world in regards to which use the metric system and which do
not.
 Myanmar and Liberia and United States are those countries that did not adapted officially
the metric system and the rest of the countries already into metric system

3. Evaluate the current Covid-19 pandemic. Examine how the 5 I’s of microbiology are
being applied in this situation. Which of the 5 I’s may be the most difficult to complete
given the situation. Explain.
 In this trying time of Covid-19 pandemic the 5I’s of microbiology are being applied
significantly in swab testing wherein first inoculation where the sample of the patient is
placed into container, second is the incubation of the sample to adjusting growth
conditions, isolation of the of the sample is done next for a better place of growth, then it
is being inspected or observed for the characteristics that will be useful for analyzation
the it can be identified to which type of microbe it belongs and in this case if the sample
is positive to the virus. The most difficult to complete in the 5 I’s is the inspection stage
for the reason that there are misinterpretation of data that may hinder the success of the
result, there are instances that the first three steps may not be done properly and so the
observation and the analyzation might lead to the wrong identification of the specimen

4. Your patient presents with a skin lesion that you believe to be impetigo, a bacterial
infection. Please list the steps you will take to identify the pathogen(s) causing this
infection.
 Steps to take in identifying pathogens causing infections:
o First is to identify the occurrence of a disease, this is knowing the epidemiology
of the outbreak
o Disease severity and duration, to examine time between the symptoms and the
appearance of the disease
o Extent of hose involvement, the interaction taking place between the pathogen
and their host
o Identification of Microbes Based on Molecular Genetics, modern nucleic acid-
based microbial detection methods make it possible to identify microbes that are
associated with a disease.
 5. Lactophenol cotton blue is utilized to stain the colorless cytoplasm of Amoeba proteus, a
common pond protozoan. Please discuss which property of microscopy is enhanced by
using this dye.
 Lactophenol cotton blue is a stain that is used to examine fungal elements, this contains
phenol that kills organisms, lactic acid which preserves fungal structures. The property of
the microscope that can be enhanced is the contrast for it helps the recognition and final
identification of fungi.

6. Which type(s) of medium would be used in each scenario?

a. Isolating the growth of Streptococcus pyogenes from a patient’s throat swab
 Basal Media
b. Isolating a pathogen from a patient’s clean-catch urine sample
 Selective Media
c. Isolating enteric bacteria such as Escherichia coliform a sample of organically grown
spinach

d. Maintaining a patient’s nasal swab specimen for further analysis and identification of
possible respiratory syncytial virus (RSV) infection


Critical Thinking
1. Can the 5 I’s of microbiology be completed for every disease?
 The 5 I’s of microbiology is not necessary to be applied and completed in every
disease where in fact some test don’t use this in performing tests.

2. When may potential mistakes be made in regards to the incorporation of the 5 I’s? How
may these mistakes affect our understanding of a disease? What can be done to reduce
these errors?
 The mistakes in the conduct using the 5 I’s will greatly affect the understanding of
a disease because it is the step four which is the inspection for the analyzation of
the disease and observing its characteristics that will lead to the last step which is
the identification of the disease. In order to reduce errors make sure to follow
properly the process and always be mindful of what you are doing, and make sure
you know what you are doing. Contamination may also affect the outcome so
make sure the cleanliness and appropriateness all the time.
3. Not all microorganisms can be grown on media. What effect does this fact have treatment
and prevention of these infectious diseases?
  There is perhaps a problem for the reason that there is hindering you in
identification to what type of microbe it belong, it will greatly affect all
individuals for they do not know their enemy and so they can’t give appropriate
prevention and treatment.

4. The Gram stain was developed in 1884. Why is this procedure still used today? What
(if any) changes have been made to the procedure in modern times?
 Gram staining helps even more up to the current for it helps a lot to identify
causing microorganisms specifically bacteria by staining and will also help the
doctor to give the proper medication.

5. How would be the identification and treatment of infectious agents change if all bacteria truly
did look and act the “same”
 The differential media is appropriate in this situation for the reason that it helps
the identification of closely related organisms or group of organisms, with a
certain dye it will produce characteristics changes or growth patterns that will
help for identification.

6. Viable but nonculturable (VBNC) bacteria have been discovered in human tissue. What are these
bacteria and what role may they play in disease and health?
 The VBNC is a unique state survival strategy of many bacteria in the environment
as a response to adverse environmental changes or conditions. The bacteria under
the VBNC are those that belongs to the very low metabolic activity and do not
divide such as E.coli present in gram negative bacteria under the subclass of
proteobacteria. This may affect the lower reproduction of women which can
eventually lead to imbalance of biota. This proteobacteria are common factor of
human disease and conditions such as inflammatory bowel disease Crohn's
disease (CD) as well as ulcerative colitis (UC) which are chronic infections that
are often associated with persistent inflammation of the intestine.