WORKSHEET # 1

MICROSCOPIC VISUALIZATION OF BACTERIA: DIFFERENTIAL AND STRUCTURAL STAINING

Name Dimayacyac, Ziara. Escala, Danica. Erni, Kyra Score _________________

Course / Year & section MEB31 Professor Ma’am Olgga Hara
Group No. 4 Date Submitted ___________

1. Give the sequence of staining with alternate washing in the Gram staining technique.

a. Smear young bacterial culture on to a clean glass slide then heat-fix them in order for the
cells to adhere to the slide.
b. Pour generous amount of crystal violet on to the smears and let it stand for 1 min.
c. Decant the excess crystal violet then gently rinse the slide with distilled water for 5 secs.
d. Next, flood the stained bacteria with Gram’s Iodine solution which is a mordant that would
enhance the stain and create a complex with the crystal violet. Let it stand for 1 min.
e. Remove excess iodine solution then gently rinse the slide with distilled water for 5 secs.
f. Few drops of a decolorizing reagent which 95% Ethanol will be added on to the stained
bacteria.
g. After 15 secs, instantly wash off the ethanol with distilled water for 5 secs.
h. Lastly, flood the bacterial smear with Safranin for 30-45 secs before washing it off again
with distilled water.
i. Prior to observation of the prepared slide, blot dry the excess water from the bacterial
smear.

2. Draw your observations from the bacterial smears that were subjected to Gram staining technique.
Use color pencils to indicate the result. D

E. coli B. subtilis Mixed bacteria

Stain Reaction: Stain Reaction: Stain Reaction:
3. What part of the bacterial cell distinguishes one group from the other with Gram staining and why?

The part of the bacterial cell that distinguishes one group from the other with Gram staining is
the cell wall. It is differentiated into two major groups which are the gram- negative and gram-
positive bacteria through their cell wall composition. By coloring these cells red or violet, the Gram
stain procedure distinguishes between Gram positive and Gram-negative groups. Gram-positive
bacteria stain violet due to the presence of a thick layer of peptidoglycan in their cell walls, which
keeps the crystal violet staining these cells. Gram negative bacteria, on the other hand, stain red,
which is due to a thinner peptidoglycan wall that does not retain the crystal violet during the
decoloring process.

4. Draw your observations from the bacterial smears that were subjected to Acid-fast staining
technique. Use color pencils to indicate the result.

S. aureus M. smegmatis
Stain Reaction: Organism stained bluish (Acid-fast Stain Reaction: Organism stained red (Acid-fast
negative) positive)

5. What is the purpose of using Ziehl’s carbol fuchsin stain with gentle heating in the acid-fast staining
technique? What cell component distinguishes acid-fast from non-acid fast reactions of the bacteria?
 D

6. Give two diseases with their causative organisms upon which the acid-fast staining technique is of
paramount importance as a diagnostic tool.

a. Tuberculosis – This disease is caused by the Mycobacterium tuberculosis bacteria in which

its presence can be detected through acid-fast staining. The sample to be analyzed often
comes from the patient’s sputum. Whenever acid-fast bacteria are confirmed, they are
generally cultured to make a positive identification.
b. Leprosy (Hansen’s Disease) – Its causative organism is the Mycobacterium leprae and the
diagnosis is confirmed by means of a skin or nerve biopsy which will be used for acid-fast
staining. Upon having the results of the tissue biopsy, various affected sites could present
typical histopathologic changes that show large numbers of foam cells. They are
considered as macrophages that have ingested Mycobacterium leprae but are unable to
digest them. In return, they multiply and utilize the macrophage as a transportation unit
in the body and cause multiple lesions among patients with leprosy.

7. Draw your observations from the bacterial smears that were subjected to endospore staining
technique. Use color pencils to indicate the result and label the structure found in this technique.

24-hr old B. subtilis 7-day old B. subtilis

8. Why is heat necessary to stain endospores?

 D

9. In the endospore staining procedure, which is the primary stain? the counterstain? In what way does
detecting endospores significant?

In endospore staining procedure, the primary stain is malachite green solution and the
counterstain is safranin. Detecting endospores are significant because endospores are bacterial
structure produces by certain species as their survival structure when growth conditions are
unfavorable. They can render the cells indefinitely but they can revert to a vegetative state when
growth conditions improve.

10. Draw your observations from the bacterial smears that were subjected to capsular staining
technique. Use color pencils to indicate the result and label the structure found in this technique.

background capsule background bacterial cell

K. pneumoniae P. vulgaris

11. How is the presence of capsules in bacterial species related to its virulence? D
Conclusion:

Preparation of bacterial smears differ depending on where it was obtained. For organisms
coming from a solid media culture that contains agar, a colony must be first mixed with water on the
glass slide while those from a broth medium can be smeared immediately upon placing it on the slide.
A liquid medium is utilized for propagation of a large number of organisms, fermentation studies, etc.
Since microorganisms cannot be seen by the naked eye, various staining techniques were established
and have their own specific purpose. With that, the steps must be performed precisely in accordance
to the said standard procedure in order to attain the expected results. The most common among
them is the gram staining technique that is differential and involves multiple steps. This was said to be
an effective method to distinguish bacteria with different types of cell walls. However, the age of the
bacterial culture is a huge factor that must be considered prior to performing this technique. Second,
acid-fast staining, which is somehow similar to gram staining, but it is necessary as a diagnostic tool. It
would enable us to differentiate two types of gram-positive cells: those that have waxy mycolic acids
in their cell walls, and those that do not. Moreover, in capsular staining, a negative staining technique
is used because capsules do not absorb most basic dyes. In this manner, the dye to be utilized will
stain the background but does not penetrate the capsules. Thus, it will appear like halos around the
borders of the cell. Lastly, endospore staining needs two stains for the procedure then it reveals the
shape and location of endospores, if they are present which appears to be green in color.

References:

Bruckner, M. Z. (2021, January 14). Gram staining. Retrieved March 20, 2021, from
https://serc.carleton.edu/microbelife/research_methods/microscopy/gramstain.html

Lumen Microbiology. (n.d). Staining Microscopic Specimens. Retrieved March 20, 2021 from
https://courses.lumenlearning.com/microbiology/chapter/staining-microscopic-specimens/

OpenStax. (n.d). Staining Microscopic Specimens. Retrieved March 20, 2021 from
https://opentextbc.ca/microbiologyopenstax/chapter/staining-microscopic-specimens/
Samanthi. (2017). Difference Between Solid and Liquid Media. Retrieved March 20, 2021 from
https://www.differencebetween.com/difference-between-solid-and-vs-liquid-media/