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Group No. 4 Section: BS BIO - 4203

Members:
Ramos, Arielle Sophia Victoria S.
Reyes, Ernel Q.
Robino, Champagne B.
Virtucio, Nickole Andriea
Sangalang, Jonah Mae N.
Yu, Dolleen Quisha B.

INTRODUCTION:

According to the American Society for Microbiology (2019), gram staining is one of the
most common laboratory tests utilized to determine the presence of bacteria in an inoculated
sample quickly. The results also determine the type of bacteria present. Generally, there are two
types of results in a gram stain: a positive and a negative stain.

A gram-positive stain pertains to a sample that is dyed purple, signifying that the sample
has a thick peptidoglycan wall, retaining the crystal violet from the staining procedure. A
gram-negative stain, however, appears to be dyed pink due to the thinness of the bacteria's
peptidoglycan wall, retaining only the safranin coloring from the staining procedure.

In this activity, an inoculated sample of unnamed bacteria was subjected to gram staining
to determine what type of bacteria it was.

OBJECTIVES:

This activity was conducted to achieve the following objectives:

● To understand the different steps involved in the gram staining procedure.
● To execute the steps in gram staining.
● To identify the sample whether it is a gram-positive or gram-negative bacteria.

MATERIALS:

The following are the materials required for the Gram Staining Laboratory activity.
Included here are necessary laboratory user needs, experiment equipment, and items for cleaning
the Laboratory area.

Laboratory Coat Gloves Mask Alcohol Detergent and

Sponge
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Glass slide Dropper Wash bottle Distilled water Matchstick or

Lighter

Alcohol Lamp Inoculating loop Culture media Forceps Crystal violet

Iodine reagent Ethanol Safranin Tissue Video

Microscope

METHODS:

The following is the procedure of Gram Staining Laboratory activity, divided into 3 stages: Pre -
Gram Staining, Gram Staining Proper, and Post - Gram Staining. Additionally, reminders of some steps
are listed here.

GRAM STAINING PROCEDURE REMINDERS

1. Prepare all necessary materials and
equipment.

2. Place a drop of distilled water in the

middle of the slide.

3. Light the alcohol lamp and sterilize the Remember: The blue flame is hotter than
inoculating loop until it's glowing red hot. the red, so it's better to use the blue part
flame for an inoculating loop

4. Prepare the culture media, balance it

out first with your three fingers then open
it near the flame using your index and
PRE-GRAM thumb fingers to avoid contamination.
STAINING
5. Let the inoculating loop cool for about If you doubt if it's hot or cold you can
10-15 seconds before transferring any test it, by tapping at the side of the culture
microbes. media. Regardless if it sizzles or not don't
put it back on the flame since the sizzles
already cool down the inoculation loop.

6. Obtain microbes from the culture Not too thick, since this could affect the
media final result.

7. After transferring the microbes to the

slide, heat the edges of the culture media
plate to prevent contamination.
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8. Spread the microbes onto the slide with Spread it widely, since we need to air dry
a drop of distilled water. it which indicates the larger the
widespread the faster it air dried.

9. By using forceps, allow the slide to air Don't let the slide stay in the flame just to
dry or gently heat it to speed up drying. be air-dried fast, just let it slide from time
Then, sterilize the loop to remove any to time.
remaining microbes.

10. Primary stain: apply crystal violet to Be generous in flooding stains onto the
cover the smear on the slide for 1 minute. slide.

11. After 1 minute, carefully rinse the This part of the procedure is called gentle
slide using a wash bottle filled with washing. Avoid direct washing of the area
distilled water. where the smear is collected.

GRAM- 13. Secondary stain: Add iodine reagent This part of the staining supports the
STAINING to the smear on the slide and let it sit for primary stain.
PROPER 1 minute. Then, rinse the slide in a wash
tank filled with distilled water, avoiding
direct washing of the smear area.

14. Decolorization: Use ethanol as the

decolorizing agent after the secondary
stain. Flood it for about 15 seconds, then
gently rinse the slide with a wash bottle
containing distilled water.

15. Counter Stain: Apply safranin to the

smear on the slide and let it sit for 1
minute. Then, rinse the slide with a wash
bottle filled with distilled water.

16. Gently pat the slide dry with tissue

POST paper, leaving the top part untouched, and
GRAM let it air dry.
STAINING
17. Once dry, observe the slide under the
microscope for the determination of its
Gram-positive, Gram-negative, or mixed
bacteria, noting their shapes at the same
time.

18. Don't forget to wash the laboratory

equipment and clean the respective area.

RESULTS AND DISCUSSION:

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Microorganisms are distinguished by their color and shape, with Gram-positive

organisms typically appearing purple or blue, while Gram-negative organisms tend to be pink or
red. Various complications and factors influenced the outcomes of the group's laboratory activity.

According to Tripathi and Sapra (2023), the interpretation of slides becomes challenging
when the microscopic smear is thick and clumped. This could be one of several reasons why the
group failed to obtain positive results. Additionally, careful monitoring of decolorization time is
essential to prevent under- or over-decolorization, especially with thicker smears requiring
longer decolorization times. Given that the group smeared the bacterial culture on the agar too
thick, it is reasonable that it should have undergone a longer decolorizing time.

Another potential factor is the age of the bacterial culture in the provided Petri dish.
Older cultures tend to lose their peptidoglycan cell walls, making Gram-positive cells appear
Gram-negative or Gram-variable. Therefore, the age of the culture must be considered, as it
could be either too young or too old.

The dilution of crystal violet, the primary stain, during the laboratory activity could also
have influenced the group's results. Inadequate exposure to crystal violet or any staining agent
could lead to altered results.

Additional factors to consider include: low concentration of crystal violet, excessive

washing between steps, prolonged decolorization, and excessive counterstaining.

ILLUSTRATION:

Figure 1. Gram Staining Slide of Group 4.

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Figure 2. Visual Results of Gram Staining Slide of Group 4.

Figure 3. Visual Results of Gram Staining Slide of Group 4.

CONCLUSION:

In conclusion, the group's gram-staining activity produced inconsistent results, most

likely due to a variety of problems and variables. Thick and clumpy microscopic smears,
impacted by variables such as incorrect decolorization and aging bacterial cultures, may have
made it impossible to discriminate between Gram-positive and Gram-negative organisms.
Furthermore, difficulties such as insufficient staining agent exposure, incorrect crystal violet
dilution, excessive washing, delayed decolorization, and extensive counterstaining may have
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hindered slide interpretation. Moving forward, paying close attention to these parameters, as well
as carefully monitoring laboratory operations, is critical for improving the accuracy and
dependability of Gram staining results.

REFERENCES:

American Society for Microbiology. Gram Stain Protocols. (2019, August 12).
https://asm.org/Protocols/Gram-Stain-Protocols

Gram staining - StatPearls - NCBI bookshelf. (2023, August 14). National Center for
Biotechnology Information. https://www.ncbi.nlm.nih.gov/books/NBK562156/