Identification and Analysis of Microbial Communities in Homemade Ice Cream

Identification and Analysis of Microbial Communities in Homemade Ice

Cream
Anna Hazel P. De Castro, John Daniel J. Esguerra, Chiara Gabrielle A. Flores, Kay U.
Gloria, Jeremae G. Jacobe, And Julianne Clarisse A. Jimenez​*
Senior High School, Aminobuddies, University of Santo Tomas, Manila, Philippines
*​
Corresponding author e-mail: julianneclarisse.jimenez.shs@ust.edu.ph

ABSTRACT
A laboratory activity with the aim of determining the presence of microbial communities in
homemade ice cream was conducted. Three microscope stained slides were prepared, 1 gram-stained ice
cream, 1 simple stained ice cream, and a control Yakult gram stained slide. Crystal violet and Safranin
were used for the gram staining of slides, and methylene blue was used for simple staining. The sample
was prepared by diluting 1 mL of ice cream to 9 mL of distilled water, obtaining a 1:10 dilution. Simple
staining results showed relatively few items of note. Gram-staining of ice cream and control obtained
significantly better results, and allowed for the determination of the nature of the bacteria found.

Keywords​: ice cream, microbes, gram staining

INTRODUCTION
Most cells and microorganisms that we observe under the microscope lack color and contrast in
their natural state, which makes it difficult to detect and analyze important cellular structures and feature.
Thus, various methods have been developed in order to increase the contrast of the cellular structures,
making it more possible identify the different parts of the specimen.​[1]
One of these methods is simple staining wherein dyes that are positively charged are used which
interact with the slightly negatively charged bacterial cell wall, therefore adhering to the cell surface.​[2]
Another one of these methods is gram staining, a differential staining procedure. It was developed by
Hans Christian Gram, a Danish microbiologist, as an effective method in distinguishing between bacteria
with various types of cell walls. It uses different stains such as crystal violet, Gram’s iodine, a
decolorizing agent, and safranin, in order to distinguish between gram positive and gram negative
bacteria.
These methods are used in microbiological analysis of consumable products, specifically food and
beverages. This includes the use of biological, biochemical, or chemical methods in order to detect,
identify, and analyze microorganisms contained in the food or beverage.​[3] This is a vital part of any food
manufacturing control strategy in order to restrict harmful microorganisms and ensure safety from
foodborne diseases. Microbiological testing can outline important information whether a sampling or
testing procedure is correctly designed and accomplished following regulatory guidelines and standards.
Nonetheless, one must still understand that this kind of testing cannot determine 100% safety from certain
pathogens since tests are only done using a small portion of the consumable products. One can mostly
achieve that no pathogens are found from the sample and/or determine the levels of sensitivity and
assurance provided by the testing procedures used.​[4]

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Identification and Analysis of Microbial Communities in Homemade Ice Cream

Microbiological testing was used in the analysis of the homemade ice cream in order to determine
its safety from foodborne illnesses, and its overall quality.
The laboratory activity aims to develop the laboratory skills involved in simple and gram staining.
Determination of the presence microbes from the samples and successfully differentiate them in terms of
size and shape, and determine whether the microbes in the ice cream samples are gram positive or gram
negative is also another objective.
The article reports that the homemade strawberry ice cream did not contain any harmful bacteria,
most likely because of its cold temperature which inhibited the growth of bacteria. With regards to the
comparison between simple and gram staining, the latter provides a clearer and more significant result,
therefore being more suitable in microbiological analysis.

MATERIALS AND METHODS

Simple Staining
Using a test tube and a pipette, 1 mL of ice cream was diluted in 9 mL of distilled water. An
inoculating loop was flame sterilized with the use of an alcohol lamp and was cooled for at least 30
seconds in order to avoid microbial death due to the loop’s high temperature. The loop was then used to
get a portion of the sample in the test tube. Additionally, the loop was not to be in contact with the inner
sides of the test tube when the samples were acquired. The sample on the loop was then spread on a dry
glass slide, creating a thin and even smear and it was air-dried and heat-fixed by running the slide quickly
through the flame until it dried to enhance the adherence of the bacterial cells to the slide. Once the
sample was dry, methylene blue was placed on the sample using a pasteur pipette and was left to stain for
three minutes. The slide was rinsed with water and was gently blotted dry using a tissue paper. It was
covered with a cover slip before it was observed under the microscope.

Gram Staining
Using a sterilized inoculating loop, a thin and even smear was made on the glass slide. It was air
dried and heat-fixed. Once it was completely dry, crystal violet solution was added on top of the sample
and left it on for one minute before it was rinsed off with water. The iodine solution was added next using
a pasteur pipette. It was left again for one minute before being washed off with water. The smear was
again washed off but using ethanol this time until the crystal violet color disappeared. Lastly, Safranin
was added and after one minute, it was washed off using water. The slide was gently blotted using tissue
paper before observing it under the microscope.

Stains on the Samples

​Two kinds of food were tested; the homemade strawberry ice cream and the yakult drink. The
homemade strawberry ice cream underwent both simple staining and gram staining. The Yakult drink on
the other hand only underwent gram staining since it served as the controlled variable in this testing.
Furthermore, Yakult drink was used as a controlled variable due to the live bacteria (​Lactobacillus casei​)
present in the said drink which also enabled us to compare its bacteria with that of the ice cream.

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Identification and Analysis of Microbial Communities in Homemade Ice Cream

RESULTS AND DISCUSSION

Staining was used for examining the bacteria in the samples, as it provided contrast. In this
experiment, a sample of ice cream and Yakult was tested for microbial organisms. Two types of staining
were used: simple and gram staining. After the ice cream samples were stained, they were observed under
the microscope. The samples were compared to a Yakult gram stain sample, our control for this
experiment.
The strawberry ice cream sample which underwent simple staining has shown a strand substance
after reacting to the positively charged stain (Fig. 1). The observations and results were expected due to
the knowledge that in a simple staining, the sample subjected to be studied will react to the stain, retaining
its color. In addition to that, utilizing simple staining harvests the cell’s size, shape and arrangement,
making the results aligned with what is supposed to be seen.

Figure 1. Ice cream simple stain under HPO

The ice cream simple stain in Figure 1 only had few findings. The major finding was a strand that
picked up the methylene blue dye.On the other hand, in gram staining, two results were expected and two
samples were subjected to the experiment. The two samples were the strawberry ice cream and Yakult,
both of which underwent gram staining. We reiterate that in gram staining, two results are expected, gram
positive which will gain a bluish-purple stain and gram negative which will gain sa red stain.
The former sample (Fig. 2), if seen under a microscope gained a pinkish-red color after the staining
which shows us that it was a gram-negative. Attaining the result of Fig.2 as positive, this leads us to state
that the substance has a thick cell membrane. As it was mentioned, a substance that has thick cell
membrane will trap the blue stain but the tap water will not be able to penetrate the cell wall of the
substance enough to remove the stain and when flooded with safranin, the colors will bind giving us the
bluish-purple stain.

Figure 2.​ I​ ce cream gram stain under HPO

In Figure 2, the ice cream gram stain showed significantly less bacteria than the Yakult sample.

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Identification and Analysis of Microbial Communities in Homemade Ice Cream

As for the latter sample, the Yakult (Fig. 3) reaped a result of dark pinkish-purple stain letting us arrive to
the statement that it is a gram positive, therefore the cell membrane of the sample is thin and is thinner
than the prior substance. When the Yakult sample were put under the gram staining, the blue stain was
captured by the sample but was easily washed off by the tap water because the cell membrane of the
sample was thin enough to be penetrated. It gained a red stain because the safranin was the last stain that
flooded the slide and there was no proceeding actions that were applied for the safranin to be removed
leaving its red stain on the sample.

Figure 3. Yakult Sample under HPO

The Yakult sample, in Figure 3, showed a large amount of bacteria under the microscope. This
was expected since there is live lactobacillus casei in Yakult. The small dots are indicators of bacteria.

CONCLUSIONS
The activity utilized different methods of staining, specifically simple staining and gram staining.
The results showed the presence of bacteria, and allowed for the determination if these were
gram-positive or gram-negative based on the color presented under the microscope. It was concluded
from this experiment that the bacterium contained in the control sample, ​Lactobacillus casei,​ was gram
positive as it exhibited a purple color, and were rod-like in shape. It was also shown from the ice cream
sample results that gram-negative bacteria was present due to the pink-red color they exhibited, although
in significantly smaller quantities compared to the control. These bacteria were slightly circular in shape.
It can be concluded that the cold temperatures in which ice cream is stored, were inhibitive to the
reproduction of bacteria. The results of simple staining was less significant compared to gram-staining,
wherein relatively few objects or organisms were of note.

REFERENCES

[1] ​O. (n.d.). Microbiology. Retrieved October 4, 2018, from

https://courses.lumenlearning.com/microbiology/chapter/staining-microscopic-specimens/
[2] ​Simple Staining: Principle, Procedure and Results. (2016).. Retrieved October 4, 2018, from
https://microbeonline.com/simple-staining-principle-procedure-results/
[3] Microbiological analysis and testing. (n.d.). Retrieved October 6, 2018, from
https://www.campdenbri.co.uk/services/microbiological-testing.php

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Identification and Analysis of Microbial Communities in Homemade Ice Cream

[4] Alam, I. I. (2016). Three Common Microbiological Testing-Methods for Food Products. Retrieved
October 6, 2018, from
https://delishably.com/food-industry/Three-Common-Methods-for-Microbiological-Testing-of-Food-Prod
ucts